EP1789528A2 - Bioreacteur pour la culture de cellules sous perfusion - Google Patents

Bioreacteur pour la culture de cellules sous perfusion

Info

Publication number
EP1789528A2
EP1789528A2 EP05796533A EP05796533A EP1789528A2 EP 1789528 A2 EP1789528 A2 EP 1789528A2 EP 05796533 A EP05796533 A EP 05796533A EP 05796533 A EP05796533 A EP 05796533A EP 1789528 A2 EP1789528 A2 EP 1789528A2
Authority
EP
European Patent Office
Prior art keywords
cell
fluid
unit
scaffold
wells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP05796533A
Other languages
German (de)
English (en)
Other versions
EP1789528A4 (fr
Inventor
Neil F. Robbins
Jon Rowley
Mark Quinto
Abel Z. Hastings
Bryan G. Towns
Bradley R. Snodgrass
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Becton Dickinson and Co
Original Assignee
Becton Dickinson and Co
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Becton Dickinson and Co filed Critical Becton Dickinson and Co
Publication of EP1789528A2 publication Critical patent/EP1789528A2/fr
Publication of EP1789528A4 publication Critical patent/EP1789528A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/10—Perfusion
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5025—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures for parallel transport of multiple samples
    • B01L3/50255—Multi-well filtration
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/12—Well or multiwell plates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices

Definitions

  • the present invention relates generally to the field of bioreactors, and, more particularly, to a system and method for culturmg cells under perfusion flow, in a single chamber or in a high throughput format.
  • Fluid flow was first established as a regulator of cellular gene expression in two-dimensional culture systems with flowing culture medium over cells adherent to glass slides. Cells respond to the fluid sheer by aligning in the direction of the force, and altering their gene expression. These two-dimensional devices are now commercially available from, for example, Flex-Cell International, as well as other vendors. Fluid flow studies have recently been translated to three-dimensional scaffolds, and it has been established that fluid sheer is another important factor in maintaining hepatocyte and bone differentiation.
  • ADMETox is an acronym for set of analyses that measure the absorption, distribution, metabolism, elimination and toxicity of a drug candidate
  • ADMETox is an acronym for set of analyses that measure the absorption, distribution, metabolism, elimination and toxicity of a drug candidate
  • tissue or organ slices offer an alternative that keeps cells in their native setting (not dissociating them from their microenvironment), while allowing for in vitro testing of xenobiotic effects on cell viability, metabolism, and other ADMET-type aspects that one desires.
  • liver slices are often utilized for measuring liver- specific drug toxicity, as well as CYP induction.
  • tissue slices In vitro culture of tissue slices also has several challenges. For example, one significant challenge is the high metabolic rates and nutrient requirements that tissue slices need in vitro. Since the tissue slices require a large nutrient load, it is necessary to culture these slices in large quantities of medium. However, the more medium that one adds to a culture increases the diffusion distance of oxygen to the extent that the rate of consumption by the tissue is greater than the diffusion of oxygen, leading to hypoxic conditions and cell death. There is, therefore, a great need for bioreactor-type devices that enhance nutrient and metabolite transport while maintaining a medium-to-high throughput parallel testing format.
  • the present invention is directed to a bioreactor system including a perfusion unit, a pumping unit in fluid communication with the perfusion unit, and a fluid source unit in fluid communication with the pumping unit.
  • the perfusion unit includes an array of cell wells configured to contain cell cultures and the fluid source unit includes an array of media wells configured to contain cell culture media.
  • the pumping unit includes an array of pumping elements in fluid communication with the cell wells and media wells and is configured to pump cell culture media from the media cells to the cell wells.
  • each of said cell wells is adapted and configured to contain a scaffold having a porous structure.
  • the scaffold is a two-dimensional scaffold.
  • the scaffold is a three-dimensional scaffold.
  • the three-dimensional scaffold may include directionally aligned pores.
  • the fluid is deliverable directly into the internal structure of said scaffold.
  • a return pathway is provided for ihe fluid to flow from the array of cell wells to the array of media wells.
  • each pathway is in fluid communication with a single cell well and a single media well.
  • each pumping element may comprise a fluid stem having a fluid port therein.
  • Each stem may be configured to extend into the cell wells.
  • each cell well may include a scaffold coupled thereto configured to receive a portion of the stem internal thereto.
