JP7759656B2 - Immortalized bovine oviduct epithelial cells and their uses - Google Patents

Description

The present invention relates to immortalized bovine oviduct epithelial cells and their uses.

In the livestock industry, in vitro embryo production and embryo transfer are carried out to efficiently increase the number of mammals, such as cattle. In recent years, with the increasing demand for livestock products, it has become essential to improve the efficiency of in vitro embryo production and fertility. From the perspectives of assisted reproductive technology and the conservation of endangered species, there is a demand for technology that can more efficiently produce embryos with the potential for high fertility rates.

Since embryos reside within the fallopian tube during fertilization and early embryonic development, a culture method that mimics the environment within the fallopian tube is effective for in vitro culture of bovine embryos. Therefore, a method was first developed in which in vitro fertilized bovine embryos were co-cultured with fallopian tube epithelial cells (Non-Patent Document 1).

Next, a method was developed to develop bovine and porcine embryos using conditioned medium prepared from oviduct epithelial cells (Patent Document 1, Non-Patent Documents 2 and 3).

It was subsequently discovered that culturing bovine embryos under low oxygen concentrations (5%) allows them to develop to the blastocyst stage with an efficiency comparable to the previous two methods. The low-oxygen method is now the mainstream method for in vitro embryo production. Furthermore, bovine oviduct epithelial cells have been reported to be immortalized by introducing the human telomere reverse transcriptase (hTERT) gene or the SV40 T antigen gene (Non-Patent Documents 4-7).

Japanese Patent Application Laid-Open No. 2003-93054

W. H. Eyestone et. al., J. Reprod. Fert. 85, 715-720. (1989) P. Mermillod et. al., Biol. Reprod. 49, 582-587.(1993) A. Van Langendonckt et. al., An overview. Reprod. Nutr. Dev. 36, 493- 502. (1996) K. Murata et. al., Tiss. Cult. Res. Commun. 25: 119-127 (2006) Murata Ken et al., Fundamental analysis of the cellular senescence mechanism using bovine oviduct epithelial cells, Abstracts of the Annual Meeting of the Japanese Society of Animal Science, Vol. 103rd, Page. 81 (2004.03.20) Murata Ken et al., Analysis of Robertsonian chromosome fusions in bovine oviduct epithelial cells, 79th Annual Meeting of the Japanese Society for Tissue Culture K. Murata et. al., In Vitro Cellular & Developmental Biology-Animal 43 7): 235-244 (2007)

In the co-culture method, isolating fallopian tube epithelial cells from bovine fallopian tubes requires approximately two hours of sterile procedures. It takes approximately one week to obtain the number of cells required for co-culture. Furthermore, because there is a limit to the number of cells that can be obtained, cells must be frequently isolated from the fallopian tube. Fallopian tube epithelial cells are made up of four types of cells, and each time the cells are isolated, their composition changes, potentially resulting in different properties for the cell population. Furthermore, with the co-culture method, not only are cell culture techniques required of the users, such as veterinarians who will actually use this technology, but the cells must also be transported frozen at temperatures below -80°C using dry ice or similar to distribute them to users.

When using conditioned medium made from fallopian tube epithelial cells, as mentioned above, it is time-consuming to isolate fallopian tube epithelial cells from the fallopian tube to prepare the conditioned medium, and because the number of cells that can be obtained is limited, the amount of conditioned medium is also limited. Therefore, as with the co-culture method, cells must be isolated frequently, and the properties of the cell population may change with each isolation, which could change the composition of the conditioned medium.

Although the hypoxic method improves embryo development rates, it also produces blastocysts with low fertility as well as highly fertile ones, so further technical improvements are needed to improve the efficiency of embryo transfer.

In addition, it has been pointed out that while immortalized cells created using genetic recombination methods exhibit high proliferation potential, they often lack differentiation potential.

