Claims
- 1. A method of generating cells derived from spheroid forming cells comprising culturing spheroid forming cells under controlled cell aggregation conditions that enable spheroid formation in liquid suspension.
- 2. The method of claim 1 wherein the conditions enable spheroid formation in liquid suspension under high cell density.
- 3. The method of claim 1 wherein the cells are kept in liquid suspension by stirring.
- 4. The method of claim 1 wherein controlled cell aggregation conditions means preventing spheroid aggregation.
- 5. The method of claim 4 wherein controlled cell aggregation conditions means permitting cell aggregation sufficient for spheroid formation and preventing aggregation between cells of different spheroids.
- 6. The method of claim 5 wherein the preventing aggregation is done by any one or more of the following means:
(a) preventing physical association between cell aggregation molecules; (b) removing inorganic salts from the cell culture; (c) blocking surface aggregation molecules with specific peptides; (d) using spheroid forming cells that are cell adhesion molecule-null spheroid forming cells; and (e) inhibiting cell adhesion molecule expression and/or activity. (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.
- 7. The method of claim 5 wherein the controlled aggregation is affected by encapsulation of spheroid forming cells.
- 8. The method of claim 7 wherein each capsule contains an initial number of spheroid forming cells sufficient to enable spheroid formation.
- 9. The method of claim 8 wherein one spheroid is formed per capsule.
- 10. The method of claim 8 wherein the initial number of cells is 1.
- 11. The method of claim 7 wherein the capsule initially comprises multiple cells of at least two different cell types.
- 12. The method of claim 11 wherein a spheroid is formed and the spheroid is a chimeric spheroid.
- 13. The method of claim 7 wherein the capsules have a capsule body comprising a matrix that permits passage of desired media constituents through the matrix to the spheroid forming cells and spheroid, but do not permit aggregation with cells outside the capsule.
- 14. The method of 7 wherein the gel microdrop method is used to encapsulate the spheroid forming cells.
- 15. The method of claim 1 wherein the cells are cultured under conditions that promote cell expansion but not cell differentiation.
- 16. The method of claim 1 wherein the cells are cultured under conditions that promote cell differentiation.
- 17. The method of claim 16 wherein the cells are cultured in an environment comprising one or more of the following: growth factors, cytokines, extracellular matrix molecules, drugs, low molecular weight molecules or compounds, retinoic acid, DMSO.
- 18. The method of claim 16 wherein differentiated spheroid forming cells of interest are selected and harvested.
- 19. The method of claim 18 wherein the cells of interest are selected on the basis of cell surface markers.
- 20. The method of claim 18 wherein the differentiated cells of interest are selected from the group consisting of: cardiac myocytes, vascular progenitor cells, neurons, hepatocytes, glial cells, kidney cells, beta cells and derivatives thereof.
- 21. 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); iii. differentiating spheroid forming cell(s) according to the method of claim 1; and iv. isolating and harvesting the specific cell-type based on the reporter gene expression.
- 22. The method for the selection according to claim 21, wherein 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/and (iv).
- 23. The method for the selection according to claim 22. wherein the antibiotic resistance gene is selected from but not limited to 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).
- 24. The method for the selection according to claim 21, wherein the reporter gene is selected from but not limited to 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).
- 25. The method for the selection according to claim 21, wherein the reporter gene is selected from but not limited to 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).
- 26. The method for the selection according to claim 21, wherein the reporter gene is selected from but not limited to 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 cell surface and the cells of interest are isolated from the cultured cells by affinity purification.
- 27. The method for the selection according to claim 21, wherein the cell type specific promoter is selected from but not limited to the group consisting of alpha-cardiac myosin heavy chain, MLC-2v, hANF, SOX2, HIP, PDX1, VE-Cadherin, human VE-Cadherin-2 and the selected cell type of interest is a cardiomyocyte, neural precursor, beta cell, endothelial cell.
- 28. The method of claim 1—wherein the spheroid forming cells are pluripotent cells.
- 29. The method of claim 28 wherein the pluripotent cells are selected from the group consisting of embryonic stem cells, embryonic germ layer cells, early primitive ectoderm-like cells, neural stem cells and adult pluripotent cells, adult mesenchymal stem cells, ductal setm cells, muscle derived stem cells, multiple adult progenitor cells.
- 30. The method of claim 29 wherein the pluripotent cells are embryonic stem cell and the spheroid is an embryoid body.
- 31. Embryonic stem cell derived cell culture obtained using the method of claim 1.
- 32. 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 claim 1 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.
- 33. 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.
- 34. A culture bioreactor for industrial production of cells derived from spheroid forming cells comprising:
c. 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 d. Inhibiting spheroid aggregation.
- 35. 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.
- 36. 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.
- 37. The bioreactor of claim 36 wherein said means for controlling cellular aggregation is means for preventing cellular aggregation.
- 38. The bioreactor of claim 36 which is seeded with at least one spheroid forming cell.
- 39. The bioreactor of claim 36 wherein at least one spheroid forming cell is encapsulated.
Priority Claims (1)
Number |
Date |
Country |
Kind |
2351156 |
Jul 2001 |
CA |
|
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application hereby claims the benefit of priority from Canadian Patent Application No. 2,351,156 filed Jul. 4, 2001 and U.S. Provisional Patent Application No. 60/302,706 filed Jul. 5, 2001, both entitled, “A Bioprocess For The Generation Of Pluripotent Cell Derived Cells And Tissues”. Both of these applications are herein incorporated by reference.
Provisional Applications (1)
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Number |
Date |
Country |
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60302706 |
Jul 2001 |
US |