By Andrew Bell, Cyrus R. Edmonds
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3. 1. Procedure Derivation of human iPSCs is most likely to be successful if attempted with early passage fibroblasts (ideally less than passage 5). Thus, early passage human fibroblasts should be frozen for future iPSC inductions should your first attempts not meet with your expectations. 2). Resuspend cell pellets in 3–4 mL of fibroblast freezing solution (usually we freeze at a concentration of about 2 × 105 cells/mL). 5 mL Nalgene cryogenic vials (#5000-1020). 36 Neely et al. Put cryo-vials into a Nalgene cell culture cryofreezing container for overnight freezing at −80°C.
Skin biopsies should be obtained only with Institutional Review Board approval. Obtain skin biopsies (about 3 mm punch biopsy) from patients using standard sterile methods. 34 Neely et al. Fig. 2. Procurement of human fibroblasts. (a) The tissue of a skin biopsy is transferred into a cell culture dish and maintained in complete DMEM media. Over the next 2 weeks, fibroblasts migrate out of the tissue and proliferate in the dish. This is an image taken 11 days after the biopsy was performed. Fibroblasts have migrated away from the skin tissue (arrow).
And Stokes W. for their contribution. References 1. Coecke S, Balls M, Bowe G, Davis J, Gstraunthaler G, Hartung T, Hay R, Merten OW, Price A, Schechtman L, Stacey G, Stokes W (2005) Guidance on good cell culture practice. Altern Lab Anim 33:261–287 2. Hartung T, Gstraunthaler G, Balls M (2000) Bologna statement on good cell culture practice (GCCP). ALTEX 17:38–39 3. OECD (2004) Organisation for Economic Co-operation and Development series on Principles of Good Laboratory Practice and Compliance Monitoring.