Patent Publication Number: US-2022234006-A1

Title: A biocompatible membrane composite

Description:
FIELD 
     The present disclosure relates generally to the field of implantable medical devices and, in particular, to a biocompatible membrane composite and uses thereof. 
     BACKGROUND 
     Biological therapies are increasingly viable methods for treating peripheral artery disease, aneurysm, heart disease, Alzheimer&#39;s and Parkinson&#39;s diseases, autism, blindness, diabetes, and other pathologies. 
     With respect to biological therapies in general, cells, viruses, viral vectors, bacteria, proteins, antibodies, and other bioactive entities may be introduced into a patient by surgical or interventional methods that place the bioactive moiety into a tissue bed of a patient. Often the bioactive entities are first placed in a device that is then inserted into the patient. Alternatively, the device may be inserted into the patient first with the bioactive entity added later. The device is formed of one or more biocompatible membranes or other biocompatible materials that permit the passage of nutrients through but prevent the passage of the cells encapsulated therethrough. 
     To maintain a viable and productive population of bioactive entities (e.g., cells), the bioactive entities must maintain access to nutrients, such as oxygen, which are delivered through the blood vessels of the host. To maximize the viability and productivity of the implanted, encapsulated cells, it is necessary to maximize access to the source of oxygen and nutrients by ensuring that the formation of blood vessels be as close as possible to the cells such that the diffusion distance and time needed for transport of the oxygen and nutrients to the implanted, encapsulated cells is minimized. 
     The implantation of external devices, such as, for example, cell encapsulation devices, into a body triggers an immune response in which foreign body giant cells form and at least partially encapsulate the implanted device. The presence of foreign body giant cells at or near the cell impermeable interface makes it difficult, if not impossible for blood vessels to form in close proximity to the encapsulated cells, thereby restricting access to the oxygen and nutrients needed to maintain the viability and health of the encapsulated cells. 
     Thus, there remains a need in the art for a material that provides the encapsulated cells sufficient immune isolation from the host&#39;s immune cells while providing an environment that is able to mitigate or tailor the foreign body response such that sufficient vascularization occurs at or near the surface of a cell encapsulation device, thereby permitting the encapsulated cells to survive and secrete a therapeutically useful substance. 
     SUMMARY 
     According to one Aspect (“Aspect 1”), a biocompatible membrane composite includes (1) a first layer has an MPS (maximum pore size) less than about 1 micron, (2) a second layer has first solid features with a majority of first solid feature spacing less than about 50 microns, where a majority of the first solid features has a representative minor axis from about 3 microns to about 20 microns, and (3) a third layer that has a pore size greater than about 5 microns in effective diameter and second solid features having a majority of a second solid feature spacing greater than about 50 microns. The second layer is positioned between the first layer and the third layer. 
     According to another Aspect (“Aspect 2”) further to Aspect 1, the first layer has a mass per area (MpA) less than about 5 g/m 2 . 
     According to another Aspect (“Aspect 3”) further to Aspect 1 or Aspect 2, the first layer has a first thickness less than about 10 microns. 
     According to another Aspect (“Aspect 4”) further to any one of Aspects 1 to 3, the second layer has a second thickness less than about 60 microns. 
     According to another Aspect (“Aspect 5”) further to any one of Aspects 1 to 4, the biocompatible membrane composite has a maximum tensile load in the weakest axis greater than 40 N/m. 
     According to another Aspect (“Aspect 6”) further to any one of Aspects 1 to 5, the first layer has a first porosity greater than about 50%. 
     According to another Aspect (“Aspect 7”) further to any one of Aspects 1 to 6, the second layer has a second porosity greater than about 60%. 
     According to another Aspect (“Aspect 8”) further to any one of Aspects 1 to 7, the biocompatible membrane composite has a measured composite z-strength greater than 100 KPa. 
     According to another Aspect (“Aspect 9”) further to any one of Aspects 1 to 8, the solid features of the second layer each includes solid features each with a representative minor axis, a representative major axis and a solid feature depth where a majority of at least two of the representative minor axis, the representative major axis, and the solid feature depth of the second layer is greater than about 5 microns. 
     According to another Aspect (“Aspect 10”) further to any one of Aspects 1 to 9, at least a portion of the first solid features in contact with the first layer are bonded solid features. 
     According to another Aspect (“Aspect 11”) further to any one of Aspects 1 to 10, the second layer has a pore size from about 1 micron to about 9 microns in effective diameter. 
     According to another Aspect (“Aspect 12”) further to any one of Aspects 1 to 11, the solid features are connected by fibrils and the fibrils are deformable. 
     According to another Aspect (“Aspect 13”) further to any one of Aspects 1 to 12, the third layer has a third thickness from about 30 microns to about 200 microns. 
     According to another Aspect (“Aspect 14”) further to any one of Aspects 1 to 13, a majority of the second solid feature spacing of the third layer is greater than about 50 microns. 
     According to another Aspect (“Aspect 15”) further to any one of Aspects 1 to 14, a majority of the second solid features in the third layer has a representative minor axis that is less than about 40 microns. 
     According to another Aspect (“Aspect 16”) further to any one of Aspects 1 to 15, at least two of the first layer, the second layer, and the third layer are intimately bonded. 
     According to another Aspect (“Aspect 17) further to any one of Aspects 1 to 16, the first layer and the second layer are intimately bonded. 
     According to another Aspect (“Aspect 18”) further to any one of Aspects 1 to 17, the third thickness of the third layer is greater than a sum of the first thickness of the first layer and the second thickness of the second layer. 
     According to another Aspect (“Aspect 19”) further to any one of Aspects 1 to 18, the third thickness of the third layer is at least two times a combined thickness of the first layer and the second layer. 
     According to another Aspect (“Aspect 20”) further to any one of Aspects 1 to 19, at least one of the first layer, the second layer, and the third layer includes a polymer, a fluoropolymer membrane, a non-fluoropolymer membrane, a woven textile, a non-woven textile, woven or non-woven collections of fibers or yarns, fibrous matrices, and combinations thereof. 
     According to another Aspect (“Aspect 21”) further to any one of Aspects 1 to 20, at least one of the first layer, the second layer, and the third layer is a polymer. 
     According to another Aspect (“Aspect 22”) further to Aspect 21, the polymer is a fluoropolymer membrane selected from an expanded polytetrafluoroethylene (ePTFE) membrane, a fluorinated ethylene propylene (FEP) membrane, and a modified ePTFE membrane. 
     According to another Aspect (“Aspect 23”) further to any one of Aspects 1 to 22, at least one of the first layer, the second layer, and the third layer is an expanded polytetrafluoroethylene membrane. 
     According to another Aspect (“Aspect 24”) further to any one of Aspects 1 to 23, the third layer is a spunbound non-woven polyester material. 
     According to another Aspect (“Aspect 25”) further to any one of Aspects 1 to 24, the second solid features of the third layer include fibers of a non-woven or a woven textile. 
     According to another Aspect (“Aspect 26”) further to any one of Aspects 1 to 25, the second solid features of the third layer includes a woven or a non-woven textile, and a second representative minor axis is a diameter of a fiber in the woven or non-woven textile. 
     According to another Aspect (“Aspect 27”) further to any one of Aspects 1 to 26, including a reinforcing component thereon. 
     According to another Aspect (“Aspect 28”) further to Aspect 27, the reinforcing component has a stiffness from about 0.01 N/cm to about 5 N/cm. 
     According to another Aspect, (“Aspect 29”) further to Aspect 27 or Aspect 28, the reinforcing component is a woven or non-woven textile. 
     According to another Aspect, (“Aspect 30”) further to any one of Aspects 1 to 29, including a first reinforcing component and a second reinforcing component. 
     According to another Aspect (“Aspect 31”) further to any one of Aspects 1 to 30 the first solid features of the second layer include a member selected from thermoplastic polymers, polyurethanes, silicones, rubbers, epoxies, and combinations thereof. 
     According to another Aspect (“Aspect 32”) further to any one of Aspects 1 to 31, including a surface coating thereon, the surface coating including one or more members selected from antimicrobial agents, antibodies, pharmaceuticals, and biologically active molecules. 
     According to another Aspect (“Aspect 33”) further to any one of Aspects 1 to 32, including a hydrophilic coating thereon. 
     According to another Aspect (“Aspect 34”) further to according to any one of Aspects 1 to 33, the biocompatible membrane composite is in the form of a cell encapsulation device. 
     According to one Aspect (“Aspect 35”) a biocompatible membrane composite includes (1) a first layer, (2) a second layer has a pore size from 1 micron to 9 microns in effective diameter, a first thickness less than about 60 microns, and first solid features with a majority of a first solid feature spacing less than about 50 microns, where a majority of the first solid features has a first representative minor axis from about 3 microns to about 20 microns, and (3) a third layer. The second layer is positioned between the first layer and the third layer. 
     According to another Aspect (“Aspect 36”) further to Aspect 35, the first layer has an MPS (maximum pore size) less than about 1 micron in diameter. 
     According to another Aspect (“Aspect 37”) further Aspect 35 or Aspect 36, the first layer has a mass per area (MpA) less than about 5 g/m 2 . 
     According to another Aspect (“Aspect 38”) further to any one of Aspects 35 to 37, the first layer has a second thickness less than about 10 microns. 
     According to another Aspect (“Aspect 39”) further to any one of Aspects 35 to 38, the biocompatible membrane composite has a maximum tensile load in the weakest axis greater than about 40 N/m. 
     According to another Aspect (“Aspect 40”) further to any one of Aspects 35 to 39, the first layer has a first porosity greater than about 50%. 
     According to another Aspect (“Aspect 41”) further to any one of Aspects 35 to 40, the second layer has a second porosity greater than about 60%. 
     According to another Aspect (“Aspect 42”) further to any one of Aspects 35 to 41, the biocompatible membrane composite has a measured composite z-strength greater than 100 KPa. 
     According to another Aspect (“Aspect 43”) further to any one of Aspects 35 to 42, the solid features of the second layer each includes a representative minor axis, a representative major axis, and a solid feature depth, where a majority of at least two of the representative minor axis, the representative major axis, and the solid feature depth of the second layer is greater than about 5 microns. 
     According to another Aspect (“Aspect 44”) further to any one of Aspects 35 to 42, the third layer has a pore size greater than about 9 microns in effective diameter. 
     According to another Aspect (“Aspect 45”) further to any one of Aspects 35 to 44, at least a portion of the first solid features in contact with the first layer are bonded solid features. 
     According to another Aspect (“Aspect 46”) further to any one of Aspects 35 to 45, the first solid features of the second layer are connected by fibrils and the fibrils are deformable. 
     According to another Aspect (“Aspect 47”) further to any one of Aspects 35 to 46, the third layer has a third thickness from about 30 microns to about 200 microns. 
     According to another Aspect (“Aspect 48”) further to any one of Aspects 35 to 47, the third layer includes second solid features with a majority of a second solid feature spacing greater than about 50 microns. 
     According to another Aspect (“Aspect 49”) further to any one of Aspects 35 to 48, a majority of the second solid features in the third layer has a representative minor axis that is less than about 40 microns. 
     According to another Aspect (“Aspect 50”) further to Aspect 48 or Aspect 49, the second solid features of the third layer include fibers of a non-woven or woven textile. 
     According to another Aspect (“Aspect 51”) further to any one of Aspects 48 to 50, the second solid features of the third layer include a woven or a non-woven textile, and where a second representative minor axis is a diameter of a fiber in a woven or non-woven textile. 
     According to another Aspect (“Aspect 52”) further to any one of Aspects 35 to 51, at least two of the first layer, the second layer, and the third layer are intimately bonded. 
     According to another Aspect (“Aspect 53”) further to any one of Aspects 35 to 52, the first layer and the second layer are intimately bonded. 
     According to another Aspect (“Aspect 54”) further to any one of Aspects 35 to 53, the third thickness of the third layer is greater than a sum of the second thickness of the first layer and the first thickness of the second layer. 
     According to another Aspect (“Aspect 55”) further to any one of Aspects 35 to 54, the third thickness of the third layer is at least two times a combined thickness of the second thickness of the first layer and the first thickness of the second layer. 
     According to another Aspect (“Aspect 56”) further to any one of Aspects 35 to 55, at least one of the first layer, the second layer, and the third layer includes a polymer, a fluoropolymer membrane, a non-fluoropolymer membrane, a woven textile, a non-woven textile, woven or non-woven collections of fibers or yarns, fibrous matrices, and combinations thereof. 
     According to another Aspect (“Aspect 57”) further to any one of Aspects 35 to 56, at least one of the first layer, the second layer, and the third layer is a polymer. 
     According to another Aspect (“Aspect 58”) further to Aspect 57, the polymer is a fluoropolymer membrane selected from an expanded polytetrafluoroethylene (ePTFE) membrane, a fluorinated ethylene propylene (FEP) membrane and a modified ePTFE membrane. 
     According to another Aspect (“Aspect 59”) further to any one of Aspects 35 to 58, at least one of the first layer, the second layer, and the third layer is an expanded polytetrafluoroethylene membrane. 
     According to another Aspect (“Aspect 60”) further to any one of Aspects 35 to 59, the third layer is a spunbound non-woven polyester material. 
     According to another Aspect (“Aspect 61”) further to any one of Aspects 35 to 60, including a reinforcing component. 
     According to another Aspect (“Aspect 62”) further to Aspect 61, the reinforcing component has a stiffness from about 0.01 N/cm to about 5 N/cm. 
     According to another Aspect (“Aspect 63”) further to Aspects 35 to 62, including an external reinforcing component and an internal reinforcing component. 
     According to another Aspect (“Aspect 64”) further to any one of Aspects 35 to 63, the first solid features of the second layer include a member selected from thermoplastic polymers, polyurethanes, silicones, rubbers, epoxies and combinations thereof. 
     According to another Aspect (“Aspect 65”) further to any one of Aspects 35 to 64, including a surface coating thereon, the surface coating is selected from antimicrobial agents, antibodies, pharmaceuticals, and biologically active molecules. 
     According to another Aspect (“Aspect 66”) further to any one of Aspects 35 to 65, including a hydrophilic coating thereon. 
     According to another Aspect (“Aspect 67”) further to any one of Aspects 35 to 66, the biocompatible membrane composite is in the form of a cell encapsulation device. 
     According to one Aspect (“Aspect 68”) a cell encapsulation device includes (1) a first biocompatible membrane composite sealed along at least a portion of its periphery to a second biocompatible membrane composite sealed along at least a portion of its periphery to define a lumen therebetween, and (2) at least one filling tube in fluid communication with the lumen, where at least one of the first and second biocompatible membranes include a first layer having an MPS (maximum pore size) less than about 1 micron, a second layer having first solid features with a majority of a first solid feature spacing less than about 50 microns, where a majority of the first solid features has a first minor axis from about 3 microns to about 20 microns, and a third layer that has a pore size greater than about 9 microns in effective diameter and second solid features where the second solid features has a majority of a second solid feature spacing greater than about 50 microns. The second layer is positioned between the first layer and the third layer. 
     According to another Aspect (“Aspect 69”) further to Aspect 68, the first layer has a mass per area (MpA) less than about 5 g/m 2 . 
     According to another Aspect (“Aspect 70”) further to any one of Aspects 68 to 69, the first layer has a first thickness less than about 10 microns. 
     According to another Aspect (“Aspect 71”) further to any one of Aspects 68 to 70, the second layer has a second thickness less than about 60 microns. 
     According to another Aspect (“Aspect 72”) further to any one of Aspects 68 to 71, the biocompatible membrane composite has a maximum tensile load in the weakest axis greater than about 40 N/m. 
     According to another Aspect (“Aspect 73”) further to any one of Aspects 68 to 72, the first layer has a first porosity greater than about 50%. 
     According to another Aspect (“Aspect 74”) further to any one of Aspects 68 to 73, the second layer has a second porosity greater than about 60%. 
     According to another Aspect (“Aspect 75”) further to any one of Aspects 68 to 74, the first solid features of the second layer each have a representative minor axis, a representative major axis and a solid feature depth where a majority of at least two of the representative minor axis, the representative major axis, and the solid feature depth of the second layer is greater than about 5 microns. 
     According to another Aspect (“Aspect 76”) further to any one of Aspects 68 to 75, at least a portion of the first solid features are bonded solid features intimately bonded to the first layer. 
     According to another Aspect (“Aspect 77”) further to Aspect 76, the first solid features of the second layer are connected by fibrils and the fibrils are deformable. 
     According to another Aspect (“Aspect 78”) further to any one of Aspects 68 to 77, the second layer has a pore size from about 1 micron to about 9 microns in effective diameter. 
     According to another Aspect (“Aspect 79”) further to any one of Aspects 68 to 78, the third layer has a third thickness from about 30 microns to about 200 microns. 
     According to another Aspect (“Aspect 80”) further to any one of Aspects 68 to 79, a majority of the second solid feature spacing of the third layer is from about 50 microns to about 90 microns. 
     According to another Aspect (“Aspect 81”) further to any one of Aspects 68 to 80, a majority of the second solid features in the third layer has a representative minor axis that is less than about 40 microns. 
     According to another Aspect (“Aspect 82”) further to any one of Aspects 68 to 81, at least two of the first layer, the second layer, and the third layer are intimately bonded. 
     According to another Aspect (“Aspect 83”) further to any one of Aspects 68 to 82, a third thickness of the third layer is greater than a sum of a first thickness of the first layer and a second thickness of the second layer. 
     According to another Aspect (“Aspect 84”) further to any one of Aspects 68 to 83, a third thickness of the third layer is at least two times a combined thickness of the first thickness of the first layer and the second thickness of the second layer. 
     According to another Aspect (“Aspect 85”) further to any one of Aspects 68 to 84, at least one of the first layer, the second layer, and the third layer includes a polymer, a fluoropolymer membrane, a non-fluoropolymer membrane, a woven textile, a non-woven textile, woven or non-woven collections of fibers or yarns, fibrous matrices, and combinations thereof. 
     According to another Aspect (“Aspect 86”) further to any one of Aspects 68 to 85, at least one of the first layer, the second layer, and the third layer is a polymer. 
     According to another Aspect (“Aspect 87”) further to Aspect 86, the polymer is a fluoropolymer membrane selected from an expanded polytetrafluoroethylene (ePTFE) membrane, a fluorinated ethylene propylene (FEP) membrane, and a modified ePTFE membrane. 
     According to another Aspect (“Aspect 88”) further to any one of Aspects 68 to 87, at least one of the first layer, the second layer, and the third layer is an expanded polytetrafluoroethylene membrane. 
     According to another Aspect (“Aspect 89”) further to any one of Aspects 68 to 88, the third layer is a spunbound non-woven polyester material. 
     According to another Aspect (“Aspect 90”) further to any one of Aspects 68 to 89, the second solid features of the third layer include fibers of a non-woven or woven textile. 
     According to another Aspect (“Aspect 91”) further to any one of Aspects 68 to 90, the second solid features of the third layer include a woven or non-woven textile, and a second representative minor axis of the first solid features is a diameter of a fiber in a woven or non-woven textile. 
     According to another Aspect (“Aspect 92”) further to any one of Aspects 68 to 91, the first solid features of the second layer include a member selected from thermoplastic polymers, polyurethanes, silicones, rubbers, epoxies, and combinations thereof. 
     According to another Aspect (“Aspect 93”) further to any one of Aspects 68 to 92, including a surface coating thereon, the surface coating includes one or more members selected from antimicrobial agents, antibodies, pharmaceuticals and biologically active molecules. 
     According to another Aspect (“Aspect 94”) further to any one of Aspects 68 to 93, including a hydrophilic coating thereon. 
     According to another Aspect (“Aspect 95”) further to Aspects 68 to 94, including a reinforcing component external to at least one of the first biocompatible membrane composite and the second biocompatible membrane composite. 
     According to another Aspect (“Aspect 96”) further to Aspects 68 to 95, including an internal reinforcing component. 
     According to another Aspect (“Aspect 97”) further to Aspect 96, the internal reinforcing component includes a filling tube. 
     According to another Aspect (“Aspect 98”) further to any one of Aspects 68 to 97, at least one of the first biocompatible membrane composite and the second biocompatible membrane composite includes both an internal reinforcing component and an external reinforcing component 
     According to another Aspect (“Aspect 99”) further to any one of Aspects 68 to 98, the reinforcing component is a woven or non-woven textile. 
     According to another Aspect (“Aspect 100”) further to any one of Aspects 68 to 99, the cell encapsulation device includes a first weld film positioned between the first biocompatible membrane composite and a first reinforcing component positioned externally on the first biocompatible membrane composite and a second weld film positioned between the second biocompatible membrane composite and a second reinforcing component positioned externally on the second biocompatible membrane composite. 
     According to another Aspect (“Aspect 101”) further to any of the preceding Aspects, a method for lowering blood glucose levels in a mammal includes transplanting a cell encapsulated device including a biocompatible membrane composite of any of the previous claims, where cells encapsulated therein include a population of PDX1-positive pancreatic endoderm cells, and where the pancreatic endoderm cells mature into insulin secreting cells, thereby lowering blood glucose. 
     According to another Aspect (“Aspect 102”) further to any of the preceding Aspects, the PDX1-positive pancreatic endoderm cells include a mixture of cells further including endocrine and/or endocrine precursor cells, where the endocrine and/or endocrine precursor cells express chromogranin A (CHGA). 
     According to another Aspect (“Aspect 103”) further to any of the preceding Aspects, a method for lowering blood glucose levels in a mammal includes transplanting a cell encapsulation device as in claim  1 , where cells encapsulated therein include a population of PDX1-positive pancreatic endoderm cells, and where the pancreatic endoderm cells mature into insulin secreting cells, thereby lowering blood glucose. 
     According to another Aspect (“Aspect 104”) further to any of the preceding Aspects, the PDX1-positive pancreatic endoderm cells include a mixture of cells further including endocrine and/or endocrine precursor cells, where the endocrine and/or endocrine precursor cells express chromogranin A (CHGA). 
     According to another Aspect (“Aspect 105”) further to any of the preceding Aspects, a method for lowering blood glucose levels in a mammal includes transplanting a cell encapsulation device including a first layer having a MPS (maximum pore size) less than about 1 micron, a second layer has first solid features with a majority of a first solid feature spacing less than about 50 microns, where a majority of the first solid features has a representative minor axis from about 3 microns to about 20 microns, and a third layer that has a pore size greater than about 5 microns in effective diameter and second solid features having a majority of a second solid feature spacing greater than about 50 microns, where the second layer is positioned between the first layer and the third layer, where at least a portion of the bonded features are intimately bonded to the first layer, and a cell population including PDX1-positive pancreatic endoderm cells, and where the pancreatic endoderm cells mature into insulin secreting cells, thereby lowering blood glucose. 
     According to another Aspect (“Aspect 106”) further to any of the preceding Aspects, the PDX1-positive pancreatic endoderm cells include a mixture of cells further including endocrine and/or endocrine precursor cells, where the endocrine and/or endocrine precursor cells express chromogranin A (CHGA). 
     According to another Aspect (“Aspect 107”) further to any of the preceding Aspects, a method for lowering blood glucose levels in a mammal includes transplanting a biocompatible membrane composite that includes a first layer having a MPS (maximum pore size) less than about 1 micron, a second layer has first solid features with a majority of a first solid feature spacing less than about 50 microns, where a majority of the first solid features has a representative minor axis from about 3 microns to about 20 microns, and a third layer that has a pore size greater than about 5 microns in effective diameter and second solid features having a majority of a second solid feature spacing greater than about 50 microns, where the second layer is positioned between the first layer and the third layer, and a cell population including PDX1-positive pancreatic endoderm cells, and where the pancreatic endoderm cells mature into insulin secreting cells, thereby lowering blood glucose. 
     According to another Aspect (“Aspect 108”) further to any of the preceding Aspects, the PDX1-positive pancreatic endoderm cells include a mixture of cells further including endocrine and/or endocrine precursor cells, where the endocrine and/or endocrine precursor cells express chromogranin A (CHGA). 
     According to another Aspect (“Aspect 109”) further to any of the preceding Aspects, an encapsulated in vitro PDX1-positive pancreatic endoderm cells include a mixture of cell sub-populations including at least a pancreatic progenitor population co-expressing PDX-1/NKX6.1. 
     According to another Aspect (“Aspect 110”) further to any of the preceding Aspects, an encapsulated in vitro PDX1-positive pancreatic endoderm cells includes a mixture of cell sub-populations including at least a pancreatic progenitor population co-expressing PDX-1/NKX6.1 and a pancreatic endocrine and/or endocrine precursor population expressing PDX-1/NKX6.1 and CHGA. 
     According to another Aspect (“Aspect 111”) further to any of the preceding Aspects, at least 30% of the population includes pancreatic progenitor population co-expressing PDX-1/NKX6.1. 
     According to another Aspect (“Aspect 112”) further to any of the preceding Aspects, at least 40% of the population includes pancreatic progenitor population co-expressing PDX-1/NKX6.1. 
     According to another Aspect (“Aspect 113”) further to any of the preceding Aspects, at least 50% of the population includes pancreatic progenitor population co-expressing PDX-1/NKX6.1. 
     According to another Aspect (“Aspect 114”) further to any of the preceding Aspects, at least 20% of the population endocrine and/or endocrine precursor population express PDX-1/NKX6.1/CHGA. 
     According to another Aspect (“Aspect 115”) further to any of the preceding Aspects, at least 30% of the population endocrine and/or endocrine precursor population express PDX-1/NKX6.1/CHGA. 
     According to another Aspect (“Aspect 116”) further to any of the preceding Aspects, at least 40% of the population endocrine and/or endocrine precursor population express PDX-1/NKX6.1/CHGA. 
     According to another Aspect (“Aspect 117”) further to any of the preceding Aspects, the pancreatic progenitor cells and/or endocrine or endocrine precursor cells are capable of maturing into insulin secreting cells in vivo. 
     According to another Aspect (“Aspect 118”) further to any of the preceding Aspects, a method for producing insulin in vivo includes transplanting a cell encapsulated device including a biocompatible membrane composite of any of the previous claims and a population of PDX-1 pancreatic endoderm cells mature into insulin secreting cells, where the insulin secreting cells secrete insulin in response to glucose stimulation. 
     According to another Aspect (“Aspect 119”) further to any of the preceding Aspects, the PDX1-positive pancreatic endoderm cells include a mixture of cells further including endocrine and/or endocrine precursor cells, where the endocrine and/or endocrine precursor cells express chromogranin A (CHGA). 
     According to another Aspect (“Aspect 120”) further to any of the preceding Aspects, at least about 30% of the population are endocrine and/or endocrine precursor population expressing PDX-1/NKX6.1/CHGA. 
     According to another Aspect (“Aspect 121”) further to any of the preceding Aspects, an in vitro human PDX1-positive pancreatic endoderm cell culture includes a mixture of PDX-1 positive pancreatic endoderm cells and at least a transforming growth factor beta (TGF-beta) receptor kinase inhibitor. 
     According to another Aspect (“Aspect 122”) further to any of the preceding Aspects, further including a bone morphogenetic protein (BMP) inhibitor. 
     According to another Aspect (“Aspect 123”) further to any of the preceding Aspects, the TGF-beta receptor kinase inhibitor is TGF-beta receptor type 1 kinase inhibitor. 
     According to another Aspect (“Aspect 124”) further to any of the preceding Aspects, the TGF-beta receptor kinase inhibitor is ALK5i. 
     According to another Aspect (“Aspect 125”) further to any of the preceding Aspects, the BMP inhibitor is noggin. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The accompanying drawings are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments, and together with the description serve to explain the principles of the disclosure. 
         FIG. 1A  is a schematic illustration depicting the determination of solid feature spacing where three neighboring solid features represent the corners of a triangle whose circumcircle has an interior devoid of additional solid features and the solid feature spacing is the straight distance between two of the solid features forming the triangle in accordance with embodiments described herein; 
         FIG. 1B  is a schematic illustration depicting the determination of non-neighboring solid features where the solid features form the corners of a triangle whose circumcircle contains at least one additional solid feature in accordance with embodiments described herein; 
         FIG. 2  is a scanning electron micrograph of the spacing (white lines) between solid features (white shapes) in an ePTFE membrane in accordance with embodiments described herein; 
         FIG. 3A  is a schematic illustration depicting the method to determine the major axis and the minor axis of a solid feature in accordance with embodiments described herein; 
         FIG. 3B  is a schematic illustration depicting the depth of a solid feature in accordance with embodiments described herein; 
         FIG. 4  is a schematic illustration of the effective diameter of a pore in accordance with embodiments described herein; 
         FIG. 5  is a scanning electron micrograph (SEM) showing a pore size in accordance with embodiments described herein; 
         FIG. 6A  is a schematic illustration of a thermoplastic polymer in the form of solid features positioned on the surface of a cell impermeable layer in accordance with embodiments described herein; 
         FIGS. 6B-6H  are schematic illustrations of sample geometries for forming solid features on a cell impermeable layer in accordance with embodiments described herein; 
         FIG. 7  is a schematic illustration of a biocompatible membrane composite having therein bonded solid features intimately bonded to the surface of the cell impermeable layer in accordance with embodiments described herein; 
         FIG. 8  is a schematic illustration of a biocompatible membrane composite where the mitigation layer has therein solid features with differing heights and widths in accordance with embodiments described herein; 
         FIG. 9  is a schematic illustration of a biocompatible membrane composite having a mitigation layer containing therein solid features that are nodes in accordance with embodiments described herein; 
         FIGS. 10A-10D  are schematic illustrations of a biocompatible membrane composites showing various locations of a reinforcing component in accordance with embodiments described herein; 
         FIG. 11A  is a schematic illustration of a cross-sectional view of a mitigation layer positioned on a cell impermeable layer where the mitigation layer is characterized at least by solid feature size, solid feature spacing, solid feature depth, and thickness in accordance with embodiments described herein; 
         FIG. 11B  is a schematic illustration of a cross-sectional view of a mitigation layer positioned on a cell impermeable layer where the mitigation layer is characterized at least by solid feature size, solid feature spacing, solid feature depth, thickness, and pore size in accordance with embodiments described herein; 
         FIG. 12  is a schematic illustration of a cross-sectional view of a biocompatible membrane composite containing a vascularization layer, a mitigation layer, and a cell impermeable layer where the vascularization layer is characterized at least by thickness, pore size, solid feature size, and solid feature spacing in accordance with embodiments described herein; 
         FIG. 13A  is a schematic illustration of a top view of a cell encapsulation device in accordance with embodiments described herein; 
         FIG. 13B  is a schematic illustration of a cross-section of the cell encapsulation device of  FIG. 13A  depicting the orientation of the layers of the biocompatible membrane composite and placement of cells in accordance with embodiments described herein; 
         FIG. 14  is a scanning electron micrograph (SEM) of the top surface of a comparable cell impermeable layer formed of an expanded polytetrafluoroethylene (ePTFE) membrane in accordance with embodiments described herein; 
         FIG. 15  is an SEM of the top surface of a vascularization layer formed of a non-woven polyester in accordance with embodiments described herein; 
         FIG. 16  is a schematic illustration of exploded view of an encapsulation device in accordance with embodiments described herein; 
         FIG. 17  is a representative histology image showing the presence of foreign body giant cells on the surface of a cell impermeable layer in Comparative Example 1 in accordance with embodiments described herein; 
         FIG. 18  is an SEM of the top surface of the mitigation layer with a discontinuous layer of fluorinated ethylene propylene (FEP) on the mitigation layer in Comparative Example 2 in accordance with embodiments described herein; 
         FIG. 19  is an SEM of the top surface of the ePTFE cell impermeable layer used in Comparative Example 2, Example 2, Example 4, and Example 5 in accordance with embodiments described herein; 
         FIG. 20  is an SEM of the top surface of the ePTFE mitigation layer used in Comparative Example 2 in accordance with embodiments described herein; 
         FIG. 21  is an SEM of the cross-section of the two layer ePTFE composite formed in Comparative Example 2 in accordance with embodiments described herein; 
         FIG. 22  is a representative histology image of foreign body giant cells forming on the cell impermeable layer of Comparative Example 2 in accordance with embodiments described herein; 
         FIG. 23  is an SEM of the top surface of the ePTFE cell impermeable layer used in Example 1 in accordance with embodiments described herein; 
         FIG. 24  is an SEM of the top surface of the ePTFE mitigation layer used in Example 1 in accordance with embodiments described herein; 
         FIG. 25  is an SEM of the cross-section of a two-layer ePTFE composite formed in Example 1 in accordance with embodiments described herein; 
         FIG. 26  is a representative histology image depicting the absence of the formation of foreign body giant cells on the cell impermeable layer of Example 1 in accordance with embodiments described herein; 
         FIG. 27  is an SEM of the top surface of the ePTFE mitigation layer with a discontinuous layer of fluorinated ethylene propylene (FEP) thereon in Example 2 in accordance with embodiments described herein; 
         FIG. 28  is an SEM of the top surface of the ePTFE mitigation layer of Example 2 in accordance with embodiments described herein; 
         FIG. 29  is an SEM of the cross-section of the two-layer ePTFE composite formed in Example 2 in accordance with embodiments described herein; 
         FIG. 30  is an SEM of the top surface of the ePTFE cell impermeable layer formed in Example 3 in accordance with embodiments described herein; 
         FIG. 31  is an SEM of the top surface of the ePTFE mitigation layer formed in Example 3 in accordance with embodiments described herein; 
         FIG. 32  is an SEM of the cross-section of the two-layer ePTFE composite formed in Example 3 in accordance with embodiments described herein; 
         FIG. 34  is an SEM of the top surface of the ePTFE mitigation layer with a discontinuous layer of FEP thereon formed in Example 4 in accordance with embodiments described herein; 
         FIG. 34  is an SEM of the top surface of the ePTFE mitigation layer formed in Example 4 in accordance with embodiments described herein; 
         FIG. 35  is an SEM of the cross-section of the two-layer ePTFE composite formed in Example 4 in accordance with embodiments described herein; 
         FIG. 36  is a representative histology image showing the absence of the formation of foreign body giant cells on the cell impermeable layer of Example 4 in accordance with embodiments described herein; 
         FIG. 37  is an SEM of the top surface of the ePTFE mitigation layer with a discontinuous layer of FEP thereon in Example 5 in accordance with embodiments described herein; 
         FIG. 38  is an SEM of the top surface of the ePTFE vascularization layer utilized in Example 5 in accordance with embodiments described herein; 
         FIG. 39  is an SEM of the cross-section of the three layer composite formed in Example 5 in accordance with embodiments described herein; 
         FIG. 40  is a schematic illustration of a top view of an insert reinforcing component in accordance with embodiments described herein; 
         FIG. 41  is a schematic illustration of an exploded view of a planar device in accordance with embodiments described herein; 
         FIG. 42  is an image of a top view of a surface of a planar device in accordance with embodiments described herein; 
         FIG. 43A  is an image of a cross-section of the planar device of  FIG. 42  taken along line A-A showing a single point bond and the lumen in accordance with embodiments described herein; 
         FIG. 43B  is an image of a cross-section the planar device of  FIG. 42  taken along line B-B showing two point bonds and the lumen in accordance with embodiments described herein; 
         FIG. 44  is a representative histology image showing the absence of the formation of foreign body giant cells on the cell impermeable layer of Example 6 in accordance with embodiments described herein; 
         FIG. 45  is a representative histology image showing the absence of the formation of foreign body giant cell on the surface of the impermeable layer of Example 2 in accordance with embodiments described herein; 
         FIG. 46  is a representative histology image showing the absence of the formation of foreign body giant cell on the surface of the impermeable layer of Example 3 in accordance with embodiments described herein; 
         FIG. 47  is a representative histology image showing the absence of the formation of foreign body giant cell on the surface of the impermeable layer of Example 5 in accordance with embodiments described herein; 
         FIG. 48  is a representative histology image showing the absence of the formation of foreign body giant cell on the surface of the impermeable layer of Example 6 in accordance with embodiments described herein; 
         FIG. 49A  is a representative histology image showing in vivo cell viability in Construct A of Example 7 in accordance with embodiments described herein; 
         FIG. 49B  is a representative histology image showing in vivo cell viability in Construct B of Example 7 in accordance with embodiments described herein; 
         FIG. 49C  is a representative histology image showing in vivo cell viability in Construct C of Example 7 in accordance with embodiments described herein; 
         FIG. 50  is a representative SEM image of the node and fibril structure of the third ePTFE membrane in Construct A of Example 7 in accordance with embodiments described herein; 
         FIG. 51  is a representative SEM image of the node and fibril structure of the third ePTFE membrane in Construct B of Example 7 in accordance with embodiments described herein; 
         FIG. 52  is a representative SEM image of the node and fibril structure of the third ePTFE membrane in Construct C of Example 7 in accordance with embodiments described herein; 
         FIG. 53  is an SEM image of the cross-section of the third ePTFE membrane of Construct A of Example 7 in accordance with embodiments described herein; 
         FIG. 54  is an SEM image of the cross-section of the third ePTFE membrane of Construct B of Example 7 in accordance with embodiments described herein; 
         FIG. 55  is an SEM image of the cross-section of the third ePTFE membrane of Construct C of Example 7 in accordance with embodiments described herein; and 
         FIG. 56  is an SEM image depicting a representative surface microstructure of the second ePTFE layer of Constructs A, B, and C having thereon FEP in accordance with embodiments described herein. 
     
