WO1997028252A1

WO1997028252A1 - Process for aggregating cells and forming sheets of mammalian tissue in a horizontally rotating bioreactor

Info

Publication number: WO1997028252A1
Application number: PCT/US1997/001732
Authority: WO
Inventors: Glenn F. Spaulding
Original assignee: Vivorx Pharmaceuticals, Inc.
Priority date: 1996-01-31
Filing date: 1997-01-30
Publication date: 1997-08-07
Also published as: AU1855397A

Abstract

A novel process is disclosed for aggregating mammalian cells and for forming sheets of mammalian cells employing a horizontally rotating bioreactor. The process is a single step process that is more efficient and effective than conventional approaches, yet allows for 3-dimensional cellular growth to achieve cell aggregates over 1 cm in size. The invention process is useful, for example, in developing tissues for transplantation. In accordance with another aspect of the invention, compositions and methods are provided for growth and proliferation of living cells using the novel combination of cell de-differentiation, followed by cell aggregation, followed by differentiation, culminating in proliferation of the original organ or cell type. De-differentiation brings particular cell types back to a less differentiated cellular state in which growth is not blocked, thus making the cells more amenable to rapid proliferation to obtain very large volumes. Cells may be de-differentiated in vessels with either adherent or non-adherent surfaces, including T-flasks, bags, multiwell plates, and petri dishes. Many types of cells may be de-differentiated for rapid proliferation (e.g., pancreatic islet cells, pancreatic duct cells, pancreatic beta cells, pancreatic acinar cells, hepatocytes, cartilage cells (chondrocytes), epithelial cells, as well as other cell types that are known in the art).

Description

Process for Aggregating Cells and Forming Sheets of Mammalian Tissue in a Horizontally Rotating Bioreactor

RELATED APPLICATIONS

This application is a continuation-in-part of United States Serial No. 60/010,879, now pending, the entire contents of which are hereby incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to the growth and proliferation of living human or mammalian cells or cell clusters in a tissue culture system. In a particular aspect, 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. In a further particular aspect, 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. BACKGROUND OF THE INVENTION

There are three general processes known in the art for aggregating tissue and forming 3-dimensional tissue constructs. One general process, static culture, involves seeding a 2-dimensional plane, such as a T-flask or sheet of biomaterial, and allowing the cells to grow and spread over the surface. Such processes are in general use in the art, and the subject of patents (e.g., U.S. Patent No. 5,032,508) . The process is slow and static, i.e., cells remain where they fall. Thus, the incidence of cell-to- cell contact is restricted to a one time event, if two or more cells happen to land in contact with one another. Cell aggregation then proceeds through the long process of cell doubling until one achieves cell confluency at the bottom of the culture vessel. Thus, cell doubling gives rise to additional cell-to-cell contact. Additional cells can later be manually seeded on to the first layer. However, in the latter instance, the process then becomes a multistep process of adding layers.

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. However, once suspended, cell concentrations are substantially diluted. To overcome the dilutional effects, 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. Moreover, 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.

It has therefore been a goal in the art to find a reliable method for extracting the insulin-producing cell clusters (islets) from a healthy pancreas and then implanting these cells into a diabetic patient to effect a cure of the disease. For example, healthy adult human pancreatic islets have been transplanted into diabetic patients to achieve independence from insulin injections. Unfortunately, however, the inadequate supplies of human islets from donors and the complications of graft rejection have necessitated the search for an improved source of islet cells.

Thus, while 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.

Transplantation of individual cells or cellular communities

(including human or porcine pancreatic islets, as well as hepatocytes, keratinocytes, chondrocytes, acinar cells, or chromaffin cells) will depend upon an inexhaustible supply of functional living cells which can be used in experimental models as well as in human therapy.

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.

Proliferation of 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.

Prior art of potential relevance to the present invention includes the following patents and, the references cited therein.

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. In contrast to Spaulding, 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. In contrast to Wolf, the present invention discloses cellular sedimentation to aggregate cells, and rotation rates from >0 to 10 revolutions per minute.

