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.
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 … Continued
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 … Continued
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 … Continued
How often do you recommend changing the culture media to maintain cells in best health and minimize stress?
PSC culture medium should be changed daily to ensure maximum cell health and minimize stress on PSCs. There are a number of weekend-free protocols that have been recently recommended for PSC media systems, which are based upon using low density seeding so that the nutrients are not depleted in the growth medium over the time … Continued
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: http://www.lifetechnologies.com/us/en/home/references/protocols/cell-culture/stem-cell-protocols/ipsc-protocols/culturing-puripotent-stem-cells-essential-8-medium.html 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. … Continued
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 … Continued
What is the optimal method through which to passage iPSCs/hESCs to minimize karyotypic anomalies from occurring?
There are a few recent articles which suggest that manual dissociation of PSCs rather than enzymatic treatment results in fewer karyotypic anomalies (Stem Cells and Development (2015) 24:653-662 & PLOS ONE (2015) 10(2)). However, in addition to the passaging method the following parameters can have a significant impact on the genomic stability of cultures, (1) … Continued
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: http://www.lifetechnologies.com/us/en/home/references/protocols/cell-culture/stem-cell-protocols/ipsc-protocols/culturing-puripotent-stem-cells-essential-8-medium.html. 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 … Continued
I am having trouble with my cells sticking to the well rim when using 96 well plates. Any recommendations?
An approach that we routinely use to eliminate edge effects, which I believe is what you are describing here, is incubation of our plates at room temperature for 15 minutes post plating prior to movement of cultures to 37 ºC incubator. This as well as ensuring that your medium is at room temperature prior to … Continued
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 … Continued
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. … Continued