The present inventors identified aggregation of embryonic stem cells and embryoid bodies (EBs) as the cause of the difficulty in generating large numbers of the embryonic stem cells (ES) cell-derived tissues. To counter this, the invention provides a novel bioprocess where aggregation of spheroid forming cells, such as embryonic stem cells and spheroids, such as EBs is controlled, such as by encapsulation of within a matrix. As a result, EBs can be generated with high efficiency and cultured in high cell density, well-mixed systems. Well-mixed conditions facilitate measurement and control of the bulk media conditions and allow for the use of scalable bioreactor systems for clinical production of tissue. Therefore, the invention enables generation of ES cell-derived tissue on a clinical scale. The invention is also applicable to any spheroid-forming cells and other types of pluripotent cells.

Description of the Invention

SUMMARY OF THE INVENTION

This patent application is based upon a novel technology that overcomes one or more of the limitations of the prior art: a) the ability to measure and control the extracellular environment, b) scalability, and c) cell density and allows for the scalable generation of cells and.or tissue derived from spheroid-forming cell, preferably pluripotent, preferably ES cells.

The present inventors have determined that ES cells cultured in stirred bioreactors fail to generate EBs in an efficient manner because the cells aggregate forming large cell clumps that results in poor cell proliferation and incomplete cell differentiation. As such, in one aspect, the present invention provides a process to control cellular and spheroid aggregation of spheroid forming cells, such as ES cells or neuronal stem cells, to enable the use of a stirred tank reactor or other scalable, well-mixed and controlled bioreactor systems such as a fluidized bed reactor. The invention can also have benefits in other systems, especially liquid systems such as LSC.

In one embodiment, the invention provides a method of culturing differentiating pluripotent cells, preferably ES cells, in a bioreactor system by controlling cellular aggregation. Although a preferred embodiment of the invention is with ES cells, the invention is not limited to ES cell derived cells and tissues. It is equally applicable to any spheroid forming cell type, preferably pluripotent cell type that can differentiate as a cell cluster or aggregate, or form spheroid bodies, such as adult pluripotent cells (Schwartz, Reyes, et al. 2002; Clarke, Johansson et al. 2000) embryonic germ cells (EG cells) (reviewed in Thomson and Odorico, 2000), early primitive ectoderm-like cells (EPL) (Pelton and Sharma, 2002), and neuronal stem cells. As such the embodiments of the invention as outlined below in relation to embryonic stem cells also apply to other pluripotent cells where controlling aggregation of the cells at various points of expansion and or differentiation can enhance the efficiency of said expansion, spheroid formation or differentiation and are intended to be encompassed within the scope of the present invention.

In one embodiment, the invention provides a method of generating pluripotent cell derived cells comprising culturing pluripotent cells, such as ES cells, EG cells, EPL cells, and adult pluripotent cells (Schwartz, Reyes, et al. 2002), while controlling cellular and spheroid aggregation. In a preferred embodiment, the method is conducted under conditions that permit cell differentiation and proliferation. In another embodiment, the pluripotent cells are encapsulated to prevent aggregation with neighboring cells, spheroids or cells contained in separate capsules. In another embodiment the aggregation between specific cell types is controlled (to enable aggregation necessary for spheroid formation, where applicable) and the aggregation between spheroids of these cells is prevented. In yet another embodiment the kinetics of aggregation is controlled.

In one embodiment, the invention provides a method of generating embryonic stem cell derived cells comprising culturing embryonic stem cells under conditions that enable embryoid body formation and embryonic stem cell differentiation while controlling cell aggregation.

In a preferred embodiment, the embryonic stem cells are encapsulated to control cell aggregation such that each capsule will transiently contain and give rise to one embryoid body. In another embodiment, each capsule contains a predetermined number of ES cells that are permitted to aggregate and together give rise to a single EB. Preferably, the undifferentiated embryonic stem cells are first encapsulated to prevent aggregation between ES cells contained within separate capsules. Encapsulated embryonic stem cells are then cultured under conditions that enable cell proliferation and differentiation, leading to embryoid body formation. In a more preferred embodiment, emulsification of cells with agar in inert silicon oil is used to encapsulate the embryonic stem cells. In a most preferred embodiment, this emulsification results in the generation of agarose microcapsules containing embryonic stem cells. In yet another embodiment the process of encapsulation allows for the control of pluripotent cells aggregation by interfering with cell surface receptor binding. In a preferred embodiment, the process of encapsulation allows for the control of ES cell aggregation by interfering with E-cadherin mediated ES cell aggregation.