U.S. Patent No. 5,057,428 issued to S. Mizutani et al. on October 15, 1991, relates to a cylindrical bioreactor tank which is rotated about a horizontal axis. There is a cylindrically shaped mesh in the chamber which defines inner and outer chambers. A pipe conveys oxygen from an air pump into the chamber and a flow path is established to flow return pipes which provide for continuous replenishment of spent media. 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 . , however, the present invention discloses perfusion by cellular or aggregate sedimentation and through horizontal rotations that move cells from the bottom to the top. In further contrast to Mizutani et al., the present invention discloses a process for cellular aggregation leading to tissue formation.

U.S. Patent No. 5,026,650 issued to R.P. Schwartz et al . on June 25, 1991, relates to a cylindrically formed cell culture chamber and system for mammalian cell growth which is rotated on a central horizontally disposed oxygenating shaft. 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. In contrast to Schwartz, 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.

U.S. Patent No. 4,948,728 issued to G. Stephanopauous et al . , on August 14, 1990, discloses a porous ceramic material with a plurality of flow passages. 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.

U.S. patent No. 4,962,033 issued to J.M. Serkes, on October 9, 1990, is an example of a cell culture roller bottle which has rigid walls that are not gas permeable.

U.S. Patent No. 4,391,912 issued to K. Yoshida et al., on July 5, 1983, is an example of a cell culture system with hollow fibers that is not rotated. BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention, multistep cell culture processes are avoided, and instead multiple cell-to-cell contacts are continuously facilitated throughout the culture process. Thus, according to the invention, cells are placed in a horizontally rotating bioreactor, instead of being suspended in static culture media (as is taught in the prior art) . According to the invention, cells are introduced into a horizontally rotating bioreactor and allowed to sediment to the bottom. The horizontally rotating bioreactor is only slowly rotated. By slowly rotating the bioreactor the cells are constrained to the bottom of the bioreactor, yet the slow rotation eventually moves cells off the bottom toward the top of the bioreactor. The movement brings the cells from the bottom towards the top in a manner similar to the way in which a clothes dryer brings wet laundry to the top to dry. In contrast, 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.

Bringing the cells slowly toward the top of the bioreactor as contemplated by the present invention enhances 3-dimensional cell perfusion and greatly increases the number of cell-to-cell contacts between different cell types. Eventually, each cell comes into contact with a cell or cellular aggregate that is predisposed to adherence thereto. Hence, aggregation occurs and continues throughout the cell culture process. Aggregates eventually become larger, and aggregate with other aggregates or cells, forming larger aggregates, which then differentiate into tissue. As described, the process maintains a low hydrodynamic shear environment while increasing the number of cell-to-cell contacts. Thus, slow rotation provides the advantages of three dimensional perfusion, increased number of cell contacts, and a 3-dimensional growth environment.

In accordance with another aspect of the present invention, the process of aggregate formation as described herein can be further constrained so as to enable the formation of sheets.

It can be appreciated that the maintenance of a normal metabolic environment, the increased cell-to-cell contact and the 3-dimensional perfusion of cells or cellular aggregates provided by the process of the present invention significantly improves the scientific capability for engineering tissues. Costs and FDA compliance are significantly improved by using disposable cell culture chambers and standard rotating devices. In a preferred aspect, finer control of rotation can be obtained employing stepper motor drive means .

BRIEF DESCRIPTION OF THE FIGURE

Figure 1 is a schematic diagram of a horizontally rotating bioreactor. In the figure, 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, and 4 illustrates the displacement of cells or cellular aggregates by slow rotation of the bioreactor.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, 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. By combining slow rotational rates with modified inner wall surfaces that allow cellular adhesion, sheets of mammalian cells can readily be formed.

In order to facilitate rapid aggregation of mammalian cells according to the invention, 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, as opposed to sedimentation, 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.

In accordance with the present invention, 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, 0₂, C0₂, glucose, and the like.

In a preferred embodiment for aggregate formation, various malignant human tumors, isolated by conventional methods as known in the art, contain the cell types of choice. In this instance, 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.

