Methods for culturing undifferentiated cells using sustained release compositions

• US 8,481,308 B2
• Filed: 07/01/2011
• Issued: 07/09/2013
• Est. Priority Date: 07/01/2010
• Status: Active Grant

• Current Assignee: Regenerative Research Foundation
• Original Assignee: Regenerative Research Foundation
• Inventors: Stern, Jeffrey, Stern, Sally Temple
• Primary Examiner(s): Carlson, Karen Cochrane
• Assistant Examiner(s): MOSS, NATALIE M
• Application Number:
  US13/175,667
• Publication Number:
  US 20120003736A1
• Time in Patent Office: 739 Days
• Field of Search: 435/325, 435/384, 435/405
• US Class Current: 435/325
• CPC Class Codes: C12N 2500/16 Magnesium; Mg chelators
• CPC Class Codes: C12N 2500/34 Sugars
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/602 Sox-2
• CPC Class Codes: C12N 2501/603 Oct-3/4
• CPC Class Codes: C12N 2501/604 Klf-4
• CPC Class Codes: C12N 2501/606 c-Myc
• CPC Class Codes: C12N 2506/1307 from adult fibroblasts
• CPC Class Codes: C12N 2531/00 Microcarriers
• CPC Class Codes: C12N 2533/40 Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0607 Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0621 Eye cells, e.g. cornea, iris pigmented cells photoreceptors C12N5/062
• CPC Class Codes: C12N 5/0623 Stem cells
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

Methods for culturing undifferentiated mammalian cells, such as stem and progenitor cells, are provided. The methods involve incubating the cell in the presence of a sustained release composition containing at least one growth factor, wherein the sustained release composition continuously releases the growth factor(s), and wherein the presence of the sustained level of growth factor maintains the cell in an undifferentiated state.

Claims

1. A method for maintaining a culture of mammalian stem or progenitor cells in an undifferentiated state, which comprises:

(a) adding to the cell culture a sustained release composition comprising at least one releasable growth factor that maintains the cells in an undifferentiated state, and

(b) repeating the step of adding the sustained release composition to maintain the cells in an undifferentiated state.

2. The method of claim 1, wherein the sustained release composition is added to the cell culture at least once a week.

3. The method of claim 2, wherein the sustained release composition is added to the cell culture not more often than every other day.

4. The method of claim 2, wherein the sustained release composition is added to the cell culture not more often than every three days.

5. The method of claim 1, wherein the growth factor is fibroblast growth factor 2 (FGF2).

6. The method of claim 1, wherein the sustained release composition comprises poly(DL-lactide-co-glycolide) (PLGA) microspheres.

7. The method of claim 1, wherein the step of adding the sustained release composition is repeated to maintain the cells in an undifferentiated state for at least about two weeks.

8. The method of claim 1, wherein the cells comprise one or more cells selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, neural stem cells, retinal pigment epithelial stem cells, hematopoietic stem cells, mesenchymal stem cells, cancer stem cells and epiblast stem cells.

9. The method of claim 8, wherein the cells comprise human embryonic stem cells.

10. The method of claim 8, wherein the cells comprise neural progenitor cells.

11. The method of claim 1, wherein the sustained release composition further comprises one or more additional growth factors.

12. The method of claim 11, wherein the one or more additional growth factors include one or more growth factors selected from the group consisting of epidermal growth factor (EGF), platelet-derived growth factor (PDGF), sonic hedgehog (Shh), leukemia inhibitor factor (LIF) and a Wnt protein.

13. A method for reducing the number of cell culture feedings required to maintain stem or progenitor cells in an undifferentiated state, which comprises:

(a) adding to the cell culture a sustained release composition comprising at least one releasable growth factor that maintains the cells in an undifferentiated state,

wherein the sustained release composition releases the growth factor into the cell culture such that a concentration of the growth factor that is about 80-100% of the starting concentration is maintained for a period of at least three days; and

(b) repeating the step of adding the sustained release composition at spaced apart time intervals to maintain the concentration of the growth factor.

14. The method of claim 13, wherein the sustained release composition is added to the cell culture at least once a week.

15. The method of claim 13, wherein the sustained release composition is added to the cell culture not more often than every other day.

16. The method of claim 13, wherein the sustained release composition is added to the cell culture not more often than every three days.

17. The method of claim 13, wherein the growth factor is fibroblast growth factor 2 (FGF2).

18. A method for culturing mammalian stem or progenitor cells with higher OCT4, Nanog or Nestin expression, which comprises:

(a) adding, to a culture of mammalian stem or progenitor cells, a sustained release composition comprising at least one growth factor that is released over a period of time and maintains a higher expression level of OCT4, Nanog or Nestin compared to when the sustained release composition is not added, and

(b) repeating the step of adding the sustained release composition such that the higher expression level is maintained.

19. The method of claim 18, wherein the sustained release composition is added to the cell culture at least once a week.

20. The method of claim 18, wherein the sustained release composition is added to the cell culture not more often than every other day.

21. The method of claim 18, wherein the sustained release composition is added to the cell culture not more often than every three days.

22. The method of claim 18, wherein the step of adding the sustained release composition is repeated such that the high expression level of OCT4, Nanog or Nestin is maintained for at least about two weeks.

23. The method of claim 18, wherein the growth factor additionally maintains a lower expression level of SOX17, Brachyury or SSEA-1 compared to when the sustained release composition is not added.

24. The method of claim 23, wherein the step of adding the sustained release composition is repeated such that the low expression level of SOX17, Brachyury or SSEA-1 is maintained for at least about two weeks.

25. The method of claim 18, wherein the growth factor is fibroblast growth factor 2 (FGF2).