    
    
     DETAILED DESCRIPTION 
     Persons skilled in the art will readily appreciate that various aspects of the present disclosure can be realized by any number of methods and apparatus configured to perform the intended functions. It should also be noted that the accompanying figures referred to herein are not necessarily drawn to scale, and may be exaggerated to illustrate various aspects of the present disclosure, and in that regard, the figures should not be construed as limiting. Directional references such as “up,” “down,” “top,” “left,” “right,” “front,” and “back,” among others are intended to refer to the orientation as illustrated and described in the figure (or figures) to which the components and directions are referencing. It is to be appreciated that the terms “biocompatible membrane composite” and “membrane composite” are used interchangeably herein. It is to be noted that all ranges described herein are exemplary in nature and include any and all values in between. In addition, all references cited herein are incorporated by reference in their entireties. 
     The present disclosure is directed to a biocompatible membrane composite. The membrane composite contains a first layer, a second layer, and a third layer. Each layer is distinct and serves a necessary function for the survival of encapsulated cells. In certain embodiments, the first layer functions as a cell impermeable layer, the second layer functions as a mitigation layer, and the third layer functions as a vascularization layer. Herein, the term “first layer” is used interchangeably with “cell impermeable layer”, the term “second layer” is used interchangeably with “mitigation layer”, and the term “third layer” is used interchangeably with “vascularization layer” for ease of convenience. Each layer is distinct, serving a unique function that supports the survival of the encapsulated cells The mitigation layer is positioned between the cell impermeable layer and the vascularization layer and reduces the formation of foreign body giant cells on the surface of the cell impermeable layer. In at least one embodiment, the mitigation layer includes solid features (e.g., nodes) that are present in the membrane forming the mitigation layer. In other embodiments, the mitigation layer includes solid features (e.g., printed solid features) that are provided and/or formed on a surface of the cell impermeable layer. In some embodiments, the cell impermeable layer and the mitigation layer are intimately bonded or otherwise connected to each other to form a composite layer having a tight/open structure. As used herein, “intimate bond” and “intimately bonded” refer to layers of the biocompatible composite or to solid features within the biocompatible composite that are not readily separable or detachable at any point on their surface. A reinforcing component may optionally be positioned on either side of the biocompatible membrane composite (i.e., external to) or within the biocompatible membrane composite (i.e., internal to) to provide support to and prevent distortion of the membrane composite. Herein, a “reinforcing component” may be further described as being external or internal to a cell encapsulation device and may be nutrient impermeable or nutrient permeable. The biocompatible membrane composite may be used in or to form a device for encapsulating biological entities and/or cell populations. It is to be appreciated that the term “about” as used herein denotes +/−10% of the designated unit of measure. 
     Biological entities suitable for use with the biocompatible membrane composite include, but are not limited to, cells, viruses, viral vectors, gene therapies, bacteria, proteins, polysaccharides, antibodies, and other bioactive entities. It is to be appreciated that if a biological entity other than a cell is selected for use herein, the bioactive component or product of the biological entity needs to be able to pass through the cell impermeable layer, but not the entity itself. For simplicity, herein the biological entity is referred to as a cell, but nothing in this description limits the biological entity to cells or to any particular type of cell, and the following description applies also to biological entities that are not cells. 
     Various types of prokaryotic cells, eukaryotic cells, mammalian cells, non-mammalian cells, and/or stem cells may be used with the biocompatible membrane composite described herein. In some embodiments, the cells secrete a therapeutically useful substance. Such therapeutically useful substances include hormones, growth factors, trophic factors, neurotransmitters, lymphokines, antibodies, or other cell products which provide a therapeutic benefit to the device recipient. Examples of such therapeutic cell products include, but are not limited to, insulin and other pancreatic hormones, growth factors, interleukins, parathyroid hormone, erythropoietin, transferrin, collagen, elastin, tropoelastin, exosomes, vesicles, genetic fragments, and Factor VIII. Non-limiting examples of suitable growth factors include vascular endothelial growth factor, platelet-derived growth factor, platelet-activating factor, transforming growth factors bone morphogenetic protein, activin, inhibin, fibroblast growth factors, granulocyte-colony stimulating factor, granulocyte-macrophage colony stimulating factor, glial cell line-derived neurotrophic factor, growth differentiation factor-9, epidermal growth factor, and combinations thereof. 
     As discussed above, the biocompatible membrane composite includes a first layer (i.e., cell impermeable layer). The cell impermeable layer serves as a microporous, immune isolation barrier, and is impervious to vascular ingrowth and prevents cellular contact from the host. Herein, layers that do not have openings large enough to allow cellular ingrowth may be referred to as “tight” layers. The pores of the cell impermeable layer are sufficiently small so as to allow the passage therethrough of cellular nutrients, oxygen, waste products, and therapeutic substances while not permitting the passage of any cells. Because the cell impermeable layer has an MPS that is sufficiently small so as to prevent vascular ingrowth, it is necessary to balance the parameters of the cell impermeable layer that could also negatively impact the mass transport and diffusion properties of the cell impermeable layer. For instance, while the MPS is small enough to prevent cell ingress or vascular ingrowth, the cell impermeable layer is sufficiently open so as to allow the passage of molecules (i.e. nutrients and therapeutic molecules) therethrough. Layers that have openings large enough to allow cellular ingrowth may be referred to as “open layers”. 
     Diffusion resistance is further minimized by keeping the cell impermeable layer thin, porous, and low in mass. It is to be appreciated that sufficient porosity of the cell impermeable layer be maintained so as to allow the passage of molecules. In certain embodiments, the porosity of the cell impermeable layer is greater than about 50%, greater than about 60%, greater than about 70%, or greater than about 80%. Additionally, the porosity may range from about 50% to about 98%, from about 50% to about 90%, from about 50% to about 80%, or from about 60% to about 90%. It is also to be appreciated that sufficient durability and strength of the cell impermeable layer be maintained so that immune isolation can be provided in vivo through an intended use by ensuring the integrity of this tight layer. It is therefore necessary to balance the tradeoffs of the competing properties of strength and diffusion resistance. In certain embodiments, the maximum tensile load of the weakest axis of the cell impermeable layer is greater than about 40 N/m, greater than about 130 N/m, greater than about 260 N/m, greater than about 600 N/m, or greater than about 1000 N/m. Additionally, the maximum tensile load of the weakest axis may range from about 40 N/m to about 2000 N/m, 40 N/m to about 780 N/m, 40 N/m to about 350 N/m, from about 130 N/m to about 2000 N/m, from about 130 N/m to about 450 N/m, or from about 260 N/m to about 2000 N/m. 
     In certain embodiments, the cell impermeable layer has a combination of tensile strengths in orthogonal directions (D1, D2) that result in a geometric mean tensile strength that is greater than about 20 MPa, greater than about 50 MPa, greater than about 100 MPa, or greater than about 150 MPa when the geometric mean tensile strength is defined by the following equation: 
     
       
         
           
             
               Geometric 
               ⁢ 
                   
               Mean 
             
             = 
             
               
                 
                   
                     
                       ( 
                       
                         Tensile 
                         ⁢ 
                             
                         
                           Strength 
                           
                             D 
                             ⁢ 
                             1 
                           
                         
                       
                       ) 
                     
                     2 
                   
                   + 
                   
                     
                       ( 
                       
                         Tensile 
                         ⁢ 
                             
                         
                           Strength 
                           
                             D 
                             ⁢ 
                             2 
                           
                         
                       
                       ) 
                     
                     2 
                   
                 
               
               . 
             