In another embodiment, the invention provides a method wherein the embryonic stem cells are cultured under conditions wherein embryoid bodies and/or differentiated embyronic stem cells can be formed. For example, some ES cell lines require aggregation of multiple ES cells to enable EB formation. Following these permissive steps, cells are transferred to conditions that prevent aggregation of embryoid bodies. In one embodiment, the method further comprises a step wherein the differentiated embryonic stem cells and/or tissues of interest are selected and harvested. In a preferred embodiment, the cells and/or tissues of interest are cardiomyocytes or cardiac tissue or hematopoietic cells or tissue or endothelial cells or tissues.

In another embodiment, the invention provides an embryonic stem cell derived cell culture obtained using the method of the invention. In a further embodiment, the invention provides embryonic stem cell derived cells and tissues obtained using the method of the invention.

In yet another embodiment the invention provides a method to identify factors, e.g. any variant, condition, substance, that affect embryonic stem cell differentiation and/or embryoid body formation, said method comprising culturing the embryonic stem cells while preventing embryoid body and embryonic stem cell aggregation, in the presence of the factor to be tested and then detecting the effect of the variant on embryonic stem cell differentiation and embryoid body formation. In a further embodiment, of said method, the effect on embryonic stem cell differentiation and embryoid body formation is compared to a control culture, preferably a negative control wherein embryonic stem cells are cultured under the same conditions except in the absence of the factor to be tested. The invention also provides a method of identifying factors that affect cell proliferation or differentiation in any cell culture by encapsulating said cells. The invention also encompassed a method of preventing cell aggegation in any cell type by encapsulation.

Other features and advantages of the present invention will become apparent from the following detailed description. For instance where reference is made to embryoid bodies or embryonic stem cells, the invention can also be applied to spheroid forming cells and spheroids. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a new method for generating spheroid-forming cell and/or pluripotent cell derived cells and tissues, such as for example cardiomyocytes, hematopoietic cells, endothelial cells, insulin producing beta cells, neuronal cells, glial cells, kidney cells, hepatocytes, vascular progenitor cells or derivatives (such as hematopoietic stem cells or endothelial cells), to name a few.

The present inventors have shown that as cell density increases, cell aggregation occurs more readily, resulting in lower cell expansion and impaired cell differentiation (also see Dang, et al. 2002). The present inventors have also hereby shown that cellular and spheroid aggregation is indeed the cause of the inability to culture spheroid-forming cells in a high density culture, such as in a liquid suspension culture (e.g. a stirred liquid bioreactor system). As such the inventors have shown that controlling cellular and/or spheroid aggregation can increase spheroid forming efficiency.

Thus in one embodiment, the invention provides a method of generating cells and/or tissues derived from spheroid forming cells, preferably pluripotent cells comprising culturing spheroid forming cells under controlled cell aggregation conditions that enable spheroid formation in liquid suspension.

In another embodiment of the method of the invention, the spheroid forming cells can be cultured under high density conditions. Although the invention will work at low cell density (even seeding the initial culture with 1 cell), it can also work with an initial cell density of about 10.sup.2 to about 10.sup.6 cells/ml, preferably about 10.sup.3 to about 10.sup.6 cells/ml, more preferably from about 10.sup.4 to about 10.sup.6 cells/ml. These cells can then be cultured under controlled aggregation conditions to yield to form spheroids. As can one start with a culture with more cells, the resulting number of spheroids formed will be greater than in cultures commenced at a lower cell density. In one embodiment, each spheroid may be cultured to about 30,000 to 35,000 cells per spheroid. It should be noted that this is but one embodiment, and the invention is not limited to such numbers. This the present method permits generation of cells at an industrial scale.