  • the fluid source unit is also removably couplable to the pumping unit.
  • the present invention is also directed to a method of growing cells, comprising pumping cell culture media from a first array of wells of a fluid source unit into a second array of wells of a perfusion unit, wherein each well of the perfusion unit is adapted and configured to house a cell adherent structure.
  • the method further comprises the step of perfusing the media into and through a scaffold.
  • the cell adherent structure comprises a two-dimensional scaffold, and in another embodiment the cell adherent structure comprises a three-dimensional scaffold.
  • the first array of wells is in fluid communication with the second array of wells for the return of media to the second array of wells.
  • each well of the first array of wells is in singular fluid communication with a corresponding well of the second array of wells.
  • the present invention is also directed to a perfusion bioreactor system, including an array of bioreactor units.
  • Each bioreactor unit includes a cell adherent structure in fluid communication with a fluid stored in a fluid reservoir and, in operation, fluid flows from the fluid source directly into and through the cell adherent structure.
  • the cell adherent structure is a three-dimensional scaffold having a porous structure, and in another variation the cell adherent structure is a two-dimensional scaffold.
  • the cell adherent structure is fluidly interconnected to the fluid reservoir by a pumping unit.
  • FIG. 1 is an exploded view of a first embodiment of a bioreactor system according to the present invention
  • FIG. 2 is a perspective view of one embodiment of a perfusion unit of the bioreactor system shown in FIG. 1 ;
  • FIG. 3 is a perspective view of one embodiment of a pumping unit of the bioreactor system shown in FIG. 1 ;
  • FIG. 4 is a cross-sectional exploded view of a bioreactor unit of the system of FIG.1;
  • FIG. 5 is a cross-sectional view of another embodiment of a bioreactor system according to the present invention.
  • FIG. 6 is a cross-sectional view of another embodiment of a bioreactor system according to the present invention.
  • FIG. 7 is a cross-sectional view of another embodiment of a bioreactor system according to the present invention.
  • FIGS. 8-10 are cross-sectional views of another embodiment of a bioreactor system according to the present invention
  • FIGS. 11-12 are cross-sectional views of another embodiment of a bioreactor system according to the present invention.
  • FIG. 13 is a graphical representation of one example of a cell biology experiment performed according to the invention showing hepatocytes growth on scaffolds with perfusion;
  • FIGS. 14-16 are graphical representations of additional examples of cell biology experiments performed according to the invention.
  • FIG. 17 is a side view of one embodiment of a system according to the present invention.
  • FIG. 18 is a perspective view of one embodiment of a carrier of the system of FIG. 17;
  • FIG. 19 is a partial perspective view of the carrier of FIG. 18 depicting a single well as seen from the bottom;
  • FIG. 20 is a partial perspective view of the carrier of FIG. 18 depicting a single well as seen from the top;
  • FIGS. 21 and 22 are partial side views of a single well of the carrier of FIG. 18 shown without and with a screen.
  • the present invention relates to bioreactors generally, and, more particularly, to a system and method for culturing cell specimens under perfusion flow, in a single chamber or in a high throughput format for the high throughput discovery of complex environments for controlling cell function and engineered tissue development.
  • the present invention may also be utilized for creating highly relevant cell cultures and systems for direct drug testing on cells in dynamic cell cultures, for drug discovery, drug testing, or ADMETox applications.
  • a preferred embodiment of a bioreactor system 5 generally includes a multi-well platform comprising an array of bioreactor units 10 wherein in each bioreactor unit, an independent cell study or experiment may be performed.
  • the bioreactor system 5 comprises a perfusion unit 12 and a fluid source unit 14 fluidly interconnected by a pumping unit or station 16.
  • perfusion unit 12 is a multi-well plate including a plurality of main chambers or wells 18 configured to house or contain a cell culture.
  • the fluid source unit 14 may comprise one or more separate multi-well plates including a plurality of fluid reservoir chambers or wells 20 to store fluid, such as cell culture media.
  • each main chamber or well 18 is in fluid communication with a corresponding individual fluid reservoir chamber 20.
  • top perfusion unit 12 and fluid source unit 14 include 24 chambers or wells, however, in alternative embodiments any number of chambers or wells may be provided.
  • the wells of top perfusion unit 12 and the fluid source unit 18 may be miniaturized to comprise 48 wells per plate, 96 wells per plate, or smaller.
