We are gearing up to run some high throughput assays using pluripotent stem cells. While clump passaging of PSCs using EDTA we have noticed significant heterogeneity in the seeding of our PSCs across the plate and also in recovery of our PSCs post-passaging from experiment to experiment. Any advice on how to consistently passage and seed PSCs?

Answer

The following are points to consider when passaging to improve recovery:

(1)Confluency at the time of harvest-The confluency at the time of harvest can have a significant impact on the recovery of cells from passaging, as well as following cryopreservation. To ensure that the health of the cells is optimal, pluripotent stem cells should be between 60-80% confluent. Cells that are overly confluent tend to result in poor recovery post-split.

(2)Clump size-The size of cell clump can have a significant impact on recovery following EDTA passaging. Too large of clumps can result in aberrant differentiation, whereas too small of clumps can result in loss of cell-cell interactions, triggering apoptosis. Therefore, avoiding too long or short of an incubation time with the passaging reagent and avoiding over-trituration are key elements to controlling clump size.

(3)Alternative Option of Single Cell Passaging-Alternatively to clump passaging approaches using EDTA, Dispase, or Collagenase, single cell passaging has become an alternative approach which minimizes the inconsistencies associated with clump size. This approach generally requires the inclusion of a ROCK inhibitor or ROCK inhibitor cocktail to improve the recovery of cells post-split, depending on the growth medium and matrix. Thermo Fisher Scientific offers a supplement for post-thaw recovery, as well as for recovery from single cell passaging − RevitaCell™ Supplement (Cat #A26445-01). This supplement contains a blend of a ROCK inhibitor with higher specificity than Y-27632 or Thiazovivin, coupled with compounds having antioxidant and free-radical scavenger properties. This cocktail minimizes the stress associated with recovery from single cell passaging and facilitates cellular attachment. Refer to the product insert for this protocol for a detailed single cell passaging protocol.