In one embodiment the invention provides a method for generating cells and./or tissues from spheroid forming and/or pluripotent cell comprising culturing the cells under conditions that control cell aggregation. In a preferred embodiment, the invention provides a method for efficient formation of spheroids or EBs and the culture of spheroid or EBs in suspension at higher cell densities. This is done by controlling cell aggregation. In one embodiment, the invention provides a scalable method for generating spheroid forming cell or pluripotent cell derived cells and tissues, such as spheroid or ES derived cells and tissues for various uses, such as for transplantation.

In another embodiment, the invention provides a method of culturing spheroid-forming cells, such as pluripotent cells in a bioreactor system where the culture conditions can be measured and controlled. In yet another embodiment, the invention provides a scalable and controllable culture of spheroid-forming cells, such as pluripotent cells by allowing them to be cultured in stirred bioreactors, such as stirred liquid bioreactors. This is done by controlling cell aggregation. This improves the efficiency of generation of differentiated cells.

In one embodiment, the bioreactor or culture system used in the invention is one that keeps the cells and/or spheroids in liquid suspension. In another embodiment, this culture system keeps the cells and/or spheroids in liquid suspension by stirring, but other methods or means can be used, such as by agitation of the system, use of various media or other environmental conditions.

The term "controlling cell aggregation" refers to the process of effectively modulating the extent and kinetics of cell aggregation such that an endpoint, not effectively attainable without such control, is achieved. As used herein in relation to cell and/or spheroid or EB aggregation, "controlling cell aggregation" means that cell aggregation can be permitted or prevented as desired. For example, in one aspect of the invention, aggregation of spheroid forming cells, such as ES cells sufficient to induce spheroid or EB formation is permitted, and aggregation between cells within a spheroid or EB is permitted. However aggregation between spheroid-forming cells or ES cells beyond that determined to induce spheroid or EB formation is prevented. Aggregation between separate EBs is also prevented.

Further, benefits of the invention (i.e. increasing efficiency of ES or pluripotent cell derived cells or tissue) can be obtained with any degree of prevention of inhibition of cellular aggregation. Controlling aggregation may, in some application consist of preventing aggregation where the term "preventing" includes any inhibition of (i.e. that may prevent some, but not necessarily all) of spheroid forming cell or pluripotent cell and/or spheroid or EB aggregation in a particular culture system. It would be appreciated by those skilled in the art upon reading the description herein that any prevention of spheroid, EB and/or spheroid-forming cell or pluripotent cell aggregation would improve cell yields in the culture system and/or cellular differentiation.

In one embodimeint, "preventing, spheroid and/or ES cell and/or spheroid or EB aggregation" means that after spheroid-forming cells or ES cells are cultured to form spheroids or embryoid bodies, there may be cellular aggregation between cells within a spheroid or embryoid body but not between cells of different spheroids or embryoid bodies or between spheroid or embryoid body themselves.

The term "embryonic stem cell derived cells and tissues" as used herein refers to any cells or tissues that are derived from embryonic stem cells. The term "pluripotent cell derived cells and tissues" as used herein refers to any cells or tissues that are derived from pluripotent cells or tissues.

Thus in one embodiment, the invention provides a method of culturing spheroid-forming cells or ES cells comprising culturing the cells under conditions that promote spheroid or EB formation, including controlled aggregation of spheroid-forming or ES cells, then preventing cell and spheroid or EB aggregation.

In another embodiment, the invention provide a method of controlling the production of spheroids from spheroid-forming cells by controlling the aggegation of such cells. In one embodiment, the spheroid forming cells are pluripotent cells such as neural stem cells which form neurospheres, or are embryonic stem cell which form EBs.

In a preferred embodiment the method of the invention does not adversely affect spheroid or EB formation, cell growth, or cell differentiation. Thus in another preferred embodiment, the method does not adversely effect the delivery of nutrients, oxygen, or cytokines. In yet another embodiment, the cells can further comprise culturing the ES cells under conditions that promote differentiation of the cells and spheroid or EB formation. In another embodiment, the cells can be cultured under conditions that does not promote cell differentiation or further differentiation. Thus the method of the invention, by adujusting culture conditions and generate differentiated cells or be used to expand the existing cell culture initially or at any point of the method. For instance, in one embodiment culture conditions can change be changed during the method to control differentiation conditions.

