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WO1997028252A1 - Procede permettant d'agreger des cellules et de former des feuilles de tissu de mammifere dans un bioreacteur a rotation horizontale - Google Patents

Procede permettant d'agreger des cellules et de former des feuilles de tissu de mammifere dans un bioreacteur a rotation horizontale Download PDF

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Publication number
WO1997028252A1
WO1997028252A1 PCT/US1997/001732 US9701732W WO9728252A1 WO 1997028252 A1 WO1997028252 A1 WO 1997028252A1 US 9701732 W US9701732 W US 9701732W WO 9728252 A1 WO9728252 A1 WO 9728252A1
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Prior art keywords
cells
bioreactor
cell
differentiated
rotating
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PCT/US1997/001732
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English (en)
Inventor
Glenn F. Spaulding
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Vivorx Pharmaceuticals, Inc.
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Application filed by Vivorx Pharmaceuticals, Inc. filed Critical Vivorx Pharmaceuticals, Inc.
Priority to AU18553/97A priority Critical patent/AU1855397A/en
Publication of WO1997028252A1 publication Critical patent/WO1997028252A1/fr

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    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0062—General methods for three-dimensional culture
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0676—Pancreatic cells
    • C12N5/0677—Three-dimensional culture, tissue culture or organ culture; Encapsulated cells

Definitions

  • the present invention relates to the growth and proliferation of living human or mammalian cells or cell clusters in a tissue culture system.
  • the present invention relates to a novel process for rapidly aggregating mammalian cells, and for the creation of sheets of mammalian tissue.
  • the invention process involves constraining the 3-dimensional tissue culture environment while concurrently providing 3- dimensional perfusion of the aggregating cells. Consequently, there is a greater incidence of cell-to-cell contact, thereby facilitating cell-to-cell interactions that are likely to lead to aggregation. It will be appreciated that the invention process is low cost and universally useful for culturing large aggregates or sheets of mammalian tissue.
  • the present invention relates to methods and compositions for the proliferation of pancreatic islets of Langerhans as a therapy for Type I and Type II diabetes mellitus.
  • Invention methods and compositions employ a complex cell- culture medium containing various nutrients and growth factors which are necessary or sufficient to promote long- term cell growth or multiplication and to avoid senescence or loss of biological function.
  • a second general process for aggregating tissue and forming 3-dimensional tissue constructs involves placing cells in a static gel matrix. Cells aggregate but soon become mass transfer limited, due to the inability to adequately perfuse cells inside the gel matrix. Aggregate size is thus limited, otherwise centers of hypoxic necrosis appear.
  • a third general process for aggregation is to suspend cells in a horizontally rotating bioreactor.
  • cell concentrations are substantially diluted.
  • higher initial cell concentrations may be used.
  • the disadvantage resides in the fact that a high cell concentration requirement precludes the use of rare cell types or small samples, e.g., human biopsies.
  • rare cell sub-populations may be lost due to the reduced incidence for contact with other nurturing cell types.
  • Insulin-dependent Type-1 diabetes mellitus is a life-threatening disease characterized by the loss of glucose-induced insulin secretion from insulin-producing beta cells in the pancreatic islets of Langerhans . Diabetes affects more than 100 million people worldwide, to whom multiple insulin injections must be given periodically throughout the day. Standard therapy has included parenteral administration of insulin (either bovine or porcine or recombinant human) by means of multiple injections or by means of an indwelling catheter-and-pump. Despite the medical improvement afforded by such injections, this therapy still cannot duplicate the precise feedback of insulin secretion provided by a normal pancreas. Indeed, such treatment can only temporarily delay the pathological complications of the disease.
  • pancreatic islet transplantation is an effective therapy for restoring glucose responsiveness
  • the limited availability of human donors requires that alternative sources of transplantable islets be developed.
