PSC Stress Management: Minimizing the Impact of Stress on Pluripotent Stem Cell Health in a Range of Workflows

Sponsored by: Thermo Fisher Scientific
Session ends: March 6th, 2015, 3:00pm MST
Answers by: Rhonda Newman, Ph.D., Life Technologies


Have you ever wished you had a safety net for your pluripotent stem cells, suffering loss of a cell line during processes such as cryopreservation, electroporation, or single cell passaging? While stem cells have a tremendous proliferative capacity, long term culture of these cells has been shown to cause an accumulation of mutations that result in genetic instability, increasing tumorigenicity and thus limiting their usefulness in research and clinical applications. Solutions to reduce the stress on pluripotent stem cells provides multiple benefits including enhanced post-thaw recovery, as well as providing support during manipulation of cells in diverse workflows including high throughput screening and gene editing. Furthermore, these solutions provide greater consistency between experiments and operators. Discover solutions to help your cells stress less – visit this week’s Ask the Expert session and submit your questions.

This session is sponsored by
Thermo Fisher Scientific

Please take advantage of the opportunity to ask our expert a question and participate in a lively discussion of Cell Culture Basics!

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Questions & Answers

It has been a few days since I thawed my iPSCs and I still see no colonies. I have been adding media daily, how long should I wait or is there anything else I should try?

Much is determined by the cell density at which the cells were cryopreserved, the cryopreservation solution used, and whether recovering feeder-dependent or feeder-free iPSCs. If recovering feeder-dependent iPSCs, then it can take 3-4 days to recover and show nice emergence of colonies. If recovering feeder-free iPSCs, then I would discontinue the culture and start again. […]» Read More

We are seeing greater than 10% differentiation in our iPSC culture. We aren’t sure why we are having these morphology changes. How would you troubleshoot or would you recommend adding to the culture?

There are a few things that I would recommend considering in your trouble shooting: (1) Stability of Culture Medium-basic fibroblast growth factor is a common component in growth medium that is essential for maintaining the pluripotency of PSCs. This component has been shown to quickly degrade at 37 ºC (Stem Cells (2006) 24:568-574). Therefore, ensuring […]» Read More

We are transitioning from feeder to feeder free culture. Can you recommend a method for this process?

Instructions for transitioning to Essential 8® Medium can be found here: To minimize the stress associated with the transfer process we generally will follow the guidance under the “Adaptation using Geltrex® LDEV-Free, hESC-Qualified Basement Membrane Matrix as an Intermediary” section. Subsequently, cells can be passaged using EDTA (i.e., Versene Solution, Cat #15040-066) and transitioned […]» Read More

What do you think is the optimal seeding density for hPSCs?

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 […]» Read More

I want to transition my iPSCs from MEFs to Matrigel. What is the best method for transition for the cells and is there anything I should add to the media?

Instructions for transitioning to Essential 8 medium on Geltrex, which is comparable to Matrigel, can be found here: under the “Adaptation using Geltrex® LDEV-Free, hESC-Qualified Basement Membrane Matrix as an Intermediary” section. In your cause you would be staying on the richer media substrate rather than moving to a leaner matrix such as rhVTN-N. […]» Read More

How many times can I passage my cells before the health suffers? Are there any products that can help extend the number of passages or time in culture?

Pluripotent stem cells, unlike primary cells, have an infinite lifespan. However, with increasing passage number there are a number of reports indicating chromosomal instability and differentiation bias that can result due to the stress associated with passaging methods and culture conditions. Therefore, we generally recommend that karyotype analysis of cultures be assessed every 10 passages […]» Read More

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?

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 […]» Read More

We have had challenges with cryopreservation and post-thaw recovery of our PSCs from cryopreservation? The post-thaw viability is high upon directly post-thaw, but the following day very few PSCs are shown to recover. Any advice for support of cells during post-thaw recovery?

While many cryopreservation solutions are shown to have high viability direct post-thaw (i.e., 80-90% viability), during the first 24 hours post-thaw significant apoptosis and necrosis occurs, resulting in substantial loss in PSC viability and thus poor post-thaw recovery. The following are points to consider to improve post-thaw recovery of your PSCs: (1) Choice of cryopreservation […]» Read More