  • each well includes a passage or hole 15 extending through the base of the well to permit the passage of fluid therethrough.
  • a triangulated post structure 17 is fixed onto a base portion of each well 18 and extends above hole 15. Post structure 17 facilitates the attachment of a cell adherent structure or scaffold 22 (shown in FIG. 4) to grow cell cultures.
  • Each well may also contain a fluid return pathway 19.
  • perfusion unit 12 and fluid source unit 14 may be made from polystyrene, polycarbonate, polypropylene, other plastic, or any other suitable material, and may be injection molded in parts or in their entirety.
  • perfusion unit 12 and fluid source unit 14 are preferably configured and dimensioned to be removably coupled to pumping unit 16. Accordingly, perfusion unit 12 and fluid source unit 14 may be interchangeable components of the system, such that a plurality of like units or plates may be exchanged or removably coupled to pumping unit 16 as desired.
  • the fluid source unit 14 is configured to be removably coupled to the pumping unit 16 such that the fluid source unit 14 may be re- usable or disposable for media addition.
  • perfusion unit 12 may be removed from one pumping unit 16 to another to associate cell cultures with different fluid/dynamic environments.
  • Pumping unit 16 comprises an array of fluid connectors and/or hardware components to fluidly connect each main chamber 18 with each fluid reservoir chamber 20.
  • pumping unit or station 16 may comprise any hardware components suitable for transferring or pumping fluid from the fluid source unit 14 to the perfusion unit 12 such as, for example, motorized pump(s), valves, tubes, pipes, or other devices or means for pumping or transferring the fluid.
  • any type of pumping mechanism may be used, including but not limited to peristaltic, centrifugal, vibrating, piezo, or an air or fluid driven pumping mechanism, or individual electronic pumps wherein each perfusion unit could be programmed with a different pumping rate.
  • pumping unit or station 16 utilizes a peristaltic pumping mechanism including an array of pumping plates 30 mounted upon driving rods 32.
  • Rods 32 are slidably mounted to housing 34 in bearings 36 and in operation are driven back and forth along the axis of rods 32 by a motor attached to coupling plate 38.
  • pumping plates 30 squeeze flexible tubing 40 against static plates 42 to pump the fluid contained in flexible tubing 40.
  • single direction valves 41 are provided on either side of flexible tubing 40 and interposed between an inlet tube 47 and an outlet tube 49 to pump or direct fluid flow in one direction from the fluid reservoir chambers 20 of fluid source unit 14 toward the main chambers 18 of perfusion unit 12.
  • a return pathway 19 is preferably built into each main chamber 18 of perfusion unit 12 which fluidly connects to return pathway 25 pumping unit 16 to provide for fluid return to the fluid source unit 14 from the perfusion unit 12, thereby creating a plurality or array of individual and separate bioreactor units 10.
  • each bioreactor unit 10 is an independent fluidly self-contained entity.
  • each bioreactor unit generally includes a single main chamber or well 18 in fluid communication with the fluid source, housed for example in a single fluid reservoir chamber or well 20.
  • a cell adherent structure or scaffold 22 is preferably housed within each main chamber 18 to facilitate high density cell culture growth.
  • the cell adherent structure is a three-dimensional scaffold, such as a porous body having a plurality of three-dimensional cell adherent surfaces, however, in alternate embodiments, the cell adherent structure may be two-dimensional, such as a slide or plate having a two- dimensional cell adherent surface.
  • the cell adherent structure may have varied shapes such as, for example, a tubular or cylindrical shape, such that a transplantable medical device/implant with a biological component may be engineered in a high throughput device.
  • cells and/or tissue may adhere or grow upon the tubular structure to grow cell or tissue containing tubes such as, for example, vascular grafts, stents, neural tubes, shunts, etc., for transplantation into the body of a patient.
  • cartilage and/or bone may be grown or engineered in a predetermined shape.
  • the cell adherent structure is coupled to the main chamber about a fluid port 44 such that the fluid flows directly into or about the cell adherent structure.
  • a three-dimensional scaffold 22 may be coupled, molded, bonded, synthesized, or otherwise attached to the main chamber 18 such that a stem or fluid port 44 extends into the central portion or interior of the scaffold when, for example, perfusion plate 12 is coupled to pumping unit 16.
