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WO1992007615A1 - Foie bio-artificiel - Google Patents

Foie bio-artificiel Download PDF

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Publication number
WO1992007615A1
WO1992007615A1 PCT/US1991/007952 US9107952W WO9207615A1 WO 1992007615 A1 WO1992007615 A1 WO 1992007615A1 US 9107952 W US9107952 W US 9107952W WO 9207615 A1 WO9207615 A1 WO 9207615A1
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Prior art keywords
cells
cell
hollow fiber
collagen
liver
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Application number
PCT/US1991/007952
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English (en)
Inventor
Russell A. Shatford
Frank B. Cerra
Scott L. Nyberg
Wei-Shou Hu
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Regents Of The University Of Minnesota
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Publication of WO1992007615A1 publication Critical patent/WO1992007615A1/fr

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    • 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
    • C12M25/10—Hollow fibers or tubes
    • C12M25/12—Hollow fibers or tubes the culture medium flowing outside the fiber or tube
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/022—Artificial gland structures using bioreactors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/14—Dialysis systems; Artificial kidneys; Blood oxygenators ; Reciprocating systems for treatment of body fluids, e.g. single needle systems for hemofiltration or pheresis
    • A61M1/16—Dialysis systems; Artificial kidneys; Blood oxygenators ; Reciprocating systems for treatment of body fluids, e.g. single needle systems for hemofiltration or pheresis with membranes
    • A61M1/1621—Constructional aspects thereof
    • A61M1/1623—Disposition or location of membranes relative to fluids
    • A61M1/1625—Dialyser of the outside perfusion type, i.e. blood flow outside hollow membrane fibres or tubes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/34—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration
    • A61M1/3472—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration with treatment of the filtrate
    • A61M1/3475—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration with treatment of the filtrate with filtrate treatment agent in the same enclosure as the membrane
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/34—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration
    • A61M1/3472—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration with treatment of the filtrate
    • A61M1/3486—Biological, chemical treatment, e.g. chemical precipitation; treatment by absorbents
    • A61M1/3489—Biological, chemical treatment, e.g. chemical precipitation; treatment by absorbents by biological cells, e.g. bioreactor
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/126—Immunoprotecting barriers, e.g. jackets, diffusion chambers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin

Definitions

  • This invention involves the use of cells cultured in a three-dimensional gel matrix within a bioreactor such as a hollow-fiber or flat-bed system. Specifically, liver cells are maintained in this bioreactor allowing this device to be used as a bioartificial liver for patients with liver failure.
  • left ventricular assist devices exist for the injured heart; dialysis units are used for kidney failure; parenteral nutrition are used for the nonfunctioning gastrointestinal tract; ventilators, extracorporeal membrane oxygenators, and veno-venous bypass techniques are employed to support lung function.
  • ventilators extracorporeal membrane oxygenators, and veno-venous bypass techniques are employed to support lung function.
  • extracorporeal membrane oxygenators are used for the nonfunctioning gastrointestinal tract
  • veno-venous bypass techniques are employed to support lung function.
  • liver performs many complex tasks necessary for survival. These tasks have been difficult to develop or maintain in mechanical systems.
  • the liver is the metabolic factory synthesizing glucose, lipids, and proteins — including albumin, enzymes, clotting factors, and carrier molecules for trace elements.
  • the liver maintains appropriate plasma concentrations of amino and fatty acids, as well as detoxifying nitrogenous wastes, drugs, and other chemicals. Waste products, such as bilirubin, are conjugated and excreted via the biliary tree.
  • Hepatic protein synthesis vastly increases the complexity of hepatic support. Culturin ⁇ Hepatocytes Systems that employ hepatocytes to provide biochemical function are problematic because hepatocytes can be difficult to maintain in culture.
  • Human heoatocellular carcinoma cell lines secrete the maior plasma proteins and hepatitis B surface antigen. Science; 1980 Jul 25; 209: 497-9.) These cell lines have the potential to transmit the transforming virus to the patient. As a result, it is doubtful that regulatory agencies would approve the use of transformed cells for humans, even if the risk of transmission were proven minimal.
  • hepatocytes have been investigated. These approaches have included adding hormones and growth factors to the culture media, adding extracellular matrix constituents, and growing the hepatocytes in the presence of another cell type.
  • Cells routinely used in co-culture work with hepatocytes are endothelial cells, or hepatic nonparenchymal cells such as Kupffer cells.
  • DMSO Dimethyl sulfoxide
  • phenobarbital also are known to prolong hepatocyte viability and function.
