Open-source hardware- and software-based cryomicroscopy system for investigation of phase transitions in cryobiological research.

The development of inexpensive equipment adapted for the study of a specific biological object is very important for cryobiology. In the presented work, we have proposed a simple system for microscopy utilising open-source platform Arduino. Testing this system showed that it had sufficient sensitivity to determine the physical processes occurring in a cryopreserved sample such as intra- and extracellular water crystallisation and salt eutectic. Utilising this system, we investigated the mechanisms of cryoprotection and cryodamage of testis interstitial cells (ICs) in cryoprotective media, which included cryoprotective agents such as dimethyl sulphoxide (Me2 SO), as well as foetal bovine serum or polymers (dextran, hydroxyethyl starch and polyethylene glycol). It was shown that a serum-/xeno-free medium that included 0.7 M Me2 SO and 100 mg/mL dextran was able to reduce intracellular water crystallisation in cells, change the structure of extracellular ice, and reduce salt eutectic and recrystallisation. All these effects correlated with better IC survival after cryopreservation in the medium. This medium is potentially less toxic as it has lower concentrations of Me2 SO compared to serum-containing media developed for cryopreservation of testicular cells. This would pave a way for the creation of nontoxic serum-free compositions that does not require removal before use of cryopreserved living cells for laboratory practice or in clinics.

Phase transitions and mechanisms of cryoprotection of serum-/xeno-free media based on dextran and dimethyl sulfoxide.

The development of serum-/xeno-free media may help avoid the drawbacks of using serum and its components, such as probable contamination, instability of composition, or difficulty in sterilization. The objectives of this research were to investigate the use of combinations of a permeating cryoprotective agent (Me2SO) and non-permeating polymers (polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, hydroxyethyl starch, dextran) for cryopreservation of interstitial cells (ICs) of rat testis, and to propose the mechanism of cryoprotection of such compositions. In the course of this study, the best combination was 100 mg/ml dextran (M.m. 40 kDa) (Dex40) with 0.7 M Me2SO in Ham's F12. The ICs were additionally cooled and warmed to different end temperatures (-30, -50, -50 and -196 °C) to determine which temperature intervals contributed most to the IC loss. Then, the cryoprotective action of this serum-/xeno-free medium was investigated in comparison with serum or albumin-containing media by differential scanning calorimetry (DSC) and thermomechanical analysis (TMA). The results showed that the medium based on Dex40 did not decrease the amount of ice formed. However, it could undergo other phase separation and phase transformation to form glassy states. Potential cell-damaging physical processes such as eutectic crystallization/melting, recrystallization of NaCl and/or Me2SO derivatives, found in serum-containing media and taking place in specific temperature intervals, were not observed in the Dex40 based media. This was in good correlation with indicators of cell survival. Additionally, the application of Dex40 allowed using Me2SO in lower concentrations (0.7 M) than required for serum-containing media (1.4 M), which may decrease the toxicity of serum-/xeno-free media.

The development of the cell cryopreservation protocol with controlled rate thawing.

Thawing in the water bath is often considered as a standard procedure. The thermal history of samples thawed in this way is poorly controlled, but cryopreservation and banking of cell-based products require standardization, automation and safety of all the technological stages including thawing. The programmable freezers allow implementation of the controlled cooling as well as the controlled thawing. As the cell damage occurs during the phase transformation that takes place in the cryoprotectant medium in the process of freezing-thawing, the choice of warming rates within the temperature intervals of transformations is very important. The goal of the study was to investigate the influence of warming rates within the intervals of the phase transformations in the DMSO-based cryoprotectant medium on the cell recovery and to develop a cryopreservation protocol with controlled cooling and warming rates. The temperature intervals of phase transformations such as melting of the eutectic mixture of the cryoprotectant solution (MEMCS), melting of the eutectic salt solution (MESS), melting of the main ice mass (MMIM), recrystallization before MEMCS, recrystallization before MESS and recrystallization before MMIM were determined by thermo-mechanical analysis. The biological experiments were performed on the rat testicular interstitial cells (TIC). The highest levels of the cell recovery and metabolic activity after cryopreservation were obtained using the protocol with the high (20 °C/min) warming rate in the temperature intervals of crystallization of the eutectics as well as recrystallizations and the low (1 °C/min) warming rate in the temperature intervals of melting of the eutectics as well as MMIM. The total cell recovery was 65.3 ± 2.1 %, the recovery of the 3-beta-HSD-positive (Leydig) cells was 82.9 ± 1.8 %, the MTT staining was 32.5 ± 0.9 % versus 42.1 ± 1.7 %; 57.4 ± 2.1 % and 24.0 ± 1.1 % respectively, when compared to the thawing in the water bath.