  • each main chamber 18 of perfusion plate 12 is configured to receive scaffolds that may be coupled, fastened, or otherwise connected to a portion of each main chamber 18 by any suitable means known to those skilled in the art.
  • scaffold 22 may be releasably plugged into or attached to main chamber 18.
  • the scaffolds can be made from any type of polymer, ceramic, metal or mixture of any type suitable for adhering cells thereto.
  • the scaffold is made from a hydrogel-based material, which may be synthesized from covalently crosslinked alginate, hyalrunic acid or a blend of the two polysaccharides at any mixing percentage as desired.
  • the mixing percentage may be tailored to achieve a desired degradation profile for the final application.
  • the scaffolds may be made of other suitable materials, such as those disclosed in U.S. Patent Publication No. 2004/0147016 entitled "Programmable scaffold and methods for making and using same", the entire contents of which are incorporated by reference.
  • the scaffold may be a porous structure having randomly aligned pores.
  • scaffolds may be used that have directionally aligned pores such that a less random pore pattern may be attained and fluid flow may be further assured of navigating or flowing through all of the pores of the scaffold.
  • the scaffolds may be modified with any number or type of cell signaling or cell interacting molecule, such as those disclosed in U.S. Patent Publication No. 2004/0147016, entitled "Programmable scaffold and methods for making and using same," the entire contents of which are incorporated by reference.
  • fluid is pumped directly into the internal scaffold structure and may perfuse or flow from the interior 46 of scaffold 22 to the exterior 48 of scaffold 22.
  • fluid is pumped at a rate ranging from about 10 to 0.1 milliliters per minute.
  • fluid may readily flow through the internal pores of the scaffold as opposed to circumventing the scaffold or flowing mainly along the exterior of the scaffold.
  • the enhanced diffusion mass transport provided by the perfused fluid flow advantageously allows metabolites and nutrients to diffuse into and out of scaffold 22.
  • perfusion culture permits long term tissue engineering experiments allowing growth of high density cell cultures to mimic tissues.
  • FIG. 5 an alternative embodiment of a main chamber 18 is shown wherein the fluid flow 50 is directed from an inlet 52 through an alternative scaffold 54 and exits the scaffold and chamber at an outlet 56 as opposed to flowing randomly throughout the scaffold.
  • main chamber 18 includes a two-dimensional cell adherent structure 6>2 with a cell adherent upper surface.
  • the cell adherent structure 62 is coupled to the chamber 18 such that fluid may flow along path 63 through fluid port 44 and a.cross the two- dimensional surface of structure 62 and returns through return pathway 64.
  • a plate 66 covers structure 62 and is spaced therefrom to contain the fluid such that the fluid flows directly over the cell adherent surface.
  • cell specimen 70 may be coupled, fastened, or otherwise connected to a portion of each main chamber 18 by any suitable means known to those skilled in the art.
  • cell specimen 70 may be a cell adherent structure or scaffold and in other embodiments cell specimen 70 may comprise portions or slices of tissue.
  • cell specimen 70 may comprise liver slices, pancreatic islets, liver spheroids, 3-D tissue models (such as those commercially available from Mattek, Inc. or Regenemed, Inc.), 3-D cancer models (such as those commercially available from Mina Bissell), cells on microcarriers or fiber disks (such as those commercially available from fibracell), or any other cellular bodies that may be grown in vitro.
  • cell specimen 70 has a cylindrical or disc shape and may " be held in place in main chamber 18, for example, between a pair of washers 72.
  • Washers 72 include a central opening to permit fluid flow therethrough. In operation, fluid may flow through port 44 and perfuse through cell specimen 70 and exit through the central opening of the top washer 72 and return via return pathway 75.
  • the present embodiment is configured to keep slices or cell specimens emerged at all times in media, while exposing the tissue or cell specimen to fluid flow similar to in vivo conditions and enhancing gas and nutrient transfer.
  • the present system facilitates the maintaining of cell viability, and the maintaining of the specimens or tissue slices in a format for drug testing.
  • the configuration of this embodiment may be advantageously utilized with, for example, tissue slices or scaffolds made of polymer or ceramic material or other materials that cannot be synthesized in place.
