Cryopreserving agent for blood nucleocyte freezing

FIELD: medicine.

SUBSTANCE: solution contains dimethylacetamide - 3.5 ml, hydroxyethylstarch (as a part of Infukol) - 46.3 ml and a phosphate buffer (NaH2PO42H2O, mole) in an amount required to provide a pH value of the solution of 6.5-7.0. The presented cryophylactic for blood nucleocyte freezing to -80C and further thawing provides the safety of 94.2-97.1% functionally active cells, for freezing to -196C and thawing provides the safety of 92.7-97.8% functionally active cells.

EFFECT: improving the solution properties.

4 ex

 

The invention relates to medicine and blood transfusion and for the creation of funds - protecting solution for cryopreservation of nuclear blood cells at low and ultralow temperatures.

Combined cryoprotective solution for freezing nuclear blood cells has the following composition: dimethylacetamide (HC) - 3.5 ml; gidroxiatilkrahmal (in the "Infocol") - 46,3 ml; phosphate buffer (NaH2PO42H2O mol) in an amount necessary to ensure the pH 6,5-7,0.

Experimental studies performed in the laboratory canning blood and tissues of Federal state budgetary institution of science "Kirov research Institute of Hematology and blood transfusion of the Federal medical-biological Agency".

As a prototype we have chosen hladograd solution "Lackritz" for the freezing and storage of cells at -196C for clinical purposes // Troshina V.M. 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), including endocellular cryoprotector dimethylacetamide, glucose, disodium salt of EDTA and water for injection.

A significant disadvantage of the prototype is that proposed his car is AMI enclosing the solution does not contain hydroxyethylamine (BSE), which has a number of positive effects: the extracellular immunologically inert and non-toxic.

The technical result of the invention is the creation of cryoconserved for freezing nuclear blood cells at -80C and -196C, providing high morphological preservation of thawed cells.

The technical result is achieved by the fact that cryoconserved additionally contains cryoprotector gidroxiatilkrahmal (in the "Infocol"), 1) with the extracellular cryoprotective effect that complements endocellular hladograd influence dimethylacetamide cells and makes the combined cryoconserved more fully, giving when cooled to -80C and -196C expressed cryobiological (technical) effect, and 2) the inclusion of non-toxic "Infocol" and dimethylacetamide in non-toxic to cells concentration in cryoconserved does not require launder before use and reduces the risk of post-transfusion complications. Cryoconserved has a pH 6,5-7,0 conducive to blood cells. Its ingredients obligatory trope nuclear blood cells and are produced in the Russian Federation.

Example preparation of cryoconserved for freezing 50 ml suspension of nuclear blood cells to -80C

In a sterile room at a temperature of +22C in a volumetric flask of 50 ml with personnel who provide 3.5 ml of dimethylacetamide (HC), to him, using a graduated pipette, add 46,3 ml "Infocol" with constant stirring. To the mixture are added dropwise phosphate buffer (NaH2RHO42H2O mol) in an amount necessary to ensure a pH of 6.5 to 7.0. The bottle with the protective solution is hermetically sealed, labeled, sterilized by autoclaving (1.2 ATM - 30 minutes).

Thus prepared protective solution in an amount of 5 ml send to sanitary and bacteriological examination. In the absence of bacterial contamination protective solution can be used for canning nuclear cells to -80C. the Prepared solution was stored in a refrigerator at +2+4C.

An example of using cryoconserved for freezing the suspension of nuclear blood cells to -80C

The preservative solution comprising dimethylacetamide (HC) at a concentration of 7%, "Infocol in concentration of 92.6%, phosphate buffer (NaH2PO42H2O mol) in an amount necessary to ensure a pH of 6.5 to 7.0, with constant stirring, mixed in a volume ratio of 1:1 with concentrated nuclear blood cells in polymeric container Compuplast", the latter is placed in a metal canister-holder. Cell suspension incubated 20 min at room temperature and placed for 24 hours in an electric freezer, cooled to -80C, dal the first storage is carried out at a specified temperature.

