Cryopreservation of primate embryonic stem cells with chemically-defined solution without Me2SO

Area of Science:

• Stem Cell Biology
• Cryobiology
• Regenerative Medicine

Background:

• Human embryonic stem cells (hESCs) possess pluripotency, making them valuable for regenerative medicine and tissue engineering.
• Efficient and reliable cryopreservation methods are essential for maintaining hESC viability and function.
• Current cryopreservation techniques may not fully preserve hESC characteristics.

Purpose of the Study:

• To develop and evaluate a highly efficient cryopreservation method for primate embryonic stem cells.
• To establish a chemically-defined, dimethyl sulfoxide (DMSO)-free medium for vitrification of hESCs.
• To assess the viability, pluripotency, and differentiation potential of cryopreserved hESCs.

Main Methods:

• Vitrification of cynomolgus and human ES cells using a serum-free, DMSO-free medium.
• The cryopreservation medium comprised Euro-Collins solution, ethylene glycol (EG), and polyethylene glycol (PEG).
• Comparison of vitrification with conventional slow-freezing methods; assessment of cell recovery, marker expression, and teratoma formation.

Main Results:

• Vitrification yielded a significantly higher recovery ratio (22.9+/-7.7%) compared to slow-freezing (0.4+/-0.2%).
• Cryopreserved hESCs maintained expression of key undifferentiated markers (Nanog, Oct-4, SSEA-4, alkaline phosphatase activity).
• Pluripotency was confirmed by successful teratoma formation in SCID mice post-transplantation.

Conclusions:

• A novel vitrification method using a DMSO-free, chemically-defined medium provides highly efficient cryopreservation of primate ES cells.
• This method ensures the maintenance of hESC viability, pluripotency, and differentiation capacity after thawing.
• The established protocol offers a reliable approach for banking and utilizing valuable hESC lines in research and clinical applications.