  • Insulin can promote some functions with an effect that varies with concentration. If only insulin is added to the medium, urea cycle enzyme expression is decreased. This negative effect can be counteracted by the addition of glucagon and dexamethasone.
  • Hormonally defined media can also prolong hepatocyte function and viability. (Jefferson, et al., 1984, supra. ) Using a serum-free hormonally defined medium, good function in baboon hepatocytes has been shown for over 70 days. This medium consisted of epidermal growth factor (100 ng/ml), insulin (lOu/ml), glucagon 4mg/ml), albumin (0.5 mg/ml), linoleic acid (5 mg/ml) , hydrocortisone —6 —7
  • One third of the extract was composed of carbohydrates and noncollagenous proteins; the other two thirds were collagens — 43% Type I, 43% Type III, and the remainder an undefined mixture of others including Type IV.
  • This mixture may not accurately reflect the local hepatocyte environment — the peri-sinusoidal space or Space of Disse.
  • Heparan sulfate proteoglycan binds both cell growth factors and cells.
  • Hepatocytes also can be cultured on Matrigel, a biomatrix produced by a sarcoma cell line (EHS) .
  • Matrigel contains Type IV collagen, laminin, entactin, and heparan sulfate.
  • EHS sarcoma cell line
  • Matrigel contains Type IV collagen, laminin, entactin, and heparan sulfate.
  • hepatocytes have been shown to maintain normal albumin synthesis for 21 days. (Bissell, et al., 1987, suprg.) Close duplication of the normal environment of the hepatocyte has also been attempted by culturing hepatocytes in a confluent monolayer on collagen. A second layer of Type I collagen is added to recreate the normal matrix "sandwich" formed on the "top” and on the "bottom” of the hepatocyte.
  • lipocytes play a key role in matrix production. Lipocytes are reported to be as numerous as Kupffer cells, and have been suggested to produce the majority of Type I collagen, Type II collagen, Type IV collagen, laminin, and proteoglycans — particularly dermatin sulfate proteoglycan and chondroitin sulfate proteoglycan. (Friedman, S.L.; Roll, F.J.; Boyles, J.; Bissell, D.M. Hepatic lipocytes: The principle collagen-producing cells of normal rat liver. PNAS; Dec 1985; 82: 8681-5.) It is of particular interest that these specific proteoglycans were those that best support gap junctions. (Spray, et al., 1987, supra. )
  • bioartificial liver systems currently being investigated for support of liver failure include extracorporeal bioreactors (Arnaout, W.S.; Moscioni, A.D.; Barbour, R.L.; Demetriou, A.A. Development of bioartificial liver: bilirubin conjugation in Gunn rats. Journal of Surgical Research; 1990; 48: 379-382; Margulis MS, Eruckhimov EA, Ahdieimann LA, Viksna LM. Temporary organ substitution bv hemoperfusion through suspension of active donor hepatocytes in a total complex of intensive therapy in patients with acute hepatic insufficiency.
  • hepatocyte cultures such as microencapsulated gel droplets (Cai, Z.; Shi, Z.; O'Shea, G.M.; Sun, A.M. Microencapsulated hepatocytes for bioartificial liver support. Artificial Organs; 1988 May; 12(5): 388-393) and spheroid aggregates (Saito, S.; Sakagami, K. ; Koide, N.; Morisaki, F.; Takasu S, Oiwa T, Orita K. Transplantation of spheroidal aggregate cultured hepatocytes into rat spleen. Transplantation Proceedings; 1989 Feb; 21(1): 2374-77.).
  • hepatocyte microencapsulation The technique for hepatocyte entrapment within microencapsulated gel droplets (hepatocyte microencapsulation) is similar to the technique successfully used for pancreatic islet encapsulation (O'Shea, G.M.; Sun, A.M. Encapsulation of rat islets of Lanoerhans prolongs xenograft survival in diabetic mice. Diabetes; 1986 August; 35: 943-46; Cai, et al., 1988, supra) . Microencapsulation allows nutrient diffusion to the hepatocytes, and metabolite and synthetic production diffusion from the hepatocytes.
  • Microencapsulation also provides intraperitoneal hepatocytes with "immuno-isolation" from the host defenses (Wong, H.; Chang, T.M.S. The viability and regeneration of artificial cell microencapsulated rat hepatocvte xenograft transplants in mice. Biomat. Art. Cells, Art. Org.; 1988; 16(4): 731-739.) Plasma protein and albumin synthesis (Sun, A.M.; Cai, Z.; Shi, Z.; Fengzhu, M. ; O'Shea, G.M.; Gharopetian, H. Microencapsulated hepatocytes as a bioartificial liver. Trans.
