Method for cryonic preservation of leukocytes with xenon

FIELD: medicine.

SUBSTANCE: invention refers to physiology, cryobiology and medicine, namely to human blood nuclear cell preservation technique with the use of inert gas. The method involves cell saturation in Compoplast 300 plasticised container placed in a metal cryobarocontainer with inert gas, xenon at pressure 0.6 atm for 20min in the room temperature environment of 21±2°C; excessive gas is eliminated, and the biological object is removed from the metal container and frozen to -28°C in ethanol, placed into an electric freezer at -80°C. The bioobject is unfrozen in a water bath at 39±1.5°C for 1.5 min.

EFFECT: implementing the invention provides the reliable cryonic preservation of leukocytes in the inert gas medium and the high level of qualitative and morphological safety of leukocyte concentrate of human blood.

1 tbl, 3 ex

 

The invention relates to the Cryobiology and medicine, in particular to the technology of canning nuclear human blood cells with a gas cryoprotectant.

There is a method of cryopreservation of organs and tissues IN SITU, taken as a prototype. The method consists in the fact that the biological object (heart, kidney, and others) is cooled in water to 0°C with simultaneous saturation of the mixture of xenon, krypton, argon in the ratio of 2.5:47,5:50 vol.%, then displace the water of a specified gas mixture and at a pressure of 1.5 ATM, lower the temperature to -43°C, then decrease the pressure of the gas medium to normal and continue cooling to -196°C [RU (11) 2268590 C1 (51) IPC A01N 1/02 (2006.01)]. The method allowed us to achieve reliable cryopreservation heart of the animal in the composition of the whole body. The transplanted graft, canned six hours, restore adequate cardiac function.

The disadvantage of the prototype is bulky, expensive gelatina technology of freezing (-196°C) and high complexity involving a large number of skilled performers, which significantly reduces the availability of the proposed method. In addition, it involves freezing the whole body of the animal, followed by extraction of specific authority and does not imply effective conservation of nucleated cells of the human body.

T the economic result of the present invention is to develop a reliable method cryosurvival leukocytes in inert gas, ensuring high safety nucleated cells.

The technical result is achieved by saturation of the cell suspensions (leucocytes concentrates) in plastic container Compuplast 300", placed in a metal tribromoethanol, the inert gas is xenon under pressure of 0.6 ATM for 20 min at room temperature with subsequent freezing down to -28°C in ethanol and deposited in elektromedizinische -80°C, the defrost is carried out in a water bath at a temperature of 39±1,5°C for 1.5 min After thawing in a water bath morphologically intact cells in different series of experiments is to 95.3% to 97,6%, resistant to the vital dye membrane has from 43,3% to 64.5% of the cells.

Practically the method is as follows.

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

Example 1.

Leucocytes concentrates blood donor volunteers volume 17±2 ml in plastic container Compuplast 300", placed in tribromoethanol, saturated with an inert gas xenon 20 minutes at room temperature (21±2°C under a pressure of 0.3 ATM. Freezing was carried out on ustopincomes program: in the first stage, 15 min, cooled to -28°C alcohol (96°) bath, in the second phase shifted in the air environment of elektronorgtechnica at -80°C where it was kept during the day. Next Compuplast 300" bioobject were removed from tribromoethanol, pre-reducing the pressure to atmospheric xenon. Was unfrozen bioobjects 10 min in 20-liter water bath at a temperature of 19.6±2,1°C.

Determined the total number of unfrozen cells, their morphological integrity and vitality of the vital dye Trypanosoma blue. The obtained data were expressed in percentage, taking the initial level of 100% (table.1).

When aggregating data to calculate the arithmetic mean value±standard deviation (M±δ). To identify the statistical significance of differences between groups was applied nonparametric test of Wilcoxon signed using a computer program for biomedical statistics "patch BIOSTAT".

Example 2.

Leucocytes concentrates in plastic container Compuplast 300", placed in tribromoethanol, saturated with an inert gas xenon 20 minutes at room temperature (21±2°C under a pressure of 0.3 ATM. Freezing was carried out by two-stage program: in the first stage, 15 min, cooled to -40°C alcohol (96°) bath, in the second phase shifted in the air environment of elektronorgtechnica at -80°C where it was kept during the su is OK. Next Compuplast 300" bioobject were removed from tribromoethanol, pre-reducing the pressure to atmospheric xenon. Was unfrozen bioobjects 10 min in 20-liter water bath at a temperature of 19.6±2,1°C. the data Obtained are presented in table.1.

Example 3.

Leucocytes concentrates blood donors-volunteers in plastic container Compuplast 300", placed in tribromoethanol, saturated with an inert gas xenon 20 minutes at room temperature (21±2°C under a pressure of 0.6 ATM, with the subsequent removal of excess xenon. Freezing was carried out in plastic container Compuplast 300" two-stage program: in the first stage cooled to -28°C alcohol (96°) bath for 15 min, the second stage is shifted in the air environment of elektronorgtechnica at -80°C where it was kept during the day. Was unfrozen bioobjects 1.5 min in 20-liter water bath at a temperature of 39±1,5°C. the data Obtained are presented in table.1.

Table 1
Morphological and functional parameters of leukocytes after daily exposure elektromedizinische -80°C
Use caseViability, %Morfologic the Skye safety, %The safety of granulocytes, %
Example 143,3±3,1to 95.3±5,651,0±14,0
Example 248,3±16,695,1±7,0of 83.6±9,9*
Example 364,5±14,4*the 97.6±2,586±11*
* - the difference with the indicator of example 1 was statistically significant (p<0,05)

The technology specified in Example 3 provides a more effective preservation of nucleated blood cells of man. The proposed method is very simple to implement, gives a stable positive result and will find wide application in medical practice.

The method for cryopreservation of cells includes the saturation of the cells in plastic container Compuplast 300", placed in a metal tribromoethanol, the inert gas is xenon under pressure of 0.6 ATM for 20 min at room temperature 21±2°C with subsequent removal of excess gas and remove it from the metal container, freezing of the biological object to -28°C in ethanol and moving in electromobiles at -80°C, PR is the thawing of the bio-object is carried out in a water bath 39±1,5°C for 1.5 minutes



 

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