           
         
       
     
     The geometric mean tensile strength of the cell impermeable layer may range from about 20 MPa to about 180 MPa, from about 30 MPa to about 150 MPa, from about 50 MPa to about 150 MPa, or from about 100 MPa to about 150 MPa. 
     In some embodiments, the cell impermeable layer has an MPS that is less than about 1 micron, less than about 0.50 microns, less than about 0.30 microns, or less than about 0.10 microns as measured by porometry. The MPS may be from about 0.05 microns to about 1 micron, from about 0.1 microns to about 0.80 microns, from about 0.1 microns to about 0.6 microns, from about 0.1 microns to about 0.5 microns, or from about 0.2 microns to about 0.5 microns as measured by porometry. 
     In addition, the cell impermeable layer has a thickness that is less than about 30 microns, less than about 20 microns, less than about 10 microns, or less than about 5 microns. The thickness may range from about 1 micron to about 30 microns, from about 1 micron to about 20 microns, from about 1 micron to about 10 microns, from about 5 microns to about 10 microns, or from about 1 micron to about 5 microns. The mass per area (MpA) of the cell impermeable layer may be less than about 25 g/m 2 , less than about 20 g/m 2 , less than about 10 g/m 2 , less than about 5 g/m 2 , or less than about 3 g/m 2 . The MpA may range from about 3 g/m 2  to about 25 g/m 2 , from about 0.5 g/m 2  to about 20 g/m 2 , from about 0.5 g/m 2  to about 10 g/m 2 , or from about 0.5 g/m 2  to about 5 g/m 2 . 
     As discussed previously, the biocompatible membrane composite includes a second layer (i.e., mitigation layer) which is sufficiently porous to permit growth of vascular tissue into the mitigation layer, and in some instances, also acts as an initial vascularization layer. The mitigation layer creates a suitable environment to minimize, reduce, inhibit, or even prevent the formation of foreign body giant cells while allowing for access to blood vessels at the cell impermeable layer. Ingrowth of vascular tissues into the mitigation layer facilitates nutrient transfer through the cell impermeable layer. Herein, layers that have openings large enough to allow vascular ingrowth may be referred to as “open” layers. Blood vessels, which are the source of oxygen and nutrients for implanted cells, need to form in the mitigation layer so that they are sufficiently close to the cell impermeable layer such that the distance for nutrient diffusion to any encapsulated cells is minimized. The thinness of the cell impermeable layer helps to reduce the distance over which diffusion must occur. 
     The ingrowth of vascular tissue through the mitigation layer up to the cell impermeable layer facilitates nutrient transfer across the cell impermeable layer. The mitigation layer creates an environment that enables a sufficient formation of blood vessels into the mitigation layer positioned adjacent to the cell impermeable layer instead of the formation of foreign body giant cells. As a result, and as shown in the Examples, foreign body giant cells do not form at the interface of the cell impermeable layer and the mitigation layer such that foreign body cells impede sufficient vascularization. It is to be noted that foreign body giant cells may individually form at the interface of the cell impermeable layer and the mitigation layer, but they do not impede or prevent the vascularization needed for growth of encapsulated cells. 
     The mitigation layer is characterized at least in part by the inclusion of a plurality of solid features that have a solid feature spacing, which is discussed in detail below. “Solid features” as used herein may be defined as three dimensional components within the mitigation layer and/or vascularization layer that are generally immovable and resistant to deformation when exposed to environmental forces, such as, but not limited to, cell movement (e.g., cellular migration and ingrowth, host vascularization/endothelial blood vessel formation). To facilitate the reduction or mitigation of the formation of a barrier of foreign body giant cells at the cell impermeable layer, the solid features abutting the surface of the cell impermeable layer adjacent to the mitigation layer help prevent the fusion of multiple macrophages into multinucleated foreign body giant cells at this interface. In some embodiments, the solid features in the mitigation layer abutting the cell impermeable layer are intimately bonded to the cell impermeable layer and are herein referred to as “bonded solid features”. “Non-bonded solid features” are those solid features within the mitigation layer that are not bonded (intimately bonded or otherwise) to the cell impermeable layer. The solid features in the mitigation layer may be formed of, for example, thermoplastic polymers, polyurethanes, silicones, rubbers, epoxies, and combinations thereof. 
     In some embodiments, the solid features of the mitigation layer project outwardly from a plane defined by the cell impermeable layer. In such embodiments, the solid features of the mitigation layer projecting from the cell impermeable layer may be intimately bonded with the cell impermeable layer and spaced such that they provide blockades or barriers to the formation of foreign body giant cells at this tight, cell impermeable interface. In some embodiments, the solid features may be a feature of the mitigation layer (e.g. nodes), and may be connected to each other, such as by fibrils or fibers. In another embodiments, the solid features may be provided and/or otherwise formed on the surface of the cell impermeable layer (e.g., printed solid features) such that the solid features project outwardly from the plane defined by a plane defined by the cell impermeable layer. 
     In embodiments where the mitigation layer has a node and fibril microstructure (e.g., formed from a fibrillated polymer), the nodes are the solid features and the fibrils are not the solid features. Indeed, in some embodiments, the fibrils may be removed, leaving only the nodes in the mitigation layer. In embodiments where the nodes within the mitigation layer are the solid features, those nodes which are bonded to the cell impermeable layer are bonded solid features. In at least one embodiment, the mitigation layer is formed of an expanded tetrafluoroethlyene (ePTFE) membrane having a node and fibril microstructure. 
     The solid features of the mitigation layer do not negatively impact the overall diffusion resistance of the biocompatible membrane composite for applications that require a rapid time course of diffusion. The solid features of the mitigation layer are of a sufficiently small size such that they do not interfere with the amount of porous area needed for diffusion across the cell impermeable layer. Also, the thickness of the mitigation layer is sufficiently thin so as to maximize mass transport of oxygen and nutrients to encapsulated cells from the interstitium during the acute period post implantation. The space between the solid features are sufficiently open to allow for easy and rapid penetration/integration of host tissue up to the cell impermeable layer (i.e., tight layer) to decrease the duration of the acute period. “Acute period” is defined herein as the time period prior to host cell/vascular infiltration. 
     The majority of the solid feature spacing of the solid features adjacent to the cell impermeable layer is less than about 50 microns, less than about 40 microns, less than about 30 microns, less than about 20 microns, or less than about 10 microns. As used herein, the term “majority” is meant to describe an amount over half (i.e., greater than 50%) of the measured values for the parameter being measured. In addition, the phrase “solid feature spacing” is defined herein as the straight-line distance between two neighboring solid features. In this disclosure, solid features are considered neighboring if their centroids represent the corners of a triangle whose circumcircle has an empty interior. As shown pictorially in  FIG. 1A , the designated solid feature (P) is connected to neighboring solid features (N) to form a triangle  100  where the circumcircle  110  contains no solid features within. Solid features (X) designate the solid features that are not neighboring solid features. Thus, in the instance depicted in  FIG. 1A , the solid feature spacing  130  is the straight distance between the designated solid features (P), (N). In contrast, the circumcircle  150  shown in  FIG. 1B  drawn from the triangle  160  contains therein a solid feature (N), and as such, cannot be utilized to determine the solid feature spacing in the mitigation layer (or the vascularization layer).  FIG. 2  is a scanning electron micrograph depicting measured distances, e.g., the white lines  200  between the solid features  210  (white shapes) in a mitigation layer formed of an expanded polytetrafluoroethylene membrane. In some embodiments, the majority of the solid feature spacing may range from about 5 microns to about 45 microns, from about 10 microns to about 40 microns, from about 10 microns to about 35 microns, or from about 15 microns to about 35 microns. 
     The solid features also include a representative minor axis, a representative major axis, and a solid feature depth. The representative minor axis of a solid feature is defined herein as the length of the minor axis of an ellipse fit to the solid feature where the ellipse has the same area, orientation, and centroid as the solid feature. The representative major axis of a solid feature is defined herein as the length of the major axis of an ellipse fit to the solid feature where the ellipse has the same area, orientation, and centroid as the solid feature. The major axis is greater than or equal to the minor axis in length. The representative minor axis and representative major axis of a layer are the respective median values of all measured representative minor axes and representative major axes of features in the layer. The minor and major axes of an ellipse  320  to fit the solid feature  310  is shown pictorially in  FIG. 3A . The representative minor axis of the solid feature  310  is depicted by arrow  300 , and the representative major axis of the solid feature  310  is depicted by arrow  330 . A majority of the solid features in the mitigation layer has a minor axis that range in size from about 3 microns to about 20 microns, from about 3 microns to about 15 microns, or from about 3 microns to about 10 microns. The solid feature depth is the length of the projection of the solid feature in the axis perpendicular to the surface of the layer (e.g., mitigation layer or vascularization layer). The solid feature depth of the solid feature  310  is shown pictorially in  FIG. 3B . The depth of the solid feature  310  is depicted by line  340 . In at least one embodiment, the depth of the solid features is equal to or less than the thickness of the mitigation layer. The solid feature depth of a layer is the median value of all measured solid feature depths in the layer. In at least one embodiment, the majority of at least two of the representative minor axis, representative major axis, and solid feature depth in a layer is greater than 5 microns. 
     In embodiments where the solid features are interconnected by fibrils or fibers, the boundary connecting the solid features creates a pore. It is necessary that these pores are open enough to allow rapid cellular ingrowth and vascularization and not create a resistance to mass transport of oxygen and nutrients. The pore effective diameter is measured by quantitative image analysis (QIA) and performed on a scanning electron micrograph (SEM) image. The “effective diameter” of a pore is defined as the diameter of a circle that has an area equal to the measured area of the surface pore. This relationship is defined by the following equation: 
     
       
         
           
             
               Effective 
               ⁢ 
                   
               Diameter 
             
             = 
             
               2 
               × 
               
                 
                   
                     Area 
                     π 
                   
                 
                 . 
               
             
           
         
       
     
     Turning to  FIG. 4 , the effective diameter is the diameter of the circle  400  and the surface pore is designated by reference numeral  420 . The total pore area of a surface is the sum of the area of all pores at that surface. The pore size of a layer is the effective diameter of the pore that defines the point where roughly half the total pore area consists of pores with diameters smaller than the pore size and half the total pore area consists of pores with diameters greater than or equal to the pore size.  FIG. 5  illustrates a pore size  500  (white in color), pores smaller in size  510  (shown in light grey), and pores larger in size  520  (shown in dark grey). Pores that intersect with the edge of the image  530  were excluded from analysis and are shown in black. 
     The pore size of the mitigation layer may range from about 1 micron to about 9 microns in effective diameter, from about 3 microns in effective diameter to about 9 microns in effective diameter, or from about 4 micron in effective diameter to about 9 microns in effective diameter as measured by quantitative image analysis (QIA) performed on an SEM image. Also, the mitigation layer has a thickness that is less than about 60 microns, less than about 50 microns, less than about 40 microns, less than about 30 microns, or less than about 20 microns. The thickness of the mitigation layer may range from about 3 microns to about 60 microns, from about 10 microns to about 50 microns, from about 10 microns to about 40 microns, or from about 15 microns to about 35 microns. In some embodiments, the mitigation layer has a porosity greater than about 60%. In other embodiments, the mitigation layer has a porosity greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 95%. In some embodiments, the porosity may be about 98% or about 99%. The porosity of the mitigation layer may range from about 60% to about 98%, from about 70% to about 98%, or from about 80% to about 98%. 
     As discussed previously, the biocompatible membrane composite also includes a third layer (i.e., vascularization layer). The vascularization layer is an “open” layer that permits additional vascular penetration from the host and rapid anchoring and attachment of the biocompatible membrane composite within the tissue of the host. Additionally, the vascularization layer provides a porous matrix to harbor the growth of a sufficient quantity of additional, new blood vessels to feed the encapsulated cells. The vascularization layer is designed such that there are solid features to enable host integration and attachment. As the vascularization layer does not meet the same criteria as the mitigation layer, the two are separate and distinct layers. The solid features of the vascularization layer have increased spacing and pore sizes therebetween compared to the solid features of the mitigation layer to facilitate a more rapid ingrowth of the tissue or blood vessels into the vascularization layer. In some embodiments, the majority of the solid feature spacing of the solid features in the vascularization layer is greater than about 50 microns, greater than about 60 microns, greater than about 70 microns, or greater than about 80 microns. A majority of the solid features in the vascularization layer has a solid feature spacing that range from about 50 microns to about 90 microns, from about 60 microns to about 90 microns, or from about 70 microns to about 90 microns. 
     The pore size and overall thickness of the vascularization layer is sufficient to provide space to harbor the necessary quantities of additional blood vessels to provide nutrients and oxygen to cells. A pore size of the vascularization layer may be greater than about 9 microns in effective diameter, greater than about 25 microns in effective diameter, greater than about 50 microns in effective diameter, greater than about 75 microns in effective diameter, greater than about 100 microns in effective diameter, greater than about 125 microns in effective diameter, greater than about 150 microns in effective diameter, greater than about 175 microns in effective diameter, or greater than about 200 microns in effective diameter as measured by QIA performed on an SEM image. In some embodiments, the pore size of the vascularization layer may range from about 9 microns in effective diameter to about 200 microns in effective diameter, from about 9 microns in effective diameter to about 50 microns in effective diameter, from about 15 microns in effective diameter to about 50 microns in effective diameter from about 25 microns in effective diameter to about 50 microns in effective diameter, from about 50 microns in effective diameter to about 200 microns in effective diameter, from about 75 microns in effective diameter to about 175 microns in effective diameter as measured by QIA performed on an SEM image. 
     Additionally, the vascularization layer may have a thickness that is greater than about 30 microns, greater than about 50 microns, greater than about 75 microns, greater than about 100 microns, greater than about 125 microns, greater than about 150 microns, or greater than about 200 microns. In addition, the thickness of the vascularization layer may range from about 30 microns to about 300 microns, from about 30 microns to about 200 microns, from about 30 microns to about 100 microns, from about 100 microns to about 200 microns, or from about 100 microns to about 150 microns. In at least one embodiment, the thickness of the vascularization layer is at least two times the combined thickness of the cell impermeable layer and the mitigation layer. In some embodiments, the thickness of the vascularization layer is greater than a sum of a thickness of the cell impermeable layer and a thickness of the mitigation layer. In addition, a majority of the solid features in the vascularization layer has a representative minor axis that is less than 50 microns, less than about 40 microns, less than about 30 microns, less than about 20 microns, less than about 10 microns, less than about 5 microns, or less than about 3 microns. In some embodiments, a majority of the solid features in the vascularization layer has a representative minor axis that ranges in size from about 3 microns to about 40 microns, from about 3 microns to about 30 microns, from about 3 microns to about 20 microns, from about 3 microns to about 10 microns, or from about 20 microns to about 40 microns. The solid features present in the vascularization layer also have a major axis that is greater in length than the minor axis and may effectively be unlimited in length, such as a continuous fiber of a non-woven. The solid features in the vascularization layer also have a depth that is less than or equal to the total thickness of the vascularization layer. 
     In some embodiments, the biocompatible membrane composite, including the cell impermeable layer, is perforated with discretely placed holes. The perforation size, number, and location can be selected to optimize cell function. As few as one (1) perforated hole may be present. The perforations are of a sufficient size to allow host vascular tissue (such as capillaries) to pass through the biocompatible membrane composite in order to support, for example, encapsulated pancreatic cell types. While the cell impermeable layer maintains its function as a microporous, immune isolation barrier in locations where no perforations are present, due to the discrete perforations where portions of the cell impermeable layer have been removed, the cell impermeable layer in its entirety is no longer cell impermeable because the discrete perforations allow vascular ingrowth and cellular contact from the host to pass through the biocompatible membrane composite. Because cell encapsulation device embodiments that contain a perforated cell impermeable layer allow for host immune cell contact with cells, the cells are no longer protected from immune rejection unless the host is immunocompromised or treated with immunosuppressant drugs. 
     An optional reinforcing component may be provided to the biocompatible membrane composite to minimize distortion in vivo so that the cell bed thickness is maintained (e.g., in an encapsulated device). This additional, optional reinforcing component provides a stiffness to the biocompatible membrane composite that is greater than the biocompatible membrane composite itself to provide mechanical support. This optional reinforcing component could be continuous in nature or it may be present in discrete regions on the biocompatible membrane composite, e.g., patterned across the entire surface of the biocompatible membrane composite or located in specific locations such as around the perimeter of the biocompatible membrane composite. Non-limiting patterns suitable for the reinforcing component on the surface of the membrane composite include dots, straight lines, angled lines, curved lines, dotted lines, grids, etc. The patterns forming the reinforcing component may be used singly or in combination. In addition, the reinforcing component may be temporary in nature (e.g., formed of a bioabsorbable material) or permanent in nature (e.g., a polyethylene terephthalate (PET) mesh or Nitinol). As is understood by one of ordinary skill in the art, the impact of component stiffness depends not just on the stiffness of a single component, but also on the location and restraint of the reinforcing component in the final device form. In order for a component (e.g., a reinforcing component) to be practically useful for adding stiffness to the biocompatible composite membrane, the reinforcement component should have a stiffness greater than about 0.01 N/cm, although a final determination of the stiffness needed will depend on location and restraint in the finished cell encapsulation device. In some embodiments, the reinforcement component may have a stiffness from about 0.01 N/cm to about 5 N/cm, from about 0.05 N/cm to about 4 N/cm, from about 0.1 N/cm to about 3 N/cm, or from about 0.3 N/cm to about 2 N/cm. 
     In at least one embodiment, the reinforcing component may be provided on the external surface of the vascularization layer to strengthen the biocompatible membrane composite against environmental forces. In this orientation, the reinforcing component has a pore size sufficient to permit vascular ingrowth, and is therefore is considered an “open” layer. Materials useful as the reinforcing component include materials that are significantly stiffer than the biocompatible membrane composite. Such materials include, but are not limited to, open mesh biomaterial textiles, woven textiles, non-woven textiles (e.g., collections of fibers or yarns), and fibrous matrices, either alone or in combination. In another embodiment, patterned grids, screens, strands, or rods may be used as the reinforcing component. The reinforcing component may be positioned on the outer surface of the biocompatible membrane adjacent to the cell impermeable layer (see, e.g.  FIG. 10C ). In this orientation, the reinforcing component may be a cell impermeable and nutrient impermeable dense layer as long as there is sufficient spacing for cells to reside between the reinforcing component and the cell impermeable layer. Additionally, the reinforcing component may be oriented within or between the composite layers at discrete regions or the composite layers themselves could also be reinforcing components (see, e.g.  FIGS. 10A, 10B, and 10D ). It is to be appreciated that the reinforcing component could be located externally, internally, or within the biocompatible membrane, or combinations thereof. 
     In at least one embodiment, the cell impermeable layer, the mitigation layer, and the vascularization layer are bonded together by one or more biocompatible adhesive to form the biocompatible membrane composite. The adhesive may be applied to the surface of one or more of the cell impermeable layer, the mitigation layer, and the vascularization layer in a manner to create a discrete or intimate bond between the layers. As used herein, the phrases “discrete bond” or “discretely bonded” are meant to include bonding or bonds in intentional patterns of points and/or lines around a continuous perimeter of a defined region. Non-limiting examples of suitable biocompatible adhesives include fluorinated ethylene propylene (FEP), a polycarbonate urethane, a thermoplastic fluoropolymer comprised of TFE and PAVE, EFEP (ethylene fluorinated ethylene propylene), PEBAX (a polyether amide), PVDF (poly vinylidene fluoride), Carbosil® (a silicone polycarbonate urethane), Elasthane™ (a polyether urethane), PurSil® (a silicone polyether urethane), polyethylene, high density polyethylene (HDPE), ethylene chlorotetrafluoroethylene (ECTFE), perfluoroalkoxy (PFA), polypropylene, polyethylene terephthalate (PET), and combinations thereof. The mitigation layer may be intimately bonded to the cell impermeable layer. The vascularization layer may be intimately or discretely bonded to the mitigation layer. In at least one embodiment, the mitigation layer is intimately bonded to the cell impermeable layer. In some embodiments, the cell impermeable layer and the mitigation layer are co-expanded as a composite layer. In embodiments where the cell impermeable layer and mitigation layer or the cell impermeable layer, mitigation layer, and vascularization layer, measured composite z-strengths may be greater than 100 kPa. Additionally, the measured composite z-strength may range from about 100 kPa to about 1300 kPa, from about 100 kPa to about 1100 kPa, from about 100 kPa to about 900 kPa, from about 100 kPa to about 700 kPa, from about 100 kPa to about 500 kPa, from about 100 kPa to about 300 kPa, or from about 100 kPa to about 200 kPa. 
     At least one of the cell impermeable layer, the mitigation layer, and the vascularization layer may be formed of a polymer membrane or woven or non-woven collections of fibers or yarns, or fibrous matrices, either alone or in combination. Non-limiting examples of polymers that may be used any one or all of the cell impermeable layer, the mitigation layer, and the vascularization layer include, but are not limited to, alginate; cellulose acetate; polyalkylene glycols such as polyethylene glycol and polypropylene glycol; panvinyl polymers such as polyvinyl alcohol; chitosan; polyacrylates such as polyhydroxyethylmethacrylate; agarose; hydrolyzed polyacrylonitrile; polyacrylonitrile copolymers; polyvinyl acrylates such as polyethylene-co-acrylic acid, polyalkylenes such as polypropylene, polyethylene; polyvinylidene fluoride; fluorinated ethylene propylene (FEP); perfluoroalkoxy alkane (PFA); polyester sulfone (PES); polyurethanes; polyesters; and copolymers and combinations thereof. 
     In some embodiments, the polymer(s) forming the polymer membrane forming the cell impermeable layer, mitigation layer, and/or vascularization layer is a fibrillatable polymer. Fibrillatable, as used herein, refers to the ability to introduce fibrils to a polymer membrane, such as, but not limited to, converting portions of the solid features into fibrils. For example, the fibrils are the solid elements that span the gaps between the solid features. Fibrils are generally not resistant to deformation upon exposure to environmental forces, and are therefore deformable. The majority of deformable fibrils may have a diameter less than about 2 microns, less than about 1 micron, less than about 0.75 microns, less than about 0.50 microns, or less than about 0.25 microns. In some embodiments, the fibrils may have a diameter from about 0.25 microns to about 2 microns, from about 0.5 microns to about 2 microns, or from about 0.75 microns to about 2 microns. 
     Non-limiting examples of fibrillatable polymers that may be used to form one or more of the cell impermeable layer, the mitigation layer, and the vascularization layer include, but are not limited to, tetrafluoroethylene (TFE) polymers such as polytetrafluoroethylene (PTFE), expanded PTFE (ePTFE), modified PTFE, TFE copolymers, polyvinylidene fluoride (PVDF), poly (p-xylylene) (ePPX) as taught in U.S. Patent Publication No. 2016/0032069 to Sbriglia, porous ultra-high molecular weight polyethylene (eUHMWPE) as taught in U.S. Pat. No. 9,926,416 to Sbriglia, porous ethylene tetrafluoroethylene (eETFE) as taught in U.S. Pat. No. 9,932,429 to Sbriglia, and porous vinylidene fluoride-co-tetrafluoroethylene or trifluoroethylene [VDF-co-(TFE or TrFE)] polymers as taught in U.S. Pat. No. 9,441,088 to Sbriglia, and combinations thereof. 
     In some embodiments, the fibrillatable polymer is a fluoropolymer membrane such as an expanded polytetrafluoroethylene (ePTFE) membrane. Expanded polytetrafluoroethylene (ePTFE) membranes (and other fibrillated polymers) have a node and fibril microstructure where the nodes are interconnected by the fibrils and the pores are the space located between the nodes and fibrils throughout the membrane. As used herein, the term “node” is meant to denote a solid feature consisting largely of polymer material. When deformable fibrils are present, nodes reside at the junction of multiple fibrils. In some embodiments the fibrils may be removed from the membrane, such as, for example, by plasma etching. In at least one embodiment, an expanded polytetrafluoroethylene membrane is used in one or more of the cell impermeable layer, the mitigation layer, and the vascularization layer. Expanded polytetrafluoroethylene membranes such as, but not limited to, those prepared in accordance with the methods described in U.S. Pat. No. 3,953,566 to Gore, U.S. Pat. No. 7,306,729 to Bacino et al., U.S. Pat. No. 5,476,589 to Bacino, WO 94/13469 to Bacino, U.S. Pat. No. 5,814,405 to Branca et al. or U.S. Pat. No. 5,183,545 to Branca et al. may be used herein. 
     In some embodiments, one or more of the cell impermeable layer, the mitigation layer, and the vascularization layer is formed of a fluoropolymer membrane, such as, but not limited to, an expanded polytetrafluoroethylene (ePTFE) membrane, a modified ePTFE membrane, a tetrafluoroethylene (TFE) copolymer membrane, a polyvinylidene fluoride (PVDF) membrane, or a fluorinated ethylene propylene (FEP) membrane. In further embodiments, the vascularization layer may include biocompatible textiles, including woven and non-woven fabrics (e.g., a spunbound non-woven, melt blown fibrous materials, electrospun nanofibers, etc.), non-fluoropolymer membranes such as polyvinylidene difluoride (PVDF), nanofibers, polysulfones, polyethersulfones, polyarlysulfones, polyether ether ketone (PEEK), polyethylenes, polypropylenes, and polyimides. In some embodiments, the vascularization layer is a spunbound polyester or an expanded polytetrafluoroethylene (ePTFE) membrane. 
     In some embodiments at least one of the mitigation layer, vascularization layer or reinforcing component is formed of a non-woven fabric. There are numerous types of non-woven fabrics, each of which may vary in tightness of the weave and the thickness of the sheet. The filament cross-section may be trilobal. The non-woven fabric may be a bonded fabric, a formed fabric, or an engineered fabric that is manufactured by processes other than weaving or knitting. In some embodiments, the non-woven fabric is a porous, textile-like material, usually in flat sheet form, composed primarily or entirely of fibers, such as staple fibers assembled in a web, sheet, or batt. The structure of the non-woven fabric is based on the arrangement of, for example, staple fibers that are typically randomly arranged. In addition, non-woven fabrics can be created by a variety of techniques known in the textile industry. Various methods may create carded, wet laid, melt blown, spunbonded, or air laid non-woven materials. Non-limiting methods and substrates are described, for example, in U.S. Patent Publication No. 2010/0151575 to Colter, et al. In one embodiment, the non-woven fabric is polytetrafluoroethylene (PTFE). In another embodiment, the non-woven fabric is a spunbound polyester. The density of the non-woven fabric may be varied depending upon the processing conditions. In one embodiment, the non-woven fabric is a spunbound polyester with a basis weight from about 0.40 to about 1.00 (oz/yd 2 ) a nominal thickness of about 127 to about 228 microns and a fiber diameter of about 0.5 microns to about 26 microns. The filament cross-section is trilobal. In some embodiments, the non-woven fabrics are bioabsorbable. 
     In some embodiments, it may be desirable for one or more of the vascularization layer and reinforcing component to be non-permeant (e.g., biodegradable). In such an instance, a biodegradable material may be used to form the vascularization layer and/or the reinforcing component. Suitable examples of biodegradable materials include, but are not limited to, polyglycolide:trimethylene carbonate (PGA:TMC), polyalphahydroxy acid such as polylactic acid, polyglycolic acid, poly (glycolide), and poly(lactide-co-caprolactone), poly(caprolactone), poly(carbonates), poly(dioxanone), poly (hydroxybutyrates), poly(hydroxyvalerates), poly (hydroxybutyrates-co-valerates), expanded polyparaxylylene (ePLLA), such as is taught in U.S. Patent Publication No. 2016/0032069 to Sbriglia, and copolymers and blends thereof. Alternatively, the vascularization layer may be coated with a bio-absorbable material or a bio-absorbable material may be incorporated into or onto the vascularization layer in the form of a powder. Coated materials may promote infection site reduction, vascularization, and favorable type 1 collagen deposition. 
     The biocompatible membrane composite may have at least partially thereon a surface coating, such as a Zwitterion non-fouling coating, a hydrophilic coating, or a CBAS®/Heparin coating (commercially available from W.L. Gore &amp; Associates, Inc.). The surface coating may also or alternatively contain antimicrobial agents, antibodies (e.g., anti-CD 47 antibodies (anti-fibrotic)), pharmaceuticals, and other biologically active molecules (e.g., stimulators of vascularization such as FGF, VEGF, endoglin, PDGF, angiopoetins, and integrins; Anti-fibrotic such as TGFb inhibitors, sirolimus, CSF1R inhibitors, and anti CD 47 antibody; anti-inflammatory/immune modulators such as CXCL12, and corticosteroids), and combinations thereof. 
     In some embodiments, the solid features of one or both of the mitigation layer and the vascularization layer may be formed by microlithography, micro-molding, machining, or printing (or otherwise laying down) a polymer (e.g., thermoplastic) onto a cell impermeable layer to form at least a part of a solid feature. Any conventional printing technique such as transfer coating, screen printing, gravure printing, ink-jet printing, patterned imbibing, and knife coating may be utilized to place the thermoplastic polymer onto the cell impermeable layer.  FIG. 6A  illustrates a thermoplastic polymer in the form of solid features  620  positioned on a cell impermeable layer  610  (after printing is complete), where the solid features  620  have a feature spacing  630 . Non-limiting examples of geometries for forming the solid features include, but are not limited to, dashed lines (see  FIG. 6B ), dots and/or dotted lines (see  FIGS. 6C, 6G ), geometric shapes (see  FIG. 6H ), straight lines (see  FIG. 6D ), angled lines (see  FIG. 6F ), curved lines, grids (see  FIG. 6E ), etc., and combinations thereof. 
     Materials used to form the solid features of the mitigation layer include, but are not limited to, polyurethane, polypropylene, polyethylene, polyether amide, polyetheretherketone, polyphenylsulfone, polysulfone, silicone polycarbonate urethane, polyether urethane, polycarbonate urethane, silicone polyether urethane, polyester, polyester terephthalate, melt-processable fluoropolymers, such as, for example, fluorinated ethylene propylene (FEP), tetrafluoroethylene-(perfluoroalkyl) vinyl ether (PFA), an alternating copolymer of ethylene and tetrafluoroethylene (ETFE), a terpolymer of tetrafluoroethylene (TFE), hexafluoropropylene (HFP) and vinylidene fluoride (THV), polyvinylidene fluoride (PVDF), and combinations thereof. In some embodiments, polytetrafluoroethylene may be used to form the pattern features. In further embodiments, the solid features may be separately formed and adhered to the surface of the cell impermeable layer (not illustrated). 
     Biocompatible membrane composite  700  is depicted in  FIG. 7 , which includes a cell impermeable layer  710 , a mitigation layer  720 , a vascularization layer  730 , and the optional reinforcement layer  740 . In the depicted embodiment, the solid features  750  are bonded to the surface of the cell impermeable layer  710  to form bonded features within the mitigation layer  720 . In some embodiments, the solid features  750  do not penetrate into the pores of the vascularization layer  730 . The solid features  750  are depicted in  FIG. 7  as being essentially the same height and width and extending between the cell impermeable layer  710  and the vascularization layer  730 , although it is to be appreciated that this an example and the solid features  750  may vary in height and/or width. The distance between solid features  750  is the solid feature spacing  760 . 
       FIG. 8  is another biocompatible membrane composite  800  that includes a cell impermeable layer  810 , a mitigation layer  820 , a vascularization layer  830 , and the optional reinforcement layer  840 . In the depicted biocompatible membrane composite, the solid features  850 ,  880  are nodes that differ in height and width, and may or may not extend the distance between the cell impermeable layer  810  and the vascularization layer  830 . The solid features  850 ,  880  are connected by fibrils  870 . In  FIG. 8 , the majority of the solid feature depth is less than the total thickness of the mitigation layer  820 . Solid features  880  are bonded solid features. 
     Turning to  FIG. 9 , an biocompatible membrane composite  900  containing a cell impermeable layer  910 , a mitigation layer  920 , a vascularization layer  930 , and an optional reinforcement layer  940  is depicted. In this embodiment, solid features within the mitigation layer  920  are the nodes of a mitigation layer  920  that are formed in an ePTFE membrane. The nodes  950 ,  980  are interconnected by fibrils  970 . Nodes  950 ,  980  are positioned within the mitigation layer  920 . Nodes  980 , however, are not only within the mitigation layer  920 , but are also in contact with, and are intimately bonded to, the cell impermeable layer  910 . 
     As discussed above, the reinforcing component may be oriented within or between the composite layers at discrete regions. In one non-limiting embodiment shown in  FIG. 10A , the reinforcing component  1030  is formed as discrete regions on the cell impermeable layer  1000  and are positioned within the mitigation layer  1010  of the biocompatible membrane composite  1050 . The vascularization layer  1020  is shown for reference only. In the embodiment depicted in  FIG. 10B , the reinforcing component  1030  is positioned on the mitigation layer  1010  as discrete regions and are positioned within the vascularization layer  1020  of the biocompatible membrane composite  1050 . The cell impermeable layer  1000  is shown for reference only. In yet another non-limiting embodiment depicted in  FIG. 10C , the reinforcing component  1030  is external to the biocompatible membrane composite  1050 . Specifically, the reinforcing component  1030  is positioned on a side of the cell impermeable layer  1000  opposing the mitigation layer  1010 . The vascularization layer  1020  is shown for reference only. Turning to  FIG. 10D , the reinforcing component  1030  is located between the mitigation layer  1010  and the vascularization layer  1020  of the biocompatible membrane composite  1050 . The cell impermeable layer  1000  is shown for reference only. 
     In the embodiments described herein, the mitigation layer  1100  may be formed by placing or otherwise depositing a polymer in a pattern (as described above) which is characterized by one or more of the following: the solid feature size (i.e., minor axis)  1110 , solid feature spacing  1120 , solid feature depth  1160 , thickness  1130 , the absence of fibrils and/or the pore size (as measured by quantitative image analysis (QIA) performed on an SEM image), as depicted generally in  FIG. 11A . A cell impermeable layer  1150  is shown for reference only. 
       FIG. 11B  depicts a mitigation layer  1200  that is formed of a polymer having a node and fibril microstructure that is characterized by one or more of the following: the solid feature size (i.e., minor axis)  1210 , solid feature spacing  1220 , solid feature depth  1270 , thickness  1230 , the presence of fibrils  1260 , and/or the pore size (as measured by quantitative image analysis (QIA) performed on an SEM image)  1240 . A cell impermeable layer  1250  is shown in  FIG. 11B  for reference only. 
     Also, in the embodiments described herein, the vascularization layer  1300  may be characterized by one or more of the following: thickness  1310 , pore size  1320 , solid feature size (i.e., minor axis)  1340 , and solid feature spacing  1330  as depicted generally in  FIG. 12 . A cell impermeable layer  1350  and a mitigation layer  1360  are shown for reference only. 
     The biocompatible membrane composite can be manufactured into various forms including, but not limited to, a housing, a chamber, a pouch, a tube, or a cover. In one embodiment, the biocompatible membrane composite forms a cell encapsulating device as illustrated in  FIG. 13A .  FIG. 13A  is a top view of a cell encapsulating device  1400  formed of two layers of the biocompatible membrane composite that are sealed along a portion of their periphery  1410 . Only the outer layer of the biocompatible membrane composite  1420  is shown in  FIG. 13A . The cell encapsulating device  1400  includes an internal chamber (not shown) for containing cells and a port  1430  that extends into the internal chamber and is in fluid communication therewith. 
       FIG. 13B  is a cross-sectional illustration of the cell encapsulation device of  FIG. 13A . As shown, a first biocompatible membrane composite  1450  is positioned adjacent to a second biocompatible membrane composite  1460 . The biocompatible membrane composites  1450 ,  1460  each include a cell impermeable layer  1470 , a mitigation layer  1480 , and a vascularization layer  1490 . The optional reinforcing component is not depicted in  FIG. 13B , although it could be utilized in this embodiment. A chamber  1435  (i.e., lumen) is located between the two membrane composites  1450 ,  1460  for the placement of cells (and/or other biological entities). 
     Having generally described this disclosure, a further understanding can be obtained by reference to certain specific examples illustrated below which are provided for purposes of illustration only and are not intended to be all inclusive or limiting unless otherwise specified. 
     Test Methods 
     Porosity 
     The porosity of a layer is defined herein as the proportion of layer volume consisting of pore space compared to the total volume of the layer. The porosity is calculated by comparing the bulk density of a porous construct consisting of solid fraction and void fraction to the density of the solid fraction using the following equation: 
     
