UA72374A - Method for vitrification of oocytes and embryos of mammals in ultrahigh heat exchange rates - Google Patents

Abstract

The method for the vitrification of oocytes and embryos of mammals in ultrahigh heat exchange rates comprises the saturation of the biological object in the equilibrating solution and the cryopreservation of the vitrifying solution. The biological object is held in the sealed stem dipped in the liquid nitrogen. The thawing of the object is performed in the water bath.

Description

The invention relates to biotechnology in animal husbandry, namely to the development of methods of cryopreservation of oocytes and embryos of mammals using vitrification.

There is a well-known method of vitrification of oocytes and embryos of mammals, which is based on the use of high heat exchange rates (V 30"С/s) and high concentrations of cryoprotectant (C-5095 and higher) (Izaspekko M.M., Igaspepko E.R.,

Oviazpko R.I., Spepspepko M.I. Onga-garia eeegipd oi gai etbguoz m/yp garia aishiop oi reppearie sguorgoiesiapiv //

Stuobiiodu.-1997.-M. 34.-R.157-164). Straws with a diameter of 2 mm are used as containers, which are filled with a cryoprotective medium that holds the biological object in the traditional way (by 9595 of its total volume) and are closed on one side with a plastic plug, and on the other - moistened with dry alcohol.

However, the use of this method does not allow obtaining a high level of preservation of devitrified embryos (8-60905). The decrease in the preservation of oocytes and embryos of mammals during cryopreservation at high rates of heat exchange is due to the toxic effect on the biological object of highly concentrated solutions of cryoprotectants and a significant expansion of the medium. In some cases, the increase in the volume of the medium during freezing and the boiling of liquid nitrogen during thawing, which penetrates into the container through the plugs during storage, lead to mechanical destruction of the containers, and, as a result, to the loss of the biological object.

The method of vitrification of oocytes and embryos of mammals at extremely high rates of heat exchange in open drawn straws with a diameter of 0.8 mm, which freeze at a speed

With 2 100"C/s by direct immersion in liquid nitrogen (Mada R. Noit apa N. SaiIezep. Orep Riiea Bigazh |(ORBI| Mipsayop:

A Mem MUau yu Nedise Sguoipisheivz oi Vomipe Oma apa Etrguoz // MoiIestsag hergodisiop apa demeiortepi.-1998.-

M.51.-R.53-58).

However, this method has disadvantages, which are: the use of a low speed of thawing relative to the speed of freezing, which leads to partial recrystallization during thawing, and relatively high concentrations of both vitrifying and equilibrating cryoprotectant solutions, which reveal a toxic and osmotic effect on the biological object; the use of open containers for the environment that holds the biological object contributes to the violation of sterility conditions. These shortcomings make the process of cryopreservation low-tech, and also prevent a high level of preservation of deconserved biological objects.

The task of the invention is to create a new method of vitrification of oocytes and embryos of mammals at extremely high rates of heat exchange, which by using closed drawn straws for a biological object, optimal ratio of the speed of thawing to freezing, optimal filling of straws with a vitrifying solution, low concentrations of cryoprotectants allows to significantly increase the level of preservation of bioobjects objects

The task is solved in such a way that in the method of vitrification of oocytes and embryos of mammals at ultra-high rates of heat exchange, which includes saturation of the bioobject in an equilibrating solution, cryopreservation of the vitrifying solution, which keeps the bioobject in the drawn straw by its direct immersion in liquid nitrogen, followed by by thawing in a water bath, it is additionally provided that a hermetically sealed straw is used, which holds the vitrifying solution with the bioobject, the thawing speed exceeds the freezing speed by at least 1.5 times, the straw is filled with the vitrifying solution with the bioobject at 1095 of its total object to him, thawing is carried out in a water bath at a temperature of 40"C with stirring, and the concentration of the vitrifying and equilibrating cryoprotectant solution is determined according to formulas 1 and 2, answered) - c (B) according to de Setip - the minimum concentration of the vitrifying cryoprotectant solution (905, IM/m)); n - the number of types of cryoprotectants included in the composition vitrifying solution; B - the rate of thawing ("S/s); C((B) - the minimum concentration of each of the types of cryoprotectants, determined at a given rate of thawing (95, |m/m|),

Sit(B)-Setip(B)-40, (2) where Sitip is the minimum concentration of the equilibrating cryoprotector solution (95, |(HMI);

In the case when the vitrifying solution consists of glycerin and sucrose, its minimum concentration is determined by the formula

Setipts(B):y(C1(8)-C2(B)), cSi(8)-57.63-1.15-805026 (А-0.998),

С2(8)-7,433.:10582-7,613-102-8--63,16 (8-0,999), where Si, Сg are the minimum concentration of glycerol and sucrose, respectively.

The use of hermetically closed drawn straws in the claimed method makes it possible to exclude direct contact between the refrigerant and the vitrifying solution that holds the biological object, which ensures sterility conditions. The traditional way to solve this problem is to add antibiotics to the vitrifying solution or to use liquid nitrogen, previously purified from bacteria and fungi using special filters with pores of size 0.2 7. However, the use of antibiotics negatively affects the level of preservation of deconserved biological objects, and the process Refrigerant cleaning makes the cryopreservation method low-tech, which increases the cost of one deconserved embryo. The proposed method is simpler to implement and does not require expensive drugs and equipment compared to traditional methods (Maya R. Noit apa N. SaIPezep. Orep RiiPei bigaj (ORBI Milpisaiop: A Mem Uuau yu Vedise Stuoipishpev oi

Vomipe Oma apa Etbrguoz // MoiIesshag gergodisiip apa demeIortepi.-1998.-M.51.-R. 53-58).