  • bioreactor system 80 is a pneumatic system comprising three disposable or reusable pieces or components: a bottom reservoir plate 82 configured to contain media, a pumping device 84 that induces the motion of the media, and a top perfusion plate 86 that mates with the bottom reservoir plate and pumping device, and is configured to maintain the position of the tissue slices or cell specimen 70 in the media flow and create a closed fluid path for the media to return to bottom reservoir plate 82.
  • Both the bottom reservoir plate and the "top perfusion plate generally include multiple wells or chambers 83 and each plate may be injection molded and may be disposable or reusable items.
  • Each well of the perfusion plate 86 generally comprises an inlet portion 88 and an outlet portion 90.
  • Each well of the bottom reservoir plate 82 generally comprises a fluid reservoir 92 and a pair of one-way valves or check valves 94.
  • the one-way valves may be molded into a one piece plate.
  • Each of the pair of one-way valves is aligned with the corresponding inlet and outlet portions 88, 90 of the perfusion plate to direct and or allow the fluid or media to flow from fluid reservoir 92 into the inlet 88 and out of the outlet 90 and return to the fluid reservoir 92.
  • cell specimen 70 may be positioned within th_e inlet portion 88 of each well of the perfusion plate 86.
  • two mesh discs and a retaining ring may be used to retain the tissue slice or cell specimen 70 in position on the perfusion plate.
  • the optimal geometry and orientation of the cell specimen may vary . depending on the tissue type. For example, the tissue may be oriented vertically or horizontally to the fluid flow.
  • the pumping device 84 of the present embodiment generally compiises a pressure chtamber 96 having an air inlet 98 and a flexible diaphragm 100 that interfaces with the bottoim reservoir plate 82.
  • air pressure is introduced through inlet 98 into the pressure chamber 96 and the flexible diaphragm 100 expands and exerts pressure on the fluid reservoirs 92 of the bottom plate 82 causing the upwaxd flow of media or fluid.
  • the diaphragm 100 contracts, releasing pressure on the fluid reservoirs 92 and drawing or inducing the downward or return flow of media or fluid.
  • the media or fluid is self-purging or actively drained as opposed to gravity-driven.
  • the pumping device 84 purges the perfusion plate well during operation.
  • the pumping device may be disposable or reusable.
  • the pumping device may be sterilized using any suitable sterilization method known to those skilled in the art.
  • Several variations of the multi-well plate pumping device may also be used, including electric, peristaltic, and other diaphragm pumping techniques known to those skilled in the art.
  • bioreactor system 110 is a pneumatic system comprising three disposable or reusable pieces or components: a bottom reservoir plate 112 configured to contain media, a pumping device 114 that induces the motion of the media, and a top perfusion plate 116 that mates with the pumping device, and is configured to maintain the position of the tissue slices or cell specimen 70 in the media flow and create a closed fluid path for the media to return to bottom reservoir plate 112.
  • Both the bottom reservoir plate 112 and the top perfusion plate 116 are substantially similar to plates 82, 86 described above and generally include multiple wells or chambers 113.
  • Each plate may be injection molded and may be disposable or reusable items. Also, the plates may be sterilized using any suitable sterilization method known to those skilled in the art.
  • Each well of the perfusion plate 116 generally comprises an inlet portion 118 and an outlet portion 120.
  • Each well of the bottom reservoir plate 112 generally comprises a fluid reservorr 122 and a pair of one-way valves or check valves 124. In one embodiment, the one-way valves may be molded into a one piece plate.
  • Each of the pair of one-way valves 124 is connected via flexible passages or tubing 132 and another pair of one-way valves 125 with the corresponding inlet and outlet portions 118, 120 of the perfusion plate to direct and or allow the fluid or media to flow from fluid reservoir 122 into the inlet 118 and out of the outlet 120 and return to the fluid reservoir 122.
  • cell specimen 70 may be positioned within the inlet portion 118 of each well of the perfusion plate 116.
  • two mesh discs and a retaining ring may be used to retain the tissue slice or cell specimen 70 in position on the perfusion plate.
  • the optimal geometry and orientation of the cell specimen may vary depending on the tissue type. For example, the tissue may be oriented vertically or horizontally to the fluid flow.
  • the pumping device 114 of the present embodiment generally comprises a pressure chamber 126 having an air inlet 128 and a plurality of flexible diaphragms 130 that surround flexible passages 132.
