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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.

Components:

• Iscove's modified Dulbecco's medium (IMDM); Gibco/Invitrogen #12440-053 for 500ml; 12440-046 for 1 L; store 4 degrees and protect from light.
• Hyclone FBS tested for ES cells; heat inactivated; 40ml aliquots; stored -20. Characterized Fetal Bovine Serum, Catalog #SH30071, Lot#ARH27145.
• OR
• Knockout SR serum replacement for ES cells (SR); Gibco 10828-028; 10ml aliquots; stored -20. DO NOT HEAT INACTIVATE.
• 100x 2-mercaptoethanol (Sigma M7522, 14.3 M stock): dilute 70 microliters into 100ml of sterile ddH2O and filter sterilize; 10ml aliquots; store 4 degrees.
• 100x MEM nonessential amino acids: Gibco/Invitrogen #11140-050 for 100ml; 10ml aliquots; store 4 degrees.
• 100x Pen/strep: Gibco/Invitrogen #15070-063 for 100ml; 10ml aliquots; store -20.
• Recombinant LIF protein, produced as per the V. Prideaux protocol; filter sterilized; put into 30 microliter aliquots; flash frozen in liquid nitrogen and stored -80; each batch tested for efficacy against older batches.
• Alternatively LIF can be purchased from Chemicon (ESGRO 10⁷ units: ESG1107; enough for 10L of medium) and supplemented at 10³units/ml.
• Gelatinized tissue culture dishes:
• .1% gelatin (typeB from bovine skin, Sigma G9382); .3g in 300ml ddH2O autoclaved; cover dishes with solution and aspirate off; allow to air dry in hood--approximately 1/2 hour (alternatively can allow to air dry overnight): use within 24 hours.
• Primary mouse embryonic fibroblast feeder layers (PMEFs), Mitomycin-C treated. Purchased from Specialty Media (now Millipore), PMEF-CF.

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

• 40ml Iscove's MDM (contains 4 mM L-glutamine and 1 mM sodium pyruvate)
• To an aliquot of IMDM add in exactly the order listed:
• 10ml FBS or SR (20% final)
• 0.5ml 100x (10 mM) 2-mercaptoethanol (.1mM final)
• 0.5ml 100x MEM nonessential amino acids (.1 mM final)
• 0.5ml 100x PEN/STREP (50 u or µg/ml final)
• 30 µl of LIF (one -80 aliquot)

 

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