In order to solve the above problems, the present invention includes any one of the following aspects.
<1> A conditioned medium obtained by culturing immortalized bovine oviduct epithelial cells, which are non-genetically modified cells.
<2> The conditioned medium according to <1>, wherein the immortalized bovine oviduct epithelial cells have a population doubling level of more than 200.
<3> The conditioned medium according to <1> or <2>, which contains a substance that precipitates when the culture of the immortalized bovine oviduct epithelial cells is subjected to ultracentrifugation.
<4> A method for preparing a conditioned medium, comprising the steps of culturing immortalized bovine oviduct epithelial cells, which are non-genetically modified cells, in a cell culture medium, and incubating the cells in a medium that can be used for both cell culture and embryo development culture.
<5> A method for culturing a mammalian embryo, comprising the step of culturing a mammalian embryo using the conditioned medium according to any one of <1> to <3>.
<6> The method for culturing mammalian embryos according to <5>, wherein the mammalian embryos are bovine embryos, and the method comprises the steps of selecting embryos that have cleaved 27 hours after insemination, selecting embryos that have reached the 2-cell stage and have uniform blastomeres 31 hours after insemination, and selecting embryos that have grown to the 8-cell stage or higher 55 hours after insemination.
<7> Immortalized bovine oviduct epithelial cells, which are non-genetically modified cells.
<8> The immortalized bovine oviduct epithelial cells according to <7>, having a population doubling level of more than 200.
<9> A method for producing immortalized bovine oviduct epithelial cells, which are non-genetically modified cells, comprising a step of subculturing bovine oviduct epithelial cells collected from the oviduct of a bovine.

The immortalized bovine oviduct epithelial cells of the present invention enable the production of large quantities of homogeneous oviduct epithelial cells, and also enable the mass production of conditioned medium containing these immortalized bovine oviduct epithelial cells and having uniform properties. Furthermore, the mammalian embryo culture method of the present invention enables the efficient production of highly fertile blastocysts.

1 shows the growth curve of immortalized bovine oviduct epithelial cells according to Example 1 of the present invention. 1 shows the blastocyst development rate on day 9 of culture in each conditioned medium concentration test group according to Example 3 of the present invention. 1 shows the percentage of embryos that satisfy the selection conditions at each time point after insemination in each conditioned medium concentration test group according to Example 4 of the present invention. 1 shows the percentage of non-selected blastocysts (those that do not satisfy the four conditions for embryo development) in each conditioned medium concentration test group according to Example 4 of the present invention. 1 shows the incidence of blastocysts that satisfy the four conditions for embryonic development and the incidence of blastocysts that do not satisfy the four conditions for embryonic development in each conditioned medium concentration test group according to Example 4 of the present invention. 1 shows the percentage of embryos that meet the selection criteria in each medium at each time point after insemination according to Example 6 of the present invention. 1 shows the percentage of non-selected blastocysts (those that do not meet the four conditions for embryo development) in each medium according to Example 6 of the present invention. 1 shows the incidence of blastocysts that satisfy the four conditions for embryo development and the incidence of blastocysts that do not satisfy the four conditions for embryo development in each medium according to Example 6 of the present invention.

[Definitions of terms, etc.]
As used herein, "immortalized cells" refers to cell lines that have vigorous proliferation ability even when their population doubling level exceeds 200. "Population doubling level" refers to the number of times that cells can divide in a culture environment. A population doubling level of more than 200 means that cells can divide more than 200 times in a culture environment. Note that normal fallopian tube epithelial cells can only divide approximately 25 to 50 times.

As used herein, "in vitro produced embryo" refers to an embryo that has undergone in vitro maturation and in vitro fertilization.

As used herein, "conditioned medium" refers to a medium in which cells or tissues are cultured, resulting in the secretion of cellular factors such as physiologically active substances into the culture solution, and the medium contains these cellular factors. When cells or embryos are cultured in the medium, the cellular factors contained in the conditioned medium support cell differentiation, proliferation, and embryo development.

As used herein, "insemination" refers to the process of placing sperm and eggs in a single fertilization medium.

1. Immortalized bovine oviduct epithelial cells
The present invention provides immortalized bovine oviduct epithelial cells, which are non-genetically modified cells.

Immortalized bovine oviduct epithelial cells are derived from bovine oviduct epithelial cells isolated from bovine oviducts.

Examples of the types of cattle used include cattle (Bos taurus), zebu (Bos indicus), and water buffalo (Bubalus bubalis). Dairy breeds, meat breeds, dual-purpose breeds, draft breeds, and dual-purpose breeds are acceptable. The types of cattle used are not particularly limited, and examples include Japanese Black cattle, Japanese Brown cattle, Japanese Shorthorn cattle, and other Wagyu breeds, Holsteins, Jerseys, and native breeds of each country.