  • Flexible passages 132 extend between the fluid reservoirs 122 of the bottom plate and a pair of one-way valves or check valves 125 aligned with the inlet and outlet portions 118, 120 of the perfusion plate 116. As best seen in FIG.
  • the pumping device 114 purges the perfusion plate well during operation.
  • the pumping device may be disposable or reusable.
  • the pumping device may be sterilized using any suitable sterilization method known to those skilled in the art.
  • any suitable sterilization method known to those skilled in the art.
  • Several variations of the multi-well plate pumping device may also be used, including electric, peristaltic, and other diaphragm pumping techniques known to those skilled in the art.
  • a hepatocyte cell may be cultured in fluid reservoir chamber 20, while an islet cell may be cultured in main chamber 18.
  • the cells are in fluid communication via the media contained within the bioreactor unit 10 of FIG. 4.
  • multiple parallel wells may be in fluid communication with each other.
  • a hepatocyte cell may be cultured in well Al of FIG. 1, while an islet cell may be cultured in well D6 of FIG. 1. All of the wells may be fluidly connected together by channels or other fluid pathways, such that after a period of time, the media from wells Al, D6, and as many of the wells in fluid communication, will mix with each other and may come to a steady state.
  • soluble and non-soluble signaling molecules consisting of growth factors, cytokines, extracellular matrix molecules, etc.
  • environments may be created utilizing a variety of parenchymal cells and non-parenchymal cells from tissues including bone marrow, vasculature, skin, pancreas, liver, bone, cartilage, smooth muscle, cardiac muscle, skeletal muscle, kidney, etc.
  • cells such as endothelial cells may be used to create vascularization with the host.
  • one skilled in the art could also create cultures consisting of several types of tissue systems for studying complex metabolic diseases such as the metabolic syndrome.
  • several cell types may be incorporated to study fluid sheer and perfusion, for example, to determine fluid flow that most likely promotes cell-type segregation for vasculorgenesis and tissue development.
  • the cells or tissue grown in the multi-well design may be used as a platform for testing drugs in a medium to high throughput format for direct drug testing on cells in dynamic cell cultures, either for drug discovery, drug testing, or ADMETox applications.
  • sensing technology may be incorporated into the bioreactor system. For example, biosensing technology for sensing important cell culture variables such as glucose, ammonia, urea, pH, or general fluorescent detectors for monitoring metabolism of fluorescent compounds may be utilized with the system.
  • a perfusion unit 12 may be used to grow cell cultures with preset conditions or particularly desirable characteristics which can then be later used for further experimentation and or discovery.
  • the modularity and interchangeability of perfusion unit 12 advantageously permits the shipment and or transfer of a plurality of cell cultures which can be easily remounted on another pumping station 16 or similar device to perform further experimentation and/or drug testing or discovery.
  • a preferred embodiment of a scaffold handling system 201 generally includes a multi-well cartridge or carrier 205 comprising an array of well units 210 wherein, in each well unit, an independent scaffold 220 may be held and a biological experiment may be performed.
  • carrier 205 of scaffold handling system 201 comprises four well units 211, 212, 213, and 214 and includes sidewalls or flanges 216 and 218 extending distally from the lateral ends of cross-member 217 to mate with a multi-well plate.
  • carrier 205 may have one well unit.
  • carrier 205 may have 8 well units.
  • carrier 205 may have 3 well units.
  • Each well unit 210 generally comprises a frustoconical or tapered body 230 exetending distally from the top of carrier 205 and includes a scaffold holding chamber 232 at the distal end 234.
  • a cell adherent structure or scaffold 220 is preferably housed or held within each well unit 210 to facilitate high density cell culture growth.
  • the cell adherent structure is coupled or loaded into to the well unit 210 about a distal end 234.
  • a three-dimensional scaffold 220 may be coupled, molded, bonded, ' synthesized, or otherwise attached to the distal chamber 232.
  • scaffold 220 may be releasably plugged into or attached to chamber 232 for example by friction fit.
  • scaffold holding chamber 232 is tapered, i.e. wider at the distal end of the well unit and narrower at the top or proximal end of the chamber.
  • This tapered feature of chamber 232 may accommodate a range of scaffold sizes.
  • chamber 232 may accommodate scaffolds with diameters ranging from about 4.8 mm to about 5.1 mm.
