Patent Publication Number: US-2020298487-A1

Title: Sterile additive manufacturing system

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     The present application claims priority to and is a National Stage application, filed under 35 U.S.C § 371, of International Application No. PCT/M2018/059476, filed on Nov. 29, 2018, which claims the benefit, under 35 USC § 119(e) of U.S. Provisional Patent Application No. 62/652,757, filed Apr. 4, 2018, and U.S. Provisional Patent Application No. 62/592,202, filed Nov. 29, 2017. Each of the foregoing applications are incorporated herein by reference for all purposes. 
    
    
     BACKGROUND OF THE DISCLOSURE 
     Additive manufacturing, or 3D printing, can generally include “printing” an object by successively depositing layers of patterned material atop one another. Additive manufacturing can, in a process termed “bioprinting,” generate biological components or structures that can include cells, proteins, or growth factors that have biological function in the produced construct. The bioprinting process may need to comply with the good manufacturing practices (GMP) guidelines to be applicable to clinical or pharmaceutical use. The printing material can be extruded through the lumen of a printing nozzle. The printing material can be extruded from the printing nozzle under pressure. Extruding the printing material under pressure can cause the formation of aerosol droplets that can contaminate the environment surrounding the printer. The production process consists of certain steps but is not limited to these steps 
     SUMMARY OF THE DISCLOSURE 
     In order to print clinical or pharmaceutical substances, a bioprinting process may need to comply with sterile process requirements and prevent the cross contamination. The present solution describes systems and methods to bioprint cellular constructs or organs in a sterile fashion that substantially prevents cross contamination during the manufacturing process. 
     During the bioprinting process a number of tests may need to be performed on the cellular construct. Some of the tests that are performed can be destructive and can result in damage to the tested cellular construct or cells therein. The present solution bioprints a plurality of appendices to the cellular construct. The appendices can be printed as removable samples of the cellular construct and can include the same cells and materials as those included within the cellular construct. At predetermined times, the appendices can be removed from the cellular construct and tested. The appendices can serve as a proxy for the cellular construct and provide insight into the health and proliferation of the cells within the cellular construct. 
     The present solution includes a three-dimensional (3D) printer designed to meet clean room requirements in biologics or pharmaceutical applications. The printer can be configured for 3D bioprinting. The printer can be used to create acellular scaffolds or organ templates, cellular scaffolds, tissue grafts, and multi-cellular organs. For example, the printer can be used to generate cellular tissues such as skin, bone, and cartilage, which can generally be referred to as cellular constructs. These tissue constructs can include biological components such as cells, growth factors, pharmaceuticals, or a combination thereof. The biological components can be mixed, solubilized, or coextruded with synthetic or natural polymers, proteins, or other biocompatible materials. The biological components can be included into the bioprinted materials, often hydrogels, by mixing them before the printing process or alternatively by introducing the biological components after the printing process as coatings or infill. Cells can be embedded in the polymer mix to form biologically functional cellular constructs, tissue grafts, or organs. Cells or other biological components can be introduced to printed scaffold structures or templates by spray coating the object or by infiltration of the biological material into the printed template. The printer can also be used to manufacture custom pharmaceutical tablets and medications. 
     The produced tissue grafts are produced in sterile environment. Mammalian (e.g., human) cells are isolated from clinical tissue biopsy. The isolated cells can be primary cells, progenitor cells, stem cells or a combination of these. The cells are isolated by mechanically and/or chemically disrupting the extracellular matrix or carrier fluid to release the cells. The collected cells are commonly expanded in monolayer or 3D cultures until a sufficient cell number is obtained. Cells can be expanded multiple weeks depending on the initial isolation cell yield and the application need. Cells can be transfected or gene edited to modify the genome prior printing to obtain desired cell function in the created tissue. These cells can then be mixed with natural or synthetic polymers for a cellular biomaterial mix. Biopolymers such as but not limited to hyaluronan, collagen, gelatin, chondroitin sulfate, alginate, gellan gum or any combination can be used to prepare the polymer mix with the cells. Synthetic polymers such as poly ethylene glycol (PEG), poloxamers, polyoxazolines, polypropylene glycol, poly (L/D)lactide, polyglycolic acid, polymethacrylate polyachrylamide or a combination or a block-copolymers of these. For example, a polymer mix of alginate and gellan gum can be used to mix the cells to for the cellular polymer mix that is suitable for a bioprinting process. 
     The cellular polymer mix such as alginate and gellan gum with chondrocytes can be loaded into a printing syringe after a mixing process to obtain homogeneous end material. The mixing process can be manual mixing, extrusion with static mixer or an active mixing process which end product is collected to the syringe. Mixing process can be performed inside the syringe or before loading the materials mix into the syringe. 
