ABSTRACT
Mouse embryonic fibroblasts (MEFs) can be used in co-culture to support generation of induced pluripotent stem cells (iPSCs) and the normal growth and proliferation of human pluripotent stem cells (hPSCs). Here, we describe the necessary steps to derive, expand, harvest, inactivate, plate, and use MEFs as feeders for iPSC generation and maintenance.
Subject(s)
Cell Culture Techniques/methods , Embryo, Mammalian/cytology , Feeder Cells/radiation effects , Fibroblasts/radiation effects , Animals , Cells, Cultured , Coculture Techniques/methods , Cryopreservation , Feeder Cells/cytology , Feeder Cells/physiology , Fibroblasts/cytology , Fibroblasts/physiology , Induced Pluripotent Stem Cells/physiology , MiceABSTRACT
The ability of human pluripotent stem cells (hPSCs) to proliferate indefinitely in culture while maintaining their pluripotent properties makes them a powerful tool for use in research, and provides tremendous potential for diagnostic testing, and therapeutic application. Success in these areas, however, is dependent on the ability to effectively expand them in long-term culture while preserving their distinct nature. Contained in this chapter are detailed protocols for the feeder-independent culture and expansion of hPSCs using mTeSR1 medium and Matrigel matrix, and guidelines for the successful transfer of those cells to alternative platforms. These protocols have been used widely by laboratories around the world to successfully expand hPSCs for long-term culture while maintaining their undifferentiated, pluripotent state.
