KR100793680B1 - Novel oil-free embryo culturing system - Google Patents

Abstract

A method for culturing an embryo is provided to improve the development of the embryo before the implantation of a mammal by using an oil-free culture medium, thereby being usefully used for developing embryos of various mammals. To culture an embryo using a container which consists of a lower portion closed microchannel, the container is one selected from the group consisting of a container, the lower portion of the microchannel having a shape of gradually being narrowed and a closing cap being formed on the upper portion of the microchannel, a container, the lower portion of the microchannel having a U-shape or a corn shape, and a container, the diameter of the microchannel being 0.1-0.2 cm and the length of the lower portion which is gradually narrowed is 1-3 cm. In the method, a culture medium is oil-free, the number of the embryo in the container is 1-8/mul, and the embryo is an embryo of mammal.

Description

New Oil-Free Embryo Culturing System

1 shows an example of a new fertilized egg culture vessel according to the present invention.

2 is a graph showing the effect of the density of the fertilized egg on the formation of the fertilized egg.

3 is a graph showing the rate of development of blastocysts according to three different culture systems at the same fertilized egg density (two fertilized eggs / μl): drop (conventional method), MTC (method according to the invention), MTC- oil (application of oil in the same manner as the existing drop as an example of the method according to the invention)

4 is a graph depicting the average cell population of mouse blastocysts in three different culture systems: drop (conventional method), MTC (method according to the invention), MTC-oil (one of the methods according to the invention), Cell mass (ICM), trophoblast (TE).

FIG. 5 is a graph showing the division of stages of blastocysts at the end of incubation of fertilized eggs: drop (conventional method), MTC (method according to the invention), MTC-oil (an example of the method according to the invention).

Figure 6 is a photograph showing the fertilized egg in culture with the medium in the fertilized egg culture vessel according to the present invention. A total of 10 fertilized eggs were cultured up to the blastocyst stage, and the U-shaped bottom provides a microenvironment suitable for embryo development, and autocrine and paracrine factors such as growth factors can accumulate around the fertilized eggs.

7 is a graph showing the limits of fertilized egg density. Unlike the existing method, it can be seen that there is no limit in the case of the method according to the present invention.

1.Erbach GT, Bhatnagar P, Baltz JM, Biggers JD (1995) Zinc is a possible toxic contaminant of silicone oil in microdrop cultures of preimplantation mouse embryos Human Reproduction 10 3248-3254

Summers MC, Biggers JD (2003) Chemically defined media and the culture of mammalian preimplantation embryos: historical perspective and current issues Human Reproduction Update 9 557-582

Taka M, Iwayama H, Fukui Y (2005) Effect of the well of the well (WOW) system on in vitro culture for porcine embryos after intracytoplasmic sperm injection Journal of Reproduction Development 51 533-537

4.Thouas GA, Jones GM, Trounson AO (2003) The 'GO' system--a novel method of microculture for in vitro development of mouse zygotes to the blastocyst stage Reproduction 126 161-169

5.Thouas GA, Korfiatis NA, French AJ, Jones GM, Trounson AO (2001) Simplified technique for differential staining of inner cell mass and trophectoderm cells of mouse and bovine blastocysts Reproductive Biomedicine Online 3 25-29

6.Vajta G, Peura TT, Holm P, Paldi A, Greve T, Trounson AO, Callesen H (2000) New method for culture of zona-included or zona-free embryos: the Well of the Well (WOW) system Molecular Reproduction and Development 55 256-264

7.Van Soom A, Mahmoudzadeh AR, Christophe A, Ysebaert MT, de Kruif A (2001) Silicone oil used in microdrop culture can affect bovine embryonic development and freezability Reproduction in Domestic Animals 36 169-76

Lane M, Gardner DK (1992) Effect of incubation volume and embryo density on the development and viability of mouse embryos in vitro Human Reproduction 7 558-562

9.Paria BC, Dey SK (1990) Preimplantation embryo development in vitro: cooperative interactions among embryos and role of growth factors Proceedings of the National Academy of Sciences of the United States of America 87 4756-4760

10.Zhu BK, Walker SK, Maddocks S (2004) Optimization of in vitro culture conditions in B6CBF1 mouse embryos Reproduction, nutrition, development 44 219-231

Miller KF, Goldberg JM, Collins RL (1994) Covering embryo cultures with mineral oil alters em bryo growth by acting as a sink for an embryotoxic substance Journal of assisted reproduction and genetics 11 342-345