     The present solution can also include printing kits. Each printing kit can include printing materials syringe and printer components for a printing run. The printing kit, and the components thereof, can be sterilized and then passed into the printer&#39;s enclosure through an airlock. Once in the enclosure, the components can be assembled to form the 3D printer&#39;s deposition head and the deposition head can be loaded with the printing materials. Multiple printing syringes and nozzles can be used during the printing process to produce multi-material or multi-cellular constructs. Multiple syringes can be used alternating the extruding syringe within the printing process. The kit can also include a sterile transportation unit into which the completed item is deposited. The transportation unit enables the item to be transferred to an incubator (or other location) while remaining in a sterile environment. The waste from the printing run can be placed back into the kit and removed from the printer, which can be sterilized after the printing run. 
     The printer can include a sterile (or clean room-like) environment for the printing of items. The printer can include an enclosure that isolates the manufacturing processes from the external environment. The enclosure can be flooded with chemical sterilizers to sterilize the enclosure between printing runs. The enclosure can also prevent the aerosol droplets (generally referred to as particles) from contaminating the external environment. All printer surfaces are compatible with chemical acid and base cleaning cycles and gassing. After printing, the isoprinter can be wiped down with acid and base detergents to remove any possible spills or solid materials preventing the gas to reach all printer surfaces. After the wiping, the gassing utilizing H 2 O 2  or similar is performed to sterilize the isoprinter. 
     The bioprinted construct, composed of liquid, semi-solid or gel-like materials is required to go through a crosslinking process to further stabilize, solidify or reinforce the structure of the created tissue graft. Multiple gelation methods including but not limited to thermal, ionic-, enzymatic, radical or chemical reactions can be used within the isolator space during or after the bioprinting process. For example gellan gum and alginate biopolymer mix can be crosslinked in the presence of mono-, di- or tri-valent cations including but not limited to Mg 2+ , Ca 2+ , Sr 2+ , Ba 2+ , Zn 2+ , Cu 2+  or Fe 3+ . 
     According to at least one aspect of the disclosure, an additive manufacturing system can include at least one pass-through chamber coupled with an enclosure. The at least one pass-through chamber can include a first portal to enable passage from an external environment to an interior of the at least one pass-through chamber and a second portal to enable passage from the interior of the at least one pass-through chamber to an interior of the enclosure. The enclosure can include a first port configured to receive a deposition head of a three-dimensional printer. The enclosure can include a first bellow configured to couple with the deposition head and a perimeter of the first port. The system can include a 3D printer. 
     The system can include a printing kit. The printing kit can include a base plate configured to receive material extruded from the deposition head. The printing kit can include a sleeve that can include a first end configured to couple with the deposition head and a second end configured to couple with the base plate to form a secluded volume within the enclosure. 
     The printing kit can include a containment bag configured to collect waste from a process of printing a biological scaffold with the 3D printer. The printing kit can include a transport unit configured to enable transportation of the biological scaffold. The printing kit can include syringe including a printing material of the 3D printer. The printing material can include at least one biopolymer and a plurality of cells. 
     The system can include a second pass-through chamber coupled with the enclosure. The system can include one or more access ports configured to enable a user to manipulate items within the enclosure. The 3D printer can include a plurality of deposition heads. Each of the plurality of deposition heads can be configured to deposit a different printing material. 
     According to at least one aspect of the disclosure, an additive manufacturing kit can include a base plate configured to receive material extruded from a deposition head of a three-dimensional printer. The kit can include a sleeve that can include a first end configured to couple with the deposition head and a second end configured to couple with the base plate to form a secluded volume. The kit can include a syringe that can include a printing material. 
     In some implementations, the printing material can include a biopolymer mix with cells. The printing kit can include serializable housing to store the base plate, the sleeve, and the syringe. 
     According to at least one aspect of the disclosure, a method can include isolating chondrocytes from a biopsy. The method can include generating a biopolymer printing material that can include at least one polymer and the chondrocytes. The method can include forming a cellular construct from the biopolymer printing material with an additive manufacturing system. The system can include at least one pass-through chamber coupled with an enclosure. The at least one pass-through chamber can include a first portal to enable passage from an external environment to an interior of the at least one pass-through chamber and a second portal to enable passage from the interior of the at least one pass-through chamber to an interior of the enclosure. The enclosure can include a first port configured to receive a deposition head of a three-dimensional printer. The enclosure can include a first bellow configured to couple with the deposition head and a perimeter of the first port. 
     The biopolymer printing material can include at least one of a gelling polysaccharide or sodium alginate. The method can include forming at least one appendix on the cellular construct from the biopolymer printing material. The method can include excising one of the at least one appendices of the cellular construct for testing. The method can include cross linking the cellular construct with a calcium chloride solution. 