  • one or more nubs or protrusions 236 may extend radially inward from the perimeter of chamber 232 to further grip or hold a scaffold therein by friction.
  • each well unit 210 defines an opening 237 to permit physical and visual access to a scaffold 220 held therein.
  • a window 238 extends through the carrier 205 adjacent the well units 210 to provide access to the bottom of the well therethrough.
  • the open top of each well unit 210 i.e. opening 237 and window 238, facilitate aspiration aspiration or pipetting within the well unit.
  • a longitudinal slot, channel, or opening 239 extends along a lateral portion of body 230.
  • Opening 239 facilitates fluid overflow and permits perfusion circulation when carrier 205 is used in combination with a perfusion bioreactor as described in more detail below.
  • a ledge 241 may be provided adjacent the distal end of body 230 to accommodate a screen to hold scaffold 220 in a longitudinal direction, entrap cells or minimize particulate flow.
  • screen 250 may be positioned and/or molded adjacent ledge 241 to prevent movement of scaffold 220 in the proximal direction while permitting fluid flow therethrough.
  • Scaffold handling system 201 and carrier 205 of FIGS. 17 and 18 are configured and dimensioned to be used with a multi-well plate having a plurality of main chambers or wells to house or contain a cell culture or cell culture experiment.
  • Multi-well plates are well known to those skilled in the art. Exemplary multi-well plates include the BD FalconTM multi-well plates, available in 24-well plates and 96-well plates.
  • carrier 205 of the present embodiment is configured and dimensioned to be inserted into and/or mate with such a 24-well plate.
  • carrier 205 may be placed across a single row of the 24-well plate with each of the well units 211, 212, 213, and 214, extending into a corresponding well of the 24-well plate so that biological experimentation may be conducted.
  • Multiple carriers 205 may be placed aver additional rows of the multi-well plate such that a scaffold may be held in each well of the multi-well plate.
  • six carriers 205 may be utilized with the 24-well plate.
  • any number of arrays and configurations may be utilized such that the entire multi-well plate may include a cell adherent scaffold.
  • Sidewalls or flanges 216, 218 of carrier 205 extend distally from the lateral sides of carrier 205 and are configured and dimensioned to extend about the lateral outside of the multi-well plate to accurately mate carrier 205 with the 24-well plate.
  • flanges 216 and 218 may have a chamfered edge 219 for easy repositioning with respect to the multi-well plate.
  • one or more nubs, locating pins, or protrusions 240 may be provided on the underside of carrier 205 to facilitate the alignment of carrier 5 with the individual wells of a multi-well plate.
  • the combination of protrusions 40, flanges 16, 18, and the geometry of carrier 205 lead to a reliable and repeatable system to hold scaffolds in place with respect to a multi-well plate.
  • scaffold handling system 201 and carrier 205 of FIGS. 17 and 18 may also be used with a multi-well plate of the aforementioned perfusion bioreactor.
  • carrier 205 of the present embodiment is configured and dimensioned to be inserted into and/or mate with such a multi-well plate of a perfusion bioreactor.
  • carrier 205 may be placed across a single row of the multi-well plate of the perfusion bioreactor in the same manner as described above with respect to a 24-well plate with each of the well units 211, 212, 213, and 214, extending into a corresponding well of the multi-well plate of the bioreactor so that biological experimentation may be conducted.
  • handling system 201 is advantageously configured to permit perfusion of cell culture media through the scaffolds.
  • the reliable and repeatable positioning of the carrier 205 is configured to hold the scaffold(s) 220 in the flow line of the perfusion bioreactor such that cell culture media flows through the scaffold from the distal end to the proximal end of each well unit 210.
  • Overflow channel or opening 239 facilitates the return flow of perfusion media out though the proximal side of the scaffold 220.
  • a scaffold 220 or multiple scaffolds may be loaded or inserted into well units 210 of carrier 205.
  • the scaffold(s) 220 may then be manipulated such as by being treated with chemicals, sterilized with ultraviolet radiation, seeded with cells, or other treatments.
  • the scaffold may be inserted into a multi-well plate with cell culture media or biological agents to conduct biological experiments.
  • media can be perfused through scaffold(s) 220.
  • carrier 205 can be easily moved to a separate or fresh dry plate for microscopy without the need to handle the scaffolds directly.