Examples of culturing conditions that promote spheroid forming cell or ES cell differentiation and spheroid or EB formation include but are not limited to conditions described in Keller et al. 1995 or O'Shea et al. 1999 or those outlined in Tables 10 or 11 (see Original Patent) and references noted therein to enhance generation of particular cell types. The spheroid-forming cell and other cells used in the invention can be genetically modified cells. The genetic modification can be for a variety of purposes including, to enhance cell selection by conferring particular identifying characteristic to the desired cell, to generate cells for gene or cell therapy. Other applications would be apparent to those skilled in the art.

Particular cell types can be selected for and/or harvested using a variety of methods, including cell surface receptors or other labeling, tagging or monitoring or selection methods known in the art.

Examples of conditions that can be used in the present invention for culturing spheroid-forming cells, such as ES cells to control spheroid or EB and/or cell aggregation include but are not limited to: (a) Methods for preventing physical association between cell aggregation molecules (i.e. encapsulation, such as described herein or Sefton et al. 1997). (b) Methods that use reduced inorganic salt concentration as described in Boraston et al. 1992 and Ko et al. 2001. (c) Methods that block surface aggregation molecules with specific peptides or other molecules, such as described in Burdsal et al. 1993 (d) Using genetically modified spheroid forming or ES cells such as E-cadherin-null ES cells such as described in Larue et al. 1996 or methods that inhibit E-cadherin expression or antagonize or inhibit E-cadherin activity. (e) Addition of agents that prevent cell aggregation such as dextran sulfate or other sulfated polyanions as described in Dee et al. 1997 (f) Methods that prevent cell aggregation by physically separating one ore more spheroid forming cells to enable spheroid formation in distinct compartments such as described in the single cell liquid suspension culture (scLSC) method in this patent without limitation to a single cell.

In one embodiment of the invention, the above methods can be used alone or in combination, ie. more than one method can be used to prevent spheroid, EB and/or spheroid-forming or ES cell and/or non-clonal spheroid-forming or non-clonal ES cell aggregation.

The selected spheroid forming or ES cell culturing strategy preferably does not affect the development and differentiation of pluripotent cells, preferably ES cells, or EBs in any adverse way.

In a preferred method of the invention, the spheroid-forming or ES cells are encapsulated. In one embodiment, the average number of cells encapsulated can be predetermined or conditions can be adjusted to get a desired number of cells per capsule. For instance, in one embodiment, the number of cells per capsule is 1-10, 1-5, 5, or 1, but the invention is not limited by such examples. The spheroid-forming or ES cell can be encapsulated using methods such as described in Weaver et al. 1988 or Turcanu et al. 2001 and then cultured under conditions that promote sperhoid or EB formation and cellular differentiation, such as described in Keller et al, 1995 or O'Shea et al. 1999.

A variety of methods can be used to encapsulate cells. Although, the Examples described herein use the Gel Microdrop Technique (GMD), patented and owned by One Cell Systems, Boston Mass. The GMD technique was designed to encapsulate cells in agarose gel for the purpose of isolating individual cells with specific protein secretion profiles; however, the present invention adapted the process for encapsulation of spehroid-forming or ES cells and controlling cell and spheroid or EB aggregation. It is important to note that the microencapsulation of spheroid-forming cells such as ES cells for the purposes of the present invention does not depend upon the GMD technique as any method of encapsulation could be used.

In one embodiment the size of the capsule can be controlled, for instance by tempurature, impeller spped or encapsulation time. Preferably the size of the capsule is such that maintains the spheroid-forming cells and/or sperhoids in the capsule until aggregation cell surface markers are no longer expressed. For instance, for ES cells, EBs that emerge after about 4 days no longer express E-cadherin and thus do not have a tendency to aggregate.

The matrix of the capsule can be composed of a variety of substances. Inone embodiment it is an agarose capsule. In another embodiment, the matrix should be biocompatible in that it should not have a detrimental effect on cell proliferation and/differention as desired. In yet another embodiment, the matrix should such that permits the passage or exposure of desired media components to the spheroid-forming cells or spheroid. This can be done by keeping certain components in the capsule and others out or by permitting the passage of certain components through the capsule matrix.