  • ASAIO 1986; 32: 39-41; Cai, et al., 1988, supra) ; cytochro e P450 activity and conjugation activity (Tompkins, R.G.; Carter, E.A. ; Carlson, J.D.; Yarmush, M.L. Enzvmatic function of alginate immobilized rate hepatocytes. Biotechnol. Bioeng.; 1988; 31: 11-18); gluconeogenesis (Miura, Y. ; Akimoto, T.; Yagi, K. Liver functions in hepatocytes entrapped within calcium aloinate. Ann. N.Y. Acad. Sci.; 1988; 542: 531-32); ureagenesis
  • Spheroid aggregate cultured hepatocytes have also been proposed for the treatment of fulminant hepatic failure.
  • Multiple techniques exist for hepatocyte aggregation into spheroids (Saito, S.; Sakagami, K. ; Koide, N.; Morisaki, F.; Takasu, S.; Oiwa, T.; Orita, K. Transplantation of spheroidal aggregate cultured hepatocytes into rat spleen. Transplanatation Proceedings; 1989 Feb; 21(1): 2374-77; Koide, N.; Shinji, T. ; Tanube, T.; Asano, K.; Kawaguchi, M. ; Sakaguchi, K. ; Koide, Y.
  • hepatocyte aggregation would improve the beneficial results of intraperitoneal hepatocyte injection therapy.
  • Such therapy has been used experimentally in the treatment of enzyme deficiency diseases, acute liver failure and hepatic cirrhosis with varying degress of success (Saito, et al., 1989, supra) .
  • Extracorporeal bioreactor designs for the purpose of artificial liver support have included perfusion of small liver cubes (Lie TS, Jung V,
  • the device consisted of a rabbit hepatocyte liquid suspension (1-2 liters) separated from the patient's blood by a cellulose acetate dialysis membrane. Each treatment used fresh hepatocytes during a single four to six hour dialysis (run). Multiple runs successfully reduced serum bilirubin and reversed metabolic encephalopathy in a single case.
  • the bioartificial device consisted of a small 20 ml cartridge filled with pig hepatocytes in liquid suspension, along with activated charcoal granules. The cartridge was perfused through a Scribner arteriovenous shunt access. Patients were treated daily for six hours. The hepatocyte suspension was changed hourly over each six hour treatment period. Improved survival was demonstrated in the treated group (63%) when compared with the standard medical therapy control group (41%). Culturing hepatocytes with a hollow fiber cartridge is another example of bioartificial liver support.
  • hepatocytes are loaded in the extracapillary space of the hollow fiber cartridge, while medium, blood or plasma is perfused through the lumen of the hollow fibers.
  • Cells may be free in suspension (Wolf, C.F.W.; Munkelt, B.E. Bilirubin conjugation bv an artificial liver composed of cultured cells and synthetic capillaries. Trans. ASAIO; 1975; 21: 16-27); attached to walls (Hager, J.C; Carman, R. ; Stoller, R.; Panol, C; Leduc, E.H.; Thayer, W.R.; Porter, L.E.; Galletti, P.M.; Calabresi, P. Trans. ASAIO; 1978; 24: 250-253); or attached to microcarriers which significantly increase the surface area within the extracapillary space (Arnaout, et al., 1990, supra) .
  • Bilirubin uptake, conjugation and excretion by Reuber hepatoma cells within a hollow fiber cartridge was reported in 1975. (Wolf, et al., 1975, supra) . Tumor cell suspensions were injected by syringe into the shell side of the compartment while bilirubin containing medium was perfused through the hollow fiber intraluminal space. This technique has not been reported clinically, possibly due to the risk of tumor seeding by hepatoma cells.
  • Another hollow fiber device developed for liver support uses hepatocytes attached to microcarriers loaded into the extracapillary cavity of a hollow fiber cartridge. In this device, blood flows through semi-permeable hollow fibers allowing the exchange of small molecules. Using this system, increased conjugated bilirubin levels have been measured in the bile of glucuronosyl transferase deficient (Gunn) rats. (Arnaout, W.S.; Mosicioni,
  • SUBSTITUTESHEET nutrients are provided by the patient's blood stream.
  • this system may require an intact in vivo biliary tree for the excretion of biliary and toxic wastes.
  • a hollow fiber bioreactor in its "conventional" configuration, may not be optimal for a bioartificial liver.
