Methods: Conventionally, single cell suspensions are required to carry out CBMN procedure. However dissociating colonies of highly sociable hESCs into single cells will disrupt stem cell niche, activate apoptosis signaling pathways and cause a dramatic decrease in hESC viability. To overcome this problem, we cultured hESCs under Matrigel-supported feeder-free condition. CBMN assay was performed directly on hESC monolayer without dissociating the cells into single cell suspension. The frequencies of scorable cells, micronucleated binuclear cells were compared between the in situ and conventional protocol.
Results: High frequency of unscorable cells with premature chromatin condensation and DNA fragmentation was observed after hESCs underwent the conventional CBMN procedure; whereas such apoptotic hallmarks remained at low level under the in situ protocol. Although we found no significant difference in micronucleus frequencies between the two protocols, efficiency of microscopic scoring was improved under the in situ protocol because of less unscorable cells and tighter spatial organization of hESCs on chamber slides.
Conclusion: The in situ protocol circumvents the technical challenge of getting highly viable hESCs to run CBMN assay and opens up possibility to efficiently perform the genotoxicity test on hESCs.