for sustaining growth. The conditioned medium can be generated by incubating
normal growth medium with feeder cells for 24 h before use.
Feeder-free culture of hESCs is often carried out on tissue culture plastics coated
withMatrigel, a substrate derived from mouse tumour and rich inextracellular
matrixproteins such aslaminin,collagenandhepran sulphate proteoglycan.Itis
also rich in growth factors such as basic fibroblast growth factor (bFGF) which
can help to sustain and promote stem cell growth whilst maintaining them in an
undifferentiated state.
Practically, dishes are coated with 5% Matrigel made up in culture medium. Just
prior to use, the Matrigel is removed and replaced with culture medium before plating
cells. The hESCs, subcultured from feeders or obtained from frozen stocks, are resus-
pended in conditioned medium supplemented often with bFGF at a concentration of
4ngml^1 before seeding. Alternatively, normal growth medium could be used but this
will require a much higher concentration usually around 100 ng ml^1 bFGF. Once
established, hESCs are fed every day with fresh growth medium. Colonies on Matrigel
tend to show a different morphology to those on feeders; they tend to be larger and
less packed initially than when cultured on feeders.Practical hints and tips in using Matrigel
All work with Matrigel, other than plating of the hESCs, should be carried out at 4C.
Thus, when coating tissue culture plastics with Matrigel, all the plates and pipette tips
should be kept on ice and used cold to prevent the Matrigel solidifying. Stock Matrigel
is usually in the solid form and should be placed on ice or in the fridge at 4C
overnight until it liquefies. Once liquefied, the Matrigel should be diluted in ice-cold
culture medium at a final concentration of 5%. Each plate should have a smooth even
layer of Matrigel and if this is not the case, the plates should be incubated at 4C until
the Matrigel liquefies and settles as a uniform layer. Once coated, Matrigel plates
should be used within 7 days of preparation.Fig. 2.12Plating of hESCs onto feeder layer.67 2.6 Stem cell culture