The conditions under which the ES cells are cultured for differentiation will depend on the type of ES cell derived cells and tissues desired. For instance, to obtain hematopoietic stem cells one would preferably culture the ES cells under certain conditions, such as described in Wiles and Johansson 1999; Rathjen et al. 1998; or Keller et al, 1993. On the other hand, if one wishes to obtain cardiomyocytes, the cells would be cultured under specific conditions, such as in the presence of specific cytokines, i.e. TGF-.beta. (Dickson et al. 1993), or using a selectable genetic marker such as described for cardiac myocytes in Klug et al (1996). In vitro, ES cells have been shown to differentiate into a variety of cell types and tissues, including beta cells, cardiomyocytes, hepatic cells, kidney cells, neuronal cells, and hematopoietic cells. Various cell types and culturing conditions are described in Zandstra and Nagy, 2001 for general approaches; Wang et al, 1992 for endothelial cells; Rohwedel et al., 1994, Fleishman et al, 1998 and Mummery et al. 2002 for cardiac and muscle cells; Bain et al, 1995, Tropepe et al, 1999 and Reubinoff et al. 2001 for neuronal cells; Palacios et al, 1995 and Kyba et al., 2002 for hematopoietic stem cells; Choi et al, 1998 and Levenberg et al. 2002 for hematopoietic and endothelial cells. Assady et al 2001 for insulin-producing cells. Since the differentiation of the ES cells results in a complex mixture of all possible cell types an efficient enrichment protocol would be necessary to obtain a homogeneous population of specific cell types or tissues to be utilized for various purposes including the generation of surrogate cells for human therapies or for drug screen testing. This enrichment could be done by the introduction of a specific construct into ES cells, comprising a cell or tissue type specific promoter controlling the expression of a marker gene. The resulting transgenic ES cell(s) can be expanded, differentiated and selected to generate the desired cell type of interest using techniques known in the art, for instance as described in Klug et al (1996) for cardiac cells (WO 95/14079, May 26, 1995; U.S. Pat. Nos. 5,733,727 and 6,015,671) or Soria et al (2000) or Soria et al (2001). The method is widely applicable to any cell type for which the tissue specific promoter is known. Table 9 (see Original Patent) provides some examples of promoters useful for the selection of specific cell lineages without limiting the number of useful promoters to that table.

The initial undifferentiated spheroid-forming or ES cells used in the method can be obtained by methods known in the art. For instance one can use ES cells obtained directly from the inner cell mass of the embryo, or one can use ES cells that were cultured, i.e. expanded under conditions that did not promote differentiation, such as described in Klug et al. 1996.

The encapsulation material chosen should prevent aggregation of cells contained in different capsules. Suitable material would include but is not limited to agarose, alginate, polymers such as poly HEMMA (Valbacka et al (2001) and others or matrices described in Weaver et al. (1988) and Sefton et al (1977).

The material selected for encapsulation should permit adequate delivery of any desired nutrients, cell culture media, growth factors, cytokines, or any other factors to promote desired ES cell growth, differentiation and EB formation, e.g., through functionalization of the encapsulation matrix, or by diffusion through the pores. The material selected may depend on the factors that are to be delivered. Example of matrices that could be used are agarose, alginate, and polymers that support cell growth. Examples of suitable matrices are described in Weaver et al. (1988) and Sefton et al (1977). However, a person skilled in the art would be familiar with other suitable matrices. In another embodiment of the invention, the size of the capsule can be controlled, which can determine the time at which the EB emerges from this capsule. In one approach, varying the rate of emulsification during the encapsulation process can control the size of the microcapsule. The different capsule sizes produced under each of the conditions tested are shown in Table 3 (see Original Patent). Other factors that may control the size of the microcapsules include the addition of surfactants to the encapsulation solution, the ratio of encapsulation gel to liquid, and others factors that would be familiar to those practiced in the art upon reading this description. The efficiency of cell production could be affected by the timing of the emerging EB, as once the EB emerges from the capsule, that particular method of prevention of EB aggregation would be gone. The effect of any subsequent EB aggregation would depend on the stage of development of the emerged EB and any limitations in size (numbers of cells) of an EB. The size of the capsule could also effect efficiency of delivery of various growth or differentiation factors or any other factors or nutrients to the cells. For instance larger capsules would have greater surface in which such factors could pass through, or provide a substrate onto which one could functionalize bioactive molecules (Irvine et al, 1998). This could also be accomplished by controlling the porosity of the encapsulation matrix. In one embodiment, the method of the invention enables the culture of differentiating ES cells in conventional commercially available bioreactor systems that include the use of standard bioprocess equipment (probes, filters, etc).

