Cryopreservation of single cell hPSCs using ROCK inhibitors 

Introduction

Biobanking is a critical step in the development of cellular products in regenerative medicine.  Cryopreservation of hPSCs is notoriously problematic in it’s inefficiency.  The ability to recover hPSCs from frozen stocks can be difficult, slow, and inefficient, as only a fraction of the cells are recovered upon thawing.  Efficient freezing and thawing protocols that yield viable, healthy cells are crucial for clinical applications of hPSCs.   

Historically, hPSCs are routinely passaged and cryopreserved as “clumps”, or small colony aggregates, in order to minimize apoptosis and damaging cell stress due to enzymatic or mechanical cell dissociation. However, freezing cultures using this method is laborious and unfortunately yields very low thawing efficiencies and slow rates of growth for those surviving cells.  Cryopreservation and passaging hPSCs as single cells holds immense benefit to hPSC culture, as it would allow for more efficient and less subjective and labor-intensive culture methods, enabling scale-up to the necessary cell numbers required for cell-based therapies.   

Benefits of freezing hPSC as single cells include a more scalable method of harvesting large quantities of cells and allowing for a more accurate cell count per vial, with much greater consistency between frozen vials and higher predictability of thawing recovery and time to first passage. Other downstream culture manipulations are also greatly enhanced by the ability to dissociate hPSCs to single cells, including transfection, flow cytometry, high-throughput screening, and differentiation assays. 

Image: Protocol example for the freezing and thawing of hPSCs using ROCK inhibitors. Adapted from Chen et. al. 2014. Current Protocols. Stem Cell Biology.

ROCK inhibitors boost recovery after cryopreservation 

In 2007, Watanabe et al. reported a selective Rho-associated coiled coil kinase (ROCK) inhibitor (Y-27632) promotes survival of dissociated hPSCs, laying the foundation for small molecule ROCK inhibitor treatments in cell cultures recovering from cryopreservation.  The ROCK pathway becomes hyperactive during single-cell dissociation of hPSCs, leading to apoptosis and anoikis and the use of ROCK inhibitors has become a routine culture supplement for dissociation during passaging and other manipulations. When freezing hPSCs as single cells, the addition of a ROCK inhibitor is vital to improve the overall survival and viability of the cells recovering from thaw.  

ROCK inhibitors are supplemented in the hPSC culture medium during the first 24 hours of the cell recovery post-thaw to significantly increase the attachment and survival of the dissociated and cryopreserved hPSCs. 

Below are a few ROCK inhibitors that are widely used in hPSC cryopreservation recovery: 

Y-27632 

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 today, Y27632 remains the most widely used ROCK inhibitor for many hPSC applications. Y27632 is used at a final concentration of 10 µM in culture media. 

Thiazovivin  

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. 

Chroman 1  

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. 

New small molecule combination provides comprehensive cytoprotection in hPSC post-thaw survival and recovery  

Captivate Bio’s new CET Cocktail Kit contains Chroman 1, Emricasan, and Trans-ISRIB (CET) powdered small molecules which has been shown to significantly improve dissociation- and stress-induced apoptosis of hPSCs through a number of biomechanical actions. The CET Cocktail pack of molecules 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. Chroman 1, Emricasan, and Trans-ISRIB (CET) were first combined with a polyamine solution to create the CEPT supplementation strategy, which provides comprehensive cytoprotection to effectively promote cell attachment and survival of dissociated cells.  

This study showed that recovery of cryopreserved dissociated hPSCs is dramatically improved with use of CEPT over other ROCK inhibitors and commercially available cryopreservation solutions. When banking hPSCs as single cells, CEPT can be used both during cryopreservation as well as in the thawing media. 

Learn More

• Introduction to ROCK inhibitors for hPSC culture
• Learn more about Captivate Bio’s small molecules for stem cell research
• Research Spotlight: Efficient single-cell cloning of hPSCs using the CEPT cocktail

References

• Watanabe et al. Nature Biotechnology. 2007; 25:681-686.
• Claassen et al. Molecular Reproduction and Development. 2009; 76(8):722-732.  
• Lin et al. Nature Methods. 2009; 6:805.       
• Chen et al. Nature Methods. 2021; 18: 528-541.