Figure 1. Selection of a cell dissociation reagent suitable for slow-freezing.

(A) hiPSC 201B7 colonies were dissociated with Pronase/EDTA, trypsin/EDTA, Dispase II, collagenase IV, or CTK, followed by cryopreservation with Formula A medium (6% HES, 5% DMSO, 4% BSA, and 50% D-MEM/F12 in saline). Recovery frequencies (rate, %) were determined by scoring the number of post-thaw ALP+ colonies at day 5 in 6-well dish for comparison against non-frozen cells at day 5 in 6-well dishes that had been passaged with the same dissociation buffer. The results of 3 independent experiments are shown with standard deviation bars [SD]). *; P<0.05. (B) Sizes of 100 randomly selected cell clumps (in µm²) after dissociation in the indicated medium. Sizes are plotted as blue dots. Red bars show each median. A photo of cell clumps after use of the indicated cell detachment reagent is shown in the lower panel. Scale bars; 500 µm. *; P<0.05.