Description: The germ-line competent cell line MESC 20 was established from the inner cell mass of a 3.5 day male pre-implantation mouse embryo (strain 129/Olac). These pluripotent cells retain the ability to participate in normal embryonic development. Differentiation of the cells is inhibited by Leukaemia Inhibiting Factor (LIF). Addition of LIF allows culture without the use of a feeder layer.

Also Known As:

Species: Mouse (129/Olac)

Tissue: embryo

Growth Properties: Adherent

Morphology: Adherent monolayer of spheroidal cells.

Growth Medium: MEF medium consists of Advanced DMEM/F12 (Invitrogen 12634010)”, 10% FBS (Perbio SH30070.03E), 2 mM Glutamine (Invitrogen 25030024) and 0.1 mM ß-mercaptoethanol (Sigma M6250). KSR medium consists of KO-DMEM (Gibco 10829), 20% Knock-Out Serum Replacer (Gibco 10828), 2 mM Glutamine (Invitrogen 25030024), NEAA (Invitrogen 11140035), 0.1 mM ß-mercaptoethanol (Sigma M6250) and LIF 1000 Units/ml (ESGRO ESG1106).

Subculturing Procedure: 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 10,000 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 10,000 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.

Release Conditions: No

Depositor: DG Brown, MRC Toxicology Unit, Hodgkin Building, Leicester LE2 4UE

Originator: Yes

References: Brown DG, Willington MA, Findlay I, Muggleton-Harris AL.1992 Criteria that optimize the potential of murine embryonic stem cells for in vitro and in vivo developmental studies. In Vitro Cell Dev Biol.28A(11-12):773-8. PMID: 1483967.

Hyperlink to ECACC Cell Line Data Sheet: 11120814