The immortalized bovine oviduct epithelial cells of this embodiment may be isolated after repeated subculture over an extended period of time. The immortalized bovine oviduct epithelial cells may be cells obtained by repeated subculture until the population doubling level reaches 200 or more, preferably 210 or more, and more preferably 220 or more. Furthermore, the immortalized bovine oviduct epithelial cells are cells obtained after subculture for a period required to achieve such a population doubling level, for example, 10 months or more, 12 months or more, or 24 months or more.

It has been pointed out that immortalized cells produced using genetic recombination techniques often exhibit high proliferation potential but lack differentiation potential. In contrast, the immortalized bovine oviduct epithelial cells of the present invention possess both high proliferation potential and high differentiation potential.

Furthermore, the immortalized bovine oviduct epithelial cells of the present invention have the ability to produce physiologically active substances for embryonic development.

One example of a physiologically active substance for embryonic development is exosomes. Exosomes are a type of extracellular vesicle with a particle diameter of approximately 100 nm, containing membrane proteins, adhesion molecules, various enzymes, miRNA, mRNA, and other components. Exosomes are secreted from cells and taken up by other cells, where they are involved in intercellular communication.

The proliferation potential of cells can be evaluated, for example, using the population doubling level as an indicator. In one embodiment, the immortalized bovine oviduct epithelial cells have a population doubling level of more than 200, more preferably more than 210, and even more preferably more than 220.

In addition, the proliferation ability of cells can be evaluated using, for example, telomerase activity as an indicator.

Immortalized bovine oviduct epithelial cells can be preserved semi-permanently if frozen. For frozen storage, it is preferable to use liquid nitrogen or similar.

2. Method for producing immortalized bovine oviduct epithelial cells
The present invention also provides a method for producing immortalized bovine oviduct epithelial cells, which comprises a step of subculturing bovine oviduct epithelial cells isolated from bovine oviducts (subculture step).

Before subculture, bovine oviduct epithelial cells are isolated and collected, for example, from the oviducts of slaughtered cows. The types of cows are as described above.

In one embodiment, the fallopian tube epithelial cells are cultured for passage for, for example, 10 months or more, 12 months or more, or 24 months or more.

The number of passages of the oviduct epithelial cells required to obtain immortalized bovine oviduct epithelial cells is preferably 70, more preferably 75, and even more preferably 80.

Culturing is carried out using a cell culture medium such as D-MEM solution supplemented with 10% fetal bovine serum. It is also preferable to maintain the culture temperature at 38.0°C during subculture.

The immortalized bovine oviduct epithelial cells of the present invention have high proliferation and differentiation capabilities, making it possible to produce and supply large quantities of oviduct epithelial cells without having to re-isolate epithelial cells from bovine oviducts.

Therefore, it is possible to efficiently obtain immortalized bovine oviduct epithelial cells without the hassle of repeatedly isolating them from bovine oviducts.

Non-genetically modified, immortalized bovine oviduct epithelial cells can be suitably used as a component of conditioned medium used in the developmental culture of mammalian embryos. Furthermore, by using non-genetically modified, immortalized bovine oviduct epithelial cells, it is possible to mass-produce conditioned medium with uniform properties, as described below. Furthermore, the resulting immortalized bovine oviduct epithelial cells have the effect of improving the rate of blastocyst development when used in conditioned medium for embryo culture in in vitro embryo production.

[3. Conditioned medium]
The present invention provides a conditioned medium obtained by culturing immortalized bovine oviduct epithelial cells, which are non-genetically modified cells.

Immortalized bovine oviduct epithelial cells include those described in [1. Immortalized bovine oviduct epithelial cells] and provide the active components of the conditioned medium.

The category of "conditioned medium obtained by culturing immortalized bovine oviduct epithelial cells, which are non-genetically modified cells" encompasses not only cases in which the conditioned medium contains the immortalized cells, but also cases in which the conditioned medium contains only one or more components contained in the cultured bovine oviduct epithelial cells. For example, the category of the conditioned medium of the present invention also encompasses conditioned medium that contains, among the substances contained in the conditioned medium containing immortalized cells, a substance that precipitates when the conditioned medium is ultracentrifuged (precipitate). In one example, the category also encompasses conditioned medium that contains exosomes as one of the components contained in the immortalized bovine oviduct epithelial cells.

