Abstract:
A device for supplying cell culture modules with nutrients has an arrangement of channels, pumps and valves in or on a plate. The valves may be pinch valves operated by deforming an elastic cover over a solid body and the pump may be a pinch valve pump. The channels may be defined, at least in part, by the plate. The pumps, channels and valves may be located within the thickness of the plate and the cover. The device may be used to supply nutrients to cell culture modules according to a perfusion operation, a re-circulation operation and/or a combination of both. A pump may comprise a generally rigid solid body and a seal. The solid body may wholly or partially define an inlet channel, a plenum and an outlet channel. A port between the inlet channel and the plenum is covered by the seal. A surface of the plenum is deformable. Deforming the surface forces liquid in the plenum to push the seal to cover the inlet channel port and to flow through the outlet channel. When the surface is returned to its original position, fluid flows into the plenum at least partially through the inlet channel displacing or deforming the seal so as to allow flow through the port.

Description:
[0001]    This application is a continuation of PCT/EP2007/009605, filed Nov. 6, 2007, which claims the benefit of U.S. Patent Application No. 60/864,678, filed Nov. 7, 2006, both of which are hereby incorporated herein in their entirety by this reference to them. 
     
    
     FIELD 
       [0002]    This specification relates to devices or processes for cultivating cells or to a pump, conduit or check valve or a method for making a pump, conduit or check valve. 
       BACKGROUND 
       [0003]    The comments in this background section are not an admission that anything discussed in this section is citable as prior art or part of the common general knowledge of persons skilled in the art in any country. 
         [0004]    Some systems for cell cultivation have been developed which provide in some way for the supply of nutrient media to, and the removal of metabolic waste products from, a cell culture. In some systems, cells have been supported on hollow plastic fibers inside of bioreactors. Literature discussing cell cultivation includes the following (1) Sauer, I. M. et al.: The Slide Reactor—a simple hollow fiber based bioreactor suitable for light microscopy; Artificial Organs 29 (3): 264-267, 2005; (2) Sauer, I. M. et al.: Development of a hybrid liver support system. Ann N Y Acad Sd 944: 308-19, (2001); Millis, J. M: et al.: Initial experience with the modified extracorporeal liver-assist device for patients with fulminant hepatic failure: system modifications and clinical impact. Transplantation 74: 1735-46; (2002); and, (4) Glockner, H. et al.: New miniaturized hollow fiber bioreactor for in vivo like cell culture, cell expansion and production of cell-derived products. Biotechnol Prog 17: 828-31 (2001). 
         [0005]    PCT Publication No. WO 2004/024303 A2, and related U.S. Publication No. 2006/0014274 A1, disclose a fiber cassette having a housing that is delimited by two congruent base surfaces and at least one circumferential surface and has an interior having at least one cavity. At least one layer of fibers is arranged in the interior of the housing essentially parallel to at least one center plane of the housing, wherein ends of the fibers are anchored fixedly in the interior of the housing. A first one of the at least one cavity defines an outer compartment that surrounds the fibers externally. The at least one center plane does not intersect the base surfaces within the outer compartment. The fibers are arranged U-shaped or essentially parallel to one another and end within the interior of the housing. The housing has at least one opening for supplying and/or removing fluids. PCT Publication No. WO 2004/024303 A2 and U.S. Publication No. 2006/0014274 are incorporated herein in their entirety by this reference to them. 
         [0006]    PCT Application No. PCT/CA2006/000739 describes a cell culture bioreactor and is incorporated herein in its entirety by this reference to it. 
       SUMMARY 
       [0007]    The following summary is intended to introduce the reader to this specification but not to define any invention. Inventions may reside in a combination or sub-combinations of the apparatus elements or process steps described below or in other parts of this document. The invention protected by this document is described in the claims. The inventors do not waive or disclaim their rights to any invention or inventions disclosed in this specification merely by not describing such other invention or inventions in the claims. 
         [0008]    The inventors have observed that prior cell cultivation systems involve a complex arrangement of transport hoses, pumps, valves, connectors and other elements for transporting nutrients through a cell culture area. This interferes with economical production of a cell cultivation system, particularly a system of multiple identical culture areas, for example for parallel production to increase output, for drug screening or other applications where it is desirable to grow multiple cultures at the same time. Space requirements of prior systems may also be large. 
