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

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 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.

(2) 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.

(3) 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.

(4) 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.

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.

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).

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 elapsed in culture prior to the subsequent media change. However, basic fibroblast growth factor (FGF2), a key component in PSC growth medium which is essential for maintenance of pluripotency of PSCs is shown to quickly degrade at 37 ºC (Stem Cells (2006) 24:568-574). Therefore, using extended feed options such as the weekend-free protocols is not recommended for optimal cell health and maintenance of pluripotency.

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. Preliminary experiments suggest that addition of RevitaCell™ Supplement (Cat #A26445-01) to the culture medium during the first 24 hours post-transition can also aid in maintenance of cell health during the transition period.

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:
https://tools.lifetechnologies.com/content/sfs/manuals/PSC_Cryopreservation_Kit_UG.pdf

https://tools.lifetechnologies.com/content/sfs/manuals/RevitaCell_Supplement_UG.pdf

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

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).

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 over onto truncated recombinant vitronectin (Cat # A14700) surface if desiring a leaner matrix.

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 plating of cells has been shown to be critical in eliminating edge effects. This paper, http://jbx.sagepub.com/content/8/5/566.long (J Biomol Screen. 2003 Oct;8(5):566-70) outlines a comparable approach with extended incubation for 1 hour at room temperature prior to moving cultures to 37 ºC incubator.

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 that, if you are using culture medium which contains FGF as a component, the medium is not repetitively warmed at 37 ºC. Additionally, ensuring that culture medium is not used past the time denoted for complete medium stability is essential (e.g., Essential 8® Medium, upon addition of the supplement to the basal medium, has been shown to remain stable at 4 ºC for 2 weeks). Furthermore, as FGF is shown to rapidly degrade at 37 ºC, daily feeding with culture medium is recommended.

(2) Split Ratios Used-Too high or too low of split ratios resulting in sparse or overcrowded cultures, respectively, can result in higher than normal differentiation of cultures.

(3) Ensuring health of cultures-Ensure that cultures are not overgrown prior to passaging, as this can result in poor cell health and survival following passaging and thus result in too sparse of a culture, which can result in substantial aberrant differentiation of cultures.

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. I would recommend using Thermo Fisher Scientific’s new RevitaCell™ Supplement (Cat #A26445-01) to assist in recovery of your cryopreserved PSCs. It is compatible with both feeder-free and feeder-dependent medium systems and should only be added for the 1st 24 hours post-thaw, with subsequent feedings with growth medium in the absence of RevitaCell™ Supplement. This supplement contains a ROCK inhibitor coupled with compounds having antioxidant and free radical scavenger properties, providing for maximum post-thaw recovery.
Here are the links to the product information and the product insert for your reference:
https://www.lifetechnologies.com/order/catalog/product/A2644501

&
https://tools.lifetechnologies.com/content/sfs/manuals/RevitaCell_Supplement_UG.pdf