microscope in a tissue culture hood. Undifferentiated colonies are identified by their
morphology and then cut into grids (see Fig. 2.11) by scoring across and perpendicular
to the first cut. Using a 1-ml pipette or pastette, the cut segments are transferred to
dishes containing fresh feeders and culture medium. The colony fragments are placed
evenly across the feeders (see Fig. 2.12) to avoid the colonies clumping together and
attaching to the dish as one mass of cells. The dishes are then carefully transferred to
a tissue culture incubator and left undisturbed for 1 day before replacing the spent
medium with fresh. Established colonies are then fed every day until subcultured.2.6.7 Feeder-free culture of hESCs
Although culture of hESCs on feeders has been extensively used, there have been
concerns over this procedure when stem cells are being considered for clinical use
in humans. One of the main drawbacks of using feeders is the concern over potential
transmission of animal pathogens to humans and the possibility of expression of
immunogenic antigens. Feeders are also inconvenient, expensive, and time-consuming
to generate and inactivate. As a result of these limitations, there has been a drive towards
developing a feeder-free culture system using feeder-conditioned media or media
supplemented with different growth factors and other signalling molecules essentialFig. 2.11Mechanically harvested hESCs.66 Cell culture techniques