         [0009]    In an apparatus described herein, a plurality of nutrient or waste transport elements are provided on or inside of, or inside the notional periphery of, a planar transport plate, alternately called a nutrient transport plate. The transport plate is an assembly of elements comprising a solid body and other components collectively adapted to assist in transporting nutrients to, or waste from, a cell culture area or module. The transport plate is planar in the sense that a set of its elements are located within an imaginary plane plus or minus 2 cm. The notional periphery of a transport plate refers to the periphery of a three-dimensional body, for example a parallelepiped, containing the transport plate. A transport plate described in relation to a set of transport elements may still be planar, and have those elements within the notional periphery of the transport plate, despite the presence of other elements attached to the transport plate and extending beyond the notional periphery. Such an apparatus may reduce one or more of the disadvantages of prior nutrient transport systems or at least provide a useful alternative to prior nutrient transport systems or bioreactors. 
         [0010]    This specification also describes an apparatus comprising one or more elements formed at least in part by a rigid solid body and arranged for one or more of the supply, removal or recirculation of nutrient media to one or more cell culture modules. The one or more elements for nutrient transport may include one or more of a transport conduit, a valve, a check valve, a connection for a fresh media container or a waste container, a pump, a connection for a cell culture module or an integrated cell culture area. A transport conduit may be formed at least in part by a surface of the solid body. A transport conduit may be a part of a nutrient supply path or a nutrient recirculation path or both. A valve or pump may be formed at least in part by a surface of the solid body. A connection may be attached to the solid body. A valve may comprise a portion of a transport conduit formed at least in part by a flexible body which may be moved into the portion of the transport conduit to prevent or inhibit flow. A pump may comprise a portion of a transport conduit formed at least in part by a flexible body between two valves or check valves. Deflecting the flexible body into the transport conduit portion causes fluid in the portion of the transport conduit portion to move out of the transport conduit portion through one valve and releasing the flexible body causes fluid to flow into the transport conduit portion through the other valve. Portions of a transport conduit that are part of a valve or pump may include a part of a resilient cover attached to the solid body. A transport conduit may have a silicone surface. 
         [0011]    An apparatus may optionally also include one or more of a sampling connection, a transducer, a sensor mount, a meter, a thermal element or a gassing element. A sensor may be positioned so as to not contact nutrient solution. A connector may be a standard, universal, or frequently used connector to facilitate the integration of an arbitrary cell culture module to the nutrient transport plate. An apparatus may be made of a sterilisable material such as a plastic. This allows an apparatus to be used as a disposable transport system, if the corresponding cell culture module is detachable or also disposable. Metals, glass, ceramics or other materials may also be used. An apparatus may be suitable for, and a process may comprise using an apparatus for, nutrient transport to modules containing or cultivating protozoa, bacteria, yeasts, fungi, plants or cells of vertebrates, for example mammals. An apparatus may be combined with a cell culture module according to WO 2004/024303 A2 or other cell culture modules which may be, for example tubular, planar, rectangular, star-shaped or other shapes. 
         [0012]    This specification also describes a process comprising providing an apparatus as described above and using the apparatus, for example by moving the flexible bodies of the apparatus, to transport nutrients through cell culture modules in perfusion, recirculation or a combination of these two operating modes. 
         [0013]    This specification also describes a transport plate wherein at least one of a conduit, a valve, a check valve or a pump comprise or essentially consist of a portion of solid body and a portion of a flexible body or cover attached to the solid body. 
         [0014]    This specification also describes a check valve or a pump. A pump may comprise a generally rigid solid body and a seal. The solid body may wholly or partially define an inlet channel, a plenum and an outlet channel. A port between the inlet channel and the plenum is covered by the seal. A cover of the plenum is deformable. The seal acts as a pair of check valves. Deforming the cover forces liquid in the plenum to push the seal to cover the inlet channel port and forces liquid to flow through the outlet channel. When the surface is returned to its original position, the seal is displaced or deformed and fluid flows into the plenum through the inlet channel. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0015]      FIG. 1  is an orthographic projection of the top, side and end of a pump. 
           [0016]      FIG. 2  shows a cross sectional elevation view of the pump of  FIG. 1  cut along line  2 - 2  of  FIG. 1 . 
           [0017]      FIG. 3  shows a plan view of the pump of  FIG. 1  cut along the line  3 - 3  of  FIG. 1 . 
           [0018]      FIG. 4  is a cross-sectional end view of the pump of  FIG. 1  cut along the line  4 - 4  of  FIG. 2 . 
