(i.e. ability to differentiate, when needed, into specialised cell types of the three germ
layers). The most common feeder cultures used are fibroblasts derived from embryos.
The methodology for this together with other techniques for successful maintenance
and propagation of hESCs are described below. Other protocols such as freezing and
resuscitation of frozen cells are similar to those already described and the reader is
therefore referred to the relevant sections above.2.6.1 Preparation of embryonic fibroblasts
Typically, fibroblasts are isolated under sterile conditions in a tissue culture cabinet
from embryos obtained from mice at 13.5 days of gestation. Each embryo is minced
into very fine pieces using sterile scissors and incubated in a cell culture incubator at
37 C with trypsin/EDTA (0.25% (w/v)/5 mM) for 20 minutes. The mixture is then
pipetted vigorously using a fine-bore pipette until it develops a sludgy consistency.
This process is repeated, returning the digest into the incubator if necessary, until
the embryos have been virtually digested. The trypsin is subsequently neutralised
with culture medium containing 10% serum ensuring that the volume of medium is at
least twice that of the trypsin used. The minced tissue is plated onto a tissue culture
flask and incubated overnight at 37C in a tissue culture incubator. The medium is
subsequently removed after 24 h and the cell monolayer washed to remove any tissue
debris and non-adherent cells. Adherent cells are cultured to 80–90% confluency
before being passaged using trypsin as described in Section 2.5.5. If needed, the
trypsinised cells could be propagated, otherwise they should be frozen as described
in Section 2.5.10 and used as stock. If the latter is preferred, ensure that cells are
frozen at no higher than passage three.Practical hints and tips in using fibroblast feeders
Mouse fibroblasts should be used as feeders for stem cell culture between passages
three and five. This is to ensure that fibroblasts support the growth of undifferen-
tiated cells. After passage five the cells may begin to senesce and could also
potentially fail to maintain stem cells in the undifferentiated state. Each batch of
feeders prepared should be tested for their ability to support cells in an undifferen-
tiated state.2.6.2 Inactivation of fibroblast cells for use as feeders
Fibroblasts isolated should be inactivated before they can be used as feeders in order
to prevent their proliferation and expansion during culture. This can be achieved
using one of two protocols which include either irradiation or treatment with the
antibiotic DNA cross-linker mitomycin C. With the former, cells in suspension are
exposed to 80 Gy of irradiation using a caesium-source gamma irradiator. This is
the dose of irradiation normally used for mouse fibroblasts; however, the radiation
dose and exposure time may vary between batches of fibroblasts. As a result, a dose62 Cell culture techniques