Articles
Share
Winter is sweeping across the United States. The cold is everywhere and the rain, snow, and power outages have brought the hustle and bustle outside almost to a standstill. We’ve been thinking about how the weather outside could be worse – at least it’s not as cold as liquid nitrogen!
We have it better than our frozen cell cultures, for sure. Ice crystals in my environment are likely not going to cause me to either burst or dehydrate while being suspended at sub-zero temperatures. Just as my coat provides protection from the elements, the ROCK inhibitor Chroman 1 can provide cytoprotection for your frozen cells which helps them survive and thrive during the freezing and thawing process.
Read on to learn more about ROCK inhibitors and their impact on your cells in the lab!
Human pluripotent stem cells (hPSCs) are notorious for poor survival after cryopreservation, generally yielding only about 1% efficiency with most cell freezing methods. The small molecule ROCK inhibitor Y27632 was the first small molecule shown to improve hPSC viability during cryopreservation in 2007. By blocking the overactive ROCK pathway, ROCK inhibitors protect cells from damaging cellular contraction and apoptosis that occurs during cryopreservation. Today, small molecule ROCK inhibitors including Y27632, Thiazovivin, and Chroman 1 are widely used in freezing and thawing media to protect hPSCs and many other sensitive cell types from apoptosis caused by the stressors of cryopreservation.
Chroman 1 is more potent than Y27632. Chroman 1 was first identified as the most potent known ROCK inhibitor by Chen et al. in 2021, and has since been the focus of multiple publications around improving the hPSC workflow. Chroman 1 is more effective at promoting cell survival than Y27632, even when used at a much lower concentration. In typical hPSC culture applications, including cryopreservation, Y27632 is used at a concentration of 10 µM in culture media. Chroman 1 shows more efficient cell survival when used at only 50 nM in culture media.
Chroman 1 is more selective than Y27632. Y27632 is known to have off-target effects, causing some concern over the potential unknown influences of supplementation with this small molecule. The high specificity of Chroman 1, along with the low dosage used, alleviates concerns of off-target interactions. Even so, Chroman 1 is still generally used in the same short 24-hour treatment strategy as Y27632.
Figure 1. Comparative potency of Y27632 and Chroman 1 was assessed against ROCK1 and ROCK2, showing high potency of Chroman 1 and relative IC50 results for each ROCK target. (Figure from Chen et al. 2021 Nature Methods)
Chroman 1 provides more effective ROCK inhibition and cytoprotection of hPSCs and other sensitive cell types than traditional Y27632 supplementation, improving cell survival of hPSCs by approximately 25% compared to Y27632.
To demonstrate the ability of Chroman 1 to maintain stable, healthy cultures, Chen et al. put Chroman 1 to the test in their groundbreaking 2021 publication: Chroman 1 was used during every passage in a long-term, stressful serial single-cell propagation of hPSCs. After supplementing the media at the recommended 50 nM concentration for 24 hours at each passage for 40+ consecutive passages, the hPSCs showed no changes in morphology, marker expression, or differentiation capacity, demonstrating that Chroman 1 can be safely used for downstream therapeutic applications.
Figure 2. Chroman 1 is a superior ROCK inhibitor to Y27632. When hPSCs were treated with either 10 µM of Y27632 or 50 nM of Chroman 1 for a 24 hour timepoint after dissociation as single cells, significantly fewer dead cells and higher numbers of live cells were detected in cultures treated with Chroman 1. After serial passaging for 40 passages in the presence of Chroman 1 at each passage (24 hour exposure at 50 nM per passage), hPSCs maintained a normal karyotype, even after extended culture in stressful conditions. (Figure from Chen et al. 2021 Nature Methods)
Learn more about how we support researchers with cell culture products, contract manufacturing and storage services, custom reagent sourcing, as well as marketing, sales, and distribution programs for our industry partners.
Learn the benefits of freezing hPSCs as single cells as well as using ROCK inhibitors including Y27632, Chroman 1, and Thiazovivin to aid in cryopreservation recovery.
Over time, ROCK inhibitors have been shown to boost hPSC viability and survival. This guide summarizes the most commonly used small molecule ROCK inhibitors for stem cell cultures.
CEPT supplementation improves hPSC single-cell passaging, differentiation, cryopreservation, cloning, and other manipulations. Meet the new CET Cocktail from Captivate Bio.