           [0019]      FIG. 5  is a cross-sectional elevation view of another pump with an actuator and controller. 
           [0020]      FIG. 6  is a top view of a transport plate. 
           [0021]      FIG. 7  shows a membrane cell culture module. 
           [0022]      FIG. 8  shows an actuator set. 
       
    
    
     DETAILED DESCRIPTION 
       [0023]    Various apparatuses or processes will be described below to provide an example of an embodiment of each claimed invention. No embodiment described below limits any claimed invention and any claimed invention may cover processes or apparatuses that are not described below. The claimed inventions are not limited to apparatuses or processes having all of the features of any one apparatus or process described below or to features common to multiple or all of the apparatuses described below. It is possible that an apparatus or process described below is not an embodiment of any claimed invention. The applicants, inventors and owners reserve all rights in any invention disclosed in an apparatus or process described below that is not claimed in this document and do not abandon, disclaim or dedicate to the public any such invention by its disclosure in this document. 
         [0024]      FIGS. 1 to 4  show a pump  10 , an actuator  12 , a controller  14  and a power supply  16 . Pump  10  comprises a rigid body  16  and a resilient body  18 . Rigid body  16  may be made, for example, of hard plastic. Resilient body  18  may be made, for example, of silicone if gas transfer is desired or rubber if not. Resilient body  18  may have a planar section  22  and one or more flap seals  20 . Planar section  22  is bonded, for example with silicone sealant or glue, to an upper surface  26  of solid body  16 . Resilient body  18  thus covers, or provides an upper surface to all or part of various elements completed by solid body  16 , such as an inlet channel  30 , a plenum  32 , an outlet channel  34 , an inlet valve body  36  and an outlet valve body  38 . Inlet channel  30  communicates with inlet valve body  36  through an inlet channel port  40 . Inlet valve body  36  communicates with plenum  32  through inlet passage  42 . Plenum  32  communicates with outlet valve body  38  through outlet passage  44  and outlet port  46 . Outlet valve body  38  communicates with outlet channel  34 . A first flap seal  20   a  sits, when not acted on by external forces, adjacent a wall of inlet valve body  36  that is pierced by inlet channel port  40 . A second flap seal  20   b  sits, when not acted on by external forces, adjacent a wall of outlet valve body  38  that is pierced by outlet port  46 . Flap seals  20  as shown are molded inserts bonded to solid body  16 , the rest of resilient body  18  or both. Alternately, flap seals  20  may be integral with resilient body  18 . Further alternately, flap seals  20  may be made of a piece of resilient sheet material folded to provide a flap part and a tab for bonding to solid body  16  or resilient body  18 . 
         [0025]    In operation, a liquid is provided in communication with inlet channel  30  and within the space between rigid body  16  and resilient body  18 . Actuator  12 , which may be for example a solenoid, is moved downward by controller  14 , powered by power supply  16 , to press a portion of resilient body  18  into plenum  32 . This displaces the liquid in plenum  32  which pushes first flap  20   a  against the wall of inlet valve body  36  and so at least partially seals, or inhibits liquid flow through, inlet channel port  40 . Pressure, or displacement of, liquid in plenum  32  also moves second flap  20   b  away from outlet port  46 . In this way, flaps  20 , and valve bodies  36 ,  38  are or function as one-way valves. Liquid flows from plenum  32  through outlet port  46  to outlet valve body  38  and out of outlet channel  34 . When actuator  12  is released, the planar section  22  of resilient member  18  returns to its at rest state. Fresh liquid flows through inlet channel  30  and inlet channel port  40 , displaces first flap  20   a , and flows into inlet valve body  36  and plenum  32 . At the same time, second flap  20   b  inhibits or prevents flow of liquid from outlet valve body  38  back into plenum  32 . Flow of liquid into plenum  32  may be caused by the force or suction pressure of resilient member  18  returning to its original position, or by a static head difference between inlet channel  30  and outlet channel  34  or by both. Reciprocating actuator  12  may move a volume of fluid through pump  10  with each depression of actuator  12 . 