  • a "conventional" hollow fiber configuration such as the two described above, cells are loaded in the extracapillary cavity (shell) while media flows through the lumen of the fibers.
  • Potential problems exist in the extracapillary space such as uncontrolled fluid flow, fluid channelling, and location dependent cell concentration and viability.
  • the present invention thus proposes a new hollow fiber bioreactor configuration, as well as a new flat-bed configuration.
  • the present invention presents a novel bioreactor configuration for cell culture, which is particularly suitable for supporting viable hepatocytes in vitro.
  • this novel bioreactor is a hollow fiber cell culture bioreactor employing cells entrapped within a fibrous and highly porous collagenous gel matrix within the hollow fiber lumen.
  • this novel bioreactor is a flat-bed bioreactor with cells entrapped within a matrix but separated from a media stream by a porous membrane.
  • This invention also relates to a cell gel matrix and a method of preparing such a cell gel matrix for cell cultivation.
  • a bioartificial liver employing this novel bioreactor for supporting hepatocyte function in a patient suffering from hepatic failure is also provided by this invention.
  • Tissue-specific function of other mammalian cells can also be supported us ng the cell gel matrix and the novel bioreactor provided by this invention, while also withdrawing desirable products or by-products therefrom.
  • Figure 3 Contraction in hepatocyte gel discs.
  • Figure 4 Bilirubin conjugation rate in spinner flasks containing hepatocyte-gel cores.
  • Figure 5 Oxygen consumption rate in the hollow fiber bioreactor over 120 hours.
  • Figure 6 HPLC analysis of bilirubin.
  • Figure 7 Bilirubin conjugation (HPLC) data.
  • Figure 8 Conjugated and unconjugated bilirubin levels (Ektachem 700XR) .
  • the stream (blood or plasma) to be detoxified flows through the shell side.
  • cells 34 such as hepatocytes
  • hepatocytes are within the hollow fiber lumen 36, entrapped in a gel matrix 38.
  • This configuration is accomplished by first suspending hepatocytes 34 in a solution of collagen or a mixture of collagen and extracellular matrix components. The pH is then adjusted to 7.4 and the cell suspension inoculated into the lumen 36 of the hollow fiber 40. A 'temperature change from 4 °C to 37°C induces collagen fiber formation. This results in cell entrapment in an insoluble fibrous and highly porous cylindrical gel 38.
  • FIG. 2 illustrates that media or blood or plasma with low molecular weight nutrients flows around hollow fibers 40 in the extraluminal shell space 32 from extraluminal inlet 42a to extraluminal outlet 42b. Molecular exchange occurs through the pores in the hollow fiber 40. Media with high molecular weight constituents flows through the hollow fiber 40 containing a contracted core of hepatocytes 34 embedded in biomatrix 38 through hollow fiber inlet 46a to hollow fiber outlet 46b.
  • This technique is useful with multiple cell lines including Chinese Hamster ovary cells, Hep2, HepG2, Vero, 293 cells, and normal diploid human cells.
  • Study of a hematoxylin and eosin (H & E) stained thin section of human hepatoblastoma (HepG2) cells within a contracted gel matrix after 7 days showed that tissue density and cytoarchitecture closely resemble in. vivo histology.
  • This bioreactor offers distinct advantages over other configurations.
  • Cells can be cultured at density close to that of tissue. At high density, cells occupy much less space, thus reducing the size of the bioreactor. Cells also obtain the benefits of close contact with minimal oxygen and nutrient limitations. Mammalian cells, at high density, may better preserve tissue specific function. This has been shown in hepatoma lines. (Kelly, J.H.;
  • This bioreactor configuration also allows manipulation of the hepatocytes' local environment.
  • Matrix constituents that support differentiated hepatocyte function can be incorporated into the gel.
  • the ability to perfuse the inner lumen provides high molecular weight growth factors at high concentrations.
  • Another advantage of such a system is that different cell types can be co-entrapped in the gel to provide possible synergistic effects which may improve tissue specific function.
  • This invention is thus capable of incorporating many factors (medium, gel matrix, co-culture, high cell density) necessary or beneficial to sustain liver specific functions. It can be used as a bioartificial liver to support patients in liver failure.
  • the new hollow fiber bioreactor 30 is illustrated in Figures 1 and 2.
  • the hollow fiber 40 cartridge allows a large surface area for oxygen and
  • Figure 1 and Figure 2 show that blood or plasma from the patient flows continuously through the extraluminal shell space 32 and semi-permeable hollow fibers 40 which separate this fluid from the hepatocytes 34.