       
         
           
             Porosity 
             = 
             
               
                 ( 
                 
                   1 
                   - 
                   
                     
                       Density 
                       Bulk 
                     
                     
                       Density 
                       
                         Solid 
                         ⁢ 
                             
                         Fraction 
                       
                     
                   
                 
                 ) 
               
               × 
               1 
               ⁢ 
               0 
               ⁢ 
               0 
               ⁢ 
               % 
             
           
         
       
     
     Thickness 
     The thickness of the layers in the composites was measured by quantitative image analysis (QIA) of cross-sectional SEM images. Cross-sectional SEM images were generated by fixing membranes to an adhesive, cutting the film by hand using a liquid-nitrogen-cooled razor blade, and then standing the adhesive backed film on end such that the cross-section was vertical. The sample was then sputter coated using an Emitech K550X sputter coater (commercially available from Quorum Technologies Ltd, UK) and platinum target. The sample was then imaged using a FEI Quanta 400 scanning electron microscope from Thermo Scientific. 
     Layers within the cross-section SEM images were then measured for thickness using ImageJ 1.51 h from the National Institutes of Health (NIH). The image scale was set per the scale provided by the SEM. The layer of interest was isolated and cropped using the free-hand tool. A number of at least ten equally spaced lines were then drawn in the direction of the layer thickness. The lengths of all lines were measured and averaged to define the layer thickness. 
     Maximum Tensile Load 
     Materials were tested for maximum tensile load using a 5500 Series Instron® Electromechanical Testing System. Samples were cut oriented in the axis of interest using a D412F or D638-V dogbone die. The samples were then loaded into the Instron® tester grips and tested at a constant rate of 20 in/min (for D412F samples) or 3 in/min (for D683-V samples) until failure. The maximum load sustained during testing was normalized by specimen gauge width (6.35 mm for D412F samples and 3.175 mm for D638-V samples) to define maximum tensile load. 
     Tensile Strength 
     Materials were tested for tensile strength using a 5500 Series Instron® Electromechanical Testing System. Unless otherwise noted, materials were testing for tensile strength prior to the application of any coatings. Samples were cut using a D412F or D638-V dogbone die. The samples were then loaded into the Instron® tester grips and tested at a constant rate of 20 in/min (for D412F samples) or 3 in/min (for D683-V samples) until failure. Maximum load was normalized by test area (defined as gauge width times material thickness) to define tensile stress. Materials were tested in perpendicular directions (D1 and D2) and the maximum stress in each direction was used to calculate the geometric mean tensile strength of the material per the below equation: 
     
       
         
           
             
               Geometric 
               ⁢ 
                   
               Mean 
             
             = 
             
               
                 
                   
                     
                       ( 
                       
                         Tensile 
                         ⁢ 
                             
                         
                           Strength 
                           
                             D 
                             ⁢ 
                             1 
                           
                         
                       
                       ) 
                     
                     2 
                   
                   + 
                   
                     
                       ( 
                       
                         Tensile 
                         ⁢ 
                             
                         
                           Strength 
                           
                             D 
                             ⁢ 
                             2 
                           
                         
                       
                       ) 
                     
                     2 
                   
                 
               
               . 
             
           
         
       
     
     Composite Bond Strength (Z-Strength) 
     Materials were tested for composite bond strength using a 5500 Series Instron® Electromechanical Testing System. Unless otherwise noted, materials were testing for tensile strength prior to the application of any coatings. Samples were fixed to a 1″×1″ steel platen using 3M 9500PC double sided tape and loaded into the Instron® with an opposing 1″×1″ steel platen with 3M 9500PC double sided tape on its surface. A characteristic compressive load of 1001 N was applied for 60 s to allow adhesive to partially penetrate the structure. After this bonding, the platens were separated at a constant rate of 20 in/s until failure. The maximum load was normalized by the test area (defined as the 1″×1″ test area) to define the composite bond. 
     Mass/Area 
     Samples were cut (either by hand, laser, or die) to a known geometry. Unless otherwise noted, materials were testing for tensile strength prior to the application of any coatings. The dimensions of the sample were measured or verified and the area was calculated in m 2 . The sample was then weighed in grams on a calibrated scale. The mass in grams was divided by the area in m 2  to calculate the mass per area in g/m 2 . 
     SEM Sample Preparation 
     SEM samples were prepared by first fixing the membrane composite or membrane composite layer(s) to an adhesive for handling, with the side opposite the side intended for imaging facing the adhesive. The film was then cut to provide an approximately 3 mm×3 mm area for imaging. The sample was then sputter coated using an Emitech K550X sputter coater and platinum target. Images were then taken using a FEI Quanta 400 scanning electron microscope from Thermo Scientific at a magnificent and resolution that allowed visualization of a sufficient number of features for robust analysis while ensuring each analyzed feature&#39;s minimum dimension was at least five pixels in length. 
     Solid Feature Spacing 
     Solid feature spacing was determined by analyzing SEM images in ImageJ 1.51 h from the National Institute of Health (NIH). The image scale was set based on the scale provided by the SEM image. Features were identified and isolated through a combination of thresholding based on size/shading and/or manual identification. In instances where the structure consists of a continuous structure, such as a nonwoven or etched surface, as opposed to a structure with discrete solid features, solid features are defined as the portion of the structure surrounding voids the their corresponding spacing extending from one side of the void to the opposing side. After isolating the features, a Delaunay Triangulation was performed to identify neighboring features. Triangulations whose circumcircle extended beyond the edge of the image were disregarded from the analysis. Lines were drawn between the nearest edges of neighboring features and measured for length to define spacing between neighboring features (see, e.g.,  FIG. 1A ). The median of all measured solid feature spacings marks the value that is less than or equal to half of the measured solid feature spacings and greater than or equal to half of the measured solid feature spacings. Therefore, if the measured median is above or below some value, the majority of measurements is similarly above or below the value. As such, the median is used as summary statistic to represent the majority of solid feature spacings. 
     Measurement of Representative Minor Axis and Representative Major Axis 
     The representative minor axis was measured by analyzing SEM images of membrane surfaces in ImageJ 1.51 h from the NIH. The image scale was set based on the scale provided by the SEM image. Features were identified and isolated through a combination of thresholding based on size/shading and/or manual identification. After isolating the features, the built in particle analysis capabilities were leveraged to determine the major and minor axis of the representative ellipse. The minor axis of this ellipse is the representative minor axis of the measured feature. The major axis of this ellipse is the representative major axis of the measured feature. The median of all measured minor axes marks the value that is less than or equal to half of the measured minor axes and greater than or equal to half of the measured minor axes. Similarly, the median of all measured major axes marks the value that is less than or equal to half of the measured major axes and greater than or equal to half of the measured major axes. In both cases, if the measured median is above or below some value, the majority of measurements is similarly above or below the value. As such, the median is used as summary statistic to represent the majority of solid feature representative minor axes and representative major axes. 
     Solid Feature Depth 
     Solid feature depth was determined by using quantitative image analysis (QIA) of SEM images of membrane cross-sections. Cross-sectional SEM images were generated by fixing films to an adhesive, cutting the film by hand using a liquid-nitrogen-cooled razor blade, and then standing the adhesive backed film on end such that the cross-section was vertical. The sample was then sputter coated using an Emitech K550X sputter coater (commercially available from Quorum Technologies Ltd, UK) and platinum target. The sample was then imaged using a FEI Quanta 400 scanning electron microscope from Thermo Scientific. 
     Features within the cross-section SEM images were then measured for depth using ImageJ 1.51 h from the National Institutes of Health (NIH). The image scale was set per the scale provided by the SEM. Features were identified and isolated through a combination of thresholding based on size/shading and/or manual identification. After isolating features, built in particle analysis capabilities were leveraged to calculate the Feret diameter and angle formed by the axis defined by the Feret diameter axis and horizontal plane for each solid feature. The Feret diameter is the furthest distance between any two points on a feature&#39;s boundary in the plane of the SEM image. The Feret diameter axis is the line defined by these two points. The projection of the Feret diameter of each solid feature in the direction of the layer thickness was calculated per the equation: 
       Projection Thickness =sin θ*Length Longest Axis .
 
     The projection of the longest axis in the direction of the layer thickness is the solid feature depth of the measured feature. The median of all measured solid feature depths marks the value that is less than or equal to half of the measured solid feature depths and greater than or equal to half of the measured solid feature depths. Therefore, if the measured median is above or below some value, the majority of measurements is similarly above or below the value As such, the median is used as summary statistic to represent the majority of solid feature depths. 
     Pore Size 
     The pore size was measured by analyzing SEM images of membrane surfaces in ImageJ 1.51 h from the NIH. The image scale was set based on the scale provided by the SEM image. Pores were identified and isolated through a combination of thresholding based on size/shading and/or manual identification. After isolating the pores, the built in particle analysis capabilities were leveraged to determine the area of each pore. The measured pore area was converted to an “effective diameter” per the below equation: 
     
       
         
           
             
               Effective 
               ⁢ 
                   
               Diameter 
             
             = 
             
               2 
               × 
               
                 
                   Area 
                   π 
                 
               
             
           
         
       
     
