RU141452U1 - DEVICE FOR VITRIFICATION OF MAMMAL OOCYtes AND EMBRYOS - Google Patents

Abstract

A device for vitrification of oocytes and embryos of mammals, consisting of an open type carrier - half straws made by cutting obliquely with a length of about 1 cm about half of a straw for cryopreservation with a volume of 0.25 ml, a standard plug for it (plug) and a protective cover made of straw for cryopreservation with a volume of 0.5 ml, characterized in that the open end of the half-straw opposite to the cut is closed with a standard plug, directly under which they pierce through the walls of the half-straw hole, and at a distance of 1-2 cm from the plug, the half-straw section is flattened in the transverse direction.

Description

Cryopreservation of embryos is widely used both in industrial cattle breeding in technology for embryo transplantation and to preserve the genetic potential of endangered and rare breeds. The objects of cryopreservation are oocytes and embryos of not only cows, but also other farm animals (pigs, sheep, horses). Cryopreservation methods are widely used when working with human oocytes and human embryos in in vitro fertilization clinics. Moreover, cow embryos are often model objects for testing methods developed for human embryos (Kuwayama et al., 2005; Huang et al., 2007).

Currently, for the purpose of cryopreservation, the method of vitrification based on ultrafast cooling of an object pre-treated in solutions with a high concentration of cryoprotectants is becoming more widespread. It is recognized that vitrification is more gentle and, therefore, more efficient than cryopreservation by the programmed slow freezing, especially when working with such highly sensitive to low temperatures objects as oocytes and cattle embryos obtained in vitro.

The possibility of vitrification is determined by three critical factors:

1. Achieving the maximum possible cooling rate of the object, about 15000-30000 ° C / min.

2. The use of a medium with a high viscosity, which is determined by the concentration and properties of individual substances, cryoprotectants (CP). The higher the concentration of CP in the medium, the higher the glass transition temperature (Tg), which reduces the chance of the formation of ice nuclei (the chance of ice nucleation) and crystallization. Different CPs have different ability to penetrate through cell membranes, cytotoxicity, Tg.

3. The minimum volume of the vitrified object, less than 1 μl.

Ultrafast cooling and subsequent heating is based on direct contact of the object in the minimum solution volume of cryoprotectants with liquid nitrogen at the stage of vitrification and with the medium at the stage of heating. To achieve these goals, several models of open-surface media have been developed, of which the most widely used devices are Cryotop®, Cryoleaf ™, cryo loops, electron microscopy nets, half-straws (hemi-straw, HS) (Saragusty, Arav, 2011), which allow control the application of a drop of a minimum volume (1 μl or less). In addition, when using these carriers, a very high cooling rate is achieved due to direct contact of the object with liquid nitrogen and a high heating rate, since the object is directly immersed in the solution for heating after extraction from liquid nitrogen. In turn, the use of minimum volumes in combination with the maximum cooling rate reduces the concentration of cryoprotectants in the vitrification solution and, accordingly, their cytotoxic effect on embryos. In general, embryo survival rates when using different carriers with an open surface are similar, there is no carrier that gives significantly better results than the rest (Elnahas et al., 2010). Therefore, the choice of carrier for vitrification often depends on its availability, cost and ease of use in a particular laboratory. Cryotop®, Cryoleaf ™, cryo loops are manufactured by manufacturers. They are available for purchase, but their cost is very high. A half-straw model (hemi-straw, HS) with a container was proposed by Vanderzwalmen et al. (2000), and then was used in several modifications (Liebermann, Tucker, 2002, 2004; Rao et al., 2012). At the same time, straws developed and used for cryopreservation of biological material, that is, non-toxic, suitable for use in conditions of extremely low temperatures, not expensive and quite affordable, were used for the manufacture of the HS system.

The objective of this utility model is to create a more reliable and easy-to-use device for vitrification of oocytes and early mammalian embryos.

PROTOTYPE OF A USEFUL MODEL

The prototype of our model for the vitrification of in vitro cattle embryos was a half-straw system, HS.