  • Intraluminal stream 46 containing high molecular weight constituents flows through hollow fibers 40 containing hepatocytes 34 in biomatrix 38.
  • the extraluminal stream 42 containing the patient's blood or plasma flows in either a counter-current, cross-current, or co-current direction to the intraluminal stream 46.
  • Molecular exchange occurs through the pores in the hollow fiber 40. It is probable that blood—particularly from a patient in liver failure—does not provide the optimal chemical environment to sustain hepatocyte function and viability.
  • Intraluminal stream 46 containing growth factors and nutrients is passed through the hollow fiber lumen. Intraluminal stream 46 can also provide toxin or metabolic product removal.
  • Our two channel hollow fiber design supplies both a "life support system" for the hepatocytes 34, and a stream of waste products.
  • the microporous hollow fibers 40 can allow diffusion of waste products, such as ammonia and bilirubin from the blood, for detoxification by the hepatocytes. Waste products are then cleared in the hollow fiber intraluminal stream 46. These conditions can result in improved hepatocyte survival and continuous function.
  • Several fundamental aspects of hepatocyte cultivation have been addressed. Prolonged hepatocyte viability and function have been demonstrated in monolayer cultures.
  • hepatocytes were cultured in three dimensional collagen gels. Dime-sized collagen "discs” and thin diameter cylindrical collagen “cores” of 0.5 or 1.1 mm in diameter were studied. Gels contained 2 gm/1 of Type I collagen in isotonic DMEM. Collagen gel discs were made by adding a mixture of collagen/DMEM and hepatocytes to empty tissue culture plates. Collagen gels have been made with other isotonic media, such as William's E medium. Media was added following gel formation. Silicone tubing was used to form thin diameter collagen gel cores. After 10 minutes of incubation at 37°, the cylindrical gel cores were extruded into media containing wells. All collagen gel experiments including bioreactor trials were done using William's E medium supplemented with 10% calf serum, insulin, L-glutamine (Modified William's E medium) or a serum-free hormonally defined media. (Lanford, supra) .
  • Collagen gel cores were used to measure metabolic activity. After formation in the silicone tubing, the gel cores were placed in spinner flasks and incubated for 30 hours. Media samples were taken for analysis at six hour intervals.
  • a hollow-fiber system assembly consisted of an Amicon HI hollow-fiber cartridge with Delrin end caps.
  • the hollow fibers are made of porous polysulfone with a 30,000 molecular weight cut-off.
  • the extracapillary space (outer shell) was perfused with Modified William's E medium.
  • the inner channel was not perfused.
  • the hollow fiber reactor was kept in a 37°C warm room following inoculation.
  • C. is the inlet oxygen concentration
  • C . is the outlet oxygen concentration
  • F is the media flow rate.
  • the oxygen uptake rate increases with increasing flow rate at low flow rates, and becomes flow independent at high flow rates.
  • a flow rate of 30 ml/min was sufficient to maintain maximum oxygen uptake without inducing the larger pressure drop seen at higher flow rates, and was used in this example.
  • Unconjugated bilirubin levels are also included on Figure 8. Judging from the appearance of conjugated bilirubin in the medium, hepatocytes cultivated in the hollow fiber bioreactor are capable of liver specific function - namely, bilirubin conjugation.
  • a system using the gel matrix concepts described herein provides constant optimal media perfusion to detoxify blood and facilitates liver cell metabolic function.
  • a device using this concept is designed such that the blood flow and media flow allow proper oxygenation, toxin transfer, and toxin-metabolite removal.
  • membrane pore size must allow proper diffusion rates for toxin removal and liver cell metabolic function.

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Abstract

Foie bio-artificiel (30) destiné à assurer les fonctions hépatiques chez les malades atteints d'insuffisance hépatique éventuellement aiguë. Le foie bio-artificiel est basé sur un bioréacteur du type qui possède deux voies d'écoulement de fluide (42, 46) séparées par un milieu perméable (38). Le bioréacteur peut être du type à fibres creuses ou du type à lit plat. Dans le premier cas, les deux voies d'écoulement de fluide correspondent à la cavité (36) entourant les fibres creuses (le compartiment extracapillaire), et aux lumières des fibres creuses proprement dites. Lesdites deux voies possèdent des orifices d'entrée et de sortie. La communication entre les deux voies d'écoulement se fait par l'intermédiaire du milieu perméable - la matière des fibres creuses. On introduit par inoculation dans les fibres creuses des hépatocytes (34) contenus dans une solution qui forme rapidement un gel à porosité élevée. Ensuite, le gel se rétrécit pour laisser ouvert un passage à l'intérieur de la fibre creuse à côté des hépatocytes enfermés au centre du gel. On peut introduire par perfusion dans ce passage des milieux nourriciers destinés aux hépatocytes.
