Method for differentiating pluripotent mammalian stem cells into a population of hepatic cells in a microchamber

Patent metadata

Field	Value
Publication number	US-10513685-B2
Application number	US-201113636855-A
Country	US
Kind code	B2
Filing date	Mar 23, 2011
Priority date	Mar 23, 2010
Publication date	Dec 24, 2019
Grant date	Dec 24, 2019

Abstract

Provided is a method that achieves control of embryoid body size and can induce differentiation in a state where the embryoid body size is controlled, by using a cell culture chamber having a plurality of microchambers formed therein. A culture method for causing differentiation of pluripotent mammalian cells uses a cell culture chamber (10) having a plurality of microchambers (11) formed on a culture surface. The cell culture chamber (10) has a culture surface formed of spaces in which the microchambers (11) have a space structure with a height of 10 μm to 500 μm and a bottom area of 100 μm2 to 1 mm2. The culture method for causing differentiation of pluripotent mammalian cells includes culturing pluripotent mammalian cells to obtain a cell population at least partially differentiated into endoderm lineage cells, by using the cell culture chambers (10).

First claim

The invention claimed is:

1. A method for differentiating pluripotent mammalian stem cells into a population of hepatic cells, the method comprising: culturing pluripotent mammalian stem cells selected from the group consisting of mouse embryonic stem cells, human embryonic stem cells, mouse induced pluripotent stem cells and human induced pluripotent stem cells, wherein the culturing is performed on a cell culture chamber comprising a plurality of microchambers formed on a culture surface, the cell culture chamber having the culture surface formed of spaces, wherein a space structure of each microchamber has a height of 10 μm to 500 μm and a bottom area of 100 μm 2 to 0.1 mm 2 ; and wherein the culturing comprises seeding the pluripotent mammalian stem cells that were not maintained on mouse fibroblasts or feeder cells into a culture medium in the cell culture chamber; replacing the culture medium with an embryoid bodies (EB) culture medium to prepare an aggregate of embryoid bodies from the pluripotent mammalian stem cells in the microchambers; and replacing the EB culture medium with culture medium comprising Activin-A to prepare a population of endoderm lineage cells from the aggregate of embryoid bodies in the microchambers; and replacing the culture medium comprising Activin-A with culture medium comprising bFGF and hBMP4 to prepare a population of hepatocyte lineage cells, then further replacing the culture medium comprising bFGF and hBMP4 with culture medium comprising hHGF to prepare a population of hepatic cells.

2. The method of claim 1 , wherein 1 to 3×10 5 pluripotent mammalian stem cells are seeded in at least one of the microchambers to obtain the population of hepatic cells.

3. The method of claim 1 , wherein the culture medium comprising Activin-A further comprises another TGF-β family member, an FGF family member, a PI3-kinase signaling pathway inhibitor, or any mixture thereof.

4. The method of claim 3 , wherein the culture medium comprises the TGF-β family member and wherein the TGF-β family member is selected from Nodal, Activin B, TGF-β, BMP2, BMP4, or any mixture thereof.

5. The method of claim 3 , wherein the culture medium comprises the FGF family member and wherein the FGF family member is selected from b-FGF, FGF-4, FGF-2, or any mixture thereof.

6. The method of claim 3 , wherein the culture medium comprises the PI3-kinase signaling pathway inhibitor and wherein the PI3-kinase signaling pathway inhibitor is selected from LY294002, rapamycin, wortmannin, lithium chloride, Akt inhibitor I, Akt inhibitor II, Akt inhibitor III, NL-71-101, or any mixture thereof.

7. The method of claim 1 , wherein the population of endoderm lineage cells partially expresses SOX17, but does not express Pdx-1.

8. The method of claim 1 , wherein the population of hepatocyte lineage cells partially expresses one of FoxA1 and FoxA2, but does not express Pdx-1.

9. The method of claim 1 , wherein the culturing is performed on a cell culture chamber comprising a plurality of microchambers formed on a culture surface in an atmosphere comprising an oxygen concentration of 4% or less.

