Method of reducing low-temperature jump of cryoprotectant solutions

FIELD: chemistry.

SUBSTANCE: method of reducing impact of low-temperature jump on cryoprotectant solution is provided at the expense of remote processing of cryoprotectant solution with cells of living organisms by ultrasonic radiation with frequency 0.50-10 before its freezing.

EFFECT: reduction of low-temperature jump of cryoprotectant solutions, which makes it possible to eliminate overcooling effect, provide continuous and smooth character of freezing and increase integrity of defrosted cells after cryoconservation.

1 dwg

 

The invention relates to the field of Cryobiology and can be used to reduce the temperature jump at the crystallization solution with the cells of living organisms.

There is a method of reducing low-temperature jump by depositing a seed for crystal formation (iodide of silver, ice crystals) (see kN. Pushkar N. S., Belous A. M. Introduction to Cryobiology. - Kiev: Naukova Dumka, 1975. - S. 21)

However, this method of ethnological and is acceptable only for small amounts of frozen material.

The closest in technical essence is a method of mechanical seeding, which is performed at temperatures (-5)C(-7C) manually by touching the tweezers or other object to the tube containing the solution with cells (see kN. Himenkov A. C., Broskow A. C. Introduction to structural Cryology. - M.: Nauka, 2005. - S. 203).

However, this method of reducing the temperature jump when cryopreservation is costly and time-consuming.

The technical objective is to create a method of reducing the low-temperature jump at the crystallization solutions of cryoprotectants that can improve the integrity deportirovannykh cells after cryopreservation.

The technical result - the reduction of low-temperature spike solutions of cryoprotectants during freezing of living cells Organismo is.

It is achieved by the fact that in the method, including freeze georaster with biological material in liquid nitrogen, before the operation of the freezing of a solution of cryoprotectants with the cells of living organisms carry out remote impact on a frozen solution of ultrasonic radiation frequency of 0.50 to 10 MHz.

The method is as follows.

Cells of living organisms diluted with cryoprotectant, put on equilibration. Before freezing the mixture of cryoprotectant with the cells of living organisms influence of ultrasonic radiation to the entire area of the solution, at a distance which depends on the power of the emitter (the bigger the radiator, the greater the distance), frequency of 0.50 to 10 MHz, since the data the parameters of the signal observed decrease in low-temperature spike solutions of cryoprotectants, which affects the increase integrity deportirovannykh cells after cryopreservation.

Example 1. Work conducted in the laboratory "Water bioresources and aquaculture" southern scientific center, Russian Academy of Sciences together with the research laboratory cryo technologies in aquaculture" Astrakhan state technical University.

Researched 4 solution - distilled water; the base solution for sturgeon (NaCl - 6.5 g/l, KCl, 0.25 g/l, CaCl20.3 g/l NaHCO3- 2 the/l, Tris-HCl to pH 7.5-8) (see Art. Burzawa-Gerard, E., Goncharov B. F., Dumas, A., Fontaine, Y. A. Further biochemical studies on carp gonadotropin, biochemical and biological comparison of c-GTH and a gonadotropine from Acipenser stellatus Pall. // Gen.Comp.Endocrinol. 1976. V. 29, No. 4. - P. 499); the base solution +10% dimethyl sulfoxide; the base solution +10% DMSO +10% egg yolk.

Containers for freezing were made of quartz glass, because through quartz glass are ultrasonic waves. Sample volume was 50 l. The thickness of a layer is 0.2 mm, the Freezing of the samples was performed in two variants. The first control, without ultrasound, the second with exposure to ultrasonic radiation (frequency 0,88 MHz with a power of 1 W/cm2at a distance of 1 cm from the ultrasonic emitter) to the entire area of the solution.

The solutions were frozen in liquid nitrogen steams under (-130)C. the Temperature was measured by thermometer ATT-2006.

Table 1 shows thermogram of freeze solution and distilled water: control option, not treated with ultrasound (---); subjected to sonication solution (-), b is a basic solution for sturgeon: control option, not treated with ultrasound (---); subjected to sonication solution (-), with a basic solution +10% dimethyl sulfoxide: control option, not treated with ultrasound (---); subjected to sonication solution (-), d - base solution +10% demeti the sulfoxide +10% egg yolk: control option not treated with ultrasound (---); subjected to sonication solution (-).

From table 1 it is seen that when the freeze control option is hypothermia (to (-2)-(-9C), and then crystallization of ice the temperature rises to 0C, which is not observed in solutions treated with non-contact ultrasonic radiation. The decrease in low-temperature jump at the crystallization solutions of cryoprotectants, to enhance the integrity defrosteranlage biological material.

Thus, remote influence on frozen solution by ultrasonic radiation, by exposure of the entire area, causing a decrease of the influence of low-temperature spike solutions of cryoprotectants, completely disappears effect of hypothermia, the rate of freezing takes continuous and smooth in nature, improving the integrity deportirovannykh cells after cryopreservation.

SOURCES of INFORMATION

1. Pushkar N. S., Belous A. M. Introduction to Cryobiology. - Kiev: Naukova Dumka, 1975. - S. 21.

2. Burzawa-Gerard, E., Goncharov B. F., Dumas, A., Fontaine, Y. A. Further biochemical studies on carp gonadotropin, biochemical and biological comparison of c-GTH and a gonadotropine from Acipenser stellatus Pall. // Gen.Comp.Endocrinol. 1976. V. 29, No. 4. - P. 499.

3. Himenkov A. N., Broskow A. C. Introduction to structural Cryology. - M.: Nauka, 2005. - S. 203. (prototype)

The way to reduce low-temperature jump is rastvorov of cryoprotectants, including the freezing of a solution of cryoprotectant with biological material in liquid nitrogen, characterized in that before the operation of the freezing of a solution of cryoprotectants with the cells of living organisms carry out remote impact on a frozen solution of cryoprotectant ultrasonic radiation frequency of 0.50 to 10 MHz.



 

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