What do you think is the optimal seeding density for hPSCs?
This question is part of the following Ask The Expert session:
PSC Stress Management: Minimizing the Impact of Stress on Pluripotent Stem Cell Health in a Range of Workflows
Company: Life Technologies
Job Title: Staff Scientist
Much of this depends upon your growth medium and matrix. For optimal cell growth in Essential 8® Medium on truncated recombinant vitronectin we recommend a cell seeding density of ~12,500 viable cells/cm2 if passaging from a proliferating culture. This seeding density will allow for cells to reach passaging confluency (i.e., ~80%) in 4-5 days. If thawing from cryopreserved cell stocks, higher cell seeding densities are required, with ~20,000 viable cells/cm2 achieving passaging confluency (i.e., ~80%) in 4-5 days. These are all guidelines provided when using RevitaCell™ Supplement inclusion in the growth medium for 24 hours at the time of passaging or for the first 24 hours post-thaw, both followed by feeding with unsupplemented medium.
Refer to the following protocols:
A few things to consider to ensure efficient cell recovery and consistency in the passaging process:
(1) Do not allow your cultures to get overly confluent as this will add additional pressure on the cells due to limiting nutrients and will result in poor cell survival.
(2) Passaging method-If using clump passaging methods, then the time of treatment with the enzyme or dissociation reagent may need to be adjusted to allow for formation of optimal size clumps. If using single cell dissociation methods, then use of laminin matrix or use of ROCK inhibitors or RevitaCell™ Supplement is often required depending on your medium/matrix system.
(3) Minimize the amount of time between passaging of cells and plating of cells