12.Lee S, Cho M, Kim E, Kim T, Lee C, Han J, Lim J (2004) Renovation of a drop embryo cultures system by using refined mineral oil and the effect of glucose and / or hemoglobin added to a serum -free medium The Journal of veterinary medical science 66 63-66

13.Canseco RS, Sparks AE, Pearson RE, Gwazdauskas FC (1992) Embryo density and medium volume effects on early murine embryo development Journal of assisted reproduction and genetics 9 454-457

14.Handy K, Spanos S (2002) Growth factor expression and function in the human and mouse preimplantation embryo Journal of Endocrinology 172 221-236

15. Bhatnagar P, Papaioannou VE, Biggers JD (1995) CSF-1 and mouse preimplantation development in vitro Development 121 1333-1339.

The present invention relates to a method of culturing oil-free fertilized eggs. The present invention also relates to a novel oil-free fertilized egg culture apparatus (container) used in the method of culturing the fertilized egg.

In vitro culture systems of mammals have been continuously developed. Specifically, various culture conditions were intended to be changed. For example, cultured embryos with epithelial cells of the fallopian tubes, co-culture systems and continuous paired media or continuous media There have been attempts to use. To date, optimization of preimplantation fertilized egg culture has been focused on nutritional components, ie, chemically confirmed media components (Summers M.C. et al., 2003). Chemically confirmed media can be remade if desired and have the advantage of no known biological activity, such as enzymes and growth factors, but are still far from in vivo systems and optimal conditions for fertilized egg development. Not suitable

There is a growing interest in the physical environment (or factor) during in vitro culture conditions. Among the physical factors, the density of the fertilized egg is considered to be an important factor in determining the development of the fertilized egg. Previous studies (Wiley et al., 1986; Paria and Dey, 1990; Lane and Gardner, 1992) report that the development of fertilized eggs in vitro increases with increasing volume ratio between fertilized eggs and culture medium. By increasing the density of the fertilized egg, growth factors can accumulate around the fertilized egg so that an effect similar to adding a growth factor to the culture medium can be obtained.

In particular, taking into account the effects of density of fertilized eggs or the volume of culture medium, several research groups have reported culture devices: free fallopian tube (GO) and well of well (WOW) systems (Thouas GA et al., 2003). Taka M. et al., 2005; Vajta G. et al., 2000). GO is a system that increases the density of fertilized eggs by reducing the volume of medium to 1 μl through glass microtubules. WOW, on the other hand, is a culture system that uses small 250 μm and 300 μm deep wells formed in a 4-well dish with the bottom melted with a heated metal rod.

On the other hand, another limiting element of the fertilized egg culture system is oil. The oil is transparent and can be monitored and is essential for microdroplet culture systems in that it prevents medium evaporation even in small amounts. However, the lipophilic factors in the medium may be absorbed into the oil layer and contaminants may be diffused into the culture medium. Recent studies have supported this theory by revealing that a small amount of medium has a detrimental effect on the development of fertilized eggs when the medium is covered with oil (Lane et al., 1992). In small amounts, oil can inhibit the development of normal fertilized eggs by delivering toxic substances to the fertilized eggs. Batch numbers and oil types such as paraffin or minerals and storage conditions of oils can lead to non-normal development of fertilized eggs (Erbach G. T. et al., 1995; Van Soom A., 2001).

In order to reduce this detrimental effect of oil, many attempts have been made to reduce the amount of oil. That is, in the GO system, the mineral oil was reduced to 0.2 μl, and in the WOW system, the physical distance between the oil and the fertilized egg was kept as far as possible to reduce the harmful effects. However, so far no reports have been made for culture systems that are completely oil free.

The inventors of the present invention devised an oil-free culture method and a culture vessel enabling the same, and compared with a conventional drop culture system under the same density and medium volume of the fertilized egg, and confirmed that it promotes fertilized egg development. And the present invention. In addition, the superiority of the present invention was confirmed by confirming the effect of oil on the development of the embryo.

As described above, an object of the present invention is to provide a new fertilized egg culture method that can improve the development rate of fertilized eggs.

More specifically, the present invention is to provide a new fertilized egg culture method and the culture apparatus for enabling the same (oil-free), which excludes the oil that inhibits the development of the fertilized egg in the culture of the fertilized egg.

In addition, the fertilized egg culture method and culture apparatus according to the present invention causes a synergistic effect on the development of the fertilized egg before implantation as compared to the conventional fertilized egg culture method.

The present invention relates to a method of culturing an oil-free fertilized egg and a culture vessel of the fertilized egg which makes it possible.

