The MESC lines can be grown without the use of mitotically inactivated feeder cells (Brown et al., 1992 PMID: 1483967). However, the cells supplied by ECACC have been grown on mitomycin treated primary mouse embryonic fibroblasts to ensure the cells are maintained in an undifferentiated state. Mouse embryonic fibroblasts, STO (ECACC 86032003) or SNL 76/7 (ECACC 07032801) can be used. At ECACC plastic ware is pre-coated with gelatine prior to plating feeder cells.Porcine gelatine (Sigma G1890) is dissolved in sterile water (0.5g/500ml) at 56°C. The 0.1% solution is sterilized by filtration (0.22µm). Add 0.1% gelatine to plastic ware to cover bottom, and incubate for 20 minutes at room temperature. Remove gelatine, wash with PBS once and replace with appropriate culture medium. The flask/dish must not be allowed to dry out.Feeder layers are prepared on the gelatinized flasks at least 24 hours in advance of being required. An ampoule is thawed in 37°C water bath and the contents quickly transferred to a 15ml centrifuge tube. MEF medium is added drop wise to 5ml. Cells are centrifuged at 150 x g for 5 minutes at Room Temperature (RT). Cells are resuspended in 5ml of MEF medium. Cells are counted and added to flasks containing the correct medium at 1-3 x 10⁴ cells/cm².An ampoule of ES cells is thawed in 37°C water bath and the contents quickly transferred to a 15ml centrifuge tube. KSR medium is added drop wise to 5ml. Cells are centrifuged at 150 x g for 5 minutes. Cells are resuspended in 5ml of KSR medium. The prepared feeder flask is washed once with PBS and KSR medium added. ES cells should be plated at 4-5 x 10⁴ cells/cm². Cultures must be incubated in a humidified 5% CO₂/95% air incubator at 37°C. A 100% media change must be performed every day and cells passaged every 2-3 days. Colonies must not be allowed to touch each other as overgrowth will result in differentiation.