     The biopolymer printing material can include at least one of differentiated progenitor cells or differentiated stem cells harvested from the biopsy. The method can include culturing the chondrocytes from the biopsy until the chondrocytes reach a predetermined cell count. The method can include sterilizing the enclosure. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The accompanying drawings are not intended to be drawn to scale. Like reference numbers and designations in the various drawings indicate like elements. For purposes of clarity, not every component may be labeled in every drawing. In the drawings: 
         FIGS. 1A and 1B  illustrate different views of an example isolation printer that can be used to manufacture cellular constructs. 
         FIG. 2  illustrates a schematic of an example kit for use with the isolation printer illustrated in  FIGS. 1A and 1B . 
         FIG. 3  illustrates a block diagram of an example method to bioprint a cartilage organ using the system illustrated in  FIGS. 1A and 1B . 
         FIG. 4  illustrates a block diagram of an example method for additive manufacturing using the isolation printer illustrated in  FIGS. 1A and 1B . 
         FIG. 5  illustrates an example cellular construct with removable appendices manufactured with the example isolation printer illustrated in  FIGS. 1A and 1B . 
     
    
    
     DETAILED DESCRIPTION 
     The various concepts introduced above and discussed in greater detail below may be implemented in any of numerous ways, as the described concepts are not limited to any particular manner of implementation. Examples of specific implementations and applications are provided primarily for illustrative purposes. 
       FIGS. 1A and 1B  illustrate an example isolation printer  100 , which can also be referred to as an isoPrinter  100 . The isoPrinter  100  can be an additive manufacturing system.  FIG. 1A  illustrates a front view of the isoPrinter  100 .  FIG. 1B  illustrates a cross-sectional side view of the isoPrinter  100 . 
     The isoPrinter  100  includes an enclosure  102  that houses a deposition head  104  of a 3D printer  152 . The deposition head  104  can also be referred to as a printing head. In some implementations, the isoPrinter  100  can include a plurality of deposition heads  104 . A printing nozzle  206  can be coupled with each of the deposition heads  104 . The different deposition heads  104  can be used to print multi-material or multi-cellular tissue grafts. For example, the isoPrinter  100  can print an internal support structure from a first material and a cellular construct around the internal support structure in a second material. 
     The isoPrinter  100  can include a base plate  106  onto which the deposition head  104  can deposit printing material. The enclosure  102  can include a plurality of airlocks  108  (or pass-through chambers  108 ). In some implementations, one airlock  108  can be used to pass materials and the kit described in relation to  FIG. 2  into the enclosure  102  and a second airlock  108  can be used to remove materials and the kit from the enclosure  102 . Each of the airlocks  108  can include one or more portals  150 . A first portal  150  can enable passage from the exterior of the system  100  to the interior of the airlock  108 . The airlock  108  can be coupled with the enclosure  102 . A second portal  150  can enable passage between the interior of the airlock  108  and the interior of the enclosure  102 . For example, the below described kit can be passed into the airlock  108  via the first portal  150  and then passed into the interior of the enclosure  102  via the second portal  150 . The enclosure  102  can also include access ports  110  that enable a user to manipulate materials and items within the enclosure  102 . 
     The enclosure  102  can isolate the manufacturing process performed by the deposition head  104  from the external environment. By isolating the manufacturing process, the isoPrinter  100  can be used in cleanroom environments. For example, the isoPrinter  100  can be used in class B-C clean rooms. The walls of the enclosure  102  can include acrylic glass or other transparent materials that can be sterilized, disinfected, and/or sanitized. In some implementations, a substantial portion of each of the walls can be transparent. In other implementations, the walls of the enclosure  102  can be constructed from metal and the walls can include transparent viewing ports. 
     The enclosure  102  can be sealed to prevent airflow between the interior and exterior of the enclosure  102 . In some implementations, the isoPrinter  100  can include a pump to positively or negatively pressurize the interior of the enclosure  102 . In some implementations, the environment inside the enclosure  102  can be controlled for desired temperature, atmospheric gas and humidity. 
     The interior of the enclosure  102  can be sterilized. The enclosure  102  can include inlet and outlet ports to enable the introduction of sterilizers by a sterilization unit. For example, after use, the interior of the enclosure  102  can be flooded with H 2 O 2  gas to sterilize the enclosure  102 . 
     The enclosure  102  can include a port for the deposition head  104 . For example, the controller and other components of the 3D printer can be positioned outside the enclosure  102 . The 3D printer&#39;s deposition head  104  can pass into the enclosure  102  through the port. The port can include a rubber bellow that couples to the deposition head  104  and forms a seal between the deposition head  104  and the perimeter of the port. The bellow can enable the deposition head  104  to freely move in an x, y, and z directions within the enclosure  102 . 
     In some implementations, the deposition head  104  can include positioning and feedback sensors. The sensors can be configured to determine the location of the deposition head  104  (and the printing nozzle) within the enclosure  102  and relative to the base plate  106  and the material already printed on the base plate  106 . The sensors can include piezoelectric sensors and laser-based distance sensors. 