  • pancreatic islets including human or porcine pancreatic islets, as well as hepatocytes, keratinocytes, chondrocytes, acinar cells, or chromaffin cells
  • hepatocytes including human or porcine pancreatic islets, as well as hepatocytes, keratinocytes, chondrocytes, acinar cells, or chromaffin cells
  • hepatocytes including human or porcine pancreatic islets, as well as hepatocytes, keratinocytes, chondrocytes, acinar cells, or chromaffin cells
  • Fetal pancreatic islets for example, contain many undifferentiated beta cells which can mature after transplantation and which are less subject to rejection by the recipient. Unfortunately, however, fetal pancreatic islets cannot be obtained in large enough amounts to be employed as part of a practical therapeutic approach.
  • pancreatic islets in large-scale tissue-culture vessels can potentially provide an unlimited self-renewing source for therapeutic use, once methods have been developed to improve the rate and reliability of this process.
  • These needed innovations are being facilitated by investigations into: the structure and function of islets at all stages of development, the mechanisms of normal or pathological regeneration, the effects of collagen or extracellular-matrix components on growth, the use of microcarrier beads for both protection and attachment in tissue-culture vessels, the co-culture of islets with duct cells or with fibroblasts, the effects of long-term glucose concentration on insulin secretion, the stimulation or inhibition of cellular activity by various exogenous factors, and the intracellular expression of enzymes which transport or derivatize glucose in connection with insulin response.
  • U.S. Patent No. 5,330,908 issued to G.F. Spaulding, on July 19, 1994, relates to a rigid gas permeable horizontally rotating bioreactor with increased 5 surface area for gas exchange.
  • the bioreactor is rotated at a rotational rate adequate to suspend cells in the cell culture media. Cells are only allowed to sediment to the bottom during feeding, then are suspended after feeding.
  • the Spaulding invention contemplates cellular aggregation during cell suspension at typically 20 - 40 revolutions per minute. Spaulding teaches against sedimentation in a horizontally rotating bioreactor.
  • the present invention discloses cellular sedimentation to aggregate cells, and rotation rates form >0 to 10 revolutions per minute.
  • U.S. Patent No. 5,253,131 issued to D.A. Wolf et . al . , on October 6, 1992, relates to having the surface area for oxygenation increased by use of a larger gas permeable membrane disposed over a screen and fixed to the rigid walls.
  • the bioreactor is rotated at a rotational rate adequate to suspend cells in the cell culture media. Cells are only allowed to sediment to the bottom during feeding, then are suspended after feeding.
  • the Wolf invention contemplates cellular aggregation during cell suspension at typically 20 -4- revolutions per minute.
  • Wolf et al. teaches against sedimentation in a horizontally rotating bioreactor.
  • the present invention discloses cellular sedimentation to aggregate cells, and rotation rates from >0 to 10 revolutions per minute.
  • Mizutani et al . teaches perfusion through a series of tubes and concentric cylinders. Mizutani, however, does not disclose facilitated cellular contact for aggregation and tissue formation. In contrast to Mizutani et al .
  • the present invention discloses perfusion by cellular or aggregate sedimentation and through horizontal rotations that move cells from the bottom to the top.
  • the present invention discloses a process for cellular aggregation leading to tissue formation.
  • the shaft has a gas permeable membrane glued to its surface which supplies oxygen to a liquid culture medium containing microcarriers and cells. Oxygenation is accomplished by forcing air through a precision milled and drilled center shaft, wherein the center shaft is partly covered with a gas permeable membrane.
  • the bioreactor is rotated at a rotational rate adequate to suspend cells in the cell culture media. Cells are only allowed to sediment to the bottom during feeding, then are suspended after feeding.
  • the Schwartz invention contemplates cellular aggregation during cell suspension at typically 20 - 40 revolutions per minute.
  • Schwartz et al . teach against sedimentation in a horizontally rotating bioreactor.
  • the present invention discloses cellular sedimentation to aggregate cells, and rotation rates from >0 to 10 revolutions per minute.
  • U.S. Patent No. 5,015,585 issued to J.R. Robinson, on May 14, 1991 discloses a bioreactor construction utilizing a single polymer in a concentric geometric configuration to add durability and reduce complexity.
