Isolation and Freezing of Primary Mouse Embryonic Fibroblasts (MEF) For Feeder Plates This protocol describes the isolation of fibroblasts from mouse embryos. Mouse embryonic fibroblast (MEF) cells are used as a feeder layer for the culture of mouse embryonic stem (ES) cells to help maintain them as pluripotent stem cells. The inhibition of ES-cell differentiation provided by the MEF feeders appears to be due to their production of leukemia inhibitory factor (LIF).
Preparation of Mouse Embryonic Fibroblast (MEF) Feeder Plates Protocol MEF feeders are prepared weekly to provide a substrate for undifferentiated embryonic stem (ES) cells. Primary MEF cells are thawed, established in culture, treated with mitomycin C to halt their proliferation so they cannot overgrow the ES cultures, and then replated onto dishes convenient for ES cell culture. This protocol can also be used to prepare feeder cells from STO fibroblast cell lines.
Preparing Mouse Embryo Fibroblasts Protocol This protocol describes the preparation of mouse embryo fibroblasts (MEFs), which can then be used as feeder cells to maintain embryonic stem (ES) cells in the undifferentiated state.
Propagation of Pluripotent Mouse Embryonic Stem (ES) Cells Protocol This protocol describes the culture of embryonic stem (ES) cells using mitotically inactivating primary mouse embryonic fibroblast (MEF) cells as a feeder layer (preparation described in Preparation of Mouse Embryonic Fibroblast (MEF) Feeder Plates). The ES culture medium is supplemented with recombinant leukemia inhibitory factor (LIF) to help maintain the cells as pluripotent stem cells. This protocol has been optimized for the ES-D3 cell line.
Protocols for the Maintenance of Human Embryonic Stem Cells in Feeder Free Conditions Includes protocols: Mouse Embryonic Fibroblasts (MEF) Preparation; Harvesting MEFs; Cryopreservation of MEFs; Thawing and maintaining MEFs; Irradiating & Plating MEFs; Culture of Human ES cells with Matrigel® and Conditioned
Medium; Preparation of Conditioned Medium (CM); Preparation of Matrigel® -coated plates; Passage of human ES cells on Matrigel®; Daily maintenance of feeder-free culture; Freezing Human ES Cells; Thawing Human ES cells; Formation of Embryoid Bodies;