Cryopreservation of Human iPS Cell Aggregates in a DMSO-Free Solution-An Optimization and Comparative Study

Area of Science:

• Regenerative Medicine
• Stem Cell Biology
• Cryobiology

Background:

• Human induced pluripotent stem cells (hiPSCs) are crucial for regenerative medicine, necessitating effective preservation methods.
• Conventional cryopreservation using dimethyl sulfoxide (DMSO) presents challenges, particularly for multicellular hiPSC aggregates.
• hiPSC aggregates are sensitive to freezing and thawing stresses, impacting their clinical utility.

Purpose of the Study:

• To develop and optimize a DMSO-free cryopreservation solution for hiPSCs.
• To investigate the freezing behavior of hiPSC aggregates in various solutions using low-temperature Raman spectroscopy.
• To enhance post-thaw cell survival and reduce sensitivity to undercooling.

Main Methods:

• Utilized low-temperature Raman spectroscopy to visualize freezing dynamics of hiPSC aggregates.
• Employed an accelerated optimization process with a differential evolution algorithm to identify optimal DMSO-free formulations.
• Tested the cryopreservation efficacy of optimized DMSO-free solutions compared to DMSO-containing solutions.

Main Results:

• hiPSC aggregates showed sensitivity to undercooling in DMSO-containing solutions.
• Developed a DMSO-free solution using non-toxic molecules that improved post-thaw cell survival and reduced undercooling sensitivity.
• The optimized DMSO-free solution demonstrated superior adaptability to different freezing conditions and deviations.

Conclusions:

• A DMSO-free cryopreservation solution can effectively preserve hiPSC aggregates, overcoming limitations of conventional methods.
• Optimization of multi-component DMSO-free solutions is key to enhancing cell protection during freezing and thawing.
• This approach holds promise for improved cryopreservation of hiPSCs and other cell types in translational regenerative medicine.