1. A half-straw model (hemi-straw, HS) was proposed by Vanderzwalmen et al. (2000), and in the author's version, it was really prepared by a longitudinal section of about one centimeter of straw for cryopreservation of 0.25 ml (mini straw) from its open end (Vanderzwalmen et al., 2003). The open inner surface of the half-straw obtained as a result of the slice made it possible to place oocytes or embryos in a minimum amount (0.3 μl) of a vitrification solution (VS). A half straw with the end embryos in VS was immediately lowered into liquid nitrogen. Then, using tweezers, HS was placed in a 0.5 ml straw (medium straw) in liquid nitrogen, as in a container, and closed with a stopper. To warm the HS embryos under nitrogen with tweezers, they pulled them out of the container straw and its end with the embryo was immediately placed in the heating solution.

2. In the version of Liebermann, Tucker (2002, 2004), the 0.25 ml open end of the straw was cut obliquely with a sharp scalpel so as to obtain a slice about 1 cm long. Oocytes or embryos in a minimum volume VS are placed on the very tip of the open inner surface of the slice straws. A straw with an embryo at the end of the slice is immediately dropped vertically into liquid nitrogen and placed in the lumen of 0.5 ml straws in liquid nitrogen, as in a container. A straw serving as a container closes with its color-coded plug.

3. Rao et al. (2012) prepared a half-straw carrier also by oblique slice length of about 1 cm of the open end of a straw per 0.25 ml. This straw was introduced into a 0.5 ml middle straw with an outer diameter of 2.8 mm so that the open end of the HS could move in and out of this sheath during vitrification and heating. Judging by the photograph, the fixation of the carrier in the lumen of the straw by 0.5 ml was achieved by flattening a small portion of the half straw, although the description is not given.

ESSENCE OF A USEFUL MODEL

A device for vitrifying mammalian oocytes and embryos was prepared from 0.25 (mini straws) and 0.5 ml (medium straws) straws designed and used for cryopreservation and storage of biological material in liquid nitrogen, and standard plugs (different colors for marking) to 0.25 ml straws.

The procedure for the manufacture of a device for vitrification of oocytes and mammalian embryos

1. A 0.25 ml straw from the open end is closed with a standard plug. In this case, the narrow part of the stopper fits tightly into the lumen of the straw by about 0.5 cm, and the wide part extends beyond the straw by about 4 cm and further serves as a handle when working with the device.

2. Holding the gag handle in one hand, use a safety razor blade to make an oblique cut of a straw with a length of about 1 cm so that about half of the straw is cut off. The cut off part of the straw with the filter is thrown away. The inner surface of the slice tip of the remaining half straw further serves to place the embryo in a minimum amount of vitrification medium.

3. Near the plugs with an injection needle with a 1 ml syringe, pierce the walls of the half-straw through and through. The syringe in this case serves as a convenient pen for the injection needle.

4. At a distance of 1-2 cm from the plug, the half-straw is flattened in the transverse direction to form a fixation block. When using the CAS SNT-200 sealer in 0.5 mode, the straw walls are flattened to a small extent, but its lumen is not sealed.

5. When using a half-straw carrier with a vitrified embryo directly in liquid nitrogen, it is inserted into a 0.5 ml straw and fixed in it due to the fixation unit — a flattened area. Thus, a 0.5 ml straw acts as a protective cover for a semi-straw carrier.

6. When assembled, the model is a device for vitrification of mammalian oocytes and embryos.

List of figures:

Figure 1 shows a diagram of a device for vitrifying mammalian oocytes and embryos.

Figure 2 shows the starting materials for assembling the device for vitrification of oocytes and mammalian embryos and the assembled device.

     Scheme for vitrification of in vitro cattle embryos using a device for vitrification of oocytes and mammalian embryos

1. A bath of liquid nitrogen is placed on the table next to the stereo microscope.

2. At the bottom of the tray, place a 0.5 ml straw, which will be used as a protective cover, and a goblet with a diameter of 13 mm (goblet; a standard plastic cylinder with a diameter of 9-13 mm used to place the straws when stored in liquid nitrogen).

3. Anatomical tweezers are placed in liquid nitrogen, leaving its handle above the level of nitrogen at the edge of the bath.

4. The embryo is equilibrated in solutions with different concentrations of cryoprotectants in accordance with the accepted protocol.