  • U.S. Patent No. 3,821,087 issued to R.A. Rnazek et al, on June 28, 1974 discloses a cell growth system where cells are grown on membranes in a nutrient medium. Nutrient fluids carrying oxygen flow through the vessel and pass through a membrane to contact the cell culture. The nutrient fluids are driven by an impeller into the culture vessel. Numerous capillaries are used to distribute oxygen and nutrients over a large area to reduce uneven distribution of resources. There is no rotation of the vessel, which is complicated to assemble and disassemble.
  • U.S. Patent No. 4,749,654 issued to D. Karrer et al . , on June 7, 1988, relates to a cell growth system using gas permeable membranes and a waste gas removal system.
  • a stirrer is used for agitation. Oxygen flows in through one side of the membrane and carbon dioxide flows out the other side.
  • a biofilm is in contact with an inner wall and a gas permeable membrane covers the outer wall.
  • An oxygen flow along the outer wall permeates the membrane and ceramic housing to reach biomaterial.
  • Nutrients flow along the inner wall in direct contact with the biofilm. There is no rotation of the vessel.
  • cells are placed in a horizontally rotating bioreactor, instead of being suspended in static culture media (as is taught in the prior art) .
  • cells are introduced into a horizontally rotating bioreactor and allowed to sediment to the bottom.
  • the horizontally rotating bioreactor is only slowly rotated.
  • the invention process differs from the action of a clothes dryer in that the present invention utilizes the media viscosity and slow rotation to buffer the effect of hydrodynamic shearing.
  • the process of aggregate formation as described herein can be further constrained so as to enable the formation of sheets.
  • FIG. 1 is a schematic diagram of a horizontally rotating bioreactor.
  • 1 designates the rotation of the bioreactor about a horizontal axis
  • 2 designates the bioreactor vessel itself
  • 3 refers to cells or cellular aggregates sedimented in the bioreactor
  • 4 illustrates the displacement of cells or cellular aggregates by slow rotation of the bioreactor.
  • processes are disclosed that facilitate rapid aggregation of mammalian cells, provide 3-dimensional perfusion of cells and cellular aggregates, constrain the 3-dimensional environment, and increase the concentration of locally released growth and other factors.
  • slow rotational rates By combining slow rotational rates with modified inner wall surfaces that allow cellular adhesion, sheets of mammalian cells can readily be formed.
  • cells are first placed in a horizontally rotating bioreactor (see, for example, Figure 1) and then allowed to sediment to the bottom. Sedimentation forces the entire cellular mass to the lowest area in the bioreactor. Cells are thus stacked one upon the other, in a multiplicity of layers, thereby facilitating adherence and aggregation. Cell-to-cell contacts are greatly increased. Slow rotation of the bioreactor is maintained to keep the cells and cellular aggregates in motion, further increasing the number of cell-to-cell contacts. Consequently, as the cells migrate, they will eventually find other cells with synergistic adhesive properties, in the form of complementary receptors or extracellular matrix expression, and other cells which are already in the form of aggregates.
  • Enhancement of cell contact becomes especially important for rare sub-populations that are unlikely to come in contact with other nurturing cell types in a static and/or diluted environment, i.e., few cell-to-cell or cell- to-extracellular matrix contacts occur under such conditions.
  • Suspending cells in cell culture media dilutes the number of cells per volume and decreases the potential number of cell-to-cell contacts per unit time. Dilution and reduction in the possible number of cell contacts impedes cellular aggregation, and reduces the likelihood of survival for rare cell sub-populations that require contact.
  • 3-dimensional perfusion of cells and cellular aggregates is accomplished by rotating the bioreactor employed in the practice of the present invention so that bottom cells or aggregates are moved toward the top of the bioreactor. Slow rotation brings cells from the bottom toward the top of the bioreactor without the accompanying hydrodynamic shear associated with suspensional rotation velocities. Once cells reach the top, media viscosity buffers the re- sedimentation and reduces shear. During re-sedimentation, cells or aggregates are 3-dimensionally perfused.
  • Rotational rates are typically set based on the metabolic requirements of the cell of interest and the density of cells. For example, the metabolic rate of cartilage is slow and may be initially started at 0.00001 revolutions per minute. Highly metabolic tumors, with high initial cell seeding densities, may require an initial setting as high as 1 revolution per minute. Rotational rates are adjusted based on the results of monitoring standard environmental parameters, e.g. pH, 0 2 , C0 2 , glucose, and the like.