     The pore areas were summed to define the total area of the surface defined by pores. This is the total pore area of the surface. The pore size of a layer is the effective diameter of the pore that defines the point where roughly half the total pore area consists of pores with diameters smaller than the pore size and roughly half the total pore area consists of pores with diameters greater than or equal to the pore size. 
     MPS (Maximum Pore Size) 
     Maximum Pore Size or MPS was measured per ASTM F316 using a Quantachrome 3 Gzh porometer from Anton Paar and silicone oil (20.1 dyne/cm) as a wetting solution. 
     Stiffness 
     A stiffness test was performed based on ASTM D790-17 Standard test method for flexural properties of unreinforced and reinforced plastics and electrical insulating material. This method was used to determine the stiffness for biocompatible membrane composite layers and/or the final device. 
     Procedure B of the ASTM method was followed and includes greater than 5% strain and type 1 crosshead position for deflection. The dimensions of the fixture were adjusted to have a span of 16 mm and a radius of support and nosepiece of 1.6 mm. The test parameters used were a deflection of 3.14 mm and a test speed of 96.8 mm/min. In cases where the sample width differed from the standard 1 cm, the force was normalized to a 1 cm sample width by the linear ratio. 
     The load was reported in N/cm at maximum deflection. 
     Integration of Biocompatible Membrane Composite into a Device Form 
     In order to evaluate the in vivo utility, various biocompatible membrane composites were manufactured into a device form suitable for use as an implantable encapsulation device for the delivery of a cell therapy. In this test form, two identical membrane composites were sealed around a perimeter region to form an open internal lumen space accessed by a fill tube or port to enable the loading of cells. 
     A thermoplastic film acted as the bonding component that created the perimeter seal around the device during the welding operation. The film used was a polycarbonate urethane film. The extruded tube had an outer diameter of 1.60 mm and an inner diameter of 0.889 mm. 
     Additionally, a reinforcing mechanical support having a suitable stiffness was added to the exterior of the encapsulation device. In particular, a polyester monofilament woven mesh with 120 microns fibers spaced approximately 300 microns from each other was positioned on the outside of both composite membranes (i.e., the exterior of the device). The stiffness of this layer was 0.097 N/cm. 
     All layers were cut to an approximate 22 mm×11 mm oval outer dimension size using a laser cutting table. The film was cut into oval ring profiles with a 2 mm width and placed in an intercalating stack up pattern on both sides of the biocompatible membrane composite as well as around the polyester mesh (reinforcing component). This intercalating stack-up pattern of the components allowed for a melted film bond around each of the composite layers as well as the mesh at a perimeter location. The layers of the biocompatible membrane composite were stacked symmetrically opposing the filling tube such that the cell impermeable tight layer of the biocompatible membrane composite was facing internally towards the inner lumen. An exploded view of the encapsulation device is shown in  FIG. 16 . As shown in  FIG. 16 , the cell encapsulation device is formed a first biocompatible membrane composite  1600  sealed along a portion of its periphery to a second biocompatible membrane composite  1610  along a portion of its periphery. An inner chamber is formed between the two biocompatible membranes  1600 ,  1610  with access through a filling tube  1630 . The cell encapsulation device may further include at least one weld film  1640  positioned at least between the first biocompatible membrane composite  1600  and a reinforcing component  1650  and between the second biocompatible membrane composite  1610  and another reinforcing component  1650 . A weld film  1640  may also be used to adhere the first biocompatible membrane composite to the second biocompatible membrane composite around the peripheries thereof. 
     An integral perimeter seal around the device was formed by using either an ultrasonic welder (Herrmann Ultrasonics) or a thermal staking welder. With both processes, thermal or vibrational energy and force was applied to the layered stack to melt and flow the thermoplastic film above its softening temperature to weld all the layers together. The device was constructed in a two step welding process where the energy or heat was applied from one side such that the first composite membrane was integrated into one side of the device followed by the second composite membrane onto the opposing side of the device. The final suitability of the weld was assessed by testing the device for integrity using a pressure decay test with a USON Sprint iQ Leak Tester at a test pressure of 5 psi. 
     In Vivo Porcine Study to Evaluate Host Tissue Response 
     Sterilized, empty encapsulation devices (i.e., no cells) were sealed at the fill tube using a radio frequency (RF) welder and implanted subcutaneously in the dorsum of swine using a trocar delivery technique. After 30 days, the animals were euthanized and devices with surrounding tissue were retrieved for histological imaging. 
     The tissue samples were processed such that the skin and subcutaneous tissue were reflected to expose the implanted encapsulation devices. The devices were identified using digital radiography (Faxitron UltraFocus System) when needed prior to removing the encapsulation device and surrounding tissue en bloc. Device orientation was marked with staples. All explanted devices and surrounding tissue were immersed in 10% neutral buffered formalin. Each device specimen was assigned a unique accession number. 
     Three cross-sections were taken from each specimen. The three sections from each device were embedded together in paraffin, cut into 5-10 microns thick sections, placed on a slide and stained with hematoxylin and eosin (H&amp;E) and Masson&#39;s Trichrome. 
     Images of the slide were captured using a Nikon DS-Fi Series camera and Nikon NIS Elements Microscope Imaging software. At least three magnification images of each slide were captured. Measurements were taken using the Nikon NIS Elements Microscope Imaging software which is calibrated using a certified microscope micrometer and scale bars are included on each image. 
     In Vitro Production of Human PDX1-Positive Pancreatic Endoderm and Endocrine Cells 
     The directed differentiation methods herein for pluripotent stem cells, for example, hES and iPS cells, can be described into at least four or five or six or seven stages, depending on end-stage cell culture or cell population desired (e.g. PDX1-positive pancreatic endoderm cell population (or PEC), or endocrine precursor cell population, or endocrine cell population, or immature beta cell population or mature endocrine cell population). 
     Stage 1 is the production of definitive endoderm from pluripotent stem cells and takes about 2 to 5 days, preferably 2 or 3 days. Pluripotent stem cells are suspended in media comprising RPMI, a TGFβ superfamily member growth factor, such as Activin A, Activin B, GDF-8 or GDF-11 (100 ng/mL), a Wnt family member or Wnt pathway activator, such as Wnt3a (25 ng/mL), and alternatively a rho-kinase or ROCK inhibitor, such as Y-27632 (10 μM) to enhance growth, and/or survival and/or proliferation, and/or cell-cell adhesion. After about 24 hours, the media is exchanged for media comprising RPMI with serum, such as 0.2% FBS, and a TGFβ superfamily member growth factor, such as Activin A, Activin B, GDF-8 or GDF-11 (100 ng/mL), and alternatively a rho-kinase or ROCK inhibitor for another 24 (day 1) to 48 hours (day 2). Alternatively, after about 24 hours in a medium comprising Activin/Wnt3a, the cells are cultured during the subsequent 24 hours in a medium comprising Activin alone (i.e., the medium does not include Wnt3a). Importantly, production of definitive endoderm requires cell culture conditions low in serum content and thereby low in insulin or insulin-like growth factor content. See McLean et al. (2007)  Stem Cells  25: 29-38. McLean et al. also show that contacting hES cells with insulin in concentrations as little as 0.2 μg/mL at Stage 1 can be detrimental to the production of definitive endoderm. Still others skilled in the art have modified the Stage 1 differentiation of pluripotent cells to definitive endoderm substantially as described here and in D&#39;Amour et al. (2005), for example, at least, Agarwal et al., Efficient Differentiation of Functional Hepatocytes from Human Embryonic Stem Cells,  Stem Cells  (2008) 26:1117-1127; Borowiak et al., Small Molecules Efficiently Direct Endodermal Differentiation of Mouse and Human Embryonic Stem Cells, (2009)  Cell Stem Cell  4:348-358; Brunner et al., Distinct DNA methylation patterns characterize differentiated human embryonic stem cells and developing human fetal liver, (2009)  Genome Res.  19:1044-1056, Rezania et al. Reversal of Diabetes with Insulin-producing Cells Derived In Vitro from Human Pluripotent Stem Cells (2014)  Nat Biotech  32(11): 1121-1133 (GDF8 &amp; GSK3beta inhibitor, e.g. CHIR99021); and Pagliuca et al. (2014) Generation of Function Human Pancreatic B-cell In Vitro,  Cell  159: 428-439 (Activin A &amp; CHIR) Proper differentiation, specification, characterization and identification of definitive are necessary in order to derive other endoderm-lineage cells. Definitive endoderm cells at this stage co-express SOX17 and HNF3f3 (FOXA2) and do not appreciably express at least HNF4alpha, HNF6, PDX1, SOX6, PROX1, PTF1A, CPA, cMYC, NKX6.1, NGN3, PAX3, ARX, NKX2.2, INS, GSC, GHRL, SST, or PP. The absence of HNF4alpha expression in definitive endoderm is supported and described in detail in at least Duncan et al. (1994), Expression of transcription factor HNF-4 in the extraembryonic endoderm, gut, and nephrogenic tissue of the developing mouse embryo: HNF-4 is a marker for primary endoderm in the implanting blastocyst,”  Proc. Natl. Acad. Sci,  91:7598-7602 and Si-Tayeb et al. (2010), Highly Efficient Generation of Human Hepatocyte-Like cells from Induced Pluripotent Stem Cells,”  Hepatology  51:297-305. 
     Stage 2 takes the definitive endoderm cell culture from Stage 1 and produces foregut endoderm or PDX1-negative foregut endoderm by incubating the suspension cultures with RPMI with low serum levels, such as 0.2% FBS, in a 1:1000 dilution of ITS, 25 ng KGF (or FGF7), and alternatively a ROCK inhibitor for 24 hours (day 2 to day 3). After 24 hours (day 3 to day 4), the media is exchanged for the same media minus a TGFβ inhibitor, but alternatively still a ROCK inhibitor to enhance growth, survival and proliferation of the cells, for another 24 (day 4 to day 5) to 48 hours (day 6). A critical step for proper specification of foregut endoderm is removal of TGFβ family growth factors. Hence, a TGFβ inhibitor can be added to Stage 2 cell cultures, such as 2.5 μM TGFβ inhibitor no. 4 or 5 μM SB431542, a specific inhibitor of activin receptor-like kinase (ALK), which is a TGFβ type I receptor. Foregut endoderm or PDX1-negative foregut endoderm cells produced from Stage 2 co-express SOX17, HNF1p and HNF4alpha and do not appreciably co-express at least SOX17 and HNF3p (FOXA2), nor HNF6, PDX1, SOX6, PROX1, PTF1A, CPA, cMYC, NKX6.1, NGN3, PAX3, ARX, NKX2.2, INS, GSC, GHRL, SST, or PP, which are hallmark of definitive endoderm, PDX1-positive pancreatic endoderm or pancreatic progenitor cells or endocrine progenitor/precursors as well as typically poly hormonal type cells. 
     Stage 3 (days 5-8) for PEC production takes the foregut endoderm cell culture from Stage 2 and produces a PDX1-positive foregut endoderm cell by DMEM or RPMI in 1% B27, 0.25 μM KAAD cyclopamine, a retinoid, such as 0.2 retinoic acid (RA) or a retinoic acid analog such as 3 nM of TTNPB (or CTT3, which is the combination of KAAD cyclopamine and TTNPB), and 50 ng/mL of Noggin for about 24 (day 7) to 48 hours (day 8). Specifically, Applicants have used DMEM-high glucose since about 2003 and all patent and non-patent disclosures as of that time employed DMEM-high glucose, even if not mentioned as “DMEM-high glucose” and the like. This is, in part, because manufacturers such as Gibco did not name their DMEM as such, e.g. DMEM (Cat. No 11960) and Knockout DMEM (Cat. No 10829). It is noteworthy, that as of the filing date of this application, Gibco offers more DMEM products but still does not put “high glucose” in certain of their DMEM products that contain high glucose e.g. Knockout DMEM (Cat. No. 10829-018). Thus, it can be assumed that in each instance DMEM is described, it is meant DMEM with high glucose and this was apparent by others doing research and development in this field. Again, a ROCK inhibitor or rho-kinase inhibitor such as Y-27632 can be used to enhance growth, survival, proliferation and promote cell-cell adhesion. Additional agents and factors include but are not limited to ascorbic acid (e.g. Vitamin C), BMP inhibitor (e.g. Noggin, LDN, Chordin), SHH inhibitor (e.g. SANT, cyclopamine, HIP1); and/or PKC activator (e.g. PdBu, TBP, ILV) or any combination thereof. 
     Alternatively, Stage 3 has been performed without an SHH inhibitor such as cyclopamine in Stage 3. PDX1-positive foregut cells produced from Stage 3 co-express PDX1 and HNF6 as well as SOX9 and PROX, and do not appreciably co-express markers indicative of definitive endoderm or foregut endoderm (PDX1-negative foregut endoderm) cells or PDX1-positive foregut endoderm cells as described above in Stages 1 and 2. 
     The above stage 3 method is one of four stages for the production of PEC populations. For the production of endocrine progenitor/precursor and endocrine cells as described in detail below, in addition to Noggin, KAAD-cyclopamine and Retinoid; Activin, Wnt and Heregulin, thyroid hormone, TGFb-receptor inhibitors, Protein kinase C activators, Vitamin C, and ROCK inhibitors, alone and/or combined, are used to suppress the early expression NGN3 and increasing CHGA-negative type of cells. 
     Stage 4 (about days 8-14) PEC culture production takes the media from Stage 3 and exchanges it for media containing DMEM in 1% vol/vol B27 supplement, plus 50 ng/mL KGF and 50 ng/mL of EGF and sometimes also 50 ng/mL Noggin and a ROCK inhibitor and further includes Activin alone or combined with Heregulin. Alternatively, Stage 3 cells can be further differentiated using KGF, RA, SANT, PKC activator and/or Vitamin C or any combination thereof. These methods give rise to pancreatic progenitor cells co-expressing at least PDX1 and NKX6.1 as well as PTF1A. These cells do not appreciably express markers indicative of definitive endoderm or foregut endoderm (PDX1-negative foregut endoderm) cells as described above in Stages 1, 2 and 3. 
     Stage 5 production takes Stage 4 PEC cell populations above and further differentiates them to produce endocrine progenitor/precursor or progenitor type cells and/or singly and poly-hormonal pancreatic endocrine type cells in a medium containing DMEM with 1% vol/vol B27 supplement, Noggin, KGF, EGF, RO (a gamma secretase inhibitor), nicotinamide and/or ALK5 inhibitor, or any combination thereof, e.g. Noggin and ALK5 inhibitor, for about 1 to 6 days (preferably about 2 days, i.e. days 13-15). Alternatively, Stage 4 cells can be further differentiated using retinoic acid (e.g. RA or an analog thereof), thyroid hormone (e.g. T3, T4 or an analogue thereof), TGFb receptor inhibitor (ALK5 inhibitor), BMP inhibitor (e.g. Noggin, Chordin, LDN), or gamma secretase inhibitor (e.g., XXI, XX, DAPT, XVI, L685458), and/or betacellulin, or any combination thereof. Endocrine progenitor/precursors produced from Stage 5 co-express at least PDX1/NKX6.1 and also express CHGA, NGN3 and Nkx2.2, and do not appreciably express markers indicative of definitive endoderm or foregut endoderm (PDX1-negative foregut endoderm) as described above in Stages 1, 2, 3 and 4 for PEC production. 
     Stage 6 and 7 can be further differentiated from Stage 5 cell populations by adding any of a combination of agents or factors including but not limited to PDGF+SSH inhibitor (e.g. SANT, cyclopamine, HIP1), BMP inhibitor (e.g. Noggin, Chordin, LDN), nicotinamide, insulin-like growth factor (e.g. IGF1, IGF2), TTNBP, ROCK inhibitor (e.g. Y27632), TGFb receptor inhibitor (e.g. ALK5i), thyroid hormone (e.g. T3, T4 and analogues thereof), and/or a gamma secretase inhibitor (XXI, XX, DAPT, XVI, L685458) or any combination thereof to achieve the cell culture populations or appropriate ratios of endocrine cells, endocrine precursors and immature beta cells. 
     Stage 7 or immature beta cells are considered endocrine cells but may or may not me sufficiently mature to respond to glucose in a physiological manner. Stage 7 immature beta cells may express MAFB, whereas MAFA and MAFB expressing cells are fully mature cells capable of responding to glucose in a physiological manner. 
     Stages 1 through 7 cell populations are derived from human pluripotent stem cells (e.g. human embryonic stem cells, induced pluripotent stem cells, genetically modified stem cells e.g. using any of the gene editing tools and applications now available or later developed) and may not have their exact naturally occurring corresponding cell types since they were derived from immortal human pluripotent stem cells generated in vitro (i.e. in an artificial tissue culture) and not the inner cell mass in vivo (i.e. in vivo human development does not have an human ES cell equivalent). 
     Pancreatic cell therapy replacements as intended herein can be encapsulated in the described herein devices consisting of herein described membranes using any of Stages 4, 5, 6 or 7 cell populations and are loaded and wholly contained in a macro-encapsulation device and transplanted in a patient, and the pancreatic endoderm lineage cells mature into pancreatic hormone secreting cells, or pancreatic islets, e.g., insulin secreting beta cells, in vivo (also referred to as “in vivo function”) and are capable of responding to blood glucose normally. 
     Encapsulation of the pancreatic endoderm lineage cells and production of insulin in vivo is described in detail in U.S. application Ser. No. 12/618,659 (the &#39;659 application), entitled ENCAPSULATION OF PANCREATIC LINEAGE CELLS DERIVED FROM HUMAN PLURIPOTENT STEM CELLS, filed Nov. 13, 2009. The &#39;659 application claims the benefit of priority to Provisional Patent Application No. 61/114,857, entitled ENCAPSULATION OF PANCREATIC PROGENITORS DERIVED FROM HES CELLS, filed Nov. 14, 2008; and U.S. Provisional Patent Application No. 61/121,084, entitled ENCAPSULATION OF PANCREATIC ENDODERM CELLS, filed Dec. 9, 2008; and now U.S. Pat. Nos. 8,278,106 and 8,424,928. The methods, compositions and devices described herein are presently representative of preferred embodiments and are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention and are defined by the scope of the disclosure. Accordingly, it will be apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. 
     Additionally, embodiments described herein are not limited to any one type of pluripotent stem cell or human pluripotent stem cell and include but are not limited to human embryonic stem (hES) cells and human induced pluripotent stem (iPS) cells or other pluripotent stem cells later developed. It is also well known in the art, that as of the filing of this application, methods for making human pluripotent stems may be performed without destruction of a human embryo and that such methods are anticipated for production of any human pluripotent stem cell. 
     Methods for producing pancreatic cell lineages from human pluripotent cells were conducted substantially as described in at least the listed publications assigned to ViaCyte, Inc. including but not limited to: PCT/US2007/62755 (WO2007101130), PCT/US2008/80516 (WO2009052505), PCT/US2008/82356 (WO2010053472), PCT/US2005/28829 (WO2006020919), PCT/US2014/34425 (WO2015160348), PCT/US2014/60306 (WO2016080943), PCT/US2016/61442 (WO2018089011), PCT/US2014/15156 (WO2014124172), PCT/US2014/22109 (WO2014138691), PCT/US2014/22065 (WO2014138671), PCT/US2005/14239 (WO2005116073), PCT/US2004/43696 (WO2005063971), PCT/US2005/24161 (WO2006017134), PCT/US2006/42413 (WO2007051038), PCT/US2007/15536 (WO2008013664), PCT/US2007/05541 (WO2007103282), PCT/US2008/61053 (WO2009131568), PCT/US2008/65686 (WO2009154606), PCT/US2014/15156 (WO2014124172), PCT/US2018/41648 (WO2019014351), PCT/US2014/26529 (WO2014160413), PCT/US2009/64459 (WO2010057039); and d&#39;Amour et al. 2005 Nature Biotechnology 23:1534-41; D&#39;Amour et al. 2006 Nature Biotechnology 24(11):1392-401; McLean et al., 2007 Stem Cells 25:29-38, Kroon et al. 2008 Nature Biotechnology 26(4): 443-452, Kelly et al. 2011 Nature Biotechnology 29(8): 750-756, Schulz et al., 2012 PLos One 7(5):e37004, and/or Agulnick et al. 2015 Stem Cells Transl. Med. 4(10):1214-22. 
     Methods for producing pancreatic cell lineages from human pluripotent cells were conducted substantially as described in at least the listed below publications assigned to Janssen including but not limited to: PCT/US2008/68782 (WO200906399), PCT/US2008/71775 (WO200948675), PCT/US2008/71782 (WO200918453), PCT/US2008/84705 (WO200970592), PCT/US2009/41348 (WO2009132063), PCT/US2009/41356 (WO2009132068), PCT/US2009/49183 (WO2010002846), PCT/US2009/61635 (WO2010051213), PCT/US2009/61774 (WO2010051223), PCT/US2010/42390 (WO2011011300), PCT/US2010/42504 (WO2011011349), PCT/US2010/42393 (WO2011011302), PCT/US2010/60756 (WO2011079017), PCT/US2011/26443 (WO2011109279), PCT/US2011/36043 (WO2011143299), PCT/US2011/48127 (WO2012030538), PCT/US2011/48129 (WO2012030539), PCT/US2011/48131 (WO2012030540), PCT/US2011/47410 (WO2012021698), PCT/US2012/68439 (WO2013095953), PCT/US2013/29360 (WO2013134378), PCT/US2013/39940 (WO2013169769), PCT/US2013/44472 (WO2013184888), PCT/US2013/78191 (WO2014106141), PCTU/S2014/38993 (WO2015065524), PCT/US2013/75939 (WO2014105543), PCT/US2013/75959 (WO2014105546), PCT/US2015/29636 (WO2015175307), PCT/US2015/64713 (WO2016100035), PCT/US2014/41988 (WO2015002724), PCT/US2017/25847 (WO2017180361), PCT/US2017/37373 (WO2017222879), PCT/US2017/37373 (WO2017222879); PCT/US2009/049049 (WO2010/002785), PCT/US2010/060770 (WO2011/079018), PCT/U S2014/042796, (WO2015/065537), P CT/U S2008/070418 (WO2009/012428); Bruin et al. 2013 Diabetologia. 56(9): 1987-98, Fryer et al. 2013 Curr. Opin. Endocrinol. Diabetes Obes. 20(2): 112-7, Chetty et al. 2013 Nature Methods. 10(6):553-6, Rezania et al. 2014 Nature Biotechnologyy 32(11):1121-33, Bruin et al. 2014 Stem Cell Res. 12(1): 194-208, Hrvatin 2014 Proc. Natl. Acad. Sci. USA. 111(8): 3038-43, Bruin et al. 2015 Stem Cell Reports. 5, 1081-1096, Bruin et al. 2015 Science Transl. Med., 2015, 7, 316ps23, and/or Bruin et al. 2015 Stem Cell Reports. 14; 4(4):605-20. 
     In one embodiment, human pluripotent cells were differentiated to PDX1-positive pancreatic endodermcells including pancreatic progenitors and endocrine precursors according to one of the preferred following conditions A and/or B. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Media Conditions for PDX1-positive 
               
               
                 Pancreatic Endoderm Cell Production 
               
            
           
           
               
               
               
            
               
                 Stage 
                 A 
                 B 
               
               
                   
               
            
           
           
               
               
               
            
               
                 1 
                 r0.2FBS-ITS1:5000 A100 W50 
                   
               
               
                   
                 r0.2FBS-ITS1:5000 A100 
               
               
                 2 
                 r0.2FBS-ITS1:1000 K25 IV 
               
               
                   
                 r0.2FBS-ITS1:1000 K25 
               
               
                   
                 r0.2FBS-ITS1:1000 K25 
               
               
                 3 
                 db-TT3 N50 
               
               
                   
                 db-TT3 N50 
               
               
                   
                 db-TT3 N50 
               
               
                 4 
                 db-N50 K50 E50 
               
               
                   
                 db-N50 K50 E50 
               
               
                   
                 db-N50 K50 E50 
               
               
                   
                 db-N50 K50 E50 --&gt; Cryopreserved 
               
            
           
           
               
               
               
            
               
                 Thaw 
                 db-N50 K50 E50 
                 db-N100 A5i (1 uM) 
               
               
                 (S5-A6) 
                 db-N50 K50 E50 
                 db-N100 A5i (1 uM) 
               
               
                   
                 db-N50 K50 E50 
                 db-N100 A5i (1 uM) 
               
               
                   
                   
                 db-N100 A5i (10 uM) 
               
               
                   
                   
                 db-A5i (10 uM) 
               
               
                   
                   
                 db-A5i (10 uM) 
               
               
                   
               
            
           
         
       
     