     The enclosure  102  can also include airlocks  108 . The airlocks  108  can enable a user to pass materials and equipment into and out of the enclosure  102  without cross contaminating the isoPrinter  100  and the environment by substantially preventing contaminants or undesirable particles from passing between the interior and exterior of the enclosure  102 . For example, an airlock  108  can include an interior and an exterior door (e.g., portals  150 ). The interior door can face the interior of the enclosure  102  and the exterior door can face the external environment. A user can first open the exterior door, with the interior door closed, and place the materials within the interior of the airlock  108 . After shutting the exterior door, the user can open the interior door (via the gloves of the access port  110 ). The airlocks  108  can include air showers to flow air over the items within the airlocks  108  to remove particles and contaminants from the items. In some implementations, the airlocks  108  can flow a gas sterilizer into the airlock  108  to sanitize or sterilize the item within the airlock  108 . 
     The access ports  110  can include openings in the wall of the enclosure  102 . Gloves can be sealed to the access ports  110  to enable a user to manipulate items within the enclosure  102  while still providing a barrier between the internal and external environment of the enclosure  102 . In some implementations, the access port  110  can include bellows that enable tools or robotic arms to be used within the enclosure  102 . 
     The deposition head  104  can include the of extruder of the 3D printer. The deposition head  104  can include a printing nozzle through which the printing material is extruded. The printing material can include plastics, metals, synthetic polymers, or other biocompatible materials. In some implementations, printing nozzle can be removable. The printing nozzle can include brass, stainless steel, hardened steel, or plastic. The printing material can be passed through the deposition head  104  under pressure and at a controlled temperature to the printing nozzle. The printing material can be extruded from the deposition head  104  through a lumen in the printing nozzle. In some implementations, the printing nozzle can be the needle of a syringe. In these implementations, a filled syringe can be inserted into the deposition head  104 . The deposition head  104  can include an actuator that presses against a plunger of the syringe and causes printing material to be extruded or a screw-based system to move material in the threads from the syringe&#39;s needle. 
     The isoPrinter  100  can include a port  151 . The port  151  can be opening in the enclosure  102  in the wall through which the deposition head  104  extends from the 3D printer  152 . The isoPrinter  100  can include a bellow  153  that can couple with a perimeter of the port  151  on a first end of the bellow  153  and the deposition head  104  on the second end of the bellow  153 . The bellow  153  can enable the deposition head  104  to move freely within the port  151 . The bellow  153  can form a seal to prevent contaminants from passing through the port  151  and into the interior of the enclosure  102 . 
     The isoPrinter  100  can include a base plate  106 . The deposition head  104  can deposit material onto the base plate  106 . The base plate  106  can be coupled to one or more actuators to enable the base plate  106  to move in the x, y, and z directions. The base plate  106  can be a component of the kit described in relation to  FIG. 2 . For example, prior to each build, a base plate  106  can be passed into the enclosure  102  and secured to the actuators. In some implementations, the base plate  106  is static and only the deposition head  104  moves. 
       FIG. 2  illustrates a schematic of an example kit  200 . The kit  200  can include the materials, tools, and other items that are used for a specific manufacturing run. In some implementations, the kit  200  can include any combination of a sleeve  202 , a syringe  204 , a printing nozzle  206 , a transport unit  208 , a base plate  106 , or a containment bag  212 . The kit  200  includes a housing to store the components of the kit. The kit housing and the components of the kit  200  can be sterilized. 
     The sleeve  202  can be a flexible bellow, tube, or skirt. A first end of the sleeve  202  can couple with the deposition head  104  and a second end of the sleeve  202  can couple with the perimeter of the base plate  106 . When sealed between the deposition head  104  and the base plate  106 , the sleeve  202  can form a secluded volume in which the printing deposition takes place. Use of the sleeve  202  can confine contaminants and particles from dispersing from the deposition head  104  and throughout the enclosure  102 . Containment of the particles can make the sterilization and cleaning of the enclosure  102  easier, quicker, and more cost effective. The sleeve  202  can be configured to enable full freedom of movement of the deposition head  104  during the manufacturing process. The sleeve  202  can be plastic-, rubber-, or silicon-based. The sleeve  202  can include a seal, such as a gasket or O-ring, at each of its ends to form a hermetical seal between the deposition head  104  and the base plate  106 . In some implementations, the kit  200  can include clips, lugs, or locks that can be used to secure the sleeve  202  to the deposition head  104  and/or the base plate  106 . 
     The kit  200  can include one or more printing nozzles  206 . Each of the different printing nozzles  206  can include different lumen diameters for the extrusion of the printing material. The smaller diameter lumens can enable the 3D printer to print with a relatively higher resolution when compared to larger diameter lumens. For each run, the printing nozzle  206  can be replaced. 
