Thermodynamic and structural characteristics of cryoprotective media for cryopreservation of cells

Despite the fact that cryopreservation methods are actively developing, when spermatozoa are frozen, cell loss is 40-60%. One of the main causes of cell damage at low temperatures is the formation of extra- and intracellular ice crystals. It is possible to influence the formation of crystals using various temperature regimes, as well as adding special components of cryoprotective media.

Study of the influence of various components and conditions of freezing on the process of crystal formation in cryoprotective media.

To obtain thermodynamic parameters, the method of adiabatic calorimetry was used, adapted for the study of liquid media. The study of the process of crystal formation was carried out on the PETRA III synchrotron (DESY, Germany). We studied a water-glycerol solution with a glycerol concentration of 12% by volume with the addition of albumin (4 mg / ml) and sucrose (0.5 M).

In the course of the work, the dependences of the heat capacity on temperature were obtained for an aqueous solution of glycerol (12%), as well as this solution with the addition of albumin and sucrose. It has been shown that the addition of sucrose reduces the area of ​​heterogeneous nucleation, which may contribute to better cell survival. The method of X-ray diffraction analysis was used to study the effect of the components of the medium on the formation of crystals in an aqueous solution of glycerol with a volume of 20 μL during vitrification. It was shown that the addition of sucrose (0.5 M) reduces the size of crystals formed upon freezing a water-glycerol solution, which is consistent with the results obtained by the method of adiabatic calorimetry. Also, the sizes of crystals were estimated for each test solution when freezing in large volumes (1.7 ml) by the method of slow freezing. It has been shown that the average size of crystals in a solution with all components is 8 ± 1 μm. In the course of the experiment, an uneven distribution of crystals within one test tube was found when placed vertically. In this regard, a horizontal method for placing cryovials was proposed. It is shown that, with such an arrangement, the formation of crystals is statistically significant and occurs more uniformly with a minimum scatter in comparison with the vertical arrangement.

In total, during the work, more than 400 solutions and 30 types of alternative frosts were studied. As a result of the analysis, 2 main methods of freezing were selected as the most effective: horizontal classical freezing, as well as automatic lowering of the sample over nitrogen vapor at a constant speed.