10. The method of claim 1 , wherein the cell culture chamber is a resin molding comprising acrylic resin, polylactic acid, polyglycolic acid, styrene resin, acrylic styrene copolymer resin, polycarbonate resin, polyester resin, polyvinyl alcohol resin, ethylene vinyl alcohol copolymer resin, thermoplastic elastomer, vinyl chloride resin, silicon resin, or any mixture thereof.

11. The method of claim 1 , wherein in a portion corresponding to 50% or more of an upper portion of each side wall formed in a height direction of the space structure of the microchambers, an angle of 80° to 90° between the bottom of the microchamber and a side surface of each side wall is formed.

12. The method of claim 1 , wherein a bottom of each microchamber has a major axis that is in a range of 1 to 1.5 times greater than a minor axis of the bottom.

13. The method of claim 1 , further comprising surface treating a region in which the microchambers are formed.

14. The method of claim 13 , wherein the surface treatment comprises coating the region with an inorganic substance.

15. The method of claim 13 , wherein the surface treatment comprises coating the region with an extracellular matrix, selected from collagen or laminin.

16. The method of claim 13 , wherein the surface treatment comprises coating the region with a synthetic material.

17. The method of claim 13 , wherein the surface treatment comprises coating the region by plasma treatment.

18. The method of claim 13 , wherein each microchamber comprises a concave-convex bottom surface, the concave-convex bottom surface having a diameter in a range of 1 nm corresponding to a cell focal adhesion to 20 μm corresponding to a cell.

19. The method of claim 2 , wherein in a portion corresponding to 50% or more of an upper portion of each side wall formed in a height direction of the space structure of the microchambers, an angle of 80° to 90° between the bottom of the microchamber and a side surface of each side wall is formed.

20. The method of claim 2 , wherein a bottom of each microchamber has a major axis that is in a range of 1 to 1.5 times greater than a minor axis of the bottom.

21. The method of claim 2 , further comprising surface treating a region in which the microchambers are formed.

22. A method for culturing pluripotent mammalian stem cells into a population of hepatic cells, the method comprising: seeding the pluripotent mammalian stem cells selected from the group consisting of mouse embryonic stem cells, human embryonic stem cells, mouse induced pluripotent stem cells, and human induced pluripotent stem cells, in a cell culture chamber having a culture surface and a plurality of microchambers formed on the culture surface; forming an aggregate of embryoid bodies from the pluripotent mammalian stem cells in the microchamber; culturing the aggregate of embryoid bodies in a culture medium in the microchamber; wherein the culture medium comprises a TGF-β family, a PI3-kinase signaling pathway inhibitor, or a combination thereof; replacing the culture medium with culture medium including Activin-A to prepare endoderm lineage cells from the aggregate of embryoid bodies in the microchamber; and replacing the culture medium including Activin-A with culture medium including bFGF and hBMP4, then further replacing the culture medium including bFGF and hBMP4 with culture medium including hHGF to prepare a population of hepatic cells; wherein each microchamber has a space structure having a height of 10 μm to 500 μm and a bottom area of 100 μm 2 to 0.1 mm 2 .

23. The method according to claim 22 , wherein the cell culture chamber is composed of a resin molding formed from at least one of polyglycolic acid, acrylic styrene copolymer resin, polyester resin, polyvinyl alcohol resin, ethylene vinyl alcohol copolymer resin, thermoplastic elastomer and vinyl chloride resin.

Assignees

• Itchoda Yoko
• Tazaki Go
• Hosoda Masaya
• Fukuda Motohiro
• Taniguchi Hideki
• Zheng Yun Wen
• Sekine Keisuke
• Corning Inc
• Public Univ Corp Yokohama City Univ

Inventors

• Itchoda Yoko
• Tazaki Go
• Hosoda Masaya
• Fukuda Motohiro
• Taniguchi Hideki
• Zheng Yun-Wen
• Sekine Keisuke

Classifications

• C12N5/00

  Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor; (plant reproduction by tissue culture techniques A01H4/00) · CPC title

• C12N2502/28

  Vascular endothelial cells · CPC title

• C12N5/0056

  Xeno-free medium · CPC title

• C12N5/0068

  General culture methods using substrates (for specific animal cell type C12N5/06) · CPC title

• C12N2535/10

  Patterned coating · CPC title