  • various malignant human tumors in a preferred embodiment for aggregate formation, contain the cell types of choice.
  • the rotational rates are initiated at about 0.1 revolutions per minute.
  • Teflon is the material of choice to reduce cell adherence to the bioreactor wall.
  • normal human primaries isolated by conventional methods as known in the art, contain the cell types of choice. In this instance, rotational rates are initiated at about 0.01 revolutions per minute. Teflon is the material of choice to reduce cell adherence to the bioreactor wall.
  • Cells are constrained to a 3-dimensional environment according to the present invention by allowing cells to sediment and then maintaining the cells under sedimentation conditions until the desired cellular aggregates are formed. Constraining the cells to the 3- dimensional space at the bottom of a horizontally rotating bioreactor increases the number of cell-to-cell contacts by reducing the volume in which the cells are distributed.
  • a constrained 3-dimensional environment provides the advantages of 3-dimensional perfusion and 3-dimensional tissue formation without the dilutional effects and rare cell population losses associated with similar cell seeding densities in larger suspension volumes.
  • Formation of sheets of mammalian cells as contemplated by the present invention is accomplished by slowly rotating cells in a horizontally rotating bioreactor, wherein the inner walls of the bioreactor are comprised of a material that encourages cellular adherence (e.g., coated with specific peptide(s) , lined with a sheet of a biomaterial, comprised of a polymer to which cells are known to adhere, and the like) .
  • a material that encourages cellular adherence e.g., coated with specific peptide(s) , lined with a sheet of a biomaterial, comprised of a polymer to which cells are known to adhere, and the like
  • By allowing the cells to sediment in a horizontally rotated bioreactor there is a high incidence of cell-to-wall contact.
  • new sets of cells come in contact with the wall. Those cells predisposed to wall adherence will adhere to the wall. The process is continuous.
  • Cells predisposed to adhere to the cells that are attached to the wall will form a second layer.
  • a process of selective adherence and layering takes place, resulting in the formation of a sheet of cells around the inner surface of the bioreactor.
  • the layering process is based on cellular predilections for other cells or surfaces, similar to the sorting process that occurs in an embryo.
  • Wall surfaces can be engineered for adhesion to a particular cell type or for a general cell type as is known in the art.
  • Horizontally rotating the bioreactor has the advantage of alleviating overgrowth of unwanted cell types, and ensures that each cell is exposed to a variety of surfaces for adherence. Eventually, most cells will come in contact with a cell or surface with which it will adhere.
  • mammalian cartilage isolated by conventional methods as known in the art contains the cell types of choice and polycarbonate is the material of choice for the inner bioreactor wall.
  • human skin isolated by conventional methods as known in the art contains the cell types of choice and polyglycolic acid biomaterial is the biopolymer of choice for lining the inner bioreactor wall.
  • rabbit cornea isolated by conventional methods as known in the art contains the cell types of choice and silicone is the material of choice for the inner wall of the bioreactor.
  • stepper motor As readily recognized by those of skill in the art, slow horizontal rotation of a bioreactor can be accomplished in a variety of ways. It is presently preferred that a stepper motor is utilized. Other standard motors require specialized gear ratios that allow for slow rotational rates. Gears, however, substantially increase cost, limit rotational velocity ranges, and increase failure rates. Stepper motors are inexpensive, provide the broadest range of rotational velocities, allow for programming of non-linear rotation (e.g., forward, backward, stepped, ramped, constant velocity) , and the like.
  • non-linear rotation e.g., forward, backward, stepped, ramped, constant velocity
  • cells can be isolated by conventional means or purchased commercially, for example from American Tissue Culture Corporation, Rockville MD.
  • the cell culture chamber is sterilized and fresh media and cells are admitted to completely fill the cell culture chamber, leaving essentially zero head space. Air bubbles can substantially increase hydrodynamic shear and are, therefore, preferably purged from the chamber.
