Theoretical approaches to determination of optimal cryopreservation regimens for cell spheroids of different cultivation terms

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Background: Three-dimensional culture systems are unique platforms for studying complex biological processes in vitro. Cell-cell and cell-extracellular matrix interactions form a communication network of biochemical and mechanical signals, bring spheroids (SP) closer to native tissues and significantly distinguish them from monolayer cultures. It is important for cell technologies to develop methods for cryopreservation of 3D cultures, that allows creating the stocks of valuable cell samples, save time and materials, and prevent the loss of cultures due to technical failures, contamination, phenotype drift and aging. Objectives: Development of approaches to cellular spheroids cryopreservation. Determination of the permeability parameters of L929 cells spheroids at different cultivation periods for the theoretical assessment of optimal freezing regimens. Materials and methods: We have used L929 cells, which form SPs of different diameters and can be maintained for a long time in 3D conditions. To determine the integral filtration Lp and permeability for DMSO kp coefficients for SP at different periods of cultivation, the volumetric method was used. The study of the changes in the spheroids volume in time was carried out with a confocal microscope LSM 510 META. The numerical values of the integral SF permeability coefficients were determined by approximating the experimental data on the change in the relative volume of the SP versus the exposure time in the test solution with theoretical curves calculated on the basis of a physical and mathematical model for passive mass transfer between the spheroid and the environment, provided that they coincide as much as possible. Prediction of the osmotic behavior of spheroids under cooling conditions was carried out based on the differential equation describing the kinetics of changes in the relative cell volume during extracellular crystallization of a cryoprotective solution, substituting determined values of integral permeability coefficients Lp and kp and activation energies EAL and EAk into the model equations. The kinetics of changes in the extracellular solution concentration during freezing was set analytically by approximating the phase melting diagram of the DMSO solution. Results: The filtration and permeability for DMSO molecules coefficients in SP were determined and their significant decrease with a cultivation duration was shown. The activation energy values for the penetration of water and DMSO molecules into the SP were calculated and their dependence on the cultivation time was determined. Proceeding from the determined parameters of permeability, the dynamic of changes in the volume of SPs for different periods of cultivation at different rates of cooling was calculated. Conclusions: The optimal cooling modes of SP from L929 cells were in theory determined: for 7 days of cultivation — 1,5-2 °C/min with cooling to -80°C and subsequent immersion in nitrogen; for 14 and 21 days of cultivation — 0.5 °C/min to -40°C and subsequent immersion in nitrogen.