In another embodiment the method of the invention enables culture of differentiating spheroid-forming or ES cells in stirred 3-dimensional reactors with scalable volume.

In yet another embodiment the invention allows for measurement and control of the culture environment by enabling the use of stirred or other well-mixed bioreactors.

As a result of being able to better control the ES cell culture environment, the method of the invention enables one to better study various conditions to obtain particular differentiated ES cells and tissue (i.e. hematopoietic cells, cardiomyocytes, nerve cells, beta cells, hepatic cells, kidney cells, any other cell types differentiated or not, known to develop from embryonic stem cells) and to better optimize these conditions. As such, the method of the invention can be used to derive a cell culture with a higher density of desired differentiated cells. The ability to do this may determine whether or not the process is economically viable and therefore is critical to the translation of these technologies from the lab scale. The method of the invention can also be used to identify factors (e.g., any variant, such as growth factors, differentiating factors, such as cytokines, or other conditions, such as pH, temperature, oxygen or factor concentration levels) and conditions that effect and preferably optimize spheroid froming cell or ES cell differentiation and spheroid or EB formation. Examples of some factors, especially for studying spheroid forming cell or ES cell differentiation to cardiomyocytes include but are not limited to fibroblast growth factors (FGFs), vascular endothelial growth factor (VEGF), cardiotrophin-1 (CT-1), leukemia inhibitory factor (LIF), endothelin-1(ET-1), stem cell factor (SF), opiod peptide (or supplemental DMSO), bone morphogenic protein (BMP) family members transforming growth factor beta (TGF-beta), retinoic acid (RA). Factors preferably used to study beta-cell differentiation include but are not limited to, glucose levels, nicotinamide, KGF (karatinocyte growth factor), EGF (epithelial growth factor, NGF (neural growth factor) and TGF-beta, the above factors, as well as others typically used on adult hematopoietic stem cells (Zandstra et al 1997) can also be used to elicit the development of hematopoietic and endothelial cells from pluripotent cells. This method would comprise culturing the spheroid forming cells or ES cells under conditions that control cell aggregation in the presence of the particular factor to be tested and then detecting the effect of the variant on cell proliferation and differentiation. Preferably a negative control is used, whereby the spehroid-forming cells or ES cells are cultured in the absence of the factor to be tested.

The methods and products of the invention can also be used to produce cells for drug therapy testing, to identify targets for gene and cell therapy, and in assays for such methods.

ES cells and the ES cell derived cells and tissues can also be used in the treatment of various cancers, leukemias, autoimmune diseases, organ failure, animal or tissue cloning, gene therapy, transgenic animals.

The ES cell-derived cells and tissues obtained from the invention can be used to provide cells and tissues for transplantation. For instance, ES cells can be grown under conditions to produce hematopoietic cells. Such cells could be used in bone marrow transplants, blood transfusions or infusions. ES cell derived cardiomyocytes can be used in tissue engineering, cell/tissue transplantation, gene therapy, and drug discovery. ES cells derived skin tissue can be used for reconstructive surgery, ie. for burn victims, cosmetic surgery to name a few.

Further examples of applications for ES derived cells and tissues can be found in Rathjen et al 1998 or Marshall E. 2000.

It should be noted that although the discussion above describes the invention in terms of ES cells, the same methods and applications and products of the invention would be applicable to pluripotent cells in general or spheroid forming cells other than ES cells.