PCT/US1991/007952 1990-10-29 1991-10-29 Foie bio-artificiel WO1992007615A1 (fr)

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Cited By (36)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997016527A1 (fr) 1995-10-30 1997-05-09 Cellex Biosciences, Inc. Materiel de culture pour foie bio-artificiel
EP0746343A4 (fr) * 1993-10-08 1997-12-03 Univ Michigan PROCEDES ET COMPOSITIONS CONCERNANT UN REIN BIOARTIFICIEL CONVENANT POUR UNE UTILISATION -i(IN VIVO) OU -i(EX VIVO)
WO1998051236A1 (fr) * 1997-05-14 1998-11-19 Cedars-Sinai Medical Center Intestin artificiel
US5955353A (en) * 1997-05-22 1999-09-21 Excorp Medical, Inc. Hollow fiber bioreactor with an extrafilament flow plug
EP0784499A4 (fr) * 1992-09-11 2000-03-29 Xenogenex Inc Foie artificiel et son fonctionnement
EP1045024A1 (fr) * 1999-04-12 2000-10-18 K.U. Leuven Research & Development Utilisation d'une lignée céllulaire d' hépatocytes humaines pour le traitement de la défaillance hépatique aigue et chronique
US6410320B1 (en) 1992-03-02 2002-06-25 The University Of Michigan Method and compositions for isolation and growth of kidney tubule stem cells, in vitro kidney tubulogenesis and ex vivo construction of renal tubules
US6653131B2 (en) 2001-08-30 2003-11-25 The Regents Of The University Of Michigan Method of treating systemic inflammatory response syndrome
US6942879B2 (en) 1996-09-30 2005-09-13 The Regents Of The University Of Michigan Bioartificial filtration device for filtering blood to mimic kidney function
US7160719B2 (en) 2002-06-07 2007-01-09 Mayo Foundation For Medical Education And Research Bioartificial liver system
US7332330B2 (en) 2001-09-11 2008-02-19 Renamed Biologics, Inc. Device for maintaining vascularization near an implant
US7442546B2 (en) 2002-03-15 2008-10-28 The Regents Of The University Of Michigan Method of modulating inflammatory response
US7615567B2 (en) 2000-11-08 2009-11-10 Research Triangle Institute Compounds and methods for promoting smoking cessation
CN102210892A (zh) * 2011-05-05 2011-10-12 浙江大学 一种集成式微囊悬浮型流化床式生物反应器
EP2931871A4 (fr) * 2012-12-11 2016-07-20 Pall Technology Uk Ltd Récipient pour la culture cellulaire
CN106139289A (zh) * 2016-07-29 2016-11-23 武汉仝干医疗科技股份有限公司 半透膜分层一体式生物反应器
GB2548167A (en) * 2016-03-07 2017-09-13 Indian Inst Of Tech An integrated hybrid bio-artificial liver bioreactor design and method thereof
US10130748B2 (en) 2009-03-13 2018-11-20 Mayo Foundation For Medical Education And Research Bioartificial liver
US10179896B2 (en) 2015-05-12 2019-01-15 Baker Group, LLP Method and system for a bioartificial organ
US10781425B2 (en) 2010-05-06 2020-09-22 Children's Hospital Medical Center Methods and systems for converting precursor cells into intestinal tissues through directed differentiation
CN111704998A (zh) * 2020-07-14 2020-09-25 兰州大学第一医院 一种竖置板恒流式生物人工肝反应器
US11053477B2 (en) 2014-05-28 2021-07-06 Children's Hospital Medical Center Methods and systems for converting precursor cells into gastric tissues through directed differentiation
US11066650B2 (en) 2016-05-05 2021-07-20 Children's Hospital Medical Center Methods for the in vitro manufacture of gastric fundus tissue and compositions related to same
WO2022005858A1 (fr) * 2020-06-30 2022-01-06 Corning Incorporated Récipients tubulaires de culture cellulaire à lit tassé, systèmes et procédés associés
EP3856204A4 (fr) * 2018-09-27 2022-06-29 Children's Hospital Medical Center Système de support du foie comprenant des organoïdes du foie et procédés de fabrication et d'utilisation de celui-ci
CN115537375A (zh) * 2022-10-21 2022-12-30 兰州大学第一医院 一种用于肝纤维化治疗的生物人工肝处理血清制备方法
US11584916B2 (en) 2014-10-17 2023-02-21 Children's Hospital Medical Center Method of making in vivo human small intestine organoids from pluripotent stem cells
US11767515B2 (en) 2016-12-05 2023-09-26 Children's Hospital Medical Center Colonic organoids and methods of making and using same
