RU2290808C2 - Cryoprotective solution for leukocyte freezing at low temperature - Google Patents

Abstract

FIELD: medicine, in particular cryobiology.

SUBSTANCE: claimed solution contains hexamethylenebistetraoxyethyl urea, dimethylsulfoxide, oxymethylethylpyridine succinate and water in specific component ratio. Leukocytes are frost at -70°C.

EFFECT: solution for storage of physiologically full-value cells without losses of physiologically active state thereof after thawing.

3 tbl, 1 ex

Description

The invention relates to cryobiology and medicine, in particular to the creation of a cryoprotective solution for freezing white blood cells, in particular, humans.

As a prototype, we have chosen a cryoprotective solution for freezing white blood cells [Patent 2184449 of the Russian Federation. Cryoprotective solution for freezing white blood cells at a moderately low temperature // Swedentsov EP et al., 2001], including hexamethylene bistetraoxyethylurea, sodium fumarate, citric acid and water.

A significant disadvantage of the prototype is that this solution is not used for freezing white blood cells at low temperatures (-70 ° C), does not contain the optimal composition of strong cryoprotectants GMBTOEM and DMSO (the latter is absent in the prototype), which stabilize extracellular as well as free and bound intracellular water upon cooling and transfer it to amorphous ice, which does not destroy intracellular organelles and the plasma membrane, and eliminate its recrystallization - the formation of large crystals (Belous A.M., Grishchenko V.I., 1994) during thawing, providing high safety and functionally active state of nucleated blood cells.

In the cryoprotective solution for freezing leukocytes at low temperature, containing cryoprotectants - hexamethylene bistetraoxyethylurea (briefly HMBTOEM) and dimethyl sulfoxide (briefly DMSO) - for the first time, a domestic substance is included - hydroxymethylethylpyridine succinate (briefly, which has a membrane-stabilizing effect that has anti-metabolic and anti-inflammatory properties), cell membranes, as well as inhibitory lipid peroxidation processes that occur during cryopreservation. It should be noted that succinate is a derivative of succinic acid (Nesmeyanov AN, Nesmeyanov NA The beginning of organic chemistry. Book 1. - M .: Chemistry, 1974. - p. 314-315, p. 436-437) - participates in the energy metabolism of the cell, ensuring its vital activity, and pyridine in the nucleic acid metabolism (German A. S. Piridin. Big Medical Encyclopedia. - M .; 1981. - T.17. - p. 1054-1055). The cold barrier solution has an optimum pH of 7.0-7.4 for leukocyte cells. All ingredients used in this solution are produced in the Russian Federation.

An example of using the tool

A cryoprotective solution containing 22% HMBTOEM, DMSO 8%, oxymethylethylpyridine succinate 0.2%, water for injection up to 100 ml and having a pH of 7.0-7.4 is mixed in a 1: 1 ratio with leukocyte concentrate in a polymer container Compoplast. The mixture, having stood for 8 min at room temperature, is cooled exponentially in an electric freezer in a 4-liter bath with ethyl alcohol 96 ° to -27 ° C for 19-20 minutes. Then the frozen leukoconcentrate is transferred to the chamber of the electric freezer, having a temperature of -70 ° C. The thermogram is recorded using a thermocouple inserted into the Compoplast container with a cryoprotective solution in a final concentration. Defrosting is carried out in a 20-liter water bath at a temperature of + 38 ° C for 30-45 seconds when the container is shaken 3-4 times a second. The temperature of the thawed leukoconcentrate in the container should be between + 2- + 4 ° С. Then determine the morphological safety of thawed leukocyte cells, their viability by vital dye (with 1% eosin solution) and the phagocytic activity of neutrophils in a sample with latex (according to Potapova SG et al., Problems of hematology and blood transfusion. - 1977. - № 9. - p. 58-59). The calculation is expressed as a percentage in comparison with similar indicators of cells before freezing.

Experimental studies were performed on the basis of blood cryophysiology laboratories at the Institute of Physiology of the Komi Scientific Center, Ural Branch of the Russian Academy of Sciences, and blood and tissue conservation at the Kirov Research Institute of Hematology and Blood Transfusion.