     The kit  200  can also include a syringe  204 . The syringe  204  can be prefilled with a biopolymer mix with or without cells or other printing material. A user can use the syringe  204  to fill the deposition head  104  with the printing material. In some implementations, the syringe  204  can be placed directly into the deposition head  104  and the syringe&#39;s needle can be used as the printing nozzle. The kit  200  can include a plurality of different syringes  204 . Each of the different syringes  204  can be filled with a different (or additional) printing material. 
     The kit  200  can include a transport unit  208 . The transport unit  208  can be sterile container into which the printed item is directly created or placed once printed. The printed item can be placed in the transport unit  208 , passed to the exterior of the enclosure  102 , via an airlock  108 , and then transported to another location where further processing can be performed on the printed item. In some implementations, when a biological scaffold, such as for an ear or other cellular construct, is printed, the printed item can be taken from the isoPrinter  100 , via the transport unit  208 , to an incubator where the cells in the biological scaffold can be incubated. In some implementations, the cells can be incorporated into the biopolymer mix and printed on to a biological scaffold. In other implementations, the cells can be seeded onto the biological scaffold after the scaffold is printed. In some implementations, the transport unit  208 , with the printed item, can be sterilized before implantation into the patient. 
     The kit  200  can also include a containment bag  212 . Once the printing run is completed, the waste and disposable items of the kit  200  can be placed in the containment bag  212  and the containment bag  212  can be sealed. For example, the sleeve  202 , syringe  204 , and printing nozzle  206  can be disposed after each run. In some implementations, the waste material can be directly placed into the housing of the kit  200  and not into a containment bag  212 . 
       FIG. 3  illustrates a block diagram of an example method  300  to bioprint a cartilage organ. For example, the method  300  can be used to manufacture an ear or a nose. The method  300  can include obtaining a biopsy (step  301 ). The method  300  can include isolating cells from the biopsy (step  302 ). The method  300  can include expanding the cells by supporting multiple cell doublings until sufficient number of cells is obtained (step  303 ). The method  300  can include generating a polymer mix (step  304 ) and adding the cells to the polymer mix (step  305 ). The method  300  can include bioprinting the cellular construct (step  306 ) and inspecting the construct&#39;s appendices (step  307 ). The method  300  can include further culturing of the construct to form matured tissue (step  308 ). The method  300  can include the implantation of the construct (step  309 ). 
     As set forth above, the method  300  can include obtaining a biopsy (step  301 ). The biopsy can be obtained from the patient into which the organ will eventually be implanted. In some implementations, the biopsy can be obtained from a donor. The biopsy can include auricular cartilage, nasal cartilage, nucleus pulposus, meniscus, trachea, nasal cartilage, rib cartilage, articular cartilage, synovial fluid, vitreous humor, brain, spinal cord, muscle, connective tissues, small intestinal submucosa, or liver tissue. For manufacturing an ear, the biopsy can be a 4-8 mm diameter, full thickness circular cartilage biopsy sample that is obtained from the ear contralateral to the microtia ear. Once resected, the biopsy can be stored in phosphate-buffered saline (PBS) with gentamicin (50 g/mL). This biopsy is not a critical size defect and will heal over time. In some instances, the tissue biopsy can be transported in 2-8° C. to enhance the cells ability to survive before cells isolation. 
     The method  300  can include isolating cells from the biopsy (step  302 ). The cells can be chondrocytes. The connective tissue or other unwanted tissue can be removed from the biopsy tissue and the sample tissue (e.g., cartilage) can be minced. The tissue can be minced to a size of between about 5 μm and about 50 μm, between about 50 μm and about 200 μm, or between about 200 μm and about 100 μm. Sterile filtered digestion medium including DMEM and Ham&#39;s F12, 10% fetal bovine serum (FBS), and collagenase 0.66 units/mL enzyme can be combined with the minced cartilage and allowed to incubate for 16-18 hours at 37° C. under static conditions. This can create a suspension of released chondrocytes. The suspension of released chondrocytes can be passed through a 100 μm cell strainer and centrifuged. The pelleted chondrocytes can be resuspended in fresh sterile DMEM+Ham&#39;s F12 supplemented with 10% FBS and 25 μg/mL ascorbic acid. In some implementations, the total number of cells are counted, and cell viability is determined via Trypan blue staining. 
     The method  300  can include expanding the cells (step  303 ). The cells (e.g., the chondrocytes) can be seeded into culture flasks at a concentration of about 3000 cells/cm′. The cells can be seeded in densities between about 100 and about 1000 cells/cm′ or between about 1000 and about 10000 cells/cm′ can be used according to a specific cell requirements. In some implementations, the cells can be cryopreserved for ease of patient scheduling or transportation. If cryopreserved, the cells can be suspended in a medium that can include DMEM and Ham&#39;s F12, 10% FBS, 10% DMSO. The cells can be cooled at a controlled rate of 2° C. per minute until −80° C. prior to storage in liquid or vaporized liquid nitrogen. Once removed from cryopreservation (e.g., once the patient is scheduled for the implantation procedure), the cells are thawed, and cell expansion is continued until approximately 70-100×10 6  cells are present. The cells can be harvested and washed with FBS-free medium. In some implementations, a portion of the cells are harvested, and the cell viability and gene expression are measured. Progenitor and stem cells can be differentiated at this stage prior mixing with biopolymers or used otherwise to guarantee target tissue formation. Differentiated, unipotent, cells can be further mixed into the biopolymers or used in the scaffold coatings. 
