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Minimize the Impact of Stress on Pluripotent Stem Cell Health – A Discussion
We recently finished our Ask the Expert discussion on PSC Stress Management. This week we had many interesting questions and helpful suggestions on topics including cryopreservation and thawing, differentiation and morphology changes, passaging and seeding cells, feeder and feeder free culture, and cell culture media. We also covered challenges with cells sticking and transitioning cells.
This Ask the Expert Session was sponsored by Thermo Fisher Scientific and hosted by Dr. Rhonda Newman. Dr. Newman joined Life Technologies in 2010 and is currently working on next generation stem cell culture and differentiation systems, enabling researchers to efficiently culture, expand, cryopreserve, and differentiate their stem cells to various cellular lineages. She received a Ph.D. at the University of Iowa from the lab of Dr. Madeline Shea, focusing on ligand induced allostery of calmodulin and its impact on regulation of the Ryanodine Receptor Type 1. Subsequently, she completed postdoctoral training in the lab of Dr. Ken Prehoda at the University of Oregon, studying the role of intramolecular interactions in regulating cell signaling cascades in the process of assymetric stem cell division
Discover solutions to help your cells stress less – visit this Ask the Expert session. Below is a sneak peek, for a full transcript of the discussion, please see – Ask the Expert – PSC Stress Management.
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) growth medium, (2) matrix, (3) confluency at the time of passaging. One of the causes of genomic instability of cultures is the generation of reactive oxygen species associated with stressful events, such as passaging, which results in generation of double strand breaks.
Thermo Fisher Scientific recently launched a supplement which can be used in post-thaw recovery, as well as for support of cells during passaging conditions-RevitaCell™ Supplement (Cat #A26445-01). NOTE: It should only be added to the culture medium for the first 24 hours post-thaw or post-split followed by feeding with unsupplemented medium for the remainder of time in culture. RevitaCell™ Supplement contains a specific ROCK inhibitor coupled with compounds having antioxidant and free radical scavenger properties. In general, addition of antioxidants to the culture medium has been shown to improve genome stability by reducing the ROS within the PSC during a range of processes (Stem Cell Reports 2:44-51, Scientific Reports 4:3779).
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 and we ensure that a working bank of earlier passage PSCs be maintained as a safety stock. The most predominant large karyotypic abnormalities that have been shown to accumulate include gain of chromosomes 12 and 17 and chromosome X. There is a very nice review that was published early last year reviewing genome maintenance of pluripotent stem cells (J. Cell Biol. 204(2): 153-163).
One of the mechanisms outlined in this review which is responsible for some of the genomic instability is the build-up of reactive oxygen species (ROS) during stressful processes, which include passaging conditions, as well as recovery from cryopreservation. ROS can result in double stranded breaks which are particularly deleterious as during the repair process point mutations, as well as non-homologous end joining can result. Thermo Fisher Scientific recently launched a supplement which can be used in post-thaw recovery as well as for support of cells during passaging conditions-RevitaCell™ Supplement (Cat #A26445-01). NOTE: It should only be added to the culture medium for the first 24 hours post-thaw or post-split followed by feeding with unsupplemented medium for the remainder of time in culture. RevitaCell™ Supplement contains a specific ROCK inhibitor coupled with compounds having antioxidant and free radical scavenger properties. In general, addition of antioxidants to the culture medium has been shown to improve genome stability by reducing the ROS within the PSC during a range of processes (Stem Cell Reports 2:44-51, Scientific Reports 4:3779).
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:
- Choice of cryopreservation solution-The cryopreservation solution is an essential component that can have a significant impact on the recovery of cells 24 hours post-thaw. While the viability of cells may appear artificially high direct post-thaw, the stress of thawing of cells and osmotic stress associated with the process of diluting the cryomedium solution itself is often not immediately evident.
- Temperature and time of exposure to cryopreservation solution-A key step in the cryopreservation process is ensuring that the cryopreservation medium is prechilled (i.e., at 2 to 8 °C) prior to addition to the PSCs. This is vital as it greatly impacts the permeation rate of the cryoprotectant into the cytosol of the cell. The quicker the influx of the solution into the cell, the greater the impact of osmotic shock on cell health. Additionally, the longer duration exposure to the cryoprotectant –commonly DMSO−at a range of temperatures, the higher the toxicity of the cryoprotectant. Therefore, the duration of exposure of cells to the prechilled cryopreservation medium, should be minimized in order to minimize the toxicity of the cryoprotectant on the cells.
- Quick Recovery-Just as incubation of the cells prior to cryopreservation is a vital step, minimizing exposure of the cells post-thaw to the cryoprotectant is also critical to achieving high post-thaw recovery.
- Additives to Support Post-Thaw Recovery-In order to support cells during post-thaw recovery, additives may be spiked into the growth medium for the first 24 hours followed by feeding of cells in growth medium alone for the remainder of culture. Traditional additives include ROCK inhibitors, antioxidants, and free radical scavengers. Through mechanisms of decreasing apoptosis and necrosis and facilitating cell attachment with the extracellular matrix, these compounds significantly improve recovery of cryopreserved pluripotent stem cells.
Thermo Fisher Scientific now offers a complete, ready-to-use kit – PSC Cryopreservation Kit (Cat #A26446-01)– for cryopreservation and recovery of clump passaged or single cell passaged PSCs. Together these solutions minimize the stress associated with cryopreservation of pluripotent stem cells, providing recovery of 50% greater cells 24 hours post-thaw than best-in-class commercial solutions.