Protective solution for preserving leukocyte at temperature -10°c

FIELD: medicine, physiology.

SUBSTANCE: invention relates to the development of agent as a protective solution used for preserving leukocytes at negative temperatures and their retaining in physiologically active state after escape from cold hypobiosis. The solution (pH 7.0-7.4) contains glycerol, hydroxymethylpyrimidine succinate and modegel taken in the optimal ratio. The protective solution provides safety of high percent of functionally active leukocyte cells.

EFFECT: valuable properties of solution.

3 tbl, 2 ex

 

The invention relates to medicine and physiology and for the creation of a protective solution for the preservation of cells at a temperature of -10°C.

As a prototype we used Wmini et al. "Using dimethylacetamide as a cryoprotectant during freezing of granulocytes" (W. Problems of Hematology and blood transfusion. M: Medicine. - 1977. No. 5. - p.50-54). A significant disadvantage of the prototype is that proposed by its authors enclosing the solution does not contain a stabilizing and antioxidant substances - oxitetraciclina succinate, which restores the impaired functions of cell membranes and inhibiting the processes of lipid peroxidation (Reference VIDAL. Drugs in Russia: a Handbook. M: Attraversare. - 2001. - S-349). In the prototype are not also part of genotype to the tissues and cells of the human body glycerin and drug modesel used for canning and resuspendable erythrocytes at positive temperature, consisting mainly of modified gelatin with a molecular mass of 13000-20000 (Agranenko VA, Tibilov NN. Preservative solutions for blood and its components. Reference "Blood substitutes, preservatives blood and bone marrow" under the editorship of the Great GN. M: Medicine. - 1997. - S-145).

The technical result of this is th invention is to provide a protective solution for the preservation of cells at a temperature of -10° C. This protective solution ensures the integrity of the high percentage of functionally active leukocyte cells and can be used in institutions cryobiological and medical profile, affluent household elektromedizinische at -10°C.

The technical result is achieved by the fact that in the proposed protective solution for the preservation of cells at a temperature of -10°enabled hladograd substances: the highest quality glycerin-derived succinic acid - oxitetraciclina succinate (briefly MAPS)with antioxidant and membrane-stabilizing effect, restores the function of cell membranes, inhibits lipid peroxidation, Succinate participates in the energy metabolism of cells (D. Metzler Biochemistry. Chemical reactions in a living cell. Vol.2. - M.: Mir, 1980. - S-319 and s-322). As a component in the Protective solution introduced by modesel is a drug consisting of modified gelatin with mm - 13000-20000, sodium hydronitrate disubstituted, sodium bicarbonate, sodium chloride and water for injection (Agranenko VA, Tibilov NN. Preservative solutions for blood and its components. Reference "Blood substitutes, preservatives blood and bone marrow" under the editorship of the Great GN. M: Medicine. - 1997. - S-145). Tread the solution has a pH of 7.0 to 7.4, blahop etny for blood cells. The ingredients in this solution obligatory trope leukocyte cells and are produced in Russia.

An example of obtaining protective solution (No. 2) for the conservation of leukocytes at -10°C.

In a sterile room at a temperature of+22°in a volumetric flask of 100 ml pour 70 ml of modeule, to him, using a graduated pipette, add 7 ml of glycerol (reagent grade.), 6 ml of a 5% solution of oxyethylenenitrilo succinate with constant stirring. To the mixture is added dropwise a 1 M solution of sodium hydroxide to achieve a pH of 7.0 to 7.4. With the help of modies bring the solution volume to 100 ml of Thoroughly mixed, produce control pH. the pH should be between 7.0 to 7.4.

Thus prepared protective solution in an amount of 5 ml send to barcontrol. All the ingredients, laboratory glassware, as well as gloves, gown, Shoe covers, mask, cap assistant must be sterile.

The bottle with the protective solution is hermetically sealed, eticaret and stored until use at room temperature in the dark place.

In the absence of bacterial contamination protective solution may be used for preservation of cells at -10°C.

EXAMPLE of USE

Protective solution comprising glycerol at a concentration of 7%, oxitetraciclina succinate 0,3%, modiel to 10 ml, sodium hydroxide 1 M solution to a pH of 7.0 to 7.4, mixed in equal amounts (1:1) with leucocytes concentrate in polymer container Compuplast". The mixture was incubated 20 min at room temperature and placed for 24 hours in an electric freezer (in a 3-liter bath with ethanol 96° alcohol, cooled to -10°). It should be noted that as the tread the mortar, and mix it with lacokanostra in the ratio of 1:1 at -10°do not freeze. The warm mixture is carried out in a 20-liter water bath, heated to +38°With in 2-3 seconds with vigorous shaking of the container. Temperature ottange of lacokanostra in the container should be within +2÷+4°C. Further investigate 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 A.C. et al., W. Problems of Hematology and blood transfusion. - 1977. - N9. With 58-59). The results are expressed in percentage in comparison with the similar indicators of formed elements to freeze.

EXPERIMENTAL STUDIES

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 flow the of blood.

The study were subjected to three options protective solution for leucocytes concentrate different concentrations included in their ingredients. The solutions were presented in table. 1.

After mixing 1:1 with lacokanostra final concentration of substances that are part of the solutions N1, N2 and N3 was 2 times lower than the initial level (table 2); pH remained in the range of 7.0 to 7.4.

With the introduction of leukocytes in cold geobios (-10°studied for their morphological integrity and functional activity (table 3).

The biological state of cells, especially neutrophils, determines their phagocytic activity. This indicator reliably characterize the biological integrity of the cells, was best after a cooling-warming when applying protective solution with an optimal ratio of its ingredients.

Optimum tread the solution is clear, without color, odorless, pH=7,0-7,4, mixes easily with leucocytes concentrate, does not cause the formation of conglomerates of cells, and ensures their high functional and morphological integrity before and after the cooling-warming. The solution shows a clear crotamiton property during cooling of leukocytes with +10&x000B0; C to -10°C.

Thus, the developed protective solution for the preservation of cells at a temperature of hypothermia assured high percentage of functionally active leukocyte cells. It can be used in institutions cryobiological and medical profile, which includes ordinary household electrocoloring with a freezer at -10°C.

Protective solution for the preservation of cells at a temperature of -10°characterized by the fact that it contains glycerin, oxitetraciclina succinate, modiel in the following ratio of ingredients, %:

The highest quality glycerin5,0-9,0
Oxitetraciclina succinateof 0.075 to 0.3
Modeselto 100 ml
Sodium hydroxide 1 M solutionto pH 7,0-7,4



 

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