     Table 1 Legend: r0.2FBS: RPMI 1640 (Mediatech); 0.2% FBS (HyClone), 1× GlutaMAX-1 (Life Technologies), 1% v/v penicillin/streptomycin; db: DMEM Hi Glucose (HyClone) supplemented with 0.5x B-27 Supplement (Life Technologies); A100, A50, A5: 100 ng/mL recombinant human Activin A (R&amp;D Systems); A5i: 1 uM, 5 uM, 10 uM ALK5 inhibitor; TT3: 3 nM TTNPB (Sigma-Aldrich); E50: 50 ng/mL recombinant human EGF (R&amp;D Systems); ITS: Insulin-Transferrin-Selenium (Life Technologies) diluted 1:5000 or 1:1000; IV: 2.5 mM TGF-b RI Kinase inhibitor IV (EMD Bioscience); K50, K25: 50 ng/mL, 25 ng/mL recombinant human KGF (R&amp;D Systems, or Peprotech); N50, N100: 50 ng/mL or 100 ng/mL recombinant human Noggin (R&amp;D Systems); W50: 50 ng/mL recombinant mouse Wnt3A (R&amp;D Systems). 
     One of ordinary skill in the art will appreciate that there may exist other methods for production of PDX1-positive pancreatic endoderm cells or PDX1-positive pancreatic endoderm lineage cells including pancreatic progenitors or even endocrine and endocrine precursor cells; and at least those PDX1-positive pancreatic endoderm cells described in Kroon et al. 2008, Rezania et al. 2014 supra and Pagliuca et al. 2014 Cell 159(2):428-439, supra. 
     One of ordinary skill in the art will also appreciate that the embodiments described herein for production of PDX1-positive pancreatic endoderm cells consists of a mixed population or a mixture of subpopulations. And because unlike mammalian in vivo development which occurs along the anterior-posterior axis, and cells and tissues are named such accordingly, cell cultures in any culture vessels lack such directional patterning and thus have been characterized in particular due to their marker expression. Hence, mixed subpopulations of cells at any stage of differentiation does not occur in vivo. The PDX1-positive pancreatic endoderm cell cultures therefore include, but are not limited to: i) endocrine precursors (as indicated e.g. by the early endocrine marker, Chromogranin A or CHGA); ii) singly hormonal polyhormonal cells expressing any of the typical pancreatic hormones such as insulin (INS), somatostatin (SST), pancreatic polypeptide (PP), glucagon (GCG), or even gastrin, incretin, secretin, or cholecystokinin; iii) pre-pancreatic cells, e.g. cells that express PDX-1 but not NKX6.1 or CHGA; iv) endocrine cells that co-express PDX-1/NKX6.1 and CHGA (PDX-1/NKX6.1/CHGA), or non-endocrine e.g., PDX-1/NKX6.1 but not CHGA (PDX-1+/NKX6.1+/CHA-); and v) still there are cells that do not express PDX-1, NKX6.1 or CHGA (e.g. triple negative cells). 
     This PDX1-positive pancreatic endoderm cells population with its mixed subpopulations of cells mostly express at least PDX-1, in particular a subpopulation that expresses PDX-1/NKX6.1. The PDX1/NKX6.1 subpopulation has also been referred to as “pancreatic progenitors”, “Pancreatic Epithelium” or “PEC” or versions of PEC, e.g. PEC-01. Although Table 1 describes a stage 4 population of cells, these various subpopulations are not limited to just stage 4. Certain of these subpopulations can be for example found in as early as stage 3 and in later stages including stages 5, 6 and 7 (immature beta cells). The ratio of each subpopulation will vary depending on the cell culture media conditions employed. For example, in Agulnick et al. 2015, supra, 73-80% of PDX-1/NKX6.1 cells were used to further differentiate to islet-like cells (ICs) that contained 74-89% endocrine cells generally, and 40-50% of those expressed insulin (INS). Hence, different cell culture conditions are capable of generating different ratios of subpopulations of cells and such may effect in vivo function and therefore blood serum c-peptide levels. And whether modifying methods for making PDX1-positive pancreatic endoderm lineage cell culture populations effects in vivo function can only be determined using in vivo studies as described in more detail below. Further, it cannot be assumed and should not be assumed that just because a certain cell type has been made and has well characterized, that such a method produces the same cell intermediates, unless this is also well characterized. 
     In one aspect, a method for producing mature beta cells in vivo is provided. The method consisting of making human definitive endoderm lineage cells derived from human pluripotent stem cells in vitro with at least a TGFβ superfamily member and/or at least a TGFβ superfamily member and a Wnt family member, preferably a TGFβ superfamily member and a Wnt family member, preferably Activin A, B or GDF-8, GDF-11 or GDF-15 and Wnt3a, preferably Actvin A and Wnt3a, preferably GDF-8 and Wnt3a. The method for making PDX1-positive pancreatic endoderm cells from definitive endoderm cells with at least KGF, a BMP inhibitor and a retinoic acid (RA) or RA analog, and preferably with KGF, Noggin and RA. The method may further differentiate the PDX1-positive pancreatic endoderm cells into immature beta cells or MAFA expressing cells with a thyroid hormone and/or a TGFb-RI inhibitor, a BMP inhibitor, KGF, EGF, a thyroid hormone, and/or a Protein Kinase C activator; preferably with noggin, KGF and EGF, preferably additionally with T3 or T4 and ALK5 inhibitor or T3 or T4 alone or ALK5 inhibitor alone, or T3 or T4, ALK5 inhibitor and a PKC activator such as ILV, TPB and PdBu. Or preferably with noggin and ALK5i and implanting and maturing the PDX1-positive pancreatic endoderm cells or the MAFA immature beta cell populations into a mammalian host in vivo to produce a population of cells including insulin secreting cells capable of responding to blood glucose. 
     In one aspect, a unipotent human immature beta cell or PDX1-positive pancreatic endoderm cell that expresses INS and NKX6.1 and does not substantially express NGN3 is provided. In one embodiment, the unipotent human immature beta cell is capable of maturing to a mature beta cell. In one embodiment, the unipotent human immature beta cell further expresses MAFB in vitro and in vivo. In one embodiment, the immature beta cells express INS, NKX6.1 and MAFA and do not substantially express NGN3. 
     In one aspect, pancreatic endoderm lineage cells expressing at least CHGA (or CHGA+) refer to endocrine cells; and pancreatic endoderm cells that do not express CHGA (or CHGA−) refer to non-endocrine cells. In another aspect, these endocrine and non-endocrine sub-populations may be multipotent progenitor/precursor sub-populations such as non-endocrine multipotent pancreatic progenitor sub-populations or endocrine multipotent pancreatic progenitor sub-populations; or they may be unipotent sub-populations such as immature endocrine cells, preferably immature beta cells, immature glucagon cells and the like. 
     In one aspect, more than 10% preferably more than 20%, 30%, 40% and more preferably more than 50%, 60%, 70%, 80%, 90%, 95%, 98% or 100% of the cells in the pancreatic endoderm or PDX1-positive pancreatic endoderm cell population (stage 4) are the non-endocrine (CHGA-) multipotent progenitor sub-population that give rise to mature insulin secreting cells and respond to glucose in vivo when implanted into a mammalian host. 
     One embodiment provides a composition and method for differentiating pluripotent stem cells in vitro to substantially pancreatic endoderm cultures and further differentiating the pancreatic endoderm culture to endocrine or endocrine precursor cells in vitro. In one aspect, the endocrine precursor or endocrine cells express CHGA. In one aspect, the endocrine cells can produce insulin in vitro. In one aspect, the in vitro endocrine insulin secreting cells may produce insulin in response to glucose stimulation. In one aspect, more than 10% preferably more than 20%, 30%, 40% and more preferably more than 50%, 60%, 70%, 80%, 90%, 95%, 98% or 100% of the cells in the cells population are endocrine cells. 
     Embodiments described herein provide for compositions and methods of differentiating pluripotent human stem cells in vitro to endocrine cells. In one aspect, the endocrine cells express CHGA. In one aspect, the endocrine cells can produce insulin in vitro. In one aspect, the endocrine cells are immature endocrine cells such as immature beta cells. In one aspect, the in vitro insulin producing cells may produce insulin in response to glucose stimulation. 
     One embodiment provides a method for producing insulin in vivo in a mammal, the method comprising: (a) loading a pancreatic endoderm cell or endocrine or endocrine precursor cell population into an implantable semi-permeable device; (b) implanting the device with the cell population into a mammalian host; and (c) maturing the cell population in the device in vivo wherein at least some of the endocrine cells are insulin secreting cells that produce insulin in response to glucose stimulation in vivo, thereby producing insulin in vivo to the mammal. In one aspect the endocrine cell is derived from a cell composition comprising PEC with a higher non-endocrine multipotent pancreatic progenitor sub-population (CHGA−). In another aspect, the endocrine cell is derived from a cell composition comprising PEC with a reduced endocrine sub-population (CHGA+). In another aspect, the endocrine cell is an immature endocrine cell, preferably an immature beta cell. 
     In one aspect the endocrine cells made in vitro from pluripotent stem cells express more PDX1 and NKX6.1 as compared to PDX-1 positive pancreatic endoderm populations, or the non-endocrine (CHGA-) subpopulations which are PDX1/NKX6.1 positive. In one aspect, the endocrine cells made in vitro from pluripotent stem cells express PDX1 and NKX6.1 relatively more than the PEC non-endocrine multipotent pancreatic progenitor sub-population 
     (CHGA−). In one aspect, a Bone Morphogenic Protein (BMP) and a retinoic acid (RA) analog alone or in combination are added to the cell culture to obtain endocrine cells with increased expression of PDX1 and NKX6.1 as compared to the PEC non-endocrine multipotent progenitor sub-population (CHGA−). In one aspect BMP is selected from the group comprising BMP2, BMP5, BMP6, BMP7, BMP8 and BMP4 and more preferably BMP4. In one aspect the retinoic acid analog is selected from the group comprising all-trans retinoic acid and TTNPB (4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid Arotinoid acid), or 0.1-10 μM AM-580 (4-[(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]benzoic acid) and more preferably TTNPB. 
     One embodiment provides a method for differentiating pluripotent stem cells in vitro to endocrine and immature endocrine cells, preferably immature beta cells, comprising dissociating and re-associating the aggregates. In one aspect the dissociation and re-association occurs at stage 1, stage 2, stage 3, stage 4, stage 5, stage 6 or stage 7 or combinations thereof. In one aspect the definitive endoderm, PDX1-negative foregut endoderm, PDX1-positive foregut endoderm, PEC, and/or endocrine and endocrine progenitor/precursor cells are dissociated and re-associated. In one aspect, the stage 7 dissociated and re-aggregated cell aggregates consist of fewer non-endocrine (CHGA−) sub-populations as compared to endocrine (CHGA+) sub-populations. In one aspect, more than 10% preferably more than 20%, 30%, 40% and more preferably more than 50%, 60%, 70%, 80%, 90%, 95%, 98% or 100% of the cells in the cell population are endocrine (CHGA+) cells. 
     One embodiment provides a method for differentiating pluripotent stem cells in vitro to endocrine cells by removing the endocrine cells made during stage 4 PEC production thereby enriching for non-endocrine multipotent pancreatic progenitor (CHGA−) sub-population which is PDX1+ and NKX6.1+. 
     In one embodiment, PEC cultures enriched for the non-endocrine multipotent progenitor sub-population (CHGA−) are made by not adding a Noggin family member at stage 3 and/or stage 4. In one embodiment, PEC cultures which are relatively replete of cells committed to the endocrine lineage (CHGA+) are made by not adding a Noggin family member at stage 3 and/or stage 4. In one aspect the Noggin family member is a compound selected from the group comprising Noggin, Chordin, Follistatin, Folistatin-like proteins, Cerberus, Coco, Dan, Gremlin, Sclerostin, PRDC (protein related to Dan and Cerberus). 
     One embodiment provides a method for maintaining endocrine cells in culture by culturing them in a media comprising exogenous high levels of glucose, wherein the exogenous glucose added is about 1 mM to 25 mM, about 1 mM to 20 mM, about 5 mM to 15 mM, about 5 mM to 10 mM, about 5 mM to 8 mM. In one aspect, the media is a DMEM, CMRL or RPMI based media. 
     One embodiment provides a method for differentiating pluripotent stem cells in vitro to endocrine cells with and without dissociating and re-associating the cell aggregates. In one aspect the non-dissociated or the dissociated and re-associated cell aggregates are cryopreserved or frozen at stage 6 and/or stage 7 without affecting the in vivo function of the endocrine cells. In one aspect, the cryopreserved endocrine cell cultures are thawed, cultured and, when transplanted, function in vivo. 
     Another embodiment provides a culture system for differentiating pluripotent stem cells to endocrine cells, the culture system comprising of at least an agent capable of suppressing or inhibiting endocrine gene expression during early stages of differentiation and an agent capable of inducing endocrine gene expression during later stages of differentiation. In one aspect, an agent capable of suppressing or inhibiting endocrine gene expression is added to the culture system consisting of pancreatic PDX1 negative foregut cells. In one aspect, an agent capable of inducing endocrine gene expression is added to the culture system consisting of PDX1-positive pancreatic endoderm progenitors or PEC. In one aspect, an agent capable of suppressing or inhibiting endocrine gene expression is an agent that activates a TGFbeta receptor family, preferably it is Activin, preferably, it is high levels of Activin, followed by low levels of Activin. In one aspect, an agent capable of inducing endocrine gene expression is a gamma secretase inhibitor selected from a group consisting of N—[N-(3,5-Diflurophenacetyl-L-alanyl)]-S-phenylglycine t-Butyl Ester (DAPT), RO44929097, DAPT (N—[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-Butyl Ester), 1-(S)-endo-N-(1,3,3)-Trimethylbicyclo[2.2.1]hept-2-yl)-4-fluorophenyl Sulfonamide, WPE-III31C, S-3-[N′-(3,5-difluorophenyl-alpha-hydroxyacetyl)-L-alanilyl]amino-2,3-dihydro-1-methyl-5-phenyl-1H-1,4-benzodiazepin-2-one, (N)—[(S)-2-hydroxy-3-methyl-butyryl]-1-(L-alaninyl)-(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one, BMS-708163 (Avagacestat), BMS-708163, Semagacestat (LY450139), Semagacestat (LY450139), MK-0752, MK-0752, YO-01027, YO-01027 (Dibenzazepine, DBZ), LY-411575, LY-411575, or LY2811376. In one aspect, high levels of Activin is meant levels greater than 40 ng/mL, 50 ng/mL, and 75 ng/m L. In one aspect, high levels of Activin are used during stage 3 or prior to production of pancreatic foregut endoderm cells. In one aspect, low levels of Activin means less than 30 ng/mL, 20 ng/mL, 10 ng/mL and 5 ng/mL. In one aspect, low levels of Activin are used during stage 4 or for production of PEC. In one aspect, the endocrine gene that is inhibited or induced is NGN3. In another aspect, Activin A and Wnt3A are used alone or in combination to inhibit endocrine expression, preferably to inhibit NGN3 expression prior to production of pancreatic foregut endoderm cells, or preferably during stage 3. In one aspect, a gamma secretase inhibitor, preferably RO44929097 or DAPT, is used in the culture system to induce expression of endocrine gene expression after production of PEC, or preferably during stages 5, 6 and/or 7. 
     An in vitro cell culture comprising endocrine cells wherein at least 5% of the human cells express an endocrine marker selected from the group consisting of, insulin (INS), NK6 homeobox 1 (NKX6.1), pancreatic and duodenal homeobox 1 (PDX1), transcription factor related locus 2 (NKX2.2), paired box 4 (PAX4), neurogenic differentiation 1 (NEUROD), forkhead box A1 (FOXA1), forkhead box A2 (FOXA2), snail family zinc finger 2 (SNAIL2), and musculoaponeurotic fibrosarcoma oncogene family A and B (MAFA and MAFB), and does not substantially express a marker selected from the group consisting of neurogenin 3 (NGN3), islet 1 (ISL1), hepatocyte nuclear factor 6 (HNF6), GATA binding protein 4 (GATA4), GATA binding protein 6 (GATA6), pancreas specific transcription factor 1a (PTF1A) and SRY (sex determining region Y)-9 (SOX9), wherein the endocrine cells are unipotent and can mature to pancreatic beta cells. 
     In Vivo Nude Rat Study to Evaluate Functional Response 
     The encapsulation devices were loaded ex vivo with 6-7×10 6  cells (or about 20 μL) of pancreatic progenitor cells as described in at least the teachings of U.S. Pat. No. 8,278,106 to Martinson, et. al. After being held in media for less than 24-96 hours, two devices were implanted subcutaneously in each male immunodeficient athymic nude rat. The pancreatic progenitor cells were allowed to develop and mature in vivo and functional performance of the grafts was measured by performing glucose stimulated insulin secretion (GSIS) assays at 12, 16, 20 and 23-24 weeks post-implant. 
     Nude Rat Explant Histology 
     At indicated time points post implant, nude rats were euthanized and devices were explanted. Excess tissue was trimmed away and devices were placed in neutral buffered 10% formalin for 6-30 hours. Fixed devices were processed for paraffin embedding in a Leica Biosystems ASP300S tissue processor. Processed devices were cut into 4-6 pieces of approximately 5 mm each and embedded together in paraffin blocks. Multiple 3-10 micron cross sections were cut from each block, place on slides and stained with hematoxylin and eosin (H&amp;E). Images of the slides were captured using a Hamamatsu Nanozoomer 2.0-HT Digital Slide Scanner. 
     GSIS Assay and Measurement of C-Peptide Secretion 
     Animals that had been implanted with encapsulated pancreatic progenitor cells underwent glucose stimulated insulin secretion assays at 12, 16, 20 and 23-24 weeks post device implantation to monitor graft function. Animals were fasted for 4-16 hours and blood samples were taken via jugular vein venupuncture prior to glucose administration at a dose of 3 g/kg body weight via intraperitoneal injection of a sterile 30% glucose solution. Blood samples were again drawn at 90 minutes, or 60 and 90 minutes, or 30 and 60 minutes after glucose administration. Serum was separated from the whole blood and then assayed for human c-peptide using a commercially available ELISA kit (Mercodia, catalog #10-1141-01, Uppsala Sweden). Beta-cells co-release c-peptide with insulin from pro-insulin in an equimolar ratio and c-peptide is measured as a surrogate for insulin secretion due to its longer half-life in blood. 
     EXAMPLES 
     Comparable Example 1 
     Manufacturing of a Membrane Composite 
     A composite was constructed having two distinct layers. The first layer (Cell Impermeable Layer) was a commercially available microporous, hydrophilic ePTFE membrane with a MPS of 0.4 micron sold under the trade name Biopore® from Millipore (Cork, Ireland). This first layer provided a tight, cell impermeable interface while still enabling mass transport of oxygen and nutrients therethrough. A representative scanning electron micrograph (SEM) of the surface of the ePTFE membrane forming the cell impermeable layer is shown in  FIG. 14 . 
     The second layer (Vascularization Layer) was a commercially available spunbound polyester non-woven material. This second layer was an open layer that provided tissue anchoring and enabled sufficient vascularization of the biocompatible membrane composite. A representative SEM of the surface of the non-woven material forming the vascularization layer is shown in  FIG. 15 . 
     The two layers (Cell Impermeable and Vascularization Layers) were assembled into a composite using a heated lamination process. The fibers of the non-woven material were heated to a temperature above their melt temperature so that they adhered to the ePTFE membrane across the entire surface area of the ePTFE membrane where the fibers of the spunbound non-woven made contact with the surface of the ePTFE membrane. Two examples of laminators used are a Galaxy Flatbed Laminator and a HPL Flatbed Laminator. The conditions were adjusted so that a sufficient pressure and temperature heated and melted the polyester fibers into the ePTFE membrane at a given run speed. Suitable temperature ranges were identified between 150° C.-170° C., nip pressures between 35 kPA and 355 kPA and run speeds of 1-3 meters per minute. 
     Characterization of the Biocompatible Membrane Composite 
     Each layer of the two-layer composite was evaluated and characterized for the relevant parameters necessary for the function of each layer. Parameters for layers are marked as “N/A” if they are not relevant for that layer&#39;s specific function. Parameters for layers are marked as “-” if they are practically unobtainable as a result of how the layers of the composite were processed. The methods used for the characterization of the relevant parameters were performed in accordance with the methods described in “Test Methods” section set forth above. The results of Comparable Example 1 are summarized in Table 2. 
     
       
         
           
               
               
             
               
                   
                 TABLE 2 
               
             
            
               
                   
                   
               
               
                   
                 Layer Function 
               
            
           
           
               
               
               
               
            
               
                   
                 Cell 
                   
                   
               
               
                   
                 Impermeable 
                 FBGC 
                 Vascularization 
               
               
                   
                 Biopore 
                 Mitigation 
                 PET Non- 
               
               
                 Description 
                 ePTFE 
                 None 
                 woven 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 MPS (μm) 
                 0.43 
                 none 
                 N/A 
               
               
                 Pore Size (μm) 
                 0.43 
                 none 
                 101.77 
               
               
                 Thickness (μm) 
                 25.7 
                 none 
                 77.4 
               
               
                 Mass (g/m 2 ) 
                 20.6 
                 none 
                 12.4 
               
               
                 Porosity (%) 
                 63.6 
                 None 
                 92.7 
               
               
                 Solid Feature Spacing (μm) 
                 N/A 
                 none 
                 77.9 
               
               
                 Solid Feature Minor Axis 
                 N/A 
                 none 
                 28.8 
               
               
                 (μm) 
               
               
                 Solid Feature Major Axis 
                 N/A 
                 none 
                 — 
               
               
                 (μm) 
               
               
                 Solid Feature Depth (μm) 
                 N/A 
                 none 
                 27.0 
               
               
                 Weakest Axis Tensile 
                 404.2 
                 none 
                 270.4 
               
               
                 Strength (N/m) 
               
               
                 Geometric Mean Tensile 
                 37.0 
                 none 
                 6.3 
               
               
                 Strength (MPa) 
               
            
           
           
               
               
            
               
                 Composite Bond (kPa) 
                 — 
               
               
                   
               
            
           
         
       
     
     Evaluation of the Composite Membrane Performance In Vivo 
     The biocompatible membrane composite was ultrasonically welded into a device form in accordance with the Integration of Biocompatible Membrane Composite into a Device Form set forth in the Test Methods section above and evaluated in vivo. 
     The host tissue response was evaluated in accordance with the In Vivo Porcine Study set forth in the Test Methods section set forth above. The host tissue response at the device interface demonstrated host tissue penetration through all layers of the device up to the cell impermeable layer. At this interface, the presence of foreign body giant cells were observed at the cell impermeable layer, creating a barrier for neovascularization. As shown in  FIG. 17 , foreign body giant cells (depicted by arrows)  1710  rest on the cell impermeable layers  1720 . 
     The functional response was evaluated in vivo in accordance with the In Vivo Nude Rat Study set forth in the Test Methods section set forth above. The results in Table 3 shows in vivo function of the grafted device in the animals at about 12, 16, 20, and 23 weeks. Human C-peptide levels at the various time points are indicative of the levels of insulin producing cells present in the device. 
     In Comparable Example 1, levels of c-peptide peaked about week  20  post-implant. The low c-peptide levels at the later time points indicate a low level of insulin producing cells present in the device. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 3 
               
             
            
               
                   
                   
               
               
                   
                 Mean Human c-peptide serum levels for each time point 
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 12 
                 16 
                 20 
                 23-24 
                 Sample size (n) for 
               
               
                   
                 weeks 
                 weeks 
                 weeks 
                 weeks 
                 each time point 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 GSIS Time (min) 
                 0 
                 90 
                 0 
                 90 
                 0 
                 90 
                 0 
                 90 
                 # animals 
                 # devices 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Comparative 
                  12** 
                 48 
                 30 
                 98 
                 29 
                 154 
                 62 
                 124 
                 5 
                 10 
               
               
                 Example 1 
               
               
                 Comparative 
                 33 
                 46.5 
                 20 
                 51 
                 n.d. 
                 n.d. 
                 n.d. 
                 n.d. 
                 6 
                 12 
               
               
                 Example 2 
               
               
                 Example 1 
                  27** 
                 196 
                 68 
                 437 
                 79 
                 420 
                 132 
                 488.8* 
                 6 
                 12 
               
               
                 Example 2 
                 26 
                 297.7 
                 43 
                 490 
                 91 
                 594.7 
                 118 
                 615 
                 6 
                 12 
               
               
                 Example 3 
                 141  
                 818 
                 90 
                 830 
                 91 
                 676.9 
                 103 
                 556 
                 7 
                 14 
               
               
                 Example 4 
                  8 
                 247 
                 34 
                 283 
                 56 
                 298 
                 35 
                 208 
                 6-7 
                 12-14 
               
               
                 Example 5 
                 21 
                 246 
                 25 
                 306 
                 51 
                 304 
                 77 
                 337 
                 5-6 
                 10-12 
               
               
                   
               
               
                 **60 min GSIS Time 
               
               
                 **rats were not fasted prior to GSIS assay 
               
            
           
         
       
     
     Comparable Example 2 
     Manufacturing of Biocompatible Membrane Composite 
     A composite was constructed with three distinct layers. A first layer of an ePTFE membrane (Cell Impermeable Layer) was formed according to the teachings of U.S. Pat. No. 3,953,566 to Gore. 
     A second ePTFE membrane (Mitigation Layer) was prepared according the teachings of U.S. Pat. No. 5,814,405 to Branca, et. al. During an initial machine direction (MD) expansion step, a fluorinated ethylene propylene (FEP) film was applied to the second ePTFE membrane. Through subsequent co-processing of the second ePTFE membrane and FEP through the machine direction (MD) expansion and transverse direction (TD) expansion, the FEP became discontinuous on the surface of the second ePTFE membrane as per the teachings of WO/94/13469 to Bacino.  FIG. 18  is a representative image of the second ePTFE layer  1800  surface with discontinuous layer of FEP  1810  thereon. 
     The second ePTFE layer including the discontinuous FEP thereon was laminated to the first layer by bringing the materials (with the FEP positioned between the two layers) into contact at a temperature above the melting point of the FEP. Both ePTFE layers were held under tension to prevent unintentional deformation during this lamination process. The composite was subsequently rendered hydrophilic per the teachings in U.S. Pat. No. 5,902,745 to Butler, et. al. The SEM image shown in  FIG. 19  is a representative image of the node and fibril structure of the first ePTFE layer (Cell Impermeable Layer). The SEM image shown in  FIG. 20  is a representative image of the node and fibril structure of the second ePTFE layer (Mitigation Layer).  FIG. 21  is an SEM image of a representative image of the cross-section structure of the two-layer composite  2100  including the first ePTFE layer  2110  (Cell Impermeable Layer) and the second ePTFE layer  2120  (Mitigation Layer). 
     The third layer (Vascularization Layer) was a commercially available spunbound polyester non-woven material. A representative surface microstructure of the third layer is shown in the SEM image in  FIG. 15 . This third layer was assembled into a composite with the first and second layers by placing the third layer on the top of the second layer and discretely welding to the composite at only a perimeter location during integration of the composite into a device form as described in the Test Methods section set forth above. 
     Characterization of the Biocompatible Membrane Composite 
     Each individual layer of the biocompatible membrane composite was evaluated and characterized for the relevant parameters necessary for the function of each layer. Parameters for layers are marked as “N/A” if they are not relevant for that layer&#39;s specific function. Parameters for layers are marked as “-” if they are practically unobtainable as a result of how the layers of the composite were processed. The methods used for the characterization of relevant parameters were performed in accordance with the methods set forth in the Test Methods section set forth above. The results are summarized in Table 4. 
     