5. The embryo is kept in a vitrification solution (VS) for 20-30 seconds

6. Take the prepared half-straw carrier, using the outer part of the gag as a handle.

7. Under a stereo microscope, the embryo in a minimum amount of VS is placed on the very tip of the slice on the inner surface of the half-straw.

8. Immediately the tip of the half straw with the embryo in the microdrop VS is dipped in liquid nitrogen.

9. With slow swaying, continue to lower the carrier vertically into liquid nitrogen to the level of the plug.

10. Using tweezers, take a 0.5 ml straw in liquid nitrogen and hold it below the nitrogen level.

11. The carrier with the embryo is inserted into the lumen of the straw cover until the stop of the gag handle into the upper edge of the straw cover.

12. Half-straw carrier in a protective case, i.e. a vitrification device, with a vitrified embryo under nitrogen, is placed in a goblet.

13. For storage, the goblet is placed in liquid nitrogen in a canister in a Dewar vessel.

The scheme for carrying out the heating of embryos vitrified using a device for vitrification of oocytes and mammalian embryos

1. A bath of liquid nitrogen is placed on the table next to the stereo microscope.

2. 2 anatomical tweezers are placed in the liquid nitrogen bath, leaving their handles above the nitrogen level at the edge of the bath.

3. A device with a vitrified embryo stored in a Dewar vessel, together with the goblet, is transferred to liquid nitrogen and placed in a liquid nitrogen bath.

4. Using tweezers, the device is removed from the goblet, keeping it below the level of liquid nitrogen.

5. Holding the stopper plug with one pair of tweezers, remove the carrier straw from the straw-case, which is held by the second tweezers.

6. Keeping the carrier straw under liquid nitrogen, translate it into a vertical position with the handle-gag up.

7. Slowly lift the carrier vertically, keeping only its lower tip with the vitrified embryo below the level of liquid nitrogen.

8. Quickly remove the carrier from liquid nitrogen completely and immediately lower its tip with a vitrified embryo into a Petri dish containing 3 ml of TS heating medium at a temperature of 39 ° C.

9. When the carrier is slightly swayed, the embryo is released into TS medium. In TS medium, the embryo is incubated for 1 minute.

10. The embryo is carried out through a system of solutions for dilution and washing from cryoprotectants in accordance with the accepted protocol.

11. The heated embryos are placed in a pre-equilibrated mSOF medium for cultivation. After 24 and 48 hours at the age of 8 and 9 D, the number of live embryos, as well as those having reached the stage of hatching and hatching blastocysts, is determined.

EXAMPLE OF USE OF A VITRIFICATION DEVICE FOR CATTLE EMBRYONS OBTAINED IN VITRO

WORKING SOLUTIONS

The media were prepared on the basis of stock solutions of components [Whittingham, 1971] stored at 4 or -20 ° C. Before use, gentamicin in an amount of 40-50 μg / ml was added to all working media and solutions. Oocyte maturation was performed in medium 199 with the addition of 0.66 mM Na pyruvate, 1 mM L-glutamine, 0.6 mM L-cysteine, 5 U / ml chorionic gonadotropin, 5 μg / ml follicle-stimulating hormone, 1 μg / ml estradiol-17β and 10% fetal cattle blood serum (FCS). Manipulations with oocytes, sperm and embryos in the air were carried out in a modified TALP-HEPES medium [Bavister, Yanagimachi, 1977] (TH). For fertilization, TALP-Fert modified medium was used [Parrish et al., 1986, 1988] (TF). The prepared TH and TF media were stored at -20 ° C. Aliquots of media were thawed once and used for a week. Aliquots of PHE solutions [penicillamine, hypotaurine, epinephrine; Bavister, 1989] and heparin were stored at -20 ° C and thawed immediately before use. Embryos were cultured in mSOF medium [Tervit et al., 1972].

VITRIFICATION AND HEATING EMBRYONS SOLUTIONS

The stock solution for vitrification of embryos was TH medium supplemented with 20% FCS, designation TH20.

1. The solution for embryo balancing (equilibration solution, ES) consisted of TH20 with the addition of 7.5% ethylene glycol (EG) and 7.5% DMSO.