         [0026]      FIG. 5  shows a second pump  100 . In second pump  100 , inlet valve body  36  and plenum  32  are replaced by a valve body plenum  102  and inlet passage  42  is deleted. All items downstream of plenum  32  in the pump  10  are replaced in pump  100  by a flow restricting outlet  104 . In operation, when actuator  12  presses a part of resilient member  18  into valve body plenum  102 , liquid in valve body plenum  102  forces flap  20   a  to at least partially seal port  40 . Liquid from valve body plenum  32  flows through restricting outlet  104  to leave second pump  100 . When actuator  12  is released, fluid enters valve body plenum  102  at least partially through inlet channel  30  and around flap  20   a , since restricting outlet  104  inhibits the return of liquid to valve body plenum  102 . 
         [0027]    Referring to  FIG. 6 , a bioreactor  210  comprises a nutrient transport plate  212  and a cell culture module  207 . Cell culture module  207  is plugged into, and optionally may be removed from, cell culture module connections  206 . Cell culture module connections  206  may be holes or grooves machined in solid body  209  optionally with fittings (not shown) inserted into them. An example of a cell culture module  207  is shown in more detail in  FIG. 7 . As shown, the cell culture module  207  has a cover removed from it that would otherwise enclose an outer compartment  216  and parts of an inner compartment  215 . Inner compartment  215  also includes the lumens of hollow fiber membranes  212 . The walls of hollow fiber membranes  212  and potting compound  213  as well as a base structure  211  and the cover (not shown), separate inner compartment  215  from outer compartment  216 . Cells may grow on the membranes  212  or otherwise in second compartment  216 . Nutrients may be supplied to the cells through the first compartment  215  and the walls of membranes  212 . In particular, a nutrient solution can be input into supply port  217 , into a first channel  219  portion of first compartment  215 , through the lumens of membranes  212 , into a second channel  220  portion of first compartment  215  and out through a waste port  218 . While traveling through this path, some nutrients, for example carbohydrates or gases, pass through the walls of membranes  212  to be consumed by cells in second compartment  216 . Some waste products released by the cells travel from second compartment  216  through the walls of membranes  212  and are carried away with the nutrient solution. Cell culture module  207  is attached to nutrient transport plate  212  by inserting supply port  217  and waste port  218  into cell culture module connections  206 . Ports  217  and  218  may be glued into connections  206  for a permanent attachment, or removably sealed together through a press in or other fit. Optionally, cell culture module  207  may have auxiliary ports  221  to allow for adding or removing substances to second compartment  216  without passing through the walls of membranes  212 . The auxiliary ports  221  can be used, for example, to extract cells or cell products, secretions, viruses, proteins or low molecular weight substances. The bioreactor  210  can thus be used for a variety of applications including, for example, growing high density cell cultures, testing or screening for the reaction of cell cultures to various substances, or harvesting products made by cells. 
         [0028]    Solid body  209  of nutrient transport plate  212  may be made from a sheet of a rigid material, for example a hard plastic, with a thickness in the range of, for example, 3 mm to 10 mm. Solid body  209  may be made by cutting the sheet of material to a selected width and length or perimeter shape, for example in the range of 3 cm to 15 cm. Transport channels  202  and other grooves or depressions may be made, for example by router, in the surface of solid body  209 . Alternately, solid body  209  may be formed, for example molded, with the required grooves or depressions. A nutrient supply is connected to the nutrient transport plate  212  through nutrient connector  201  which may comprise a fitting slipped into a transport channel  202  or a hole in rigid body  209 . Waste solution may leave transport plate  212  through a waste connector  224  which may be made as described for nutrient connector  201 . Samples may be extracted from a sample valve  208  which may comprise, for example, a true valve, an opening with a removable cap or, as shown, a plug of material forming a self sealing septum. 
         [0029]    The nutrient transport plate  212  provides two basic flow paths. A first flow path starts at the nutrient connector  201  and ends at waste connector  224  after passing through an area containing waste nutrient, for example the first compartment  215  of cell culture module  207  or an integrated cell culture area or a part of the second flow path described below. A second flow path travels in a loop through the nutrient transport plate  212  from a first cell culture module connection  206  to the other and then through the first compartment  215  of cell culture module  207  back to the first cell culture module connection  206 , or through a similar path involving an integrated cell culture area. While nutrient is flowing through the second flow path, a connection to nutrient connector  201  may be left open so that nutrient can be drawn in to replace nutrient consumed by the cells. Between the two flow paths, fresh nutrient solution can flow from a nutrient source to membranes  212 , old nutrient solution can flow out to a waste container or drain, or a nutrient solution can re-circulate though the membranes  212 . Further, a bioreactor  210  can be operated cyclically with, for example a period of flow through the first flow path, then a period of flow through the second flow path repeated in cycles. Control and propulsion through these flow paths may be provided as described below. 