A conditioned medium may contain all or some of the components contained in known cell culture media. The conditioned medium may also contain components of media that can be used for both cell culture and embryo development culture. Examples of media that can be used for both cell culture and embryo development culture include KSOM medium, WM medium, and CZB medium.

Other ingredients may include various amino acids, vitamins, inorganic salts, antibiotics, fetal bovine serum, etc.

4. Method for preparing conditioned medium
The present invention provides a method for preparing a conditioned medium, which includes a step of culturing immortalized bovine oviduct epithelial cells in a cell culture medium (cell culture step), and a step of incubating the cells in a medium that can be used for both cell culture and embryo development culture (incubation step).

The cell culture medium used in the cell culture process includes D-MEM solution supplemented with 10% fetal bovine serum. Culturing is carried out in the desired culture vessel, such as a culture dish.

The cell culture temperature may be any suitable temperature for cell culture, for example, 38.0°C.

The cell culture period is preferably until the cells become confluent, for example, between 3 and 4 days.

In one embodiment, the method further includes a step of removing the culture supernatant after the cell culture step and before the incubation step. The culture supernatant is removed by aspirating, centrifugation, filtering, or the like.

Media that can be used for cell culture in the incubation process and embryo development culture include KSOM medium, WM medium, and CZB medium.

The incubation temperature is, for example, 38.0°C, and the incubation period is, for example, at least one day and not more than two days.

Furthermore, in one embodiment, after the incubation step, the method further includes a step of removing contaminating cells or dead cells, etc., and sterilizing the incubated cell culture by subjecting the culture to light centrifugation, filtration, etc.

In one example of a method for preparing conditioned medium, immortalized bovine oviduct epithelial cells are cultured in a culture dish using D-MEM supplemented with 10% fetal bovine serum as the cell culture medium until they become confluent. After removing the culture supernatant, the cells are incubated for two days in KSOM medium, a medium that can be used for both cell culture and embryo development culture. The resulting preparation is referred to as conditioned medium.

In another example of a method for preparing conditioned medium, the cell culture obtained after incubation using the method described above is ultracentrifuged, and the resulting sediment is suspended in a medium that can also be used for embryo development and culture, such as KSOM medium. The preparation obtained in this manner can also be used as conditioned medium. For example, the pellet-down method using an ultracentrifuge can be used to ultracentrifuge the conditioned medium. Furthermore, the centrifugal force may be any force suitable for separating exosomes. For example, it is preferable to perform the ultracentrifugation at 100,000 g or higher. In this example, centrifugation is performed at 24,700 rpm for 2 hours.

By using the material that precipitates when the above-mentioned cell culture is ultracentrifuged as a component of the conditioned medium, embryo development media other than the medium originally used for cell culture (e.g., KSOM medium) (including embryo development media for species other than bovine, such as porcine) can also be used as the medium for suspending the precipitated material.

Furthermore, the sediment obtained by ultracentrifugation of the above-mentioned cell culture has a smaller volume than the conditioned medium itself containing immortalized cells, so it does not take up much space during storage or transportation. For example, it can be concentrated at a concentration rate of several tens to several hundred times and transported. Therefore, it is suitable as an additive for medium preparation.

Since the fallopian tube epithelium secretes various factors involved in cell proliferation, the conditioned medium created using immortalized fallopian tube epithelial cells also mimics the environment inside the fallopian tube due to the factors secreted by the fallopian tube epithelial cells. Therefore, it is thought that it can support early development.

As described above, the conditioned medium of the present invention does not require the isolation of fallopian tube epithelial cells when preparing the conditioned medium, eliminating the need for cell culture and making it extremely easy to prepare. Furthermore, the conditioned medium can be stored refrigerated, allowing for easy distribution to users. Furthermore, since the conditioned medium is prepared from immortalized cells using the method for preparing the conditioned medium of the present invention, it is possible to produce large quantities of homogeneous conditioned medium.

5. Mammalian Embryo Culture Method
The present invention also provides a method for culturing mammalian embryos, which comprises a step of culturing mammalian (mammalian) embryos using the above-mentioned conditioned medium (embryo culturing step).

