Automated Mechanical Passaging: A Novel and Efficient Method for Human Embryonic Stem Cell Expansion

There is a need for more standardized methods of maintenance and propagation of human embryonic stem cell (hESC) cultures. Enzymatic passaging currently represents the most widely used method for expansion of hESCs. Although rapid and straightforward, this technique results in variable-sized cell clusters and significant cellular trauma, which may apply selective pressure in long-term culture. Mechanical passaging has the potential advantages of defined colony fragment sizes, reduced cellular trauma, and the possibility of selecting undifferentiated colonies for transfer. However, manual dissection of individual colonies is a prohibitively time-consuming process unsuitable for maintaining large numbers of hESCs without the use of additional chemical means. In this study we report an efficient automated method for mechanically passaging hESCs. We have used this method exclusively to maintain hESCs in long-term undifferentiated culture without the use of enzymatic digestion for longer than 100 days. This automated technique can thus be used routinely to culture hESCs in the laboratory.