More specifically, the fertilized egg culture vessel of the present invention is an improvement of an existing fertilized egg culture vessel made of microtubules, and has a shape in which the lower part of the microtubules is gradually narrowed in the microtubule of the hollow tube form. The lower part of the microtubule includes a narrowing U-shape or a cone or pyramid shape. For example, tubes for PCR can be used.

The structure of the culture vessel of the present invention allows the fertilized eggs to be incubated with a small amount of medium in a narrow space to increase the density of the fertilized eggs. In addition, the gradually narrowing subtypes of the U- or horn shape provide a microenvironment suitable for the development of the fertilized egg. In particular, the round bottom of the U-type facilitates the fertilization of the fertilized egg and the walls of the microtubules are autocrine or prevent further dispersal of paracrine factors.

Meanwhile, the fertilized egg culture vessel may be made of a synthetic resin material such as polypropylene, a paraffin material, or glass in consideration of ease of use, and may be made of latex, silicone rubber, or poly-based.

A cap for sealing may be formed at the top of the microtubules in one piece or with a microtubule. That is, the fertilized egg culture vessel of the present invention is closed, thereby improving gas holding capacity and prolonging buffering time as compared to the existing fertilized egg culture system. As a result, the fertilized egg culture vessel of the present invention maintains the acid-base state more stably, thereby reducing the stress that the fertilized egg will receive during the culture.

At this time, the sealing cap may be any shape as long as it can seal the container, and the material is not limited thereto, but may have a material (latex, silicone rubber, poly series, etc.) matching the container.

In addition, the diameter of the bottom of the microtubule bottom of the fertilized egg storage container of the present invention is preferably about 0.2mm, and the length of the gradually narrowing portion of the lower portion of the microtubules is 1 to 3cm.

Figure 1 shows an example of a fertilized egg culture vessel according to the present invention. Capillary-shaped fertilized egg culture vessel may be formed integrally with a cap 10 for sealing in the upper portion 20, the lower portion 30 of the vessel has a form that is gradually narrowed. The bottom of the bottom has a U-shape.

In another aspect, the present invention relates to a new oil-free fertilized egg culture method. More specifically, by using the fertilized egg culture vessel according to the present invention, it is possible by culturing the culture medium and the desired fertilized egg culture in the fertilized egg culture vessel.

In addition, the fertilized egg culture method of the present invention preferably comprises that the number of the fertilized egg in the fertilized egg culture vessel 1 / μL to 8 / μL. In particular, the fertilized egg culture method of the present invention comprises culturing the fertilized egg with a medium in a space of 0.2mm or less diameter of the culture vessel.

Fertilized eggs that may be used in the present invention include mammalian derived fertilized eggs. Examples include, but are not limited to, fertilized eggs of mice, humans, cattle, pigs, dogs, and the like.

Hereinafter, the present invention will be described in more detail with reference to the following Examples and Experimental Examples. However, the following Examples and Experimental Examples are only for illustrating the present invention, but the scope of the present invention is not limited to these.

Unless otherwise stated, all inorganic and organic compounds used in the present invention were obtained from Sigma-Aldrich (USA). All media used herein are based on CZB and KSOM media.

Example  1: recovery of eggs, activation of fertilized eggs and in vitro culture

Hyperovulation was induced by intraperitoneal injection of 7.5 IU eCG and 7.5 IU hCG at 48 hour intervals in 7-8 week old B6D2F1 mice. Hyperovulated mice were sacrificed with cervical dislocation 16 hours after hCG injection. The oviduct was removed and transferred to a Petri dish containing 2 ml of Hepes-buffered CZB medium (HCZB) added with 300 IU / ml hyaluronidase. An oocyte bulge was opened to obtain an egg surrounded by cumulus cells. After exposure to medium for 2-3 minutes, the oocyte-free eggs were washed twice in HCZB prior to activation.

The resulting eggs were then immediately exposed to an activation medium containing 10 mM SrCl ₂ added with 5 μg / ml cytocalasin B for 5 hours in calcium-free CZB and 5% CO _{2 in} air. Incubated for 115 hours in KSOM in the environment of.

Example  2: fractional dyeing

To determine the number of inner cell mass (ICM) and trophoblast (TE) cells, see Thouas et al. The blastocysts were fractionally stained using DNA-specific fluorescent dyes according to the method described in (2001).