     The method  300  can include generating a polymer mix (step  304 ). The polymer mix can be prepared by mechanically mixing gellan gum (35 mg/mL) and sodium alginate (25 mg/mL) with dextran solution (osmolarity  300  mOsmol) at 90° C. The polymer mixture can be aseptically stored at room temperature in syringes for later use with cells. In some implementations, other gelling polysaccharides can be used for the gellan gum or alginate. For example, guar gum, cassia gum, konjac gum, Arabic gum, ghatti gum, locust bean gum, xanthan gum, xanthan gum sulfate, carrageen, carrageen sulfate, or any mixture thereof can be used. In some implementations, the polymer mix can include a bioresorbable polymer, such as PLA (polylactic acid or polylactide), DL-PLA (poly(DL-lactide)), L-PLA (poly(L-lactide)), polyethylene glycol (PEG), PGA (polyglycolide), PCL (poly-ecaprolactone), PLCL (Polylactide-co-e-caprolactone), dihydrolipoic acid (DHLA), chitosan. In some implementations, the polymer can include a synthetic polymer, such as, polyethylene glycol, polypropylene glycol, polaxomers, polyoxazolines, polyethylenimine, polyvinyl alcohol, polyvinyl acetate, polymethylvinylether-co-maleic anhydride, polylactide, poly-N-isopropylacrylamide, polyglycolic acid, polymethylmethacrylate, polyacrylamide, polyacrylic acid, and polyallylamine. In some implementations, the polymer can include any combination of the above. 
     The method  300  can include adding the cells to the polymer mix (step  305 ). A suspension including the cells is added to the polymer mixture generated at step  205  at a 1:10 ratio (cell medium:polymers). The cells can be added via static mixing that is connected directly to a printing syringe to obtain a cell concentration of 6-9×10 6  cells/ml in the biopolymer mix. 
     The method  300  can include bioprinting the cellular construct (step  306 ). The method to bioprint the cellular construct is discussed further in relation to  FIG. 4 . As an overview, a printing syringe filled with the biopolymer mix formed in step  305  can be brought to the printer via pass box as part of the prepared printing kit. The printing syringe can be attached to or inserted in the deposition head or syringe holder of the printer. In some implementations, the printing syringe can be used to fill a reservoir in the deposition head with the biopolymer mix. In an additive fashion, the biopolymer can be extruded form the printing syringe for form the cellular construct. Secondary polymer mix can be extruded from a parallel syringe for three-dimensional support structures. The transient support structures can be removed after the construct finish by physical or chemical process such as but not limited to hydrolytic dissolving, pH change or temperature shift. 
     The method  300  can include inspecting the cellular construct&#39;s appendices (step  307 ). The inspection of the appendices can include removing one or more of the appendices at predetermined intervals. Inspecting the appendices can include performing tests on the excised appendix. The testing can be destructive or non-destructive. The tests can be tests of mechanical and/or biological properties such as, but not limited to, cell viability, gene expression and cell distribution in the cellular construct. The removal and testing of the appendices can occur during a distinct phase of the method  300  or can also occur during the maturation of the construct (step  308 , below). For example, the appendices can be periodically removed and tested during the maturation of the construct to determine when the construct is ready for release to the patient. 
     The method  300  can include construct maturation (step  308 ). For example, after the cellular construct is manufactured, the cellular construct can be removed from the printer to avoid any possible cross contamination issues. The cellular construct can be cross-linked by applying a calcium chloride solution to the cellular construct. The calcium chloride solution can be applied for 240 minutes while cellular construct is positioned on an agitator plate to prevent local concentration gradient formation in the cross-linking. In some implementations, the cross-linking agent can be a monovalent, divalent and trivalent cation, enzyme, hydrogen peroxide, horseradish peroxidase, radiation polymerizable monomers such as lithium phenyl-2,4,6-trimethylbenzoylphosphinate. Polymers crosslinking via photoinitiators can be crosslinked layer-by-layer during the printing process to allow more homogeneous layer exposure. After crosslinking, despite the technique, the cellular construct can be cultured for about 2-5 weeks in culture medium containing 10 ng/mL of recombinant human transforming growth factor beta three (rhTGF-β3) or other mitogenic growth factor known to affect positively to the used cells. Maturation culture can be done in an incubator or a specific bioreaction can be used to stimulate the maturation via mechanical, chemical or biological stimulation. After the tissue maturation the cellular construct can be washed three times with rhTGF-β3-free medium. 
