Modeling cell osmotic behavior for improvement of cryopreservation protocol
DOI:
https://doi.org/10.15407/cryo35.04.196Keywords:
serum-free media, xeno-free media, dextran, penentration coefficient, activation energy, dimethyl sulfoxideAbstract
Cryopreservation of cell suspensions, such as testicular interstitial cells (ICs), which include cells differing in size, volume, and membrane composition, is a very challenging task. Cryobiology requires the determination of several parameters to optimize the cryopreservation procedure, such as membrane water permeability (or hydraulic conductivity) (Lp) and its activation energy (Ea0), the permeability of the cell plasma membrane to dimethyl sulfoxide (DMSO) (Ps) and its activation energy (Ea1), and the osmotically inactive cell volume. This study aims to measure these key cryopreservation parameters for ICs. Additionally, it seeks to evaluate how quickly ICs become saturated with DMSO and to determine the optimal exposure time to cryoprotective media before cooling. By analyzing cell volume changes in different DMSO-based solutions, the research intends to optimize the cryopreservation process, potentially eliminating unnecessary exposure steps while maintaining cell viability. The kinetics of ICs volume changes in cryoprotective media with different DMSO concentrations (0.7—2.8 M), which additionally included 100 mg/ml dextran 40 (0.7DMSO + D40) or 10% FBS (1.4DMSO + FBS), were analyzed using the modified Kedem–Katchalsky model to determine the membrane transport properties. The calculated parameters clearly showed that cell saturation with DMSO occurred within a few minutes after its addition and could reach equilibrium before the estimated start of water crystallization in the samples. Thus, additional exposure of cells to DMSO-containing media was unnecessary. It could potentially lower the metabolic activity of ICs. Accordingly, the cryopreservation procedure for ICs can be shortened by removing the exposure step without reducing viability of ICs in 0.7DMSO + D40 and 1.4DMSO + FBS.
Probl Cryobiol Cryomed. 2025; 35(4): 194—207
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