Improved and reproducible cell viability in the superflash freezing method using an automatic thawing apparatus

Area of Science:

• Biotechnology
• Cell Biology
• Cryobiology

Background:

• Cell cryopreservation preserves cells but can cause damage via cryoprotecting agents (CPAs).
• Previous CPA-free inkjet methods suffered from devitrification during thawing, reducing cell viability.
• Addressing CPA-induced cell injury and thawing-related damage is crucial for effective cryopreservation.

Purpose of the Study:

• To develop an automated, rapid thawing apparatus for CPA-free cryopreservation.
• To improve cell viability and reproducibility compared to existing methods.
• To overcome limitations of CPA-free methods that involve atmospheric exposure during thawing.

Main Methods:

• Developed an automatic thawing apparatus utilizing a spring hinge for rapid cell transfer.
• Employed high-speed camera observation to analyze cell transport time (within 20 ms).
• Conducted heat-transfer simulations to ensure cells remained below the glass-transition temperature during thawing.
• Validated the apparatus using mouse fibroblast 3T3 cells.

Main Results:

• The apparatus rapidly transports vitrified cells into a prewarmed medium within 20 milliseconds.
• Heat-transfer simulations confirmed cells stayed below the glass-transition temperature during transit.
• Evaluation with 3T3 cells demonstrated improved cell viability and enhanced reproducibility.
• The rapid thawing process effectively circumvented issues associated with CPA cytotoxicity.

Conclusions:

• The developed automatic thawing apparatus significantly improves cell viability in CPA-free cryopreservation.
• Rapid thawing, combined with superflash freezing, offers a promising alternative to conventional CPA-based methods.
• This technology enhances the reliability and effectiveness of preserving cells for research and clinical applications.