In a further embodiment, the invention provides a method for the selection of specific cell types from spheroid forming cells comprising the steps of: i. introducing a reporter gene expressing vector into at least one spheroid forming cell whereby a cell-type specific promoter is combined with an reporter gene, such that the reporter gene is expressed under the control of the cell-type specific promoter; ii. culturing the spheroid forming cell(s) comprising the method of claim 1 iii. differentiating spheroid forming cell(s) iv. isolating and harvesting the specific cell-type based on the reporter gene expression In one embodiment, the reporter gene is an antibiotic resistance gene and the cell-type of interest is isolated by the addition of an appropriate antibiotic in step (iii) or (iv).

In yet another embodiment, the method for the selection according to the reporter gene is selected from the group consisting of the Hygro mycin resistence gene (hph), the Zeocin resistence gene (Sh ble), the Puromycin resistance gene (pacA), and the Gentamycin of G418 resistance gene (aph).

In yet another embodiment, in the method for the selection according to claim 23, wherein the reporter gene is selected from the group consisting of luciferase, green fluorescence protein, red fluorescence protein, and yellow fluorescence protein and the cells of interest are selected by fluorencent activated cell sorting (FACS).

In yet another embodiment, in the method for the selection according to the reporter gene is selected from the group consisting of luciferase, green fluorescence protein, red fluorescence protein, yellow fluorescence protein, or a his-, myc-, or flag-tag ligated to a heterologous gene, or any heterologous gene which when expressed is inserted into the cellsurface and the cells of interest is isolated from the cultured cells by affinity purification.

In yet another embodiment, in the method for the the cell type specific promoter is selected from those listed in Table 9 (see Original Patent) for cell types listed therein.

The present invention also includes the Embryonic stem cell derived cell culture obtained using the method of the invention.

In yet another embodiment the invention is directed to a method to identify factors that effect cell proliferation, differentiation and/or spheroid formation, said method comprising culturing the cells as per the method of the invention in the presence of the factor to be tested and then monitoring the effect of the factor on cell proliferation, differentiation and/or spheroid formation.

In another aspect, the invention provides a method of generating cells derived from spheroid forming cells in accordance with claim 1 wherein the spheroid forming cells are cultured under conditions that enable spheroid formation, said conditions comprising culturing said spheroid forming cells in liquid suspension under non-aggregating conditions.

In another aspect the invention provides a culture bioreactor for industrial production of cells derived from spheroid forming cells comprising: a. culturing spheroid forming cells in accordance with the method of claim 1, wherein the cells are cultured in a spheroid forming cell suitable media under conditions that promote spheroid formation; while b. Inhibiting spheroid aggregation.

In another embodiment the invention provides a method to prevent aggregation between cells comprising encapsulating a cell or group of cells and thus preventing aggregation of said cells with cells not within said capsule.

In another embodiment, the invention provides a bioreactor for generating cells from spheroid forming cells comprising a means for controlling conditions suitable for spheroid formation, such conditions comprising a means for controlling cellular aggregation and means for maintaining said cells and generated cells in suspension, such as means for preventing cellular aggregation.

Further the invention also encompassed the use of encapsulating cells, as described herein to control the micro environment of a cell to be cultured. This is not limited to spheroid-forming or ES cells.
 

Claim 1 of 22 Claims

1. A bioprocess for controlling aggregation of embryoid bodies during differentiation, comprising: a) monitoring E-cadherin expression over time in a differentiating embryoid body; b) determining a time when E-cadherin is down-regulated sufficiently in said embryoid body to prevent aggregation of embryoid bodies; c) encapsulating one or more embryonic stem cells expressing E-cadherin in a biodegradable matrix so as to form a cell capsule, wherein the capsule is of a size to enable an embryoid body formed within the capsule to emerge from the capsule after down regulation of E-cadherin; d) introducing the encapsulated cells into a cell culture environment comprising cell culture media, growth factors, and other factors to promote cell growth; e) culturing the encapsulated cells from d) so as to form an embryoid body, said culturing being for a time as determined in b), such that the embryoid body formed within the capsule will emerge from the capsule and will not aggregate with other embryoid bodies upon emergence from the capsule; f) optionally harvesting differentiated cells derived from the embryoid body that emerged from the capsule from the culture in step e).

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