2. The solution for vitrification of VS embryos consisted of TH20 supplemented with 15% EG, 15% DMSO and 0.5 M sucrose.

3. The solution for warming the embryos (thawing solution, TS) consisted of TH20 with the addition of 1 M sucrose.

4. The solution for dilution 1 (dilution solution 1, DS1) was TN20 medium containing 0.5 M sucrose

5. The dilution solution 2 (DS2) was TH20 medium containing 0.25 M sucrose.

6. As the washing solution (WS), the base medium TH20 was used.

Production of Cattle Embryos in Vitro

Cattle ovaries were obtained at a meat processing plant and delivered to the laboratory at a temperature of 26-30 ° C for 3-5 hours. Oocyte maturation was carried out in a CO ₂ incubator at a temperature of 38.5 ° C for 22-23 hours. For fertilization, a cryopreserved bull seed was used. The swim up method was used to isolate the fraction of motile spermatozoa free of seminal plasma and components of dilution and cryopreservation media. Joint incubation of oocytes and sperm cells was carried out for 18-20 hours in a CO ₂ incubator at a temperature of 38.5 ° C. The obtained zygotes were freed from cumulus cells on Vortex in a hyaluronidase solution and put on cultivation in mSOF medium in an atmosphere of 5.6% CO ₂ , 5.7% O ₂ , 88.7% N ₂ for 7-12 days. The day of fertilization was considered zero (D 0).

VITRIFICATION OF CATTLE EMBRYONS OBTAINED IN VITRO

A bath of liquid nitrogen was placed on a table next to a stereo microscope. For vitrification, 7-day-old embryos at the blastocyst and expanded blastocyst stages were sequentially carried out through solutions at room temperature:

1. Laundering 1 (WS) 3-5 minutes

2. Laundering 2 (WS) 3-5 minutes

3. Equilibration (ES) 3-5 minutes

4. Vitrification (VS) 20-30 seconds

The embryo in a minimum volume VS was placed on the tip of the carrier and immediately immersed in liquid nitrogen. With slow swaying, the carrier was continued to be lowered vertically into liquid nitrogen to the stopper level. At the same time, liquid nitrogen fills the half-straw, displacing the air that exits through the holes made by the needle in the walls of the half-straw under the plug. Then the carrier was placed in a straw-protective cover located in liquid nitrogen, holding it with tweezers. The obtained complex for vitrification of oocytes and mammalian embryos with a vitrified cattle embryo was placed in a goblet and transferred to a Dewar vessel for storage.

HEATING VERIFIED EMBRYOS

A goblet with a device for vitrifying oocytes and mammalian embryos with a vitrified cattle embryo was removed from a Dewar vessel and transferred to a liquid nitrogen bath in a thermos with liquid nitrogen, located on a table next to a stereo microscope. Directly in liquid nitrogen, tweezers removed the protective cover from the floor of the carrier straw. Holding the stopper plug, the carrier half-straw was slowly lifted vertically, leaving only its tip with the verified embryo below the level of liquid nitrogen. In this case, the lumen of the straw is freed from liquid nitrogen and is filled with air, which enters through the holes in the walls under the plug, while the verified embryo is still in liquid nitrogen. Then the carrier was quickly removed from the liquid nitrogen completely, and immediately its tip with the vitrified embryo was lowered into a Petri dish containing 3 ml of TS heating medium at a temperature of 39 ° C. In this solution, the embryo was kept for 1 minute, after which it was successively carried out through solutions for dilution and washing from cryoprotectants at room temperature. General scheme for warming verified cattle embryos:

1. Heating (TS) 1 minute

2. Dilution 1 (DS1) 3-5 minutes

3. Dilution 2 (DS2) 3-5 minutes

4. Laundering 1 (WS) 3-5 minutes

5. Laundering 2 (WS) 3-5 minutes

Heated embryos were placed in a pre-equilibrated mSOF medium for cultivation. After 24 and 48 hours at the age of 8 and 9 D, respectively, the number of live embryos, as well as those reaching the stage of hatching and hatching blastocysts, was determined.

