CultureSure™ CEPT Cocktail(1,000×)

• Concentration (HPLC): Passed
• Appearance: Liquid
• Endotoxin: Less than 3 EU/mL
• Tested for sterility
• Tested for negative mycoplasma contamination

Add 1/1,000 volume of this product to the culture medium and mix thoroughly before use.

Precautions for Use

• This product should be stored frozen (-20°C). To avoid repeated freezing and thawing, it is recommended to aliquot in small volumes and freeze.
• Some precipitates may be visible when dissolving. This will not affect the quality of the product, and it can be used as it is.
  (It is possible to dissolve the precipitates by incubating at 37°C for 1~2 hours, but repeated freezing and heating should be avoided.)

Samples: human ES cells, human iPS cells

Applications:

Colony Formation Test

The human iPS cell 201B7 strain was seeded in StemSure^® hPSC Medium Δ containing bFGF supplemented with CEPT or Y-27632 at 37°C under 5% CO₂ conditions (6-well plates, 1 well= 9.5 cm²).
After at least 16 hours from the start of culture, the medium was replaced with the medium without CEPT or Y-27632, and the culture was continued with media changes every 2 to 3 days.
After checking the cells on day 8, they were fixed with 4% paraformaldehyde, stained with ALP, and colonies were counted.

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Single-cell Cloning Test

The human iPS cell 201B7 strain was seeded in StemSure^® hPSC Medium Δ containing bFGF supplemented with CEPT or Y-27632 at 37°C under 5% CO₂ conditions (96-well plates).
After at least 16 hours from the start of culture, the medium was replaced with the medium without CEPT or Y-27632, and the culture was continued with media changes every 2 to 3 days.
After checking the cells on day 13, they were fixed with 4% paraformaldehyde, stained with ALP, and the number of wells with and without colonies were counted. The colony formation rate was calculated from the number of wells with colonies relative to the total number of wells.

[Result]
When the number of cells seeded was small, more colonies were formed in the CEPT-supplemented cultures than in the Y-27632-supplemented cultures.

Undifferentiated State Maintenance

• Cells: human iPS cell 201B7 strain
• Medium: StemSure^® hPSC Medium Δ + 35 ng/mL bFGF
• Red: rBC2LCN-635 (human iPSC membrane stain)
• Blue: DAPI (nuclear stain)
• Note: BC2LCN is a recombinant lectin with high affinity for cell surface glycans of human ES cells and human iPS cells.

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[Result]
No difference in cell morphology was observed between the addition of CEPT and Y-27632. The undifferentiated state was also maintained.

1. Chen, Y. et al. : Nat. Methods, 18(5), 528 (2021).
   A versatile polypharmacology platform promotes cytoprotection and viability of human pluripotent and differentiated cells.
2. Tristan, C.A. et al. : Stem Cell Reports, 16(12), 3076 (2021).
   Robotic high-throughput biomanufacturing and functional differentiation of human pluripotent stem cells.
3. Tristan, C.A. et al. : Nat. Protoc., 18(1), 58 (2022).
   Efficient and safe single-cell cloning of human pluripotent stem cells using the CEPT cocktail.
4. Deng, T. et al. : Stem Cell Reports, 18(4), 1030 (2023).
   Scalable generation of sensory neurons from human pluripotent stem cells.
5. Takeshi, W. et al. : Regen. Med., 18(3), 219 (2023).
   International evaluation study of a highly efficient culture assay for detection of residual human pluripotent stem cells in cell therapies.