In another preferred embodiment for aggregate formation, 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.

Slow rotation of the bioreactor employed in the practice of the present invention improves the proximity of cells and increases the concentration of locally expressed growth and other factors. Under sedimentation conditions the cells are either in direct contact or in close proximity to other cells. Growth and other factors released by the cells are in high concentration in the microenvironment of the releasing cell. Thus, under sedimentation conditions, cells in close proximity to the microenvironment of the releasing cell are exposed to high factor concentrations. Predisposed cells then respond by triggering the release of their own factors and by adherence. Eventually, adherence, growth factor release, extracellular matrix expression, and differentiation leads to tissue formation suitable for transplantation. Those of ordinary skill in the art will appreciate that the above- described culture process can begin at relatively low seeding densities, which is especially important for difficult to obtain normal human primary cells useful in transplantation.

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) . By allowing the cells to sediment in a horizontally rotated bioreactor, there is a high incidence of cell-to-wall contact. As the bioreactor rotates, 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. Thus, 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.

In a preferred embodiment for sheet formation, 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.

In another preferred embodiment for sheet formation, 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. In yet another preferred embodiment for sheet formation, 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.

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. Those of ordinary skill in the art can visually determine rotational sequences that can preselect for specific shear sensitive cells (e.g., through increasing or decreasing hydrodynamic shear with abrupt rotational changes) , and for the 3-dimensional formation of aggregates (e.g., by increasing the rotational velocities to obtain spheroids or decreasing the velocities for strings of aggregates) .

For processing mammalian or non-mammalian animal cells, 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.

In accordance with another embodiment of the present invention, there are provided 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.

Thus, in accordance with this aspect of the present invention, 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. As the cells aggregate, the aggregates increase in numbers, and the increased aggregates undergo differentiation.

Further in accordance with the present invention, 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) .

Further in accordance with the present invention, 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) .

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.

In view of the teachings of the prior art, it was completely unexpected that the process of de-differentiation, followed by aggregation of the de-differentiated cells, would result in a proliferated organ construct that regained function.

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. Example 1

As an example of a general process to aggregate cells, 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.

Initially 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

18 microns to 3 cm. As the cells aggregate, they form an extracellular matrix upon which cells adhere, differentiate and become 3-dimensional tissue. Three dimensional aggregates display many of the biochemical and morphological features found in the primary tissue from which the cell isolates were derived.

To form sheets of mammalian tissue, 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) . Various surface preparations and coatings are known by those of ordinary skill in the art. Cellular rotation rates and metabolic monitoring for adjusting rotation rates are as described in the art for cell aggregation processes.

Example 2

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. As readily recognized by those of skill in the art, a wide variety of 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.

Once de-differentiation has been established, the de-differentiated cells are removed from the cell culture vessel and placed in a condition which promotes aggregation of the de-differentiated cells. Examples of 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.

In a presently preferred embodiment of the present invention, 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) . As the de-differentiated cells aggregate, 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.

Example 3

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.

Claims

That which is claimed is :

1. A method for aggregating mammalian cells, said method comprising: introducing a primary cell culture into a horizontally rotating bioreactor, allowing said cells to sediment to the bottom of said bioreactor, and slowly rotating said bioreactor at a rate sufficient to bring cells from the bottom toward the top without significant level of hydrodynamic shear.

2. A method for aggregating mammalian cells, said method comprising: allowing the cells in a primary cell culture in a horizontally rotating bioreactor to sediment to the bottom of said bioreactor, and slowly rotating said bioreactor at a rate sufficient to bring cells from the bottom toward the top without significant level of hydrodynamic shear.

3. A method for aggregating mammalian cells, said method comprising: slowly rotating a horizontally rotatable bioreactor containing a primary cell culture at a rate sufficient to bring cells from the bottom toward the top without significant level of hydrodynamic shear, wherein said cells have been allowed to sediment to the bottom of said bioreactor prior to rotating.