  • the cell culture chamber is then horizontally rotated at >0 to 10 revolutions per minute, allowing aggregation and 3- dimensional tissue growth. When the nutrients are depleted, the rotation is stopped. Cell free nutrient depleted media is withdrawn through a port and replaced with fresh media. After replenishment with fresh media, the bioreactor is again slowly rotated.
  • compositions and methods for growth and proliferation of living cells using a novel combination of de-differentiation, followed by cell aggregation and differentiation, culminating in organ or cell proliferation For example, cells from a pancreas can adhere to similar cell types, ultimately forming islets of beta cells.
  • de-differentiation is employed to bring particular cell types back to a less differentiated cellular state in which growth is not blocked.
  • the cells are thereby rendered more amenable to rapid proliferation to obtain very large volumes, as a result of enhanced mass transfer.
  • the aggregates increase in numbers, and the increased aggregates undergo differentiation.
  • de-differentiation of cells is carried out in vessels with either adherent or non-adherent surfaces (e.g., T-flasks, bags, multiwell plates, petri dishes, and the like) .
  • adherent or non-adherent surfaces e.g., T-flasks, bags, multiwell plates, petri dishes, and the like.
  • the de-differentiated cells are reaggregated employing the above-described process utilizing a horizontally rotated bioreactor as described hereinabove for enhanced cell-to- cell contact.
  • the de-differentiated cells or the aggregated cells prepared according to invention methods can be differentiated back into particular cell types suitable for therapeutic transplantation (such as by transferring the de-differentiated cells or the aggregated cells into a moving culture vessel) , or de-differentiated cells or aggregated cells prepared according to invention methods can be directly employed for therapeutic purposes by microencapsulation (e.g., within alginate beads) .
  • Invention methods of de-differentiation allow rapid proliferation to be successfully extended to many types of cells which can not be proliferated in sufficient quantities to support transplantation (e.g., pancreatic islet cells, pancreatic duct cells, pancreatic beta cells, pancreatic acinar cells, hepatocytes, cartilage cells, epithelial cells, as well as other primary cell types) .
  • transplantation e.g., pancreatic islet cells, pancreatic duct cells, pancreatic beta cells, pancreatic acinar cells, hepatocytes, cartilage cells, epithelial cells, as well as other primary cell types
  • Differentiation back into particular cell types suitable for therapeutic transplantation may be accomplished by subjecting the de-differentiated cells to conditions that support aggregation and proliferation.
  • Such aggregating conditions include moving vessels and microencapsulation in alginate beads.
  • the present invention discloses a process for aggregating cells and forming sheets of mammalian cells. It will be appreciated by those of ordinary skill in the art that the process is simple and effective. The invention will now be described in greater detail with reference to the following non-limiting examples.
  • Ham's FIO media can be sterilized and placed into the cell culture chamber.
  • a hypodermic syringe can be used to inject media inoculated with isolated mammalian cells into the chamber.
  • the cell culture chamber can include horizontally rotating bioreactors with solid walls, solid porous walls or gas permeable walls.
  • the cell culture chamber is completely filled with cells and media, with zero head space .
  • Suitable cells contemplated for use herein are commercially available, e.g., Baby Hamster Kidney (BHK) cells which can be obtained from American Tissue Culture Corporation (ATCC, Rockville, Maryland) . After cells are injected into the cell culture chamber, excess bubbles are removed. Cell culture chamber volumes can range from less than 1 ml to several liters. To provide the necessary ambient environment, the cell culture system is placed into a conventional incubator where it rotates and supports the growth of 3-dimensional cellular aggregates.
  • BHK Baby Hamster Kidney
  • Rotation rates begin at 0.01 revolutions per minute, for typical mammalian cells.
  • the rotation rates are adjusted based on the maintenance of one or more physiological parameters, i.e., rates are decreased for slowly metabolizing cells and increased for faster metabolism.
  • Physiological parameters, and the appropriate maintenance level are known by those of ordinary skill in the art and include oxygen, carbon dioxide, pH, glucose, and the like. Rotation rates can range from >0 to 10 revolutions per minute.
  • Microaggregates typically form immediately upon sedimentation, and soon thereafter larger aggregates will form and coalesce.
  • Cellular aggregates can range from 50 97/28252 PC17US97/01732
  • the inner wall is made suitable for cellular adhesion.
