2.6.5 Enzymatic subculture of hESCs
As with standard cell culture, hESCs can be passaged using enzymes but in this case
an enzyme that does not disperse clusters of cells into single cells is preferred. This is
because hESCs need to grow in colonies since single cells may not adhere to the
feeders and may differentiate easily.
One of the most commonly used enzymes for subculturing hESCs iscollagenase.
When employed, hESC colonies are washed with phosphate-buffered saline and
then incubated for 8–10 min with collagenase IV made up in serum-free medium at
a concentration of 1 mg cm^3. Curled up colonies can then be dislodged with gentle
pipetting using a 5-ml pipette to break large clumps. Alternatively, colonies can be
fragmented using glass beads. These are then washed with culture medium to remove
the enzyme which may otherwise impair the attachment and growth of the cells,
thus reducing the plating efficiency. hESCs can be washed by allowing the colonies
to sediment slowly over 5–10 mins, leaving any residual feeder cells in the super-
natant which are removed by aspiration. The colonies are subsequently resuspended
in growth medium and are usually plated at a ratio of between 1 : 3 and 1 : 6.
Alternatively, fragmented colonies could be frozen as described in Section 2.5.10
and stored for later use.2.6.6 Mechanical subculture of hESCs
An alternative to the enzymatic method of subculturing hESCs is to manually cut
colonies into appropriate size fragments using a fine-bore needle or a specially
designed cutter such as the STEMPRO®EZPassageTMdisposable stem cell passaging
tool from Invitrogen. To do this, the dish of hESCs is placed under a dissectingFig. 2.10Partially differentiated hESCs on mouse feeder cells.65 2.6 Stem cell culture