Cell Culture Guides
Share
In 2007, Watanabe et al. reported that inhibition of Rho-associated coiled coil kinase (ROCK) activity promotes survival of dissociated hPSCs, laying the foundation for small molecule ROCK inhibitor treatments in cell cultures undergoing stress, such as single-cell cloning, reprogramming, differentiation, or recovering from cryopreservation. The ROCK pathway becomes hyperactive during single-cell dissociation of hPSCs, leading to apoptosis and anoikis. The use of ROCK inhibitors has become a routine culture supplement for dissociation during passaging and other manipulations.
ROCK inhibitors are supplemented in the hPSC culture medium during the first 24 hours after dissociation to significantly increase the attachment, survival, and viability of the hPSCs.
The most commonly used small molecule ROCK inhibitors in hPSC culture, reprogramming, cloning, differentiation, and cryopreservation are Y27632, Thiazovivin, and Chroman 1. The small molecules in the CET Cocktail is based on the NIH published CEPT small molecule supplementation strategy, optimizing the use of Chroman 1.
The discovery that a small molecule ROCK inhibitor treatment of Y27632 in hPSC cultures improved survival from single-cell dissociation and cryopreservation was a groundbreaking discovery in 2007, and Y27632 remains the most widely used ROCK inhibitor used in many hPSC applications today.
Y27632 is used at a final concentration of 10 µM in culture media. Limited use of Y27632 is recommended, due to known off-target inhibition at the standard 10 µM concentration.
First published in 2009 to enhance hPSC reprogramming efficiencies, Thiazovivin is a selective ROCK inhibitor alternative to Y27632. Thiazovivin is typically used at a final concentration 5-fold lower than Y27632, at 2 µM in culture media.
The addition of Thiazovivin has been shown to also create more effective conditions for the reprogramming of cord blood mononuclear cells into iPSCs.
Chroman 1 is a potent and selective ROCK inhibitor, generally used at a final concentration of only 50 nM in culture media. In 2021, extensive studies on hPSC culture optimization for single-cell dissociation by Chen et al. highlighted Chroman 1’s superior cytoprotective capacity without off-target effects or impact to cells’ karyotype or pluripotency with routine use.
Chroman 1 is a highly potent and selective ROCK inhibitor with greater effectiveness at lower doses than Y27632.
Captivate Bio’s new CET Cocktail pack provides comprehensive cytoprotection to improve hPSC survival and recovery under stressful conditions. The CET Cocktail pack is based on the specific ROCK inhibition provided by Chroman 1. Emricasan, a pan-caspase inhibitor, synergistically enhances hPSC survival with Chroman 1. Trans-ISRIB is an integrated stress response inhibitor that further improves cell attachment and survival when used in combination with Chroman 1 and Emricasan.
In the groundbreaking 2021 paper from Chen et al. the small molecules Chroman 1, Emricasan, and Trans-ISRIB (CET) were first combined with a polyamine solution to create the CEPT supplementation strategy, an innovative combination that provides comprehensive cytoprotection to effectively promote cell attachment and survival of dissociated cells.
Image source: Morphology of hESCs grown on feeders after sorting with application of Y-27632. Emre, N. et al., PLOS ONE 5(8): e12148.
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.
Chroman 1 is a more effective ROCK inhibitor shown to improve cell survival and recovery of hPSCs after cryopreservation. Learn how Chroman 1 compares to Y-27632 in cultures in this short blog post.
In this interview, we talk to EverCell Bio’s Philip Manos about the challenges with hPSC cultures and how ROCK Inhibitors including Captivate Bio's CET Cocktail compares to traditional methods.