Briefly, blastocysts were reacted for 5 seconds in 1 ml of Solution 1 (100 μg / ml propidium iodide and 1% Triton X-100 in PBS), followed by 500 μl of Solution 2 (25 μg / ml of bisbenzimide (Hoechst). 33258) was added to 100% ethanol fixed solution) immediately and reacted at 4 ℃. After staining the whole blastocyst was raised in glycerol on a glass slide and flattened under the coverslide. To measure the number of blastocyst ICM and TE cells, slides were used with a fluorescence microscope (Zeiss, Germany) equipped with UV illumination and an excitation filter (460 nm for blue and red fluorescence, 560 nm for red only). To visualize.

Example  3: experiment design

In Experiment 1, the optimal blastocyst formation rate was measured at 5 and 10 μl drop under traditional drop culture system. In Experiment 2, eggs activated under conditions of 5 and 10 μl volumes were incubated in drop and PCR tubes, ie, oil-free microvessel culture (MTC), at the same fertilized egg density (2 fertilized eggs / μl), respectively. In Experiment 3, an oil-covered MTC system (MTC-oil) was introduced at the same fertilized egg density at a volume of 10 µl to evaluate the effect of mineral oil in an in vitro culture system.

        < Comparative example >

1.Optimum Egg Density

Optimal fertilized egg density was confirmed under a conventional drop culture system (FIG. 2). The development rate of blastocysts gradually increased to 4 fertilized eggs / μl for 10 μl drop and to 5 fertilized eggs / μl for 5 μl drop. As the volume of medium increased (5-10 μl) the optimal fertilized egg density to form blastocysts was reduced (5-4 fertilized eggs / μl). Overall blastocyst-forming rate was higher at 10 μl than 5 μl. At the same fertilized egg density (2 fertilized eggs / μl), the average blastocyst formation rate varied with the volume of the medium (60.0% for 5 μl, 78.3% for 10 μl and 60.8% for 20 μl, data not shown). .

2. Fertilized egg development

One fertilized egg density per two μL was used to compare fertilized egg development rates between the traditional drop system and the oil-free microtubule culture (MTC) system. At 10 μl of medium volume, blastocyst formation rate (89.2%) under MTC system was higher than drop culture system (78.3%). Interestingly, the blastocyst formation rate was 81.0% in the MTC system at a volume of 5 μl with only 60% development rate under the drop culture system, confirming the superiority of the MTC system.

The oiled MTC system was used to find the reason for the higher development rate in the MTC system. Under the same conditions, 10 μl mineral oil used in the existing drop culture system was applied on KSOM medium. As a result, the development rate in oil-covered MTC was lower than oil-free MTC in both 5 and 10 μl media volumes (development rate: 6.4% for 5 μl, 81.3% for 10 μl) (FIG. 3).

The above results explain why the development rate is higher in MTC system than in traditional drop culture system, which can be interpreted as removing the harmful oil effect during the culture period.

3. Cell population counting

In order to measure the ratio of endocytic mass (ICM) and trophoblast cell (TE) numbers in blastocysts, ICM and TE numbers were calculated in three different culture systems: drop culture system, oil-free MTC and oil-covered MTC system.

In the same 10 μl volume culture, more TE cell populations were observed in MTC blastocysts (70.5) compared to blastocysts (53.8) cultured in drop culture systems. However, the ICM cell population did not differ significantly between the drop (20.9) and MTC (21.5) systems.

Similar results were obtained in 5 μl culture: the number of ICM cells was not significantly different (15.8 and 17.3 for drop and MTC, respectively), but the MTC system (64.1) had a much higher number of TE cells than the drop culture system (41.8). It became.

When comparing the development rate of blastocysts, the MTC system occupied most of the total blastocysts in the middle and expansion stages in which development was more advanced than the initial blastocysts (FIG. 5). These results suggest that a large number of TE cells due to faster development in the MTC system.

4. Closed system of MTC system

An example of the present invention was to determine whether the PCR tube is a closed system. If the MTC system of the present invention is a fully closed system, no gas inflow will occur.

In order to prove this judgment, the pH of KSOM medium containing phenol red, which is an acid-base indicator, was increased to 8.0 level, and 10 µl of the prepared culture was added to each culture system (drop, oil-free MTC and oil covered MTC). The change of acidity of the culture solution by carbon dioxide gas was observed under the environment of 5% CO ₂ .

As a result, it was confirmed that the microtube used in the present invention is close to the closed system. That is, the MTC system of the present invention was found to have more gas retention capacity than drop culture.

Specifically, while the drop and open culture systems had a gas retention time of less than 1 hour, the MTC of the present invention exhibited a gas retention and buffer time of 6 hours or more.