It was found experimentally that it is optimal to fill drawn straws with a vitrifying solution with a biological object at 1095 of its total volume. In this case, the effect of volume expansion of the medium during the vitrification process on the level of preservation of oocytes and mammalian embryos is reduced compared to traditional methods in which straws are filled with a vitrifying solution with a biological object by 9595 of its total volume (Morozova I.A. , Gorbunova N.Y., Gorbunov L.V. The effect of volumetric expansion of media containing bioobjects on the safety of deconserved sperm cells and animal embryos at ultrahigh freezing rates// Rybne horodstvo: Interdiv. subject of science coll. - K.: Agrarian science.-2002. - issue 61.- b. 37-41).

If the speed of thawing is exceeded in relation to the speed of freezing by at least 1.5 times, the possibility of recrystallization processes during thawing is reduced. It also allows to reduce the concentration of both equilibrating and vitrifying solutions of cryoprotectants by 595 (Gorbunov L.V., Bezugliy M.D., Morozova

IA. Determination of the critical zone of crystal formation in the cycle of freezing-thawing of a biological object // Scientific and technical bulletin Me75 IZH UAAS.-1998.-Kharkiv, pp. 92-98).

The stated excess of the thawing speed in relation to the freezing speed is achieved by thawing the biological object contained in the straw in a water bath at 40"C under stirring conditions.

Through experimental studies using the method of regression analysis, the minimum concentrations of glycerol and sucrose were determined from the rate of thawing.

The claimed method is carried out as follows. Vitrifying and equilibrating solutions are prepared on the basis of Dulbecco's solution (FSB) with the addition of 1095 fetal calf serum. Saturation of the biological object is carried out in the equilibrating cryoprotectant solution at a temperature of 20:22 C for 10 minutes. The exposure time of a saturated biological object in the vitrifying solution is 1-1.5 minutes at the same temperature. Immediately after exposure in the vitrifying solution, the bioobject is frozen in a closed, elongated straw by direct immersion in liquid nitrogen.

Thawing of straws is carried out in a water bath at a temperature of 40"C using a magnetic stirrer. After thawing, the cryoprotectant is removed from the biological object. The level of viability of biological objects before freezing and preservation after thawing is determined by morphological indicators immediately after the removal of the cryoprotectant and after cultivation. The obtained results statistics are processed according to well-known methods.

The invention is illustrated by an example.

Example 1

The implementation of the proposed method was carried out on a model biological object - mouse embryos (table 1).

Table 1

The influence of the concentration of equilibrating and vitrifying solutions of cryoprotectants on the preservation of mouse embryos cryopreserved at high and ultrahigh freezing-thawing rates

Concentration of cryoprotectant Soo Conservation 5 (t), 95 . MI speed (M)

The diameter of the straw freezes i - tt (thaw) equilibrium After withdrawal after

All (glycerin) Glycerin | sucrose | cryoprotector of short-term paralysis of ip migo

Note: Different superscripts define values that are significantly different from each other with a probability of at least 0.95.

The traditional method of vitrification was used as a control. The research was carried out on embryos of good and excellent quality, which were obtained at the stage of late morula or early blastocyst from animals previously processed for superovulation according to a well-known technique. In the main experiment and in the control, 6028 embryos were frozen.

In the main experiment, the equilibrating solution contains 1095 glycerol, and the vitrifying solution contains 3095 glycerol and 0.58 M sucrose. Bioobjects processed in equilibrating and vitrifying solutions were frozen in closed plastic straws with a diameter of 1 mm and 1.8 mm by direct immersion in liquid nitrogen. A 0.6 M sucrose solution was used to remove the cryoprotectant.

The obtained results indicate that the proposed method of vitrification of oocytes and mammalian embryos, developed on the basis of the use of closed drawn straws with a diameter of 1 mm, allows to increase the level of preservation of deconserved biological objects by approximately 1295 in comparison with existing methods of vitrification, provided that its implementation is relatively simple and reliable .

The claimed method ensures preservation of the biological object up to 7895 times, which is 1295 times higher than the generally known results.

Claims (2)

1. The method of vitrification of oocytes and embryos of mammals at ultra-high rates of heat exchange, which includes saturation of the biological object in an equilibrating solution, cryopreservation of the vitrifying solution, which keeps the biological object in an elongated straw by its direct immersion in liquid nitrogen, followed by thawing in a water bath, which differs in that a hermetically sealed straw is used, which holds a vitrifying solution with a biological object, the speed of thawing exceeds the freezing speed by at least 1.5 times, the straw is filled with a vitrifying solution with a biological object by 10 95 of its total volume, thawing is carried out in a water bath at a temperature of 40"C while stirring, and the concentration of the vitrifying and equilibrating solution of cryoprotectants is calculated according to the formulas: (95, (m/m|); P - the number of types of cryoprotectants included in the composition of the vitrifying solution; B (handiness from melting ("S/s); ) - the minimum concentration of each of the types of cryoprotectants at a given rate of thawing, determined by the method of regression analysis (95, (mM/M|): Spip (B) - Column (B) - 40, where, C tip (B) and . No - the minimum concentration of the equilibrating cryoprotectant solution. 2. The method according to claim 1, which differs in that the minimum concentration of the vitrifying solution from glycerin and sucrose is determined by the formula S.(B)-57,63-115. ooo S.(8)-7,433.107.87 -7,613.107. B--6316, boss 17772 - the minimum concentration of glycerol and sucrose, respectively.