       
         
           
               
               
             
               
                   
                 TABLE 4 
               
             
            
               
                   
                   
               
               
                   
                 Layer Function 
               
            
           
           
               
               
               
               
            
               
                   
                 Cell 
                 FBGC 
                   
               
               
                   
                 Impermeable 
                 Mitigation 
                 Vascularization 
               
               
                   
                 ePTFE Tight 
                 ePTFE Open 
                 PET Non- 
               
               
                 Description 
                 Layer 
                 Layer 
                 woven 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 MPS (microns) 
                 0.20 
                 — 
                 N/A 
               
               
                 Pore Size (microns) 
                 0.38 
                 9.74 
                 101.77 
               
               
                 Thickness (microns) 
                 8.4 
                 95.7 
                 77.4 
               
               
                 Mass (g/m 2 ) 
                 4.2 
                 6.6 
                 12.4 
               
               
                 Porosity (%) 
                 77.4 
                 96.9 
                 92.7 
               
               
                 Solid Feature Spacing 
                 N/A 
                 63.2 
                 77.9 
               
               
                 (microns) 
               
               
                 Solid Feature Minor Axis 
                 N/A 
                 4.2 
                 28.8 
               
               
                 (microns) 
               
               
                 Solid Feature Major Axis 
                 N/A 
                 24.6 
                 — 
               
               
                 (microns) 
               
               
                 Solid Feature Depth 
                 N/A 
                 24.5 
                 27.0 
               
               
                 (microns) 
               
            
           
           
               
               
               
            
               
                 Weakest Axis Tensile 
                 799.3 
                 270.4  
               
               
                 Strength (MPa) 
               
               
                 Geometric Mean Tensile 
                 12.6 
                  6.3 
               
               
                 Strength (MPa) 
               
               
                 Composite Bond (kPa) 
                 251.4 
                 — 
               
               
                   
               
            
           
         
       
     
     Evaluation of the Biocompatible Membrane Composite In Vivo 
     The biocompatible membrane composite was thermally welded into a device form in accordance with the Integration of Biocompatible Membrane Composite into a Device Form set forth in the Test Methods section above and evaluated in vivo. 
     The host tissue response was evaluated in accordance with the In Vivo Porcine Study set forth in the Test Methods section set forth above. The host tissue response at the device interface demonstrated host tissue penetration through all layers of the device up to the cell impermeable ePTFE tight layer. At this interface foreign body giant cells were still visible at the cell impermeable layer, creating a barrier for neovascularization as seen in Comparable Example 1.  FIG. 22  is a representative histology image of the observation of foreign body giant cells (indicated by arrows  2210 ) abutting cell impermeable layers  2220 . 
     The functional response was evaluated in vivo in accordance with the In Vivo Nude Rat Study set forth in the Test Methods section set forth above. The results are shown in Table 3. The low levels of c-peptide indicate a low level of insulin producing cells present in the device. There was no marked increase in function as compared to Comparative Example 1. 
     Example 1 
     Manufacturing of Biocompatible Membrane Composite 
     A biocompatible membrane composite having three distinct layers was constructed. First, a two-layer ePTFE composite was prepared by layering and then co-expanding a first ePTFE layer consisting of a dry, biaxially-expanded membrane (Cell Impermeable Layer) prepared according to the teachings of U.S. Pat. No. 3,953,566 to Gore and a second ePTFE layer consisting of a paste extruded calendered tape (Mitigation Layer) prepared according to the teachings of U.S. Pat. No. 3,953,566 to Gore. The two-layer ePTFE composite was biaxially expanded and then rendered hydrophilic according to the teachings of U.S. Pat. No. 5,902,745, to Butler, et al. The first ePTFE layer provided a tight, cell impermeable interface while still enabling mass transport of oxygen and nutrients. A representative surface microstructure of the first layer is shown in the SEM image of  FIG. 23 . The second ePTFE membrane (Mitigation Layer) reduced the formation of foreign body giant cells at the interface of the first ePTFE layer. A representative surface microstructure of the second ePTFE membrane is shown in  FIG. 24 . A representative cross-section showing the microstructure of the composite  2500  including the first ePTFE membrane  2510  (Cell Impermeable Layer) and the second ePTFE membrane  2520  (Mitigation Layer) is shown in the SEM image of  FIG. 25 . 
     The third layer (Vascularization Layer) was a commercially available spunbound polyester non-woven material. A representative surface microstructure of the third layer is shown in the SEM image in  FIG. 15 . This third layer was assembled into a composite with the first and second layers by placing the spunbound polyester non-woven on the top of the second layer and discretely welding the spunbound polyester non-woven to the composite at only the perimeter during integration of the composite into a device form as described in the Method section set forth above. 
     Characterization of the Biocompatible Membrane Composite 
     Each individual layer of the biocompatible membrane composite was evaluated and characterized for the relevant parameters necessary for the function of each layer. Parameters for layers are marked as “N/A” if they are not relevant for that layer&#39;s specific function. Parameters for layers are marked as “-” if they are practically unobtainable as a result of how the layers of the composite were processed. The methods used for this characterization of relevant parameters were performed in accordance with the methods described in the Test Methods section set forth above. The results are summarized in Table 5. 
     
       
         
           
               
               
             
               
                   
                 TABLE 5 
               
             
            
               
                   
                   
               
               
                   
                 Layer Function 
               
            
           
           
               
               
               
               
            
               
                   
                 Cell 
                 FBGC 
                   
               
               
                   
                 Impermeable 
                 Mitigation 
                 Vascularization 
               
               
                   
                 ePTFE Tight 
                 ePTFE Open 
                 PET Non- 
               
               
                 Description 
                 Layer 
                 Layer 
                 woven 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 MPS (microns) 
                 0.35 
                 — 
                 N/A 
               
               
                 Pore Size (microns) 
                 0.51 
                 4.87 
                 101.77 
               
               
                 Thickness (microns) 
                 6.6 
                 24.7 
                 77.4 
               
               
                 Mass (g/m 2 ) 
                 2.3 
                 2.2 
                 12.4 
               
               
                 Porosity (%) 
                 83.8 
                 95.9 
                 92.7 
               
               
                 Solid Feature Spacing 
                 N/A 
                 24.4 
                 77.9 
               
               
                 (microns) 
               
               
                 Solid Feature Minor Axis 
                 N/A 
                 4.2 
                 28.8 
               
               
                 (microns) 
               
               
                 Solid Feature Major Axis 
                 N/A 
                 7.5 
                 — 
               
               
                 (microns) 
               
               
                 Solid Feature Depth 
                 N/A 
                 5.2 
                 27.0 
               
               
                 (microns) 
               
            
           
           
               
               
               
            
               
                 Weakest Axis Tensile 
                 210.9 
                 270.4  
               
               
                 Strength (N/m) 
               
               
                 Geometric Mean Tensile 
                 38.1 
                  6.3 
               
               
                 Strength (MPa) 
               
               
                 Composite Bond (kPa) 
                 170.2 
                 — 
               
               
                   
               
            
           
         
       
     
     Evaluation of the Composite Membrane Performance 
     The biocompatible membrane composite was thermally welded into a device form in accordance with the Integration of Biocompatible Membrane Composite into a Device Form set forth in the Test Methods section above and evaluated in vivo. 
     The host tissue response was evaluated in accordance with the In Vivo Porcine Study set forth in the Test Methods section set forth above. The host tissue response at the device interface demonstrated host tissue penetration through the polyester woven mesh reinforcing component, the polyester non-woven vascularization layer, and open ePTFE mitigation layer up to the tight ePTFE cell impermeable layer. While foreign body giant cells were present within the polyester woven mesh (reinforcing component) and polyester non-woven layer (Vascularization Layer), there was no observation of foreign body giant cells along the tight, ePTFE layer (Cell Impermeable Layer). The histology image shown in  FIG. 26  is a representative image of this observation, with arrows  2610  indicating the location of the foreign body giant cells in relation to each layer of the biocompatible membrane composite  2600 . Additionally, as shown in  FIG. 26 , foreign body giant cells (indicated by arrows  2610 ) did not form on the surface of the cell impermeable layer  2620 . 
     It was concluded that the biocompatible membrane composite  2600  formed of the cell impermeable layer, the mitigation layer, and the vascularization layer described in this Example reduced the formation of foreign body giant cells (indicated by arrows  2610 ) on the surface of the cell impermeable layers  2620 . 
     The functional response was evaluated in vivo in accordance with the In Vivo Nude Rat Study set forth in the Test Methods section set forth above. The results shown in Table 3 demonstrate a step change in functional response as compared to the comparative examples, indicating a significant increase in viability of insulin producing cells. At 23 weeks after implantation, the c-peptide blood serum concentration was measured in response to glucose stimulated insulin secretion and was, on average, 488.8 pM, which is 3.9× greater that of Comparative Example 1 where no mitigation layer is present. 
     Example 2 
     Manufacturing of Biocompatible Membrane Composite 
     A composite was constructed with three distinct layers. A first ePTFE membrane (Cell Impermeable Layer) was formed according to the teachings of U.S. Pat. No. 3,953,566 to Gore. 
     A second ePTFE membrane (Mitigation Layer) was prepared according to the teachings of U.S. Pat. No. 5,814,405 to Branca, et al. During machine direction (MD) expansion processing, a fluorinated ethylene propylene (FEP) film was applied to the second ePTFE membrane. Through subsequent co-processing of the second ePTFE membrane and FEP through machine direction (MD) expansion and transverse direction (TD) expansion, the FEP became discontinuous on the second ePTFE membrane as per the teachings of WO/94/13469 to Bacino. The SEM image shown in  FIG. 27  is a representative image of the second ePTFE membrane surface  2700  with the discontinuous layer of FEP  2710  thereon. 
     The second ePTFE layer including the discontinuous layer of FEP thereon was laminated to the first ePTFE layer by bringing the materials (with the FEP positioned between the two ePTFE membranes) into contact at a temperature above the melting point of the FEP. The two ePTFE layers were left unrestrained in the transverse direction during lamination. The laminate was then transversely expanded above the melting point of polytetrafluoroethylene (PTFE) such that each ePTFE layer was returned to its width prior to any necking sustained through lamination. The composite was subsequently rendered hydrophilic per the teachings of U.S. Pat. No. 5,902,745, to Butler, et al. The SEM image shown in  FIG. 19  is a representative image of the node and fibril structure of the first ePTFE membrane (Cell Impermeable Layer). The SEM image shown in  FIG. 28  is a representative image of the node and fibril structure of the second ePTFE membrane (Mitigation Layer). The SEM image shown in  FIG. 29  is a representative image of the cross-section structure of the two-layer composite  2900  (i.e., the first ePTFE membrane  2910  (Cell Impermeable Layer) and the second ePTFE membrane  2920  (Mitigation Layer)). 
     The third layer (Vascularization Layer) was a commercially available spunbound polyester non-woven material. A representative surface microstructure of the third layer is shown in the SEM image of  FIG. 15 . This third layer was assembled into a composite with the first and second layers by placing the spunbound polyester non-woven material on the top of the second ePTFE layer and discretely welding the spunbound polyester material at a perimeter location during integration of the composite into a device form as described in the Test Methods section set forth above. 
     Characterization of the Biocompatible Membrane Composite 
     Each individual layer of the biocompatible membrane composite was evaluated and characterized for the relevant parameters necessary for the function of each layer. Parameters for layers are marked as “N/A” if they are not relevant for that layer&#39;s specific function. Parameters for layers are marked as “-” if they are practically unobtainable as a result of how the layers of the composite were processed. The methods used for the characterization of relevant parameters were performed in accordance with the methods set forth above. The results are summarized in Table 6. 
     
       
         
           
               
               
             
               
                   
                 TABLE 6 
               
             
            
               
                   
                   
               
               
                   
                 Layer Function 
               
            
           
           
               
               
               
               
            
               
                   
                 Cell 
                 FBGC 
                   
               
               
                   
                 Impermeable 
                 Mitigation 
                 Vascularization 
               
               
                   
                 ePTFE Tight 
                 ePTFE Open 
                 PET Non- 
               
               
                 Description 
                 Layer 
                 Layer 
                 woven 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 MPS (microns) 
                 0.18 
                 — 
                 N/A 
               
               
                 Pore Size (microns) 
                 0.34 
                 8.06 
                 101.77 
               
               
                 Thickness (microns) 
                 6.1 
                 44.6 
                 77.4 
               
               
                 Mass (g/m 2 ) 
                 3.8 
                 6.2 
                 12.4 
               
               
                 Porosity (%) 
                 71.7 
                 93.7 
                 92.7 
               
               
                 Solid Feature Spacing 
                 N/A 
                 24.2 
                 77.9 
               
               
                 (microns) 
               
               
                 Solid Feature Minor Axis 
                 N/A 
                 4.7 
                 28.8 
               
               
                 (microns) 
               
               
                 Solid Feature Major Axis 
                 N/A 
                 31.9 
                 — 
               
               
                 (microns) 
               
               
                 Solid Feature Depth 
                 N/A 
                 11.5 
                 27.0 
               
               
                 (microns) 
               
            
           
           
               
               
               
            
               
                 Weakest Axis Tensile 
                 768.8 
                 270.4  
               
               
                 Strength (N/m) 
               
               
                 Geometric Mean Tensile 
                 22.8 
                  6.3 
               
               
                 Strength (MPa) 
               
               
                 Composite Bond (kPa) 
                 1231.9 
                 — 
               
               
                   
               
            
           
         
       
     
     Evaluation of the Composite Membrane Performance 
     The biocompatible membrane composite was thermally welded into a device form in accordance with the Integration of Biocompatible Membrane Composite into a Device Form set forth in the Test Methods section above and evaluated in vivo. 
     The host tissue response was evaluated in accordance with the In Vivo Porcine Study set forth in the Test Methods section set forth above. The host tissue response at the device interface demonstrated host tissue penetration through the polyester woven mesh reinforcing component, the polyester non-woven vascularization layer, and open ePTFE mitigation layer up to the tight ePTFE cell impermeable layer. While foreign body giant cells were present within the polyester woven mesh (reinforcing component) and polyester non-woven layer (Vascularization Layer), there was no observation of foreign body giant cells along the tight, ePTFE layer (Cell Impermeable Layer). The histology image shown in  FIG. 45  is a representative image of this observation, with arrows  4510  indicating the location of the foreign body giant cells in relation to each layer of the biocompatible membrane composite  4500 . Additionally, as shown in  FIG. 45 , foreign body giant cells (indicated by arrows  4510 ) did not form on the surface of the cell impermeable layer  4520 . It was concluded that the biocompatible membrane composite  4500  formed of the cell impermeable layer, the mitigation layer, and the vascularization layer described in this Example reduced the formation of foreign body giant cells (indicated by arrows  4510 ) on the surface of the cell impermeable layer  4520 . 
     The functional response was evaluated in vivo in accordance with the In Vivo Nude Rat Study set forth in the Test Methods section set forth above. The results shown in Table 3 demonstrate a step change in functional response as compared to the Comparative Examples, which indicated a significant increase in viability of insulin producing cells. At 24 weeks after implantation, the c-peptide blood serum concentration measured in response to glucose stimulated insulin secretion was, on average, 615 pM, which is significantly greater than that of Comparative Example 1 where no mitigation layer is present. It was concluded that in order to achieve such an increase in the degree of functional response, the mitigation layer was able to successfully mitigate the formation of the formation of foreign body giant cells at the cell impermeable interface. 
     Example 3 
     Manufacturing of Biocompatible Membrane Composite 
     A biocompatible membrane composite having three distinct layers was constructed. First, a two-layer ePTFE composite was prepared by layering and then co-expanding a first ePTFE membrane consisting of a dry, biaxially-expanded membrane (Cell Impermeable Layer) prepared according to the teachings of U.S. Pat. No. 3,953,566 to Gore and a second ePTFE layer consisting of a paste extruded calendered tape (Mitigation Layer) prepared according to the teachings of U.S. Pat. No. 3,953,566 to Gore. The two-layer ePTFE composite was biaxially expanded and then rendered hydrophilic according to the teachings of U.S. Pat. No. 5,902,745 to Butler, et al. The first ePTFE membrane provided a tight, cell impermeable interface that still enabled mass transport of oxygen and nutrients. A representative surface microstructure of the first ePTFE membrane is shown in the SEM image of  FIG. 30 . A representative surface microstructure of the second ePTFE membrane is shown in  FIG. 31 . A representative cross-section of the two-layer ePTFE composite  3200  containing the first ePTFE membrane  3210  (Cell Impermeable Layer) and the second ePTFE membrane  3220  (Mitigation Layer) is shown in the SEM image shown in  FIG. 32 . 
     The third layer (Vascularization Layer) was a commercially available spunbound polyester non-woven material. A representative surface microstructure of the spunbound polyester non-woven material is shown in the SEM image of  FIG. 15 . This third layer was assembled into a composite with the two-layer composite by placing the spunbound polyester non-woven material on the top of the second ePTFE membrane of the two-layer composite and discretely welding at a perimeter location during integration of the composite into a device form as described in the Test Methods section set forth above. 
     Characterization of the Biocompatible Membrane Composite 
     Each individual layer of the biocompatible membrane composite was evaluated and characterized for the relevant parameters necessary for the function of each layer. Parameters for layers are marked as “N/A” if they are not relevant for that layer&#39;s specific function. Parameters for layers are marked as “-” if they are practically unobtainable as a result of how the layers of the composite were processed. The methods used for the characterization of relevant parameters were performed in accordance with the methods described in the Test Methods section set forth above. The results are summarized in Table 7. 
     
       
         
           
               
               
             
               
                   
                 TABLE 7 
               
             
            
               
                   
                   
               
               
                   
                 Layer Function 
               
            
           
           
               
               
               
               
            
               
                   
                 Cell 
                 FBGC 
                   
               
               
                   
                 Impermeable 
                 Mitigation 
                 Vascularization 
               
               
                   
                 ePTFE Tight 
                 ePTFE Open 
                 PET Non- 
               
               
                 Description 
                 Layer 
                 Layer 
                 woven 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 MPS (microns) 
                 0.33 
                 — 
                 N/A 
               
               
                 Pore Size (microns) 
                 0.51 
                 5.18 
                 101.77 
               
               
                 Thickness (microns) 
                 5.6 
                 16.3 
                 77.4 
               
               
                 Mass (g/m 2 ) 
                 2.0 
                 1.9 
                 12.4 
               
               
                 Porosity (%) 
                 83.9 
                 94.7 
                 92.7 
               
               
                 Solid Feature Spacing 
                 N/A 
                 9.2 
                 77.9 
               
               
                 (microns) 
               
               
                 Solid Feature Minor Axis 
                 N/A 
                 2.6 
                 28.8 
               
               
                 (microns) 
               
               
                 Solid Feature Major Axis 
                 N/A 
                 4.3 
                 — 
               
               
                 (microns) 
               
               
                 Solid Feature Depth 
                 N/A 
                 4.8 
                 27.0 
               
               
                 (microns) 
               
            
           
           
               
               
               
            
               
                 Weakest Axis Tensile 
                 208.1 
                 270.4  
               
               
                 Strength (N/m) 
               
               
                 Geometric Mean Tensile 
                 47.1 
                  6.3 
               
               
                 Strength (MPa) 
               
               
                 Composite Bond (kPa) 
                 307.5 
                 — 
               
               
                   
               
            
           
         
       
     
     Evaluation of the Composite Membrane Performance 
     The biocompatible membrane composite was thermally welded into a device form in accordance with the Integration of Biocompatible Membrane Composite into a Device Form set forth in the Test Methods section above and evaluated in vivo. 
     The host tissue response was evaluated in the In Vivo Porcine Study_set forth in the Method section set forth above. The host tissue response at the device interface demonstrated host tissue penetration through the polyester woven mesh reinforcing component, the polyester non-woven vascularization layer, and open ePTFE mitigation layer up to the tight ePTFE cell impermeable layer. While foreign body giant cells were present within the polyester woven mesh (reinforcing component) and polyester non-woven layer (Vascularization Layer), there was no observation of foreign body giant cells along the tight, ePTFE layer (Cell Impermeable Layer). The histology image shown in  FIG. 46  is a representative image of this observation, with arrows  4610  indicating the location of the foreign body giant cells in relation to each layer of the biocompatible membrane composite  4600 . Additionally, as shown in  FIG. 46 , foreign body giant cells (indicated by arrows  4610 ) did not form on the surface of the cell impermeable layer  4620 . It was concluded that the biocompatible membrane composite  4600  formed of the cell impermeable layer, the mitigation layer, and the vascularization layer described in this Example reduced the formation of foreign body giant cells (indicated by arrows  4610 ) on the surface of the cell impermeable layer  4620 . 
     The functional response was evaluated in vivo in accordance with the In Vivo Nude Rat Study set forth in the Test Methods section set forth above. The results shown in Table 3 demonstrate a step change in functional response as compared to the comparative examples and indicated a significant increase in viability of insulin producing cells. At 24 weeks after implantation, the c-peptide blood serum concentration measured in response to glucose stimulated insulin secretion was, on average, 556 pM, which is 4.5x greater than that of Comparative Example 1 where no mitigation layer is present. In order to achieve this degree of functional response, it was concluded that the mitigation layer was able to successfully mitigate the formation of foreign body giant cells at the cell impermeable interface. 
     Example 4 
     Manufacturing of Biocompatible Membrane Composite 
     A biocompatible membrane composite was constructed with three distinct layers. A first layer consisting of an ePTFE membrane (Cell Impermeable Layer) was formed according to the teachings of U.S. Pat. No. 3,953,566 to Gore. 
     A second ePTFE membrane (FBGC Mitigation Layer) was prepared according the teachings of U.S. Pat. No. 5,814,405 to Branca, et al. During the initial machine direction (MD) expansion step, a fluorinated ethylene propylene (FEP) film was applied to the second ePTFE membrane. Through subsequent co-processing of the second ePTFE membrane and FEP through machine direction (MD) expansion and transverse direction (TD) expansion, the FEP became discontinuous on the second ePTFE membrane as per the teachings of WO/94/13469 to Bacino. The SEM image shown in  FIG. 33  is a representative image of the surface or the second ePTFE membrane  3300  having thereon discontinuous FEP  3310 . 
     The second ePTFE layer that included the discontinuous FEP layer was laminated to the first ePTFE layer by bringing the two ePTFE membranes materials into contact (with the FEP positioned between the two ePTFE membranes) at a temperature above the melting point of the FEP. Both ePTFE layers were held under tension to prevent unintentional deformation during this lamination process. The laminate was subsequently rendered hydrophilic per the teachings of U.S. Pat. No. 5,902,745 to Butler, et al. The SEM image shown in  FIG. 19  is a representative image of the node and fibril structure of the first ePTFE layer (Cell Impermeable Layer). The SEM image shown in  FIG. 34  is a representative image of the node and fibril structure of the second ePTFE membrane (Mitigation Layer). The SEM image shown in  FIG. 35  is a representative image of the cross-section structure of the two layer ePTFE laminate  3500  having the first ePTFE membrane  3510  (Cell Impermeable Layer) and the second ePTFE membrane  3520  (Mitigation Layer). 
     The third layer (Vascularization Layer) was a commercially available spunbound polyester non-woven material. A representative surface microstructure of the third layer is shown in the SEM image of  FIG. 15 . The third layer and the ePTFE laminate was assembled into a biocompatible membrane composite with the first and second ePTFE layers by placing the spunbound polyester non-woven material on the top of the second ePTFE membrane and discretely welding the spunbound polyester non-woven material to the second ePTFE membrane of the two-layer ePTFE composite at the perimeter during integration of the biocompatible membrane composite into a device form as described in the Method section set forth above. 
     Characterization of the Biocompatible Membrane Composite 
     Each individual layer of the biocompatible membrane composite was evaluated and characterized for the relevant parameters necessary for the function of each layer. Parameters for layers are marked as “N/A” if they are not relevant for that layer&#39;s specific function. Parameters for layers are marked as “-” if they are practically unobtainable as a result of how the layers of the composite were processed. The methods used for the characterization of relevant parameters were performed in accordance with the Test Methods section set forth above. The results are summarized in Table 8. 
     