US12037572B2 (en) 2019-02-05 2024-07-16 Corning Incorporated Packed-bed bioreactor systems and methods of using the same
US12116556B2 (en) 2019-11-05 2024-10-15 Corning Incorporated Fixed bed bioreactor and methods of using the same
US12281334B2 (en) 2017-04-14 2025-04-22 Children's Hospital Medical Center Multi donor stem cell compositions and methods of making same
US12297457B2 (en) 2017-10-10 2025-05-13 Children's Hospital Medical Center Esophageal tissue and/or organoid compositions and methods of making same
US12379372B2 (en) 2017-12-21 2025-08-05 Children's Hospital Medical Center Digitalized human organoids and methods of using same
US12414967B2 (en) 2016-11-04 2025-09-16 Children's Hospital Medical Center Compositions and methods of treating liver disease
US12421500B2 (en) 2018-07-26 2025-09-23 Children's Hospital Medical Center Hepato-biliary-pancreatic tissues and methods of making same
US12428622B2 (en) 2018-09-12 2025-09-30 Children's Hospital Medical Center Organoid compositions for the production of hematopoietic stem cells and derivatives thereof

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US4923598A (en) * 1987-06-23 1990-05-08 Fresenius Ag Apparatus for the treatment of blood in particular for hemodialysis and hemofiltration
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Cited By (47)

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Publication number Priority date Publication date Assignee Title
US6410320B1 (en) 1992-03-02 2002-06-25 The University Of Michigan Method and compositions for isolation and growth of kidney tubule stem cells, in vitro kidney tubulogenesis and ex vivo construction of renal tubules
EP0784499A4 (fr) * 1992-09-11 2000-03-29 Xenogenex Inc Foie artificiel et son fonctionnement
EP0746343A4 (fr) * 1993-10-08 1997-12-03 Univ Michigan PROCEDES ET COMPOSITIONS CONCERNANT UN REIN BIOARTIFICIEL CONVENANT POUR UNE UTILISATION -i(IN VIVO) OU -i(EX VIVO)
WO1997016527A1 (fr) 1995-10-30 1997-05-09 Cellex Biosciences, Inc. Materiel de culture pour foie bio-artificiel
US6942879B2 (en) 1996-09-30 2005-09-13 The Regents Of The University Of Michigan Bioartificial filtration device for filtering blood to mimic kidney function
WO1998051236A1 (fr) * 1997-05-14 1998-11-19 Cedars-Sinai Medical Center Intestin artificiel
US5993406A (en) * 1997-05-14 1999-11-30 Cedars-Sinai Medical Center Artificial gut
US5955353A (en) * 1997-05-22 1999-09-21 Excorp Medical, Inc. Hollow fiber bioreactor with an extrafilament flow plug
EP1045024A1 (fr) * 1999-04-12 2000-10-18 K.U. Leuven Research & Development Utilisation d'une lignée céllulaire d' hépatocytes humaines pour le traitement de la défaillance hépatique aigue et chronique
US7615567B2 (en) 2000-11-08 2009-11-10 Research Triangle Institute Compounds and methods for promoting smoking cessation
US6653131B2 (en) 2001-08-30 2003-11-25 The Regents Of The University Of Michigan Method of treating systemic inflammatory response syndrome
US7332330B2 (en) 2001-09-11 2008-02-19 Renamed Biologics, Inc. Device for maintaining vascularization near an implant
US7442546B2 (en) 2002-03-15 2008-10-28 The Regents Of The University Of Michigan Method of modulating inflammatory response
US7160719B2 (en) 2002-06-07 2007-01-09 Mayo Foundation For Medical Education And Research Bioartificial liver system
US8785117B2 (en) 2002-06-07 2014-07-22 Mayo Foundation For Medical Education And Research Method for treating blood or plasma using hepatocyte spheroids
US9650609B2 (en) 2002-06-07 2017-05-16 Mayo Foundation For Medical Education And Research Bioartificial liver system
US10792410B2 (en) 2009-03-13 2020-10-06 Mayo Foundation For Medical Education And Research Bioartificial liver
US10130748B2 (en) 2009-03-13 2018-11-20 Mayo Foundation For Medical Education And Research Bioartificial liver