Three variants of a cryoprotective solution for leukoconcentrate, differing in the concentration of the ingredients included in them, were studied. The composition of the solution options is presented in table 1.

Table 1
Characterization of the composition of various variants of a cold-enclosing solution for freezing white blood cells at low temperature, per 100 g of dry matter (g). Variant number of refrigerant solution The composition of the cooling solution GMBTOEM, in% DMSO, in% OMEPS, in% Water for injection, in ml. one 24 6 0.30 Up to 100 2 22 8 0.20 Up to 100 3 twenty 10 0.10 Up to 100

The final concentration of the substances included in the solutions of N1, N2 and N3 after mixing 1: 1 with leukoconcentrate turned out to be 2 times lower than the initial (table 2), the pH of the medium did not change, remaining in the range of 7.0-7.4.

Table 2.
The final concentration of the ingredients in various versions of the cold barrier solution after mixing with leukocyte concentrate. Variant number of refrigerant solution The final concentration of the ingredients in various versions of the cold barrier solution after mixing with the suspension GMBTOEM, in% DMSO, in% OMEPS, in% Water for injection, in ml. one 12 3 0.15 Up to 200 2 eleven four 0.10 Up to 200 3 10 5 0.05 Up to 200

Cooling is carried out according to an exponential program: a plastic container with a leukoconcentrate is immersed in a 4-liter alcohol bath cooled to -70 ° C, after 19-20 minutes the container with a frozen bioobject is removed from the bath and transferred to the chamber of an electric freezer with a temperature of -70 ° C for storage . Freezing is controlled by a thermogram recorded by a sensor located in a container with a simulator-solution of cryopreservative with a final concentration of its ingredients. Defrosting is carried out in a 20-liter water bath at a temperature of + 38 ° C for 30-45 seconds, shaking the container 3-4 times per second. The leukoconcentrate is heated to + 2 ° C - + 4 ° C. Leukocyte studies were performed on morphological safety and functional activity (table 3).

Table 3.
The results after freezing to -70 ° C and thawing leukocytes with various variants of the proposed coolant solution Variant of a cooling solution White blood cell viability Morphological safety,% Phagocytic activity of neutrophils,% The upper limit of the dosage of the ingredients of the solution 82.8 ± 13.41 55.0 ± 7.21 * n = 6 n = 6 The optimal ratio of the ingredients of the solution 94.5 ± 6.89 75.5 ± 8.50 n = 6 n = 6 The lower limit of the dosage of the ingredients of the solution 68.2 ± 18.34 57.0 ± 4.00 * n = 6 n = 6 * - significant difference (p <0.05)

The biological usefulness of leukocytes, primarily neutrophils, is determined by their phagocytic activity. This test, which most reliably characterizes the physiological properties of cells, was the best after freezing-heating using a cold-enclosing solution with the optimal ratio of its ingredients.

The ready-made optimal solution is transparent, with a slight yellowish tint, odorless, pH = 7.0-7.4, mixes well with leukocyte concentrate in all ratios, does not cause conglomeration of cells and ensures functional and morphological preservation at a positive temperature until freezing. The solution exhibits a pronounced cold-protective property when the white blood cells are cooled according to the exponential program from + 20 ° С to -70 ° С.

Thus, the developed cryoprotective solution when freezing leukoconcentrate to -70 ° C ensured the preservation of a high percentage of physiologically active leukocyte cells. It can be used in medical and biological institutions, where there is the necessary refrigeration equipment.

Claims (1)

A cryoprotective solution for freezing white blood cells containing hexamethylene bistetraoxyethylurea, oxymethylethylpyridine succinate and water for injection, characterized in that the white blood cells are frozen to -70 ° C, and the solution additionally contains dimethyl sulfoxide, in the following ratio of ingredients,%: Hexamethylene bistetraoxyethylurea 20.0-24.0 Dimethyl sulfoxide 6.0-10.0 Oxymethylethylpyridine Succinate 0.3-0.1 Water for injections up to 100 ml