4. A method for creating sheets of mammalian tissue, said method comprising: introducing a primary cell culture into a horizontally rotating bioreactor, wherein said inner walls of said bioreactor are comprised of materials which promote cell adherence thereto, allowing said cells to sediment to the bottom of said bioreactor, and slowly rotating said bioreactor at a rate sufficient to bring cells from the bottom toward the top without significant level of hydrodynamic shear.

5. A method for creating sheets of mammalian tissue, said method comprising: allowing the cells in a primary cell culture to sediment to the bottom of a horizontally rotating bioreactor, wherein the inner walls of said bioreactor are comprised of materials which promote cell adherence thereto, and slowly rotating said bioreactor at a rate sufficient to bring cells from the bottom toward the top without significant level of hydrodynamic shear.

6. A method for creating sheets of mammalian tissue, said method comprising: slowly rotating a horizontally rotating bioreactor containing a primary cell culture at a rate sufficient to bring cells from the bottom toward the top without significant level of hydrodynamic shear, wherein said cells have been allowed to sediment to the bottom of said bioreactor prior to rotating, and wherein the inner walls of said bioreactor are comprised of materials which promote cell adherence thereto.

7. A method for forming 3-dimensional tissue constructs, said method comprising: introducing a primary cell culture into a horizontally rotating bioreactor, allowing said cells to sediment to the bottom of said bioreactor, and slowly rotating said bioreactor at a rate sufficient to raise cells off of the bottom thereof without significant level of hydrodynamic shear, wherein said cells are constrained to the bottom portion of said bioreactor.

8. A method for forming 3-dimensional tissue constructs, said method comprising: allowing the cells in a primary cell culture in a horizontally rotating bioreactor to sediment to the bottom of said bioreactor, and slowly rotating said bioreactor at a rate sufficient to raise cells off of the bottom thereof without significant level of hydrodynamic shear, wherein said cells are constrained to the bottom portion of said bioreactor.

9. A method for forming 3-dimensional tissue constructs, said method comprising: slowly rotating a horizontally rotatable bioreactor containing a primary cell culture at a rate sufficient to raise cells off of the bottom thereof without significant level of hydrodynamic shear, wherein said cells are constrained to the bottom portion of said bioreactor, and wherein said cells have been allowed to sediment to the bottom of said bioreactor prior to rotating.

10. A method for growth and proliferation of differentiated cells, said method comprising: de-differentiating differentiated cells under static conditions, wherein said de-differentiated cells increase in number, and culturing said de-differentiated cells, once increased in number, under cell aggregating culture conditions .

11. A method according to claim 10 wherein said cells are de-differentiated in a vessel with adherent surfaces .

12. A method according to claim 11 wherein said vessel is a T-flask, a bag, a multiwell plate or a petri dish.

13. A method according to claim 10 wherein said cells are de-differentiated in a vessel with non-adherent surfaces .

14. A method according to claim 13 wherein said vessel is a T-flask, a bag, a multiwell plate or a petri dish.

15. A method according to claim 10 wherein de- differentiation brings particular cell types back to a less differentiated cellular state in which growth is not blocked.

16. A method according to claim 10 wherein differentiation back into particular cell types is accomplished by transferring the de-differentiated cells into a horizontally rotating culture vessel.

17. A method according to claim 10 wherein differentiation back into particular cell types is accomplished by microencapsulating said de-differentiated cells within alginate beads.

18. A method according to claim 10 wherein the cells are pancreatic islet cells.

19. A method according to claim 10 wherein the cells are pancreatic duct cells.

20. A method according to claim 10 wherein the cells are pancreatic beta cells.

21. A method according to claim 10 wherein the cells are pancreatic acinar cells.

22. A method according to claim 10 wherein the cells are hepatocytes.

23. A method according to claim 10 wherein the cells are cartilage cells (chondrocytes) .

2 . A method according to claim 10 wherein the cells are epithelial cells.

25. A cellular aggregate produced by the method of claim 3.

26. A tissue construct produced by the method of claim 6.

27. A tissue construct produced by the method of claim 9.