  • FIG. 8 one example of a cell biology experiment performed according to the invention is shown wherein primary rat hepatocytes were seeded onto alginate scaffolds in the perfusion chamber, and cultured with Hepatostim media under perfusion flow. The same cells were also seeded onto matrigel substrates (typically known to maintain basal CYP 3 Al activity for rat hepatocytes), and passive coated collagen type 1 substrate were used as a negative control (typically known to decrease basal CYP 3Al activity for rat hepatocytes). After 48 hours, 10OuM cortexolone was added to the media to induce CYP 3Al expression.
  • the basal 3Al activity was monitored by testosterone metabolism into 6B-hydroxytestosterone using HPLC analysis.
  • the level of 6B-hydroxytestosterone in the culture is therefore indicative of CYP 3Al expression and activity.
  • the collagen cultures did not allow for CYP 3 Al expression, and the matrigel cultures helped the hepatocytes maintain CYP 3Al expression for 3 weeks, at which point expression decreased.
  • Hepatocytes cultured under fluid flow on aligned scaffolds also maintained elevated CYP 3Al activity, but instead of decreasing at 4 weeks, the activity increased dramatically compared to the matrigel.
  • This example demonstrates that cells may be grown in this device configuration and also suggests that the novel culture conditions allowed for extended and higher expression of differentiation-specific cell function for primary rat cells. Under perfusion flow, important p450 3 Al function is maintained for 4 weeks, longer than industry standard matrigel cultures.
  • FIG. 14 one example of a cell biology experiment performed according to the invention is shown wherein mouse osteoblastic cells (MC3T3s) were seeded on calcium phosphate scaffolds, and cultured with Gibco Alpha media under perfusion flow. The same cells were also seeded on calcium phosphate scaffolds in a static condition. The metabolic activity of the cells was studied via absorbance under static and perfusion conditions for a period of 15 days. After 15 days, the cells under perfusion show a statistically significant increase of 34% in metabolic activity over the cells in the static condition.
  • M3T3s mouse osteoblastic cells
  • FIG. 15 one example of a cell biology experiment performed according to the invention is shown wherein human mesenchymal stem cells (MSCs) were seeded on calcium phosphate scaffolds, and cultured with Osteogenesis media under perfusion flow. The same cells were also seeded on calcium phosphate scaffolds in a static condition. The metabolic activity of the cells was studied via absorbance under static and perfusion conditions for a period of 10 days. After 10 days, the cells under perfusion show a statistically significant increase of 42% in metabolic activity over the cells in the static condition.
  • MSCs mesenchymal stem cells
  • FIG. 16 one example of a cell biology experiment performed according to the invention is shown wherein rat liver slices were cultured with Gibco media under perfusion flow. The slices were also cultured in a static condition. The metabolic activity of the cells was studied via absorbance under static and perfusion conditions for a period of 5 days. After 5 days, the slices under perfusion show a statistically significant increase of 136% in metabolic activity over the slices in the static condition.

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Abstract

L'invention porte sur un système de bioréacteur comprenant une plate-forme multi-puits présentant une matrice d'unités de bioréacteurs. Le système de bioréacteur comprend un module de perfusion et une source de fluide raccordée, pour une communication fluidique, par une unité de pompage. Le module de perfusion comprend une plaque multi-puits comportant une pluralité de chambres principales configurées pour loger ou contenir une culture cellulaire, des expériences ou études indépendantes des cellules pouvant être effectuées dans chaque unité de bioréacteur.
EP05796533A 2004-09-16 2005-09-16 Bioreacteur pour la culture de cellules sous perfusion Withdrawn EP1789528A4 (fr)

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US94189204A 2004-09-16 2004-09-16
US69984905P 2005-07-18 2005-07-18
PCT/US2005/032818 WO2006033935A2 (fr) 2004-09-16 2005-09-16 Bioreacteur pour la culture de cellules sous perfusion

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EP1789528A4 (fr) 2011-07-20
WO2006033935A3 (fr) 2006-10-26
CA2579680A1 (fr) 2006-03-30
JP2008513013A (ja) 2008-05-01
AU2005287162A1 (en) 2006-03-30
WO2006033935A9 (fr) 2006-05-26
AU2005287162B2 (en) 2010-12-23
JP4914835B2 (ja) 2012-04-11
WO2006033935A2 (fr) 2006-03-30

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