     In some implementations, once the cellular construct is cross-linked, the cellular construct can be further measured with reference to the 3D model of the expected cellular construct shape and size. The above procedure can also be performed on the remaining test samples. The test samples can then be tested for cell viability, PCR gene expression, and mechanical properties. Sterility, endotoxin, and Mycoplasma assays are performed on the test samples. The cellular construct can be packed in a sterile container with nutrient medium (e.g., DMEM and Ham&#39;s F12 alone), and then shipped to the clinic in a specially designed container that guarantees sterility and nutrition supply for the shipped living construct. 
     The method  300  can include construct implantation (step  309 ). The method  300  can include shipment of the construct to clinical site before implantation. The construct can be shipped to the clinical site in a transport unit. The transport unit can maintain a sterile environment until the construct is removed for implantation into a patient. 
       FIG. 4  illustrates a block diagram of an example method  400  for additive manufacturing that can be used in the above-described method  300 . The method  400  can include inputting a 3D model into the isoPrinter (step  402 ). The method  400  can include sterilizing the kit (step  404 ). The method  400  can include passing the kit into the isoPrinter (step  406 ). The method  400  can include assembling the deposition head (step  408 ). The method  400  can include printing the item (step  410 ). The method  400  can include removing the printed item from the isoPrinter (step  412 ). The method  400  can include disposing of the waste (step  414 ) and sterilizing the isoPrinter (step  416 ). 
     Also referring to  FIGS. 1 and 2 , and as set forth above, the method  400  can include inputting a 3D computer model into the isoPrinter  100  (step  402 ). The computer model can be generated via a computer aided design (CAD) program. In some implementations, the computer model can be generated by optically scanning a physical model to generate a digital model. The file, including the 3D geometry of the item to be printed item, can be loaded into the isoPrinter  100  via a direct connection (e.g., with a flash drive) or over a network. 
     The method  400  can include sterilizing the kit  200  (step  404 ). As discussed above, the kit  200  can include printing materials and disposable or reusable components of the deposition head  104 . For example, the kit  200  can include a sleeve  202 , one or more syringes  204  and printing nozzles  206 , a transport unit  208 , a base plate  106 , and a containment bag  212 . The kit  200  can also include printing filament and/or the syringe  204  can be loaded with printing material, such as a bioink or biopolymer mix. Once the kit  200  is assembled for the printing run, the kit  200  can be sterilized. The kit  200 , and the components therein, can be sterilized with heat sterilization, chemical sterilization, radiation sterilization, or any combination thereof. 
     The method  400  can include passing the kit  200  into the isoPrinter  100  (step  406 ). The kit  200  can be passed into the enclosure  102  via an airlock  108 . Once in the enclosure  102 , the components of the kit  200  can be used to assemble the deposition head  104  (step  408 ). Assembling the deposition head  104  can include loading the syringe  204  or printing material into the deposition head  104 . The printing nozzle  206  can also be applied to the deposition head  104 . The base plate  106  can be secured to the floor (or actuators in the floor) of the enclosure  102 . The sleeve  202  can be coupled with the deposition head  104  and the base plate  106  to form a secluded volume where the additive manufacturing process is performed. In some implementations, the method  400  does not include the use of the sleeve  202 . The 3D printer can then print the item (step  410 ). 
     Once the print run is complete, the printed item can be removed from the isoPrinter  100  (step  412 ). In some implementations, before removing the printed item from the isoPrinter  100 , the printed item can be placed in a transport unit  208 . The transport unit  208  can maintain the printed item in a sterile environment until the printed item is further processed (e.g., placed in an incubator or crosslinked) or implanted into a patient. In some implementations, the transport unit  208  and printed item can be re-sterilized before implantation or further processing. The printed item can be removed from the enclosure  102 , in the transport unit  208 , via one of the enclosures airlocks  108 . 
     Once the run is complete, the user can dispose of the waste (step  414 ). The waste can include the used sleeve  202 , syringe  204 , base plate  106 , and printing nozzle  206 . The user can place the used items into the containment bag  212 . The user can pass the containment bag  212  out of the isoPrinter  100  via an airlock  108 . The used components can then be discarded or cleaned, sterilize, and reused. For example, sleeve  202  can be steam cleaned and reused in a subsequent printing run. The interior of the enclosure  102  can then be sterilized (step  416 ). The interior of the enclosure  102  can be chemically sterilized or heat sterilized with, for example, steam. In an example of chemical sterilization, the enclosure  102  can be flooded with ethylene oxide or hydrogen peroxide gas. In some implementations, the isoPrinter  100  can be re-calibrated for the next print run. 
