Feeder Free Culturing

3.3.1. Routine Culture of hESCs in Feeder-Free Conditions

1. Passage hESCs (see Subheading 3.1.1.) every 4-6 d directly on fibronectin-covered plates (see Subheading 2.3., item 1).

2. Change the medium (see Subheading 2.3., item 2) on a daily basis.

3. Scrape differentiating colonies every five to seven passages.

4. Freeze the cells and thaw as described in Subheadings 3.1.2. and 3.1.3., respectively.

4. Notes

1. The freezing medium should be kept at 4-8°C for no more than 5 d.

2. If the cells are cultured for research purposes only, FBS can be used instead of human serum or serum replacement in order to reduce the costs.

3. All culture media (but those mentioned) should be kept at 4-8°C for no more than 2 wk.

4. Gelatin-coated plates can be prepared in advance and stored in a clean place at room temperature or in a 37°C incubator.

5. If a serum-free culture system is desired, human serum can be replaced with 30% SR.

6. If desirable, bovine fibronectin can be used. In this case it is highly recommended to add 1000 U/mL leukemia inhibitory factor to the culture medium (see Subheading 2.3., item 2).

7. It is recommended to leave the fibronectin in the culture plate and not to collect it before plating the hESCs. Our experience shows that this improves the plating efficiency.

8. We recommend using human recombinant growth factor as medium supplement.

9. Do not fracture the cells into small clumps.

10. Adding the medium in this stage drop by drop is highly important. If the medium is added all at once, the survival rates decrease dramatically.

11. In our experience, the use of Nalgene special freezing boxes increases cell survivability.

12. It is not recommended to leave the vials at -80°C for less than 24 h or more than a few days.

13. Foreskin fibroblasts are also commercially available (ATCC collection, for example); the commercial lines were found suitable for supporting hESC culture (5).

14. Foreskin donated from adults can also be used. The tissue can be kept at 2-8°C for up to 48 h before deriving the line.

15. The crowding of the culture influences fibroblasts. The first passage after the derivation of foreskin fibroblast is done after about 20 d of culture. Well-established lines are split every 5-7 d. If the growth rate is decreased, uniting two culture flasks into one may recover the culture.

References

1 Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., et al. (1998) Embryonic stem cell lines derived from human blastocysts. Science 282, 1145-1147.

2 Richards, M., Fong, C. Y., Chan, W. K., Wong, P. C., and Bongso, A. (2002) Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat. Biotechnol. 20, 933-936.

3 Amit, M., Carpenter, M. K., Inokuma, M. S., et al. (2000) Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev. Biol. 227, 271-278.

4. Amit, M., Margulets, V., Segev, H., et al. (2003). Human feeder layers for human embryonic stem cells. Biol. Reprod. 68, 2150-2156.

5 Hovatta, O., Mikkola, M., Gertow, K., et al. (2003). A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum. Reprod. 18, 1404-1409.

6 Cheng, L., Hammond, H., Ye, Z., Zhan, X., and Dravid, G. (2003). Human adult marrow cells support prolonged expansion of human embryonic stem cells in culture. Stem Cells 21, 131-142.

7. Xu, C., Inokuma, M. S., Denham, J., et al. (2001) Feeder-free growth of undifferentiated human embryonic stem cells. Nat. Biotechnol. 19, 971-974.

8. Amit, M., Shariki, C., Margulets, V., and Itskovitz-Eldor, J. (2004). Feeder and serum free culture system for human embryonic stem cells. Biol. Reprod. 70, 837-845.

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