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CEPT small molecules as cell culture reagents
A long-standing challenge to the successful use of stem cells for both basic research and therapeutic applications is the sensitivity of cells to stress, leading to extensive cell death. CEPT Cocktail is a poly-pharmacological strategy that uses a combination of small molecules to significantly improve survival of pluripotent stem cells (PSCs) at all stages of the stem cell workflow. The combination has been shown to be effective in cryopreservation, routine long-term passaging, single-cell cloning, gene editing, and differentiation, as well as embryoid body and organoid formation. CEPT Cocktail provides improved cytoprotection in the stem cell workflow compared with currently available reagents, by reducing cellular stress and DNA damage during passaging, and promoting cell adhesion. CEPT Cocktail therefore has broad potential for improving the manufacture of cells for disease modeling, drug screening, regenerative medicine, and cell therapy.
CEPT Cocktail Products
| Product Name | Catalog # | Product Action |
|---|---|---|
| CEPT Cocktail Kit | 7991 | Easy-to-use kit for the preparation of 5 L of CEPT Cocktail media |
| Chroman 1 | 7163 | Highly potent and selective ROCK 2 inhibitor; improves cell survival after cryogenesis |
| Emricasan | 7310 | Potent pan-caspase inhibitor; promotes survival of hPSCs in combination with other small molecules |
| Polyamine Supplement x1000 | 7739 | Media supplement to boost cell growth; comprises a lyophilized mixture of Putrescine, Spermine and Spermidine |
| Trans-ISRIB | 5284 | Integrated stress response (ISR) inhibitor; supports protein synthesis and cell attachment during cell passaging |
Single-cell dissociation and the establishment of clonal cell lines for basic and translational research have been major challenges when working with human pluripotent stem cells (hPSCs). The schematic above highlights the use of CEPT cocktail in conjunction with the microfluidics-based cell sorting instrument Hana (from Namocell, a Bio-Techne brand), for scalable single-cell cloning of hPSCs and generation of clonal cell lines. CEPT maintains pluripotency and improves single-cell cloning efficiency, and Hana uses fully automated gentle (<2 psi) cell dispensing to minimize cell damage and mechanical stress. This combination provides superior results overall compared with FACS and other culture reagents.
DMSO, Cell Cryopreserve Grade (# 7726)
DMSO, Cell Cryopreserve Grade (# 7726)
Our DMSO, Cell Cryopreserve Grade is a cryoprotective agent that can be used to protect cells from dehydration and shrinking during freezing and prevents the formation of ice crystals.
ExCellerate™ iPSC Expansion Medium
ExCellerate™ iPSC Expansion Medium
- Animal component-free
- Complete formulation – no growth factor supplementation required
- Stable cell integrity over long term culture
Fast, Gentle & Easy Single Cell Isolation
Hana and Pala, Namocell’s single cell sorting instruments, make single cell sorting fast and easy. These benchtop instruments combine microfluidics, flow cytometry and direct liquid dispensing to accomplish gentle cell sorting and dispensing in one simple step.
Stem Cell Research Product Guide
This product guide provides a background to the use of small molecules in stem cell research and lists over 200 products for use in self-renewal, differentiation, reprogramming, organoid generation and Ancillary Material Grade products.
Organoids Wall Poster
Organoids are emerging as powerful new platforms for exploring human organ development, modeling disease, screening for drug toxicity, and investigating personalized medicine. Request this poster to decorate your lab with some of the visually stunning organoid images that have been generated by cell and gene therapy scientists at Bio-Techne.
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.
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Chen et al. (2021) A versatile polypharmacology platform promotes cytoprotection and viability of human pluripotent and differentiated cells Nat.Methods. 5:528. PMID: 33941937.
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Tristan et al. (2021) Robotic high-throughput biomanufacturing and functional differentiation of human pluripotent stem cells Stem Cell Reports 12:3076. PMID: 34861164.
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Tristan et al. (2023) Efficient and safe single-cell cloning of human pluripotent stem cells using the CEPT cocktail Nat.Protoc. 1:58. PMID: 36261632.
