Hladograd solution for freezing of cells in submarine-low temperature

 

(57) Abstract:

The invention relates to medicine and physiology, concerns the creation hladograd solution for freezing of cells and store them in a physiologically active state after thawing. Hladograd solution for freezing of cells in a ratio of 1:1 at moderately low temperatures and pH 7.0 to 7.4, containing hexamethylenetetramine and water for injection, with frozen leukocytes to -20°C, and the solution further contains oxytetracyline succinate in the following ratio of ingredients, %: hexamethyltriethylenetetramine 26,0-30,0, oxytetracyclin succinate 0.1 to 0.2, the water for injection to 100 ml of the Technical result of the claimed invention - hladograd solution at the freezing lacokanostra to -20°ensures the safety of the high percentage of physiologically active leukocyte cells. 3 table.

The invention relates to physiology, medicine, namely to create hladograd solution for freezing of cells, in particular human.

As a prototype we have chosen createsite solution for freezing of cells [Patent 2184449 of the Russian Federation. Kriseman what would be hexamethyltriethylenetetramine, fumarate sodium, citric acid and water.

A significant disadvantage of the prototype is that the sodium fumarate, applied in solution to cryosurvival leukocytes, has no membrane-stabilizing action, not restores the structure and function of membranes, does not inhibit the processes of lipid peroxidation, as well as the specified solution for freezing of cells to -20°C is not used, because this method has not been developed.

The technical result of the present invention is to provide hladograd solution for freezing of cells at temperatures up to -20°C.

The technical result is achieved by the fact that the solution for freezing of cells in submarine-low temperature (down to -20°C), including cryoprotector-hexamethyltriethylenetetramine (BTOAM) and water further comprises oxitetraciclina succinate (AMAPS). Oxitetraciclina succinate (MAPS) has antioxidant and membrane-stabilizing effect, restores the structure and function of cell membranes and inhibits the processes of lipid peroxidation, which take place when the cryoconservation. It should be noted that the - n-317-319, S. 321-322] are involved in energy metabolism of cells, providing livelihoods, and the pyridine - nucleotide exchange [Herman A. C. Pyridine. Great medical encyclopedia. - M., 1981. - So 17. - S. 1054-1055]. Hladograd solution is optimal for leukocyte cell pH 7,0-7,4. All the ingredients used in this solution, obligatory trope leukocyte cells and are produced in the Russian Federation.

EXAMPLE OF USE

Hladograd solution containing BTOAM at a concentration of 28%, oxitetraciclina succinate to 0.15%, water for injection to 100 ml and having a pH of 7.0 to 7.4, mixed in the ratio of 1:1 concentrate of cells in the polymeric container Compuplast”. The mixture stand for 20 min at room temperature, cooled in an exponential program in electrical freezer in 4 litre tub with ethyl 96° alcohol having a temperature of -28°C to -20°C for 9-14 minutes. Then the frozen lacokanostra transferred into the chamber of elektronorgtechnica, in which temperature -20°C. thermogram recorded by thermocouple, is introduced into the container Compuplast” with hladograd solution at a final concentration. Defrosting is carried out in a 20-liter water bath at t is concentrate in the container should be in the range from +2 to +4°C. Then determine the morphological preservation thawed leukocyte cells, their viability by vital dye (1% solution of eosin) and phagocytic activity of neutrophils in the sample with latex (Potapova, S. et al.//Problems of Hematology and blood transfusion. - 1977. - N9. - S. 58-59). The calculation is expressed in percentage in comparison with the same indicators of the cells prior to freezing.

Experimental investigations carried out on the basis of the laboratory of criticially blood Institute of physiology, Komi science centre, Ural branch of RAS and preservation of blood and tissue SE Kirov research Institute of Hematology and blood transfusion health Ministry.

Studied three options hladograd solutions for lacokanostra different concentrations of their constituent ingredients. The solutions were presented in table 1.

The final concentration of the substances included in the composition of the solutions N1, N2 and N3 after mixing 1:1 with lacokanostra was 2 times lower than the initial level (table 2), pH of the medium was not changed, remaining within 7,0-7,4.

When conducting research on the introduction of leukocytes in the cold-suspended animation (-20°C) defined by their morphological integrity and functional actively the focus of their phagocytic activity. This test is most reliably characterize the physiological properties of cells, was better after freezing - thawing using hladograd solution with the optimal ratio of its ingredients.

Ready the optimal solution is transparent, slightly yellowish, odorless, pH 7.0 to 7.4, well miscible in all proportions with leucocytes concentrate, does not cause a conglomeration of cells and provides the functional and morphological integrity at positive temperature before freezing. The solution exhibits a pronounced hladograd property during freezing of cells in exponential program with +20°C to -20°C.

Thus, the developed hladograd solution at the freezing lacokanostra to -20°With assured high percentage of physiologically active leukocyte cells. It can be applied in health care, where there is the necessary refrigeration equipment.

Hladograd solution for freezing of cells in a ratio of 1:1 when merenneito temperature and pH 7.0 to 7.4, containing hexamethylenetetramine and water for injection, characterized in that the same applies to the ratio of ingredients, %:

Hexamethyltriethylenetetramine 26,0-30,0

Oxitetraciclina succinate 0,1-0,2

Water for injection To 100 ml



 

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