Making New ES Lines

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Growing de-novo mouse embryonic stem-cell lines (general guidelines)

These cells were derived using the basic protocol described in Bryja, et al. [An efficient method for derivation of mouse embryonic stem cells. Stem Cells 2006; 24:844-849.], with modification. This method requires alternating the media employed. All trypsinization steps employ standard FBS containing ES cell culture medium (see below) onto primary mouse embryonic fibroblast feeder layers (PMEFs). After setting down overnight the medium is changed to SR-medium, with the FBS replaced 1:1 with Serum Replacement (SR). Cells are fed daily thereafter with SR-medium until confluent, then trypsinized using FBS medium for all passaging and cryopreservation steps.

For the initial thawing, passaging, and cryopreservation employ only the TMF-house FBS that I will provide. A standard vial is the contents of a confluent 35mm dish on PMEFs. It should be thawed 1:1 onto PMEFs as follows:

(1) Thaw vial rapidly in water bath and put into 8mls of FBS containing ES cell culture medium.

(2) Recover cells by centrifugation and plate out onto PMEFs with ES cell culture medium.

(3) The next day replace medium with SR-medium and feed daily thereafter until confluent.

(4) Once confluent, passage cells 1:3 or 1:4. Alternate the media as appropriate.

(5) Once the passaged cells are confluent the majority can be cryopreserved with FBS medium. However, passage a small aliquot of cells using your own serum to verify that the cells will accept different serum. Assuming they hold up in your own serum you can stop using the TMF-house FBS.


ES cell culture medium or SR-medium/50 ml:


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