METHOD FOR CULTURING STEM CELLS

• US 20100197013A1
• Filed: 11/09/2009
• Published: 08/05/2010
• Est. Priority Date: 11/07/2008
• Status: Active Grant

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Mohr, Jeffrey C., Palecek, Sean P., Depablo, Juan J., Zhang, Jianhua, Kamp, Timothy J.
• Granted Patent: US 8,956,867 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/0603 Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies

Abstract

A three-dimensional microwell system that supports long term pluripotent cell culture and formation of homogeneous embryoid bodies (EBs) is described. Microwell-cultured pluripotent cells remain viable and undifferentiated for several weeks in culture and maintain undifferentiated replication when passaged to Matrigel®-coated, tissue culture-treated polystyrene dishes. Microwell-cultured pluripotent cells maintain pluripotency, differentiating to each of the three embryonic germ layers. Pluripotent cell aggregates released from microwells can be passaged for undifferentiated replication or differentiated to monodisperse EBs. The ability to constrain pluripotent cell growth in three dimensions advantageously provides for more efficient, reproducible culture of undifferentiated cells, high-throughput screening, and the ability to direct pluripotent cell differentiation by generating monodisperse EBs of a desired size and shape. Cardiomyocyte-rich EBs are obtained from pluripotent cells cultured in microwells of defined size and shape.

Claims

1. A method for culturing pluripotent stem cells to obtain a population of embryoid bodies, the method comprising the steps of:

harvesting substantially undifferentiated pluripotent stem cells from a dimension-constrained microwell, the cells occupying the microwell in all dimensions; and

differentiating the harvested pluripotent stem cells to form the embryoid bodies.

2. A method as claimed in claim 1, wherein the population of embryoid bodies have a substantially uniform size and shape.

3. A method as claimed in claim 1, wherein each embryoid body comprises a substantially uniform number of cells.

4. A method as claimed in claim 1, wherein the undifferentiated pluripotent stem cells are selected from the group consisting of a pluripotent stem cell colony and an aggregate of cells detached from a pluripotent stem cell colony.

5. A method as claimed in claim 1, wherein the microwell is rectangular and has a lateral dimension between about 50 microns and about 600 microns.

6. A method as claimed in claim 1, wherein the microwell has a depth between about 10 microns and about 1000 microns.

7. A method as claimed in claim 1, wherein the harvesting step comprises shearing the pluripotent stem cells from the microwell.

8. A method as claimed in claim 1, wherein the dimension-constrained microwell has cell-attracting and cell-repulsing portions.

9. An embryoid body wherein at least about 50% of the cells in the embryoid body express at least one cardiac-specific protein.

10. An embryoid body of claim 9 produced by the steps of harvesting an aggregate or colony of substantially undifferentiated pluripotent stem cells from a dimension-constrained microwell having a lateral dimension between about 100 microns and about 300 microns;

and

differentiating the harvested pluripotent stem cells to form the embryoid body.

11. A method of producing an embryoid body, the method comprising the steps of

harvesting an aggregate or colony of substantially undifferentiated pluripotent stem cells from a dimension-constrained microwell having a lateral dimension between about 100 microns and about 300 microns;

and

differentiating the harvested pluripotent stem cells to form the embryoid body,

wherein at least about 50% of the cells in the embryoid body express at least one cardiac-specific protein.

12. An array comprising a substrate having a polymer coating thereupon, the coating defining a microwell on a surface thereof, the microwell having side walls and a bottom and having a cell-attractive portion and a cell-repulsing portion.

13. An array as recited in claim 12, wherein the microwells have rectangular lateral dimensions between about 50 microns and about 600 microns and a depth between about 10 microns and about 1000 microns.

14. An array as recited in claim 12, wherein the cell-attractive portion is coated with a cell-attractive material selected from the group consisting of Matrigel®

, gelatin, laminin, collagen, fibronectin or a combination of the above.

15. An array as recited in claim 12, wherein the cell-repulsive portion is coated with a cell-repulsing alkanethiol material having a terminal moiety selected from the group consisting of poly-ethylene glycol containing three to six ethylene glycol groups and tri-ethylene glycol.

16. A population of undifferentiated human pluripotent stem cell colonies or aggregates having a substantially uniform size and shape.

17. The undifferentiated human pluripotent stem cell colonies as recited in claim 16, wherein the colonies or aggregates have rectangular lateral dimensions between about 50 microns and about 600 microns and depth between about 10 microns and about 1000 microns.

18. A population of pluripotent stem cell-derived embryoid bodies having a substantially uniform size and shape.

19. A method of cryopreserving pluripotent stem cells, comprising the step of:

freezing microwell-cultured, pluripotent stem cells in microwells.

20. A method as recited in claim 19, wherein the microwells are rectangular and have a depth between about 10 microns and about 1000 microns with lateral dimensions between about 50 microns and about 600 microns.

21. A method for culturing pluripotent stem cells, the method comprising the step of

culturing substantially undifferentiated pluripotent stem cells in a dimension-constrained microwell without subculture for at least about three weeks in a medium that does not promote differentiation, wherein greater than about 90% of the pluripotent stem cells remain undifferentiated after about three weeks.