This means that the MTC system is more stable in acid-base conditions than the drop culture system and can prevent stress factors such as pH fluctuations in the medium that can be caused by frequent incubator openings.

5. Conclusion

As above, it was confirmed that the oil-free microtubule culture (MTC) system of the present invention enhances the development of pre-implantation mouse fertilized eggs. A large number of fertilized eggs developed into more blastocyst stages with a greater number of TE cells.

When the volume of the medium was reduced to 5 [mu] l, fertilized egg development was inhibited under the traditional micro droplet culture system, but in the same volume of the medium, the MTC system maintained a high development rate. Taken together these results, the MTC system of the present invention showed an excellent effect than the traditional drop culture system.

Previous studies have shown similar effects to growth factor supplementation by increasing the density of fertilized eggs (Paria CB and Dey SK, 1990). Growth factors and cytokines can have different effects on the development of preimplantation embryos such as fertilization rate, fertilization rate to blastocyst stage, blastocyst cell number, metabolism and apoptosis (K Hardy and S Spanos, 200).

Growth factors such as CSF-1 specifically increase TE cell numbers in mice (Bhatnagar et al., 1995). In addition, certain growth factors derived from embryos and / or gonads are involved in preimplantation fertilized egg development and blastocyst function in an autocrine / paracrine fashion.

In the MTC system of the present invention, the fertilized egg was placed in a narrow space (0.2 mm diameter or less) with a small amount of medium, which was able to obtain an effect similar to increasing the density of the fertilized egg. The PCR tube and U-shaped bottom could provide a microenvironment suitable for fertilized egg development, the round bottom could easily collect the fertilized egg in the middle, and the walls of the PCR tube could prevent further dispersion of autocrine and pacrine elements (Fig. 6).

On the other hand, when oil is introduced into the MTC system of the present invention, the development of the fertilized egg was slightly reduced but still maintained a high development rate compared to the drop culture. These results suggest that not only the oil effect but also other factors are involved in and assist the development of the fertilized egg.

In the MTC system, a large number of fertilized eggs (40 fertilized eggs / 10 μl) developed very normally with high development rate. In general, it was considered that there was a limit to increasing the density of fertilized eggs. However, this limitation was overcome in the MTC system (FIG. 7). Mouse or human fertilized eggs are relatively self-sustaining and can survive to the blastocyst stage in vitro in simple salt solutions supplemented with only pyruvate and albumin (Whitten and Biggers 1968; Devreker et al., 1998). Since the MTC system is stable and does not change in physiological conditions, the efficiency of medium use can be maximized in a short distance.

In conclusion, the MTC system could be an effective alternative to the culture apparatus for the development of preimplantation mouse embryos. As an alternative to the existing fertilized egg culture apparatus, the MTC system provided stable and oil-free culture conditions. This may lead to a synergistic effect on preimplantation mouse fertilized egg development.

As described above, the inventor of the present invention improves the development of the fertilized egg culture by providing an oil-free fertilized egg culture method that adversely affects the development of the fertilized egg culture and the fertilized egg culture vessel enabling this method Increased.

Fertilized egg culture method and culture vessel of the present invention can be applied to improve the fertilized egg development rate by applying to the development of fertilized egg of various mammals, including humans or mice.

Claims (10)

delete delete delete A fertilized egg culture vessel having a closed lower microtubule, wherein the lower part of the microtubule is gradually narrowed in shape, and an upper cap of the microtubule is formed with a cap for sealing integrally or separately with the microtubule. Fertilized egg culture vessel, fertilized egg culture vessel, characterized in that the lower portion of the microtubules or U-shaped, or the diameter of the microtubule is 1 to 0.2 cm in diameter and the length of the gradually narrowing portion of the lower microtubules 1 to 3 cm, characterized in that for culturing the fertilized egg in any of the fertilized egg culture container of the fertilized egg culture container. 5. The method of claim 4, wherein the culture medium is free of oil. 5. The method of claim 4, wherein the number of fertilized eggs in the fertilized egg culture vessel is 1 / μL to 8 / μL. The method for culturing fertilized eggs according to claim 4, wherein the culture medium is 1 to 15 µl. The fertilized egg culture method according to claim 4, wherein the fertilized egg is cultured together with a medium in a space of 0.2 mm or less in diameter of the culture vessel. The method of claim 4, wherein the fertilized egg is a mammalian fertilized egg. 10. The method of claim 9, wherein the mammal is human or mouse culture method of fertilized egg.

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