CEPT Cocktail

Stem cells are highly sensitive to environmental perturbations in vitro, so there is an urgent need to develop a safe and robust strategy for long-term culturing of PSCs and their differentiated progeny to ensure their viability and functionality.

Chen et al. (2021) screened over 15,000 compounds with full dose–response curves (more than 300,000 samples) and devised a combination of four small molecules that enhances the viability of human pluripotent and differentiated cells. Chroman 1 was discovered to be a potent and selective ROCK1/2 inhibitor that significantly improves cell survival alone or in combination with other small molecules. From dose–combination matrix screening Emricasan, a pan-caspase inhibitor, was found to synergize with Chroman 1 to reduce apoptosis of PSCs. To further improve cell viability at ultralow cell density, which is critical for single cell cloning, the combination of Trans-ISRIB (an integrated stress response inhibitor) with Chroman 1 and Emricasan reverses the ER stress response and supports cell attachment during cell passaging. Polyamines are essential cations that support cell growth and stabilize both cellular structures and macromolecules, as well as mediate functions such as transcription, translation, the cell cycle and stress responses. The strongest synergistic effects were observed when Chroman 1, Emricasan, Polyamines and Trans-ISRIB were used together, leading to improved cell attachment and protein translation, as well as further improvements in cell survival.

CEPT demonstrated improved cell survival and clone formation after electroporation and genome editing using CRISPR–Cas9. Compared with other media additives, CEPT used for embryoid body (EB) and organoid formation produces higher numbers of EBs, continuous cell growth and elevated expression of lineage-specific markers, it also improves neural and kidney organoid formation with lower cell numbers plated per well. Cryopreservation and thawing of hPSCs can be challenging and inefficient. The viability of five hPSC lines (WA01, WA09, GM23476, GM25256 and GM26107), iPSC-derived cardiomyocytes, hepatocytes, astrocytes, and motor neurons were tested with CEPT versus other reagents, such as RevitaCell, CloneR, or SMC4 for cryopreservation and thawing; CEPT treatment resulted in the highest cell recovery percentage and the lowest amount of DNA double-strand breaks.