Defrost concentrate cells is carried out in a 20-liter water bath, heated to +38C for 45-60 seconds with vigorous shaking of the container. The temperature of the thawed concentrate nuclear cells in the container should be +2+4C. Further investigate the morphological preservation thawed nuclear 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. - N 9. - S. 58-59). The results are expressed in percentage in comparison with the similar indicators of formed elements to freeze.

Example preparation of cryoconserved for freezing 50 ml suspension of nuclear blood cells to -196C

In a sterile room (laminar flow Cabinet) at a temperature of +22C in a volumetric flask of 50 ml pour 3.5 ml of dimethylacetamide (HC), to him, using a graduated pipette, add 46,3 ml "Infocol" with constant stirring. To the mixture are added dropwise phosphate buffer (NaH2PO42H2Oh mol) in an amount necessary to ensure a pH of 6.5 to 7.0. The bottle with the protective solution is hermetically sealed, labeled, sterilized by autoclaving (1.2 ATM - 30 minutes). Thus prepared protective solution is examined for bacterial sagra is out. In the absence of bacterial contamination protective solution can be used for canning nuclear cells to -196C.

An example of using cryoconserved for freezing the suspension of nuclear blood cells to -196C

The preservative solution comprising dimethylacetamide (HC) at a concentration of 7%, "Infocol in concentration of 92.6%, phosphate buffer (NaH2RHO42H2Oh mol) in an amount necessary to ensure a pH of 6.5 to 7.0, with constant stirring, mixed in a volume ratio of 1:1 with concentrated nuclear blood cells in polymeric container Compuplast", the latter is placed in a metal canister-holder. Cell suspension incubated 20 min at room temperature and placed in a software freezer cell suspensions. At the first stage of freezing the biomaterial is produced at a rate of 1C/min to -80C, after which the holder with cells transferred into biogenesis with liquid nitrogen (temperature -196C). Defrost concentrate cells is carried out in a 20-liter water bath, heated to 38C for 90 seconds with vigorous shaking of the container. The temperature of the thawed concentrate cells in the container should be +2+4C. Further investigate the morphological preservation thawed nuclear cells, their viability by vital dye (1% solution easy the (a) and phagocytic activity of neutrophils in the sample with latex (Potapova YEAR et al., W. Problems of Hematology and blood transfusion. - 1977. - N 9. - S. 58-59). The results are expressed in percentage in comparison with the similar indicators of formed elements to freeze.

Experimental investigations carried out on the basis of laboratory preservation of blood and tissue Ekaterinburg, Russia microstructure Kirov research Institute of Hematology and blood transfusion of the FMBA of Russia.

The ready solution of cryoconserved transparent, no color, no smell, its pH is 6.5-7.0mm. Ready chriopractic well mixed with the concentrate of nuclear blood cells, does not cause formation of conglomerates of cells and provides them functional and morphological integrity during exposure. Cryoconserved manifests expressed hladograd properties after freezing nuclear blood cells as -80C and -196C.

Developed chriopractic for freezing nuclear blood cells at a temperature of -80C ensured the preservation of a 94.2 97.1 per cent of functionally active cells. The same cryoconserved at a temperature of -196C ensured the safety of 92.7-97.8% of functionally active cell.

Thus, the new cryoconserved can be used in medical institutions and cryobiological profile where there are conditions for the freezing and storage of blood cells at low and ultralow temperatures.

Cryoconserved for freezing nuclei is s blood cells, containing dimethylacetamide, characterized in that it additionally contains gidroxiatilkrahmal (in the "Infocol") and phosphate buffer (NaH2PO42H2Oh mol) in the following ratio of initial components:

dimethylacetamide (HC)3.5 ml
gidroxiatilkrahmal (in the "Infocol")46,3 ml

phosphate buffer (NaH2PO42H2O mol) in an amount necessary to ensure a pH of 6.5 to 7.0.



 

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