       FIG. 5  illustrates an example construct  50  manufactured with removable appendices  52 . The construct  50  can include a body  51  and one or more appendices  52 . The body  51  can include the part of the construct  50  that forms the final part that is implanted into the patient. For example, the body  51  can include the ear, nose, or other part that is delivered to the patient. The appendices  52  can be coupled with or can extend from the body  51 . 
     The construct  50  can be manufactured with one or more appendices  52 . The appendices  52  can be distributed across one or more edges of the construct  50 . For example, the appendices  52  can be placed at locations around the edge of the body  51  where removal of the appendices  52  will do minimal or no physical or cosmetic damage to the body  51  when the appendices  52  are removed. In some implementations, the appendices  52  can be printed onto the print platform (on which the body  51  is printed) and are not coupled to the body  51 . The appendices  52  can be manufactured from the same material as the body  51 . The appendices  52  can include internal support structures or other components that are incorporated into body  51 . The appendices  52  can be configured to match the mechanical and biological properties of the body  51 . Being manufactured from the same material as the body  51  (and having the same properties as the body  51 ), the appendices  52  can act as proxies for the body  51  during pre-release testing. 
     For example, the construct  50  can be printed from a polymer matrix that includes a mixture of cells. Once the construct  50  is 3D printed, the construct  50  can be incubated to enable the cells to mature and multiply. The appendices  52  can be sequentially removed from the body  51  at different time points along the maturation process. At each of the different time points, the removed appendix 52 can be tested to determine, for example, cell differentiation, cellular density, mechanical properties of the cells, cell viability, gene expression, cell distribution in the cellular construct  50 , drug interaction testing, or any combination thereof. 
     The appendices  52  can have a surface area between about 10 mm 2  and about 500 mm 2 , between about 50 mm 2  and about 1000 mm 2 , between about 100 mm 2  and about 750 mm 2 , between about 200 mm 2  and about 500 mm 2 , or between about 300 mm 2  and about 500 mm 2 . 
     While operations are depicted in the drawings in a particular order, such operations are not required to be performed in the particular order shown or in sequential order, and all illustrated operations are not required to be performed. Actions described herein can be performed in a different order. 
     The separation of various system components does not require separation in all implementations, and the described program components can be included in a single hardware or software product. 
     Having now described some illustrative implementations, it is apparent that the foregoing is illustrative and not limiting, having been presented by way of example. In particular, although many of the examples presented herein involve specific combinations of method acts or system elements, those acts and those elements may be combined in other ways to accomplish the same objectives. Acts, elements and features discussed in connection with one implementation are not intended to be excluded from a similar role in other implementations or implementations. 
     The phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including” “comprising” “having” “containing” “involving” “characterized by” “characterized in that” and variations thereof herein, is meant to encompass the items listed thereafter, equivalents thereof, and additional items, as well as alternate implementations consisting of the items listed thereafter exclusively. In one implementation, the systems and methods described herein consist of one, each combination of more than one, or all of the described elements, acts, or components. 
     As used herein, the term “about” and “substantially” will be understood by persons of ordinary skill in the art and will vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” will mean up to plus or minus 10% of the particular term. 
     Any references to implementations or elements or acts of the systems and methods herein referred to in the singular may also embrace implementations including a plurality of these elements, and any references in plural to any implementation or element or act herein may also embrace implementations including only a single element. References in the singular or plural form are not intended to limit the presently disclosed systems or methods, their components, acts, or elements to single or plural configurations. References to any act or element being based on any information, act or element may include implementations where the act or element is based at least in part on any information, act, or element. 
     Any implementation disclosed herein may be combined with any other implementation or embodiment, and references to “an implementation,” “some implementations,” “one implementation” or the like are not necessarily mutually exclusive and are intended to indicate that a particular feature, structure, or characteristic described in connection with the implementation may be included in at least one implementation or embodiment. Such terms as used herein are not necessarily all referring to the same implementation. Any implementation may be combined with any other implementation, inclusively or exclusively, in any manner consistent with the aspects and implementations disclosed herein. 
     The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” 
     References to “or” may be construed as inclusive so that any terms described using “or” may indicate any of a single, more than one, and all of the described terms. For example, a reference to “at least one of ‘A’ and ‘B’” can include only ‘A’, only ‘B’, as well as both ‘A’ and ‘B’. Such references used in conjunction with “comprising” or other open terminology can include additional items. 
     Where technical features in the drawings, detailed description or any claim are followed by reference signs, the reference signs have been included to increase the intelligibility of the drawings, detailed description, and claims. Accordingly, neither the reference signs nor their absence have any limiting effect on the scope of any claim elements. 
     The systems and methods described herein may be embodied in other specific forms without departing from the characteristics thereof. The foregoing implementations are illustrative rather than limiting of the described systems and methods. Scope of the systems and methods described herein is thus indicated by the appended claims, rather than the foregoing description, and changes that come within the meaning and range of equivalency of the claims are embraced therein.