       
         
           
               
               
             
               
                   
                 TABLE 8 
               
             
            
               
                   
                   
               
               
                   
                 Layer Function 
               
            
           
           
               
               
               
               
            
               
                   
                 Cell 
                 FBGC 
                   
               
               
                   
                 Impermeable 
                 Mitigation 
                 Vascularization 
               
               
                   
                 ePTFE Tight 
                 ePTFE Open 
                 PET Non- 
               
               
                 Description 
                 Layer 
                 Layer 
                 woven 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 MPS (microns) 
                 0.18 
                 — 
                 N/A 
               
               
                 Pore Size (microns) 
                 0.38 
                 2.40 
                 101.77 
               
               
                 Thickness (microns) 
                 8.7 
                 44.5 
                 77.4 
               
               
                 Mass (g/m 2 ) 
                 4.1 
                 3.3 
                 12.4 
               
               
                 Porosity (%) 
                 78.3 
                 96.6 
                 92.7 
               
               
                 Solid Feature Spacing 
                 N/A 
                 12.0 
                 77.9 
               
               
                 (microns) 
               
               
                 Solid Feature Minor Axis 
                 N/A 
                 3.2 
                 28.8 
               
               
                 (microns) 
               
               
                 Solid Feature Major Axis 
                 N/A 
                 32.9 
                 — 
               
               
                 (microns) 
               
               
                 Solid Feature Depth 
                 N/A 
                 12.5 
                 27.0 
               
               
                 (microns) 
               
            
           
           
               
               
               
            
               
                 Weakest Axis Tensile 
                 867.9 
                 270.4  
               
               
                 Strength (N/m) 
               
               
                 Geometric Mean Tensile 
                 24.1 
                  6.14 
               
               
                 Strength (MPa) 
               
               
                 Composite Bond (kPa) 
                 288.6 
                 — 
               
               
                   
               
            
           
         
       
     
     Evaluation of the Composite Membrane Performance 
     The biocompatible membrane composite was thermally welded into a device form in accordance with the Integration of Biocompatible Membrane Composite into a Device Form set forth in the Test Methods section above and evaluated in vivo. 
     The host tissue response was evaluated in accordance with the In Vivo Porcine Study set forth in the Method section set forth above. The host tissue response at the device interface demonstrated host tissue penetration through the polyester woven mesh reinforcing component, the polyester non-woven vascularization layer, and open ePTFE mitigation layer up to the tight ePTFE cell impermeable layer. While foreign body giant cells were present within the polyester woven mesh (reinforcing component) and polyester non-woven layer (Vascularization Layer), there was no formation of foreign body giant cells observed along the tight, ePTFE layer (Cell Impermeable Layer). The histology image of  FIG. 36  is a representative image of this observation, with arrow  3610  indicating the location of a foreign body giant cell in relation to each layer of the biocompatible membrane composite. Additionally, as shown in  FIG. 36 , foreign body giant cells  3610  did not form on the surface of the cell impermeable layer  3620 . It was concluded that the biocompatible membrane composite formed of the cell impermeable layer, the mitigation layer, and the vascularization layer described in this Example reduced the formation of foreign body giant cells on the surface of the cell impermeable layer. 
     The functional response was evaluated in vivo in accordance with the In Vivo Nude Rat Study set forth in the Test Methods section set forth above. The results in Table 3 demonstrate a step change in functional response as compared to the comparative examples, indicating a significant increase in viability of insulin producing cells. At 24 weeks after implantation, the c-peptide blood serum concentration was measured in response to glucose stimulated insulin secretion and was, on average, 208 pM, which is significantly greater than that of Comparative Example 1 where no mitigation layer was present. 
     Example 5 
     Manufacturing of Biocompatible Membrane Composite 
     A biocompatible membrane composite having three distinct layers was constructed. A first layer formed of an ePTFE membrane (Cell Impermeable Layer) was formed according to the teachings of U.S. Pat. No. 3,953,566 to Gore. 
     A two-layer composite consisting of a second ePTFE membrane (Mitigation Layer) and a third ePTFE layer (Vascularization Layer) was formed The second ePTFE membrane was prepared according to the teachings of U.S. Pat. No. 5,814,405 to Branca, et al. The ePTFE tape precursor of the second ePTFE layer was processed per the teachings of U.S. Pat. No. 5,814,405 to Branca, et al. through the below-the-melt MD expansion step. During the below-the-melt MD expansion step of the second ePTFE tape precursor, an FEP film was applied per the teachings of WO/94/13469 to Bacino. The ePTFE tape precursor of the third ePTFE layer was processed per the teachings of U.S. Pat. No. 5,814,405 to Branca, et al. through an amorphous locking step. During the first below-the-melt MD expansion step of the third ePTFE tape precursor, an FEP film was applied per the teachings of WO/94/13469 to Bacino. The expanded ePTFE tape precursor of the third ePTFE membrane was laminated to the expanded ePTFE tape precursor of the second ePTFE membrane such that the FEP side of the third ePTFE tape was in contact with the PTFE side of the ePTFE tape precursor of the second ePTFE membrane. The two layer composite was then co-expanded in the machine direction and transverse direction above the melting point of PTFE. A representative surface microstructure of the second ePTFE layer  3700  having thereon FEP  3710  is shown in the SEM image of  FIG. 37 . 
     The two-layer composite consisting of the second ePTFE membrane (Mitigation Layer) and third ePTFE membrane (Vascularization Layer) was laminated to the first ePTFE membrane (Cell Impermeable Layer). The side of the second ePTFE membrane comprising a discontinuous layer of FEP thereon was laminated to the first ePTFE layer by first bringing two-layer ePTFE composite into contact with the third ePTFE layer (with the FEP positioned between the two layers) at a temperature above the melting point of the FEP with the ePTFE membranes unrestrained in the transverse direction. The laminate was then transversely expanded above the melting point of PTFE so each layer was returned to its width prior to any necking sustained through lamination. The composite was subsequently rendered hydrophilic per the teachings of U.S. Pat. No. 5,902,745 to Butler, et al. The SEM image shown in  FIG. 19  is a representative image of the node and fibril structure of the first ePTFE membrane (Cell Impermeable Layer). The SEM image shown in  FIG. 38  is a representative image of the node and fibril structure of the third ePTFE membrane (Vascularization Layer). The SEM image shown in  FIG. 39  is a representative image of the cross-section structure  3900  of the three layer biocompatible membrane composite including the first ePTFE membrane  3910  (Cell Impermeable Layer), the second ePTFE membrane  3920  (Mitigation Layer) and the third ePTFE membrane  3930  (Vascularization Layer). 
     Characterization of the Biocompatible Membrane Composite 
     Each individual layer of the biocompatible membrane composite was evaluated and characterized for the relevant parameters necessary for the function of each layer. Parameters for layers are marked as “N/A” if they are not relevant for that layer&#39;s specific function. Parameters for layers are marked as “-” if they are practically unobtainable as a result of how the layers of the composite were processed. The methods used for the characterization of relevant parameters were performed in accordance with the methods described in the Test Methods section set forth above. The results are summarized in Table 9. 
     
       
         
           
               
               
             
               
                   
                 TABLE 9 
               
             
            
               
                   
                   
               
               
                   
                 Layer Function 
               
            
           
           
               
               
               
               
            
               
                   
                 Cell 
                 FBGC 
                   
               
               
                   
                 Impermeable 
                 Mitigation 
                 Vascularization 
               
               
                   
                 ePTFE Tight 
                 ePTFE Open 
                 ePTFE Open 
               
               
                 Description 
                 Layer 
                 Layer 
                 Layer 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 MPS (microns) 
                 0.19 
                 — 
                 — 
               
               
                 Pore Size (microns) 
                 0.34 
                 8.06 
                 10.15 
               
               
                 Thickness (microns) 
                 6.7 
                 42.8 
                 80.5 
               
               
                 Mass (g/m 2 ) 
                 3.0 
                 4.8 
                 5.5 
               
               
                 Porosity (%) 
                 79.3 
                 94.9 
                 96.9 
               
               
                 Solid Feature Spacing 
                 N/A 
                 24.2 
                 58.4 
               
               
                 (microns) 
               
               
                 Solid Feature Minor Axis 
                 N/A 
                 4.7 
                 8.6 
               
               
                 (microns) 
               
               
                 Solid Feature Major Axis 
                 N/A 
                 31.9 
                 83.7 
               
               
                 (microns) 
               
               
                 Solid Feature Depth 
                 N/A 
                 16.3 
                 11.7 
               
               
                 (microns) 
               
            
           
           
               
               
            
               
                 Weakest Axis Tensile 
                 814.7 
               
               
                 Strength (N/m) 
               
               
                 Geometric Mean Tensile 
                 13.3 
               
               
                 Strength (MPa) 
               
               
                 Composite Bond (kPa) 
                 235.0 
               
               
                   
               
            
           
         
       
     
     Evaluation of the Composite Membrane Performance 
     The biocompatible membrane composite was thermally welded into a device form in accordance with the Integration of Biocompatible Membrane Composite into a Device Form set forth in the Test Methods section above and evaluated for functional performance in vivo. 
     The host tissue response was evaluated in the In Vivo Porcine Study_set forth in the Method section set forth above. The host tissue response at the device interface demonstrated host tissue penetration through the polyester woven mesh reinforcing component, the open ePTFE vascularization layer, and open ePTFE mitigation layer up to the tight ePTFE cell impermeable layer. While foreign body giant cells were present within the polyester woven mesh (reinforcing component) and there was no observation of foreign body giant cells along the tight, ePTFE layer (Cell Impermeable Layer). The histology images shown in  FIG. 47  is are representative images of this observation, with arrows  4710  indicating the location of the foreign body giant cells in relation to each layer of the biocompatible membrane composite  4700 . Additionally, as shown in  FIG. 47 , foreign body giant cells (indicated by arrows  4710 ) did not form on the surface of the cell impermeable layer  4720 . It was concluded that the biocompatible membrane composite  4700  formed of the cell impermeable layer, the mitigation layer, and the vascularization layer described in this Example reduced the formation of foreign body giant cells (indicated by arrows  4710 ) on the surface of the cell impermeable layer  4720 . 
     The functional response of the device loaded with cells was evaluated in vivo in accordance with the In Vivo Nude Rat Study set forth in the Test Methods section set forth above. The results in Table 3 demonstrate a step change in functional response as compared to the comparative examples, which indicated a significant increase in viability of insulin producing cells. At 24 weeks after implantation, the c-peptide blood serum concentration measured in response to glucose stimulated insulin secretion was, on average, 337 pM, which is 2.7x greater than that of Comparative Example 1 where no mitigation layer was present. It was concluded that in order to achieve this increased degree of functional response, the mitigation layer was able to successfully mitigate the formation of foreign body giant cells at the cell impermeable surface. 
     Example 6 
     Manufacturing of Biocompatible Membrane Composite 
     A biocompatible membrane composite as described in Example 5 was made and formed into a planar device  4100  that included a reinforcing component  4130 , shown generally in  FIG. 41 . The planar device described in this Example differs from the previously described devices (i.e., the devices in Examples 1-5) in that the planar device is based on a reinforcing component, depicted in  FIG. 40 , that is located adjacent to the cell impermeable layers of the biocompatible membrane composites. The reinforcing component is located within the lumen of the planar device (e.g., endoskeleton) as opposed to the external reinforcing component that was provided by the woven polyester mesh in the previous Examples. The reinforcing component  4000  includes a reinforcing component  4010  and an integrated filling tube  4020  with a flow through hole  4030  to access both sides of the reinforcing component  4000 . 
     The planar device  4100  is shown generally in  FIG. 41  (in an exploded view). As shown in  FIG. 41 , the planar device  4100  includes a first biocompatible membrane composite  4110 , a second biocompatible membrane composite  4140 , a reinforcing component  4130  that includes a reinforcing component  4120  and an integrated filling tube  4150  with a flow through hole  4160  to access dual internal lumens (not shown) formed on both sides of the reinforcing component  4130  when the biocompatible membranes  4110 ,  4140  are integrated into a final device form. 
     The reinforcing component was constructed by placing a sheet of a fluorothermoplastic terpolymer of TFE, HFP, and VDF into a mold cavity and compressing the terpolymer in an heated press (Wabash C30H-15-CPX) set at a temperature above the softening temperature of the polymer so that it conforms to a final dimension and shape. The resulting reinforcing component had a thickness of approximately 270 microns and a stiffness of 0.7 N. 
     Two biocompatible membrane composites were cut to approximately 1″×2″ (2.54 cm×5.08 cm) and arranged on both sides of the reinforcing component with the Cell Impermeable Layer of each membrane composite facing inwardly towards the lumen and the planar reinforcing component. An exploded view of the individual components of the planar device  4100  is shown in  FIG. 41 . 
     The planar device is shown in  FIG. 42 . To create the planar device  4200 , a weld was formed by compressing the material stack shown in  FIG. 41  using an impulse welder along the perimeter  4210  and applying a temperature and pressure such that the reinforcing component thermoplastic softened enough to form a bond into each composite membrane. Internal points of the reinforcing component were bonded to each membrane composite surface by applying light manual pressure with a thermal head to create internal point bonds  4220  of approximately 1 mm diameter spaced at least 1.45 mm apart at 12 locations on each side. The integrity of the welds were evaluated for suitability by testing for the presence of leaks visually detected as a stream of bubbles when submerged in isopropyl alcohol at an internal pressure of 5 psi. The internal geometry of the reinforcing component  4310  and internal lumen  4330  is shown in  FIGS. 43A and 43B .  FIG. 43A  depicts a cross-section of the planar device  4200  taken along line A-A showing a single point bond  4320  and the lumen  4330 .  FIG. 43B  is a cross-section image of the planar device  4200  taken along line B-B showing two point bonds  3620  and the lumen  3630 . The finished planar device shown in  FIG. 42  was filled with a low viscosity silastic to allow for better visualization and imaging of the reinforcing component  4210  shown in  FIGS. 42A and 42B . 
     Evaluation of Composite Membrane Performance In Vivo 
     The biocompatible composite membrane integrated into the planar device described above was evaluated for functional performance in the In Vivo Porcine Study to Evaluate Host Tissue Response set forth in the Method section set forth above. The host tissue response at the planar device interface with the host&#39;s tissue demonstrated host tissue penetration through the open ePTFE vascularization and mitigation layers up to the tight ePTFE cell impermeable layer. There were very few instances of foreign body giant cells observed in the membrane composite. The histology image shown in  FIG. 44  is a representative image of an observation where there is no host penetration through the ePTFE vascularization layer  4430  and the ePTFE mitigation layer  4420 , and no obvious observations of foreign body giant cell formation in or around the membrane composite, including at the cell impermeable interface  4410 . It was concluded that the biocompatible membrane composite  4400  formed of the cell impermeable layers  4410 , the mitigation layers  4420 , and the vascularization layers  4430  described in this Example reduced the formation of foreign body giant cells on the surface of the cell impermeable layer  4410 . The lumen  4440  is also shown for reference. 
     The functional performance of planar device  4200  loaded with cells was evaluated in accordance with the Nude Rat Explant Histology set forth in the Test Methods section above. A representative histology image of a cross-section of the device is shown in  FIG. 48 . From the evaluation of the histology images, it can be concluded that the inclusion of an internal reinforcing component positioned in the lumen of planar device  4200  successfully enabled in vivo cell viability at 24 weeks as evidenced by viable cells  4810  in  FIG. 48 . 
     Example 7 
     Manufacturing of Biocompatible Membrane Composite 
     Three different composite membranes having three layers were used to construct the cell encapsulation device form described in Example 6. The first layer (Cell Impermeable Layer) and second layer (Mitigation Layer) were similar across all three constructs. However, the third layer (Vascularization Layer) was different across the three constructs. These constructs will be referred to as construct A, construct B, and construct C in this section. 
     For all three constructs, a first layer formed of an ePTFE membrane (Cell Impermeable Layer) was formed according to the teachings of U.S. Pat. No. 3,953,566 to Gore. 
     For all three constructs, a two-layer composite consisting of a second ePTFE membrane (Mitigation Layer) and a third ePTFE layer (Vascularization Layer) was formed. The second ePTFE membrane was prepared according to the teachings of U.S. Pat. No. 5,814,405 to Branca, et al. The ePTFE tape precursor of the second ePTFE layer was processed per the teachings of U.S. Pat. No. 5,814,405 to Branca, et al. through the below-the-melt MD expansion step. During the below-the-melt MD expansion step of the second ePTFE tape precursor, an FEP film was applied per the teachings of WO 94/13469 to Bacino. The ePTFE tape precursor of the third ePTFE layer was processed per the teachings of U.S. Pat. No. 5,814,405 to Branca, et al. through an amorphous locking step and above-the-melt MD expansion. Each construct&#39;s third layer was subjected to different process conditions during processing prior to layering to achieve the desired microstructure in the third layer of construct A, construct B, and construct C. During the first below-the-melt MD expansion step of the third ePTFE tape precursor, an FEP film was applied per the teachings of WO 94/13469 to Bacino. The expanded ePTFE tape precursor of the third ePTFE membrane was laminated to the expanded ePTFE tape precursor of the second ePTFE membrane such that the FEP side of the third ePTFE tape was in contact with the PTFE side of the ePTFE tape precursor of the second ePTFE membrane. The two layer composite was then co-expanded in the machine direction and transverse direction above the melting point of PTFE. A representative surface microstructure of the second ePTFE layer of Construct A, Construct B, and Construct C having thereon FEP  5620  is shown in the scanning electron micrograph (SEM) image of  FIG. 56 . 
     The two-layer composite consisting of the second ePTFE membrane (Mitigation Layer) and third ePTFE membrane (Vascularization Layer) was laminated to the first ePTFE membrane (Cell Impermeable Layer). The side of the second ePTFE membrane comprising a discontinuous layer of FEP thereon was laminated to the first ePTFE layer by first bringing two-layer ePTFE composite into contact with the third ePTFE layer (with the FEP positioned between the two layers) at a temperature above the melting point of the FEP with the ePTFE membranes unrestrained in the transverse direction. The laminate was then transversely expanded above the melting point of PTFE so each layer was returned to its width prior to any necking sustained through lamination. The composite was subsequently rendered hydrophilic per the teachings of U.S. Pat. No. 5,902,745 to Butler, et al. The SEM image shown in  FIG. 19  is a representative image of the node and fibril structure of the first ePTFE membrane (Cell Impermeable Layer). The SEM images shown in  FIG. 50 ,  FIG. 51 , and  FIG. 52  are each a representative image of the node and fibril structure of the third ePTFE membrane in each of Construct A, B, and C (Vascularization Layers). The SEM images shown in  FIG. 53 ,  FIG. 54 , and  FIG. 55  are representative images of the cross-section structures of the three layer biocompatible membrane composite including the first ePTFE membrane  5320 ,  5420  and  5520  (Cell Impermeable Layer), the second ePTFE membrane  5340 ,  5440 , and  5540  (Mitigation Layer) and the third ePTFE membrane  5360 ,  5460 , and  5560  (Vascularization Layer). 
     Characterization of the Biocompatible Membrane Composites 
     Each biocompatible membrane composite was evaluated and characterized for the relevant properties for each layer. Parameters for layers are marked as “N/A” if they are not relevant for that layer&#39;s specific function. Parameters for layers are marked as “-” if they are practically unobtainable as a result of how the layers of the composite were processed. The methods used for the characterization of the relevant properties were performed in accordance with the methods described in the Test Methods section set forth above. 
     Table 10 illustrates three (3) different biocompatible membrane composites. All three biocompatible membrane composites had the same Cell Impermeable Layer and FBGC Mitigation Layer but the Vascularization Layer was varied across Construct A (Vascularization A), Construct B (Vascularization B), and Construct C (Vascularization C). The properties of the components of the three biocompatible membrane composites are shown in Table 10. 
     
       
         
           
               
               
             
               
                   
                 TABLE 10 
               
             
            
               
                   
                   
               
               
                   
                 Construct ID 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 All 
                 All 
                 Construct A 
                 Construct B 
                 Construct C 
               
            
           
           
               
               
            
               
                   
                 Layer Function 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Cell Impermeable 
                 FBGC Mitigation 
                 Vascularization A 
                 Vascularization B 
                 Vascularization C 
               
               
                 Description 
                 ePTFE Tight Layer 
                 ePTFE Open Layer 
                 ePTFE Open Layer 
                 ePTFE Open Layer 
                 ePTFE Open Layer 
               
               
                   
               
               
                 MPS (μm) 
                 0.21-0.31 
                 — 
                 — 
                 — 
                 — 
               
               
                 Pore Size 
                 0.34 
                  8.06 
                  16.38 
                  19.69 
                 18.96 
               
               
                 (μm) 
               
               
                 Thickness 
                  8.2-12.0 
                 32.3-44.4 
                 43.1 
                 63.1 
                 30.2  
               
               
                 (μm) 
               
               
                 Mass (g/m 2 ) 
                 Pending 
                 Pending 
                 Pending 
                 Pending 
                 Pending 
               
               
                 Porosity 
                 Pending 
                 Pending 
                 Pending 
                 Pending 
                 Pending 
               
               
                 (%) 
               
               
                 Solid 
                 N/A 
                 24.2 
                 69.4 
                 163.3  
                 86.0  
               
               
                 Feature 
               
               
                 Spacing 
               
               
                 (μm) 
               
               
                 Solid 
                 N/A 
                  4.7 
                  7.5 
                 18.5 
                 8.8 
               
               
                 Feature 
               
               
                 Minor Axis 
               
               
                 (μm) 
               
               
                 Solid 
                 N/A 
                 31.9 
                 24.6 
                 38.7 
                 54.2  
               
               
                 Feature 
               
               
                 Major Axis 
               
               
                 (μm) 
               
               
                 Solid 
                 N/A 
                 10.3-19.2 
                 18.1 
                 16.5 
                 7.3 
               
               
                 Feature 
               
               
                 Depth (μm) 
               
               
                 Geometric 
                 N/A 
                 N/A 
                 Pending 
                 Pending 
                 Pending 
               
               
                 Mean 
               
               
                 Tensile 
               
               
                 Strength 
               
               
                 (MPa)* 
               
               
                 Composite 
                 N/A 
                 N/A 
                 Pending 
                 Pending 
                 Pending 
               
               
                 Bond (kPa)* 
               
               
                   
               
            
           
         
       
     
     Note that the values listed under each Construct for these properties are for the bulk values of all three layers in each construct and not just the third layer (Vascularization Layer) 
     Evaluation of Composite Membrane Performance In Vivo 
     The three biocompatible membrane composites were integrated into cell encapsulation devices as described in Example 6. 
     The functional performances of the devices (Constructs A, B and C) loaded with cells were evaluated in accordance with the Nude Rat Explant Histology set forth in the Test Methods section above. Representative histology images of cross-sections of the devices with varied vascularization layers Constructs A, B and C are shown in  FIGS. 49A, 49B and 49C , respectively. From the evaluation of the histology images, it can be observed that the formation of foreign body giant cells (FBGC) on the cell impermeable layer were mitigated and that the inclusion of an internal reinforcing component positioned in the lumen of planar devices Construct A  4900 , Construct B  4910 , and Construct C  4930  successfully enabled in vivo cell viability as evidenced by viable cells  4920 ,  4940 , and  4960  in  FIGS. 49A, 49B, and 49C . 
     The invention of this application has been described above both generically and with regard to specific embodiments. It will be apparent to those skilled in the art that various modifications and variations can be made in the embodiments without departing from the scope of the disclosure. Thus, it is intended that the embodiments cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.