US12258584B2 (en) 2010-05-06 2025-03-25 Children's Hospital Medical Center Methods and systems for converting precursor cells into intestinal tissues through directed differentiation
US10781425B2 (en) 2010-05-06 2020-09-22 Children's Hospital Medical Center Methods and systems for converting precursor cells into intestinal tissues through directed differentiation
CN102210892A (zh) * 2011-05-05 2011-10-12 浙江大学 一种集成式微囊悬浮型流化床式生物反应器
EP2931871A4 (fr) * 2012-12-11 2016-07-20 Pall Technology Uk Ltd Récipient pour la culture cellulaire
US12241090B2 (en) 2014-05-28 2025-03-04 Children's Hospital Medical Center Methods and systems for converting precursor cells into gastric tissues through directed differentiation
US11053477B2 (en) 2014-05-28 2021-07-06 Children's Hospital Medical Center Methods and systems for converting precursor cells into gastric tissues through directed differentiation
US11584916B2 (en) 2014-10-17 2023-02-21 Children's Hospital Medical Center Method of making in vivo human small intestine organoids from pluripotent stem cells
US10179896B2 (en) 2015-05-12 2019-01-15 Baker Group, LLP Method and system for a bioartificial organ
US10245368B2 (en) 2016-03-07 2019-04-02 Indian Institute Of Technology, Kanpur Integrated hybrid bio-artificial liver bioreactor design and method thereof
GB2548167B (en) * 2016-03-07 2020-03-04 Indian Institute Of Tech An integrated hybrid bio-artificial liver bioreactor design and method thereof
GB2548167A (en) * 2016-03-07 2017-09-13 Indian Inst Of Tech An integrated hybrid bio-artificial liver bioreactor design and method thereof
US11066650B2 (en) 2016-05-05 2021-07-20 Children's Hospital Medical Center Methods for the in vitro manufacture of gastric fundus tissue and compositions related to same
CN106139289A (zh) * 2016-07-29 2016-11-23 武汉仝干医疗科技股份有限公司 半透膜分层一体式生物反应器
US12414967B2 (en) 2016-11-04 2025-09-16 Children's Hospital Medical Center Compositions and methods of treating liver disease
US11767515B2 (en) 2016-12-05 2023-09-26 Children's Hospital Medical Center Colonic organoids and methods of making and using same
US12281334B2 (en) 2017-04-14 2025-04-22 Children's Hospital Medical Center Multi donor stem cell compositions and methods of making same
US12297457B2 (en) 2017-10-10 2025-05-13 Children's Hospital Medical Center Esophageal tissue and/or organoid compositions and methods of making same
US12379372B2 (en) 2017-12-21 2025-08-05 Children's Hospital Medical Center Digitalized human organoids and methods of using same
US12421500B2 (en) 2018-07-26 2025-09-23 Children's Hospital Medical Center Hepato-biliary-pancreatic tissues and methods of making same
US12428622B2 (en) 2018-09-12 2025-09-30 Children's Hospital Medical Center Organoid compositions for the production of hematopoietic stem cells and derivatives thereof
EP3856204A4 (fr) * 2018-09-27 2022-06-29 Children's Hospital Medical Center Système de support du foie comprenant des organoïdes du foie et procédés de fabrication et d'utilisation de celui-ci
US12037572B2 (en) 2019-02-05 2024-07-16 Corning Incorporated Packed-bed bioreactor systems and methods of using the same
US12180451B2 (en) 2019-02-05 2024-12-31 Corning Incorporated Packed-bed bioreactor systems and methods of using the same
US12116556B2 (en) 2019-11-05 2024-10-15 Corning Incorporated Fixed bed bioreactor and methods of using the same
CN115768864A (zh) * 2020-06-30 2023-03-07 康宁股份有限公司 管式填装床细胞培养容器、系统和相关方法
WO2022005858A1 (fr) * 2020-06-30 2022-01-06 Corning Incorporated Récipients tubulaires de culture cellulaire à lit tassé, systèmes et procédés associés
CN111704998A (zh) * 2020-07-14 2020-09-25 兰州大学第一医院 一种竖置板恒流式生物人工肝反应器
CN115537375B (zh) * 2022-10-21 2023-10-03 兰州大学第一医院 一种用于肝纤维化治疗的生物人工肝处理血清制备方法
CN115537375A (zh) * 2022-10-21 2022-12-30 兰州大学第一医院 一种用于肝纤维化治疗的生物人工肝处理血清制备方法

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