2.6.4 Culture of human embryonic stem cells
Once the feeders are ready, hESCs can be plated directly by depositing the suspension
of hESC onto the feeder layer. The dishes are placed in a cell culture incubator and
the cells allowed to attach and establish over a 24-h period. Any non-adherent cells
are removed during the first culture medium change. The cells are monitored and
fed on a daily basis until the colonies are ready to be passaged. Depending on the
conditions of growth, this can usually take up to 6 days.
As with the feeders, frozen stocks of hESCs should be resuscitated and diluted in
fresh growth medium as described in Section 2.5.11.Practical hints and tips in hESC culture
It is important to ensure that the colonies do not grow too large and to the point where
adjacent colonies touch each other as this will initiate their differentiation. Similarly,
the seeding density should be high enough to sustain growth otherwise sparsely plated
colonies will grow very slowly and may never establish fully.
Colonies should be plated on healthy feeders that are not more than 4 days old.
More importantly, only tightly packed colonies containing cells with the typical hESC
morphology should be passaged (see Fig. 2.9). Any colony that has a less defined
border (see Fig. 2.10) at the periphery, with loose cells spreading out or cells with
atypical morphology, should not be passaged because these characteristics are
evidence of cell differentiation. Should cells differentiate, these should be excised
or aspirated before passaging the undifferentiated cells. Alternatively, if the majority
of the colonies appear differentiated and no colonies display the characteristic
morphology of undifferentiated cells, then it is advisable to discard the cultures and
start with a new batch of undifferentiated hESCs.Fig. 2.9Undifferentiated hESCs on mouse feeder cells.64 Cell culture techniques