  • the wall can be made of a compatible polymer (for example polycarbonate) , coated with a polypeptide or protein (for example fibronectin) , or lined with a biopolymer (for example polyglycolic acid) .
  • a compatible polymer for example polycarbonate
  • a polypeptide or protein for example fibronectin
  • a biopolymer for example polyglycolic acid
  • Islets isolated from a donor by methods known in the art are placed in cell culture media, for example, cell culture media as can be purchased from Sigma, St. Louis, MO.
  • the mixture of cells and media are placed in a cell culture vessel.
  • cell culture vessels can be employed in the practice of the invention, e.g., petri dishes, non-adherent petri dishes, t-flasks, non-adherent t-flasks, cell culture bags comprised of polymeric materials or Teflon or silicone, cell culture vessels with cell adherent matrices disposed to the vessel, static perfused cell culture vessels, and the like.
  • Culture vessels are maintained in standard incubators under standard environmental conditions as are generally known in the art. Islet isolates are tested prior to de-differentiation for insulin response by such methods as perifusion and glucose stimulation, as are well known in art. The responsivity of the islet isolates prior to de-differentiation becomes the baseline. During islet culture under de-differentiating conditions, representative cell samples are collected from the culture vessels .
  • De-differentiation is defined by a substantial reduction in insulin release in response to glucose stimulation. Cells are allowed to continue de- differentiation, along with the constitutive doubling of the cell populations, until sufficient new cell mass is derived which is suitable for transplantation. Culture periods can range from about 10 up to about 150 days to generate suitable quantities, depending on the cell density and viability of the starting material.
  • the de-differentiated cells are removed from the cell culture vessel and placed in a condition which promotes aggregation of the de-differentiated cells.
  • aggregating vessels include petri dishes, non-adherent petri dishes, t- flasks, non-adherent t-flasks, cell culture bags comprised of polymeric materials or Teflon or silicone, alginate, cell culture vessel with cell adherent matrices disposed to the vessel, static perfused cell culture vessels, and the like.
  • Such culture vessels must maintain an oxygen mass transfer rate that supports cell aggregation without causing hypoxic necrosis in the center of the cell aggregates.
  • Methods to augment mass transfer to the core of the cell aggregated include gentle agitation of the vessel so as to not culture the aggregates under static culture conditions, horizontal rotation of the cell culture vessel, and the like.
  • cell aggregates are placed in a cell culture bag with high gas mass transfer characteristics.
  • the culture bag is placed on a rocker in an incubator or disposed to a horizontally rotating motor and placed in an incubator.
  • Representative cell culture samples are withdrawn and tested for insulin release in response to glucose challenge (as previously described and known in the art) .
  • glucose challenge as previously described and known in the art
  • the aggregation and enhanced oxygen mass transfer promotes differentiation. Aggregation in an enhanced mass transfer environment leads to differentiation, resulting in intercellular attachments which augment cell doubling of the differentiated adherent sub-populations.
  • Such sub-populations mature and begin to express the functional phenotype of the donor precursors, and therein are defined by insulin release in response to a glucose challenge (as determined utilizing assays known in the art) .
  • Such proliferated sub-populations also are characterized by their constitutive release of other factors that characterize the donor islet, e.g.; glucagon, so atostatin, and pancreatic peptide.
  • Hapatocytes, chrondrocytes, osteocytes, epithelial cells, endothelial cells, and other normal human primary cells can be derived from human donor explants employing methods which are well known in the art. Such cells can be de-differentiated in accordance with the present invention. Markers for the differentiated phenotype are known in the art, and therefore can be selected to follow the de-differentiation transition. Once de-differentiated and resultant expansion in the cell mass occurs, de-differentiation can be established by the loss of the differentiated phenotype or function. The de-differentiated cells can then be aggregated as previously described. Cellular aggregates are sampled for differentiation as determined by the re-expression of the lost phenotype or function. It will be apparent to those skilled in the art that various changes may be made in the invention without departing from the spirit and scope thereof, and therefore the invention is not limited by that which is disclosed in the drawings and specification but only as indicated in the appended claims.