EXAMPLES OF USING A VITRIFICATION DEVICE FOR CATTLE EMBRYONS OBTAINED IN VITRO

Example 1. To test the device, vitrification and heating of the blastocysts was first performed without placing the verified embryos in storage in a Dewar vessel. Six extended seven-day good-quality blastocysts were selected and vitrified by placing them one on a half-straw carrier. After exposure to liquid nitrogen for 10-15 minutes, the embryos were passed through a system of solutions for heating and washing and put on cultivation in mSOF medium. After 24 hours of cultivation, out of 6 verified blastocysts, 4 (66.7%) were alive, of which 3 blastocysts emerged from the lustrous membrane, one more was at the hatching stage. Thus, 2/3 of the embryos successfully transferred the vitrification and warming procedure, while all surviving embryos developed normally during cultivation.

Example 2. In the following experiment, 6 embryos at the age of 7 days were selected for vitrification: 3 at the stage of expanded blastocyst, 1 at the stage of blastocyst, 2 at the stage of early blastocyst. Since all embryos were of the same age, it can be assumed that their quality decreased from the stage expanded to the early blastocyst stage. After vitrification and storage for three days in liquid nitrogen, the embryos were warmed up and put on cultivation. After 24 hours, all 6 embryos were alive (100%). Moreover, by this time all embryos reached the stage of hatching blastocysts, including 1 blastocyst emerged from the shiny coat. At the age of 9 days, 5 embryos remained alive (83.3%), including 2 blastocysts emerged from the luster, the remaining 3 embryos stopped developing at the stage of hatching blastocysts.

Example 3. In the following 2 experiments, 15 blastocysts were vitrified, which were warmed after storage in liquid nitrogen for 4 to 11 days. Twenty-four hours after warming, 100% vitrified blastocysts were alive (15/15). At the age of 9 days, 86.7% of embryos (13/15) remained alive, of which 69% of blastocysts (9/13) reached the stage of the blastocyst that emerged from the shiny membrane.

Thus, when using the developed device for vitrification of oocytes and mammalian embryos, embryos are not lost during vitrification, storage in liquid nitrogen and heating. 24 hours after warming and culturing, up to 100% of vitrified cattle embryos obtained in vitro are alive. During subsequent cultivation, about 61% of the surviving embryos reach the stage of the blastocyst hatching from the shiny membrane.

CONCLUSION

The device for vitrification of oocytes and mammalian embryos is simple to manufacture and convenient to use. For the manufacture of the device, straws with a volume of 0.25 (a French mini straw) and 0.5 ml (a French medium straw) used to store biological material in liquid nitrogen, and standard plugs (plugs, different colors for marking) to straws with a volume of straws are used 0.25 ml. The standard plug for a 0.25 ml straw with a narrow part, whose length is about 6 mm, fits tightly and is very firmly held in the lumen of the straw. The protruding part of the plug is durable, has a length of about 4 cm, diameter 3 mm. Due to this, the outer part of the plug is a convenient handle, both in the manufacture of the carrier and at all stages of vitrification and heating of the embryos, including immersion and removal of the carrier from liquid nitrogen. An all-plastic plug is about 3 times heavier than a hollow straw (0.3 g versus 0.1 g, respectively), and the density of its material exceeds the density of liquid nitrogen (a cooled plug is immersed at the bottom of a container with liquid nitrogen). As a result, the plug, located on top, prevents the entire device from floating up in liquid nitrogen when it is stored in the goblet. The combination of different colors of the gag and goblet can serve as quickly distinguishable markers.

Through holes made in the walls of the carrier under the plug allow liquid nitrogen to freely fill the entire half of the straw at the stage of vitrification when the carrier with the embryo is immersed vertically in liquid nitrogen, which reduces its buoyancy in liquid nitrogen. The same openings provide fast, quiet (without turbulence) and complete leakage of liquid nitrogen from the lumen of the half-straw when it is removed with the handle-plug upwards vertically from liquid nitrogen at the stage of heating. Obtaining the full flow of liquid nitrogen from a half-straw at this stage is very important, since then the tip of the carrier with the embryo should be instantly moved from under the nitrogen into the Petri dish with a solution for heating at 39 ° C.

At the stage of vitrification, the carrier under liquid nitrogen gently enters the 0.5 ml straw until the outer wide part of the half-straw plug stops at the edges of the straw cover. In this case, the flattened portion of the carrier, abutting from the inside against the walls of the straw-case of a larger diameter, acts as a retainer and holds the carrier in its lumen. When using even the simplest sealing machine, it is easy to choose a mode that provides the same type of flattening of the walls of a half-straw carrier, necessary and sufficient for fixing in a straw-case.