         [0030]    A portion of some or all of transport channels  202  may be part of a valve body  204  formed as described for the plenum  32  of  FIGS. 2 and 3 . The valve body  204  works with an actuator  12  as described previously. The actuator  12  is operable to push in portion of cover  200  so as to fully or partially close the flow path through valve body  204  and prevent or inhibit nutrient flow and thereby provide a  204  valve. Cover  200 , although only a portion is shown in  FIG. 6 , as represented by the wiggly lines in the lower left corner of the bioreactor  210 , covers the entire upper surface of solid body  209  or as much of it as required to enclose valve bodies  204  and cover the otherwise open grooves in the solid body  209  so as to complete transport channels  202 . Cover  200  may be made of a resilient material and may further be made of an oxygen permeable material such as silicone. Cover  200  may be bonded, glued, welded or otherwise attached to the upper surface of solid body  209 . When valve body  204   aii  is closed and valve body  204   b  (and optionally valve body  204   aii ) is open, the second flow path is provided. When valve body  204   b  is closed and valve bodies  204   a  are open, the first flow path is provided. Flow through the first flow path can also be provided by having valve bodies  204   ai  and  204   b  closed and valve bodies  204   aii  open while pump  10  is operated (actuator  12  is moved into plenum  32 ) as described earlier, closing valve body  204   aii  and opening valve bodies  204   ai  and  204   b  while valve body  204   a  remains pinched, then removing the actuator  12  from plenum  32  of pump  10 . According to that operation, valve bodies  204   ai  and  204   b  are operated simultaneously and may be activated by one actuator  12  to be described further below. 
         [0031]    Nutrient transport plate  212  has a pump  10  described earlier. When using pump  10  parts of transport channels  202 , may swell temporarily to take up a volume of nutrient solution displaced by the pump  10 . The bioreactor  210  may also be operated by alternating between the flow paths. For example, pump  10  may be operated one or more times while the first flow path is open to expel old nutrient fluid containing waste products and intake fresh nutrient solution. Thereafter, pump  10  may be operated one or more times to recirculate nutrient fluid. Then these two steps above are repeated so as to cycle back and forth between refreshing and recirculating the nutrient solution. For example, a cycle may comprise operating pump  10  once to refresh nutrient, followed by operating pump  10  for 2 to 10 times to recirculate the nutrient solution. Alternately, an additional check valve may be inserted into nutrient connector  201  to allow nutrient solution to enter into but not exit from nutrient connector  201 . Then valve bodies  204   ai  and  204   b  may be left open while valve body  204   a  is occluded but left slightly open. In this configuration, operating pump  10  causes a continuous recirculation but with a continuous bleed of waste and feed of fresh nutrient solutions. Check valves may be optionally be replaced by other valves, for example pinch valves, operated so as to mimic the action of check valves. 
         [0032]    Bioreactor  210  may also have various optional additional components for process implementation, monitoring or control. If cover  200  is made of a gas permeable material, for example silicone, it acts as a gassing element. Temperature elements may heat or cool the cell culture module  207 . A temperature sensor may be inserted into a hole drilled into the edge of solid body  209  intersecting a transport channel  202 . A temperature sensor may measure nutrient solution temperature and may be connected to a temperature element in a control or feedback loop to maintain a desired temperature. One or more optical sensors may be inserted into holes drilled into the edge of solid body  209  so as to be near, but not fluidly connected to a transport channel  202 . Optical sensors may comprise a pH sensitive transducer or a dissolved oxygen level sensitive transducer. 
         [0033]      FIG. 8  shows an assembled cell cultivation system  311  having one or more stacked nutrient transport plates  212 . Nutrient transport plates  212  are connected in parallel to a nutrient container  42  and a waste container  43 . Actuators  12 , comprising a base  46  able to produce electric fields in locations below various stacked magnets  44 , are situated over the nutrient transport plates so as to be able to deflect covers  200  to provide valve operations and pumping. The system  311  is optionally located within a controlled environment chamber  208 . Control of actuators  12  may be linked through a programmable logic controller or other controller  47  to sensors or other controlled devices. 
         [0034]    The invention or inventions which are currently claimed in this document are described in the following claims.