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Abstract

Procédé nouveau permettant d'agréger des cellules de mammifère et de former des feuilles de cellules de mammifère au moyen d'un bioréacteur à rotation horizontale. Il s'agit d'un procédé en une seule étape plus rentable et plus efficace que les méthodes classiques, permettant cependant une croissance cellulaire tridimensionnelle donnant des agrégats de cellules de plus de 1 cm de dimension. Le procédé décrit est utile, par exemple, au développement des tissus pour la transplantation. Selon un autre aspect de l'invention, on décrit des compositions et des méthodes permettant la croissance et la prolifération des cellules vivantes au moyen d'un enchaînement original de dédifférenciation des cellules, suivie d'agrégation de cellule, suivie de différenciation, pour aboutir à la prolifération du type d'organe ou de cellule original. La dédifférenciation ramène certains types de cellules particuliers à un état cellulaire moins différencié, dans lequel la croissance n'est pas bloquée, ce qui rend les cellules plus sujettes à la prolifération rapide si l'on souhaite obtenir des volumes très importants. Les cellules peuvent être dédifférenciées dans des récipients présentant soit une surface adhérente, soit une surface non adhérente, tels que flacons en T, sacs, plateaux multi-puits et boîtes de Petri. De nombreux types de cellules peuvent être dédifférenciées pour une prolifération rapide, par exemple, cellules d'îlot pancréatique, cellules de conduit pancréatique, cellules bêta pancréatiques, cellules acineuses pancréatiques, hépatocytes, cellules de cartilage (chondrocytes), cellules épithéliales, ainsi que d'autres types de cellules connues dans cette technique.
PCT/US1997/001732 1996-01-31 1997-01-30 Procede permettant d'agreger des cellules et de former des feuilles de tissu de mammifere dans un bioreacteur a rotation horizontale WO1997028252A1 (fr)

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Cited By (5)

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EP1162261A1 (fr) * 2000-06-07 2001-12-12 Japan ,represented by Secretary of Agency of Industrial Science and Technology Monocouche continue d'hépatocytes primaires
WO2007076865A1 (fr) * 2005-12-30 2007-07-12 Drugmode Aps Bioréacteur pour cultures de cellules et de tissus
WO2008155072A1 (fr) * 2007-06-20 2008-12-24 Fraunhofer Gesellschaft zur Förderung der angewandten Forschung e.V. Procédé et dispositif de formation d'agrégats de cellules biologiques
WO2011016423A1 (fr) * 2009-08-02 2011-02-10 学校法人 東京女子医科大学 Feuillet de cellules de langerhans, procédé de production associé, et application associée
EP4015622A1 (fr) 2020-12-17 2022-06-22 abc biopply ag Dispositif d'ensemencement de cellules

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* Cited by examiner, † Cited by third party
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EP1162261A1 (fr) * 2000-06-07 2001-12-12 Japan ,represented by Secretary of Agency of Industrial Science and Technology Monocouche continue d'hépatocytes primaires
WO2007076865A1 (fr) * 2005-12-30 2007-07-12 Drugmode Aps Bioréacteur pour cultures de cellules et de tissus
WO2008155072A1 (fr) * 2007-06-20 2008-12-24 Fraunhofer Gesellschaft zur Förderung der angewandten Forschung e.V. Procédé et dispositif de formation d'agrégats de cellules biologiques
US8304237B2 (en) 2007-06-20 2012-11-06 Fraunhofer-Gesellschaft Zur Foerderung Der Angewandten Forschung E.V. Method and device for forming biologic cell aggregates
WO2011016423A1 (fr) * 2009-08-02 2011-02-10 学校法人 東京女子医科大学 Feuillet de cellules de langerhans, procédé de production associé, et application associée
JP5717253B2 (ja) * 2009-08-02 2015-05-13 学校法人東京女子医科大学 膵島細胞シート、製造方法及びその利用方法
EP4015622A1 (fr) 2020-12-17 2022-06-22 abc biopply ag Dispositif d'ensemencement de cellules
EP4015621A1 (fr) 2020-12-17 2022-06-22 abc biopply ag Dispositif d'ensemencement de cellules

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