The use of 0.5 ml straws as a cover makes it possible to reliably protect a carrier with a verified embryo placed on an open surface from possible accidental mechanical influences.

For storage of embryos, the device for vitrification of oocytes and mammalian embryos is placed in vessels with liquid nitrogen, similar to conventional straws during cryopreservation of biological material, while occupying a minimum volume.

Literature

Bavister B.D. A consistently successful procedure for in vitro fertilization of golden hamster eggs. Gamete Res. 1989; 23: 139-158.

Bavister B.D., Yanagimachi R. The effects of sperm extract and energy sources on the motility and acrosome reaction of hamster sperm in vitro. Biol. Reprod. 1977; 16: 228-237.

Elnahas K., Palapelas V., Diedrich K., Al-Hasani S. Vitrification of human oocytes and different development stages of embryos: An overview. Middle East Fertil. Soci. J. 2010; 15: 2-9.

Huang J., Chung J-T., Tan S. L., Chian R-C. High survival and hatching rates following vitrification of embryos at blastocyst stage: a bovine model study. Reprod. Biomed. Online 2007; 14: 464-470.

Kuwayama M., Vajta G., Kato O., Leibo S.P. Highly efficient vitrification method for cryopreservation of human oocytes. Reprod. Biomed. Online 2005; 11: 300-308.

Liebermann J., Tucker M.J. Effect of carrier system on the yield of human oocytes and embryos as assessed by survival and developmental potential after vitrification. Reproduction. 2002; 124: 483-489.

Liebermann J., Tucker M.J. Vitrifying and warming of human oocytes, embryos, and blastocysts. From: Methods in Molecular Biology, vol. 254: Germ Cell Protocols, Volume 2: Molecular Embryo Analysis, Live Imaging, Transgenesis, and Cloning. Edited by: H. Schatten © Humana Press Inc., Totowa, NJ. 2004, pp. 345-364.

Parrish J.J., Susko-Parrish J.L., Leibfried-Rutledge M.L. et al. Bovine in vitro fertilization with frozen-thawed semen. Theriogenology. 1986; 25: 591-600.

Parrish J.J., Susko-Parrish J.L., Winer M.A., First N.L. Capacitation of bovine sperm by heparin. Biol. Reprod. 1988; 38: 1171-1180.

Rao B.S., Mahesh Y.U., Charan K.V., Suman K., Sekhar N., Shivaji S. Effect of vitrification on meiotic maturation and expression of genes in immature goat cumulus oocyte complexes. Cryobiology. 2012; 64: 176-184.

Saragusty J., Arav A. Current progress in oocyte and embryo cryopreservation by slow freezing and vitrification. Reproduction. 2011; 141: 1-19.

Tervit H.R., Whittingham D.G., Rowson L.E.A. Successful culture in vitro of sheep and cattle ova. J. Reprod. Fertil. 1972; 30: 493-497.

Vanderzwalmen P., Bertin G., Debauche C. H., Standaert V., Schoysman R. In vitro survival of metaphase II oocytes and blastocysts after vitrification in an Hemi Straw (HS) system. Fertil. Steril. 2000; 74 (Suppl.): 215-216.

Vanderzwalmen P., Bertin G., Debauche Ch., Standaert V., Bollen N., van Roosendaal E., Vandervorst M., Schoysman R., Zech N. Vitrification of human blastocysts with the Hemi-Straw carrier: application of assisted hatching after thawing. Hum. Reprod. 2003; 18: 1504-1511.

Whittingham D.G. Culture of mouse ova. J. Reprod. Fertil. 1971 ;. Suppl. 14: 7-21.

Claims (1)

A device for vitrification of oocytes and embryos of mammals, consisting of an open type carrier - half straws made by cutting obliquely with a length of about 1 cm about half of a straw for cryopreservation with a volume of 0.25 ml, a standard plug for it (plug) and a protective cover made of straw for cryopreservation with a volume of 0.5 ml, characterized in that the open end of the half-straw opposite to the cut is closed with a standard plug, directly under which they pierce through the walls of the half-straw hole, and at a distance of 1-2 cm from the plug, the half-straw section is flattened in the transverse direction.

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