There are no limitations on the species or breed of animal from which mammalian embryos are derived. In the case of bovine embryos, they may be from the same species or breed as the bovine from which the immortalized bovine oviduct epithelial cells used to prepare the conditioned medium were derived, or they may be from a different species or breed. Examples of embryos include in vitro-produced embryos, and more specifically, include embryos that have been matured and fertilized in vitro, embryos produced by techniques such as intracytoplasmic sperm injection, and nuclear transfer embryos.

When using in vitro fertilized embryos as the embryos to be subjected to the embryo culture process, for example, in vitro fertilization can be performed using known in vitro fertilization techniques. For example, immature eggs can be cultured in a known in vitro maturation culture medium such as M199 solution, and then in vitro fertilization can be performed using a known in vitro fertilization medium such as BO solution and frozen-thawed sperm. In vitro fertilized embryos obtained by this can be used. Embryos immediately after insemination are preferred.

Types of mammalian embryos include human embryos, embryos of livestock animals (pig, horse, goat, sheep, cow, etc.), embryos of pet animals (cat, dog, hamster, rabbit, guinea pig, etc.), and embryos of laboratory animals (rodents such as mouse and rat, monkey, etc.), with bovine embryos being particularly preferred. In one example, the embryo is an embryo of a mammal other than a human embryo. In another example, the embryo is an embryo of an endangered animal.

The embryo culture temperature during the embryo culture process may be any temperature appropriate for embryo culture, such as 38.5°C.

In addition, other conditions, such as culture medium, culture time, carbon dioxide concentration, and oxygen concentration, can be selected appropriately to create culture conditions suitable for culturing embryos in vitro.

In one example of the embryo culture process, developmental culture is carried out under 5% hypoxia. More specifically, embryo culture is carried out under conditions of, for example, 5% oxygen, 5% carbon dioxide, and 90% nitrogen. By culturing under 5% hypoxia, the efficiency of blastocyst development can be further improved.

In the embryo culture step, embryo culture is preferably carried out in a medium containing conditioned medium at a concentration of 80% or more per medium volume, more preferably 90% or more per medium volume, and most preferably 100% per medium volume of conditioned medium.

The embryo culture period using conditioned medium is, for example, four days, from day 0 to day 3 after insemination.

Then, on the third day after insemination, discard half of the medium used between days 0 and 3 after insemination, and replenish with the same amount of embryo development culture medium such as KSOM medium, and continue culturing under the same temperature and environmental conditions. It is preferable to continue culturing until the seventh day after insemination, more preferably until the eighth day after insemination, and even more preferably until the ninth day after insemination.

When the mammalian embryo culture method of the present invention is used with the above-mentioned livestock embryos, it can be used for a wide range of embryo production in the fields of livestock farming and reproductive engineering. Furthermore, when human embryos are used, it can be suitably used in human assisted reproductive technology.

In the case of bovine embryos, there are several criteria for judging the quality of the blastocysts obtained as a result of the development and culture of bovine embryos, but it is known that if the rate of cleavage in the early stages of development is inappropriate, the conception rate after embryo transfer will be low. In other words, if the rate of cleavage in the blastocysts obtained as a result of the development and culture of bovine embryos is too fast or too slow, the conception rate after embryo transfer will be low.

Embryos that develop when the following four conditions (1) to (4) regarding cleavage rate are met are expected to have a high rate of conception when transferred to the uterus, and are one of the indicators for embryo selection. On the other hand, blastocysts that develop without meeting these four conditions, that is, blastocysts that develop at an inappropriate cleavage rate, where the cleavage rate is either too fast or too slow, are expected to have a low rate of conception when transferred to a bovine uterus.

(1) Embryos that have cleaved 27 hours after insemination. (2) Embryos at the 2-cell stage 31 hours after insemination. (3) Embryos with uniform blastomeres 31 hours after insemination. (4) Embryos at the 8-cell stage or higher 55 hours after insemination. Specifically, it is preferable to observe the cleavage of the embryos 27 hours, 31 hours, and 55 hours after insemination and select only embryos with an appropriate developmental speed.

Therefore, one embodiment of the bovine embryo culture method further includes the steps of selecting embryos that have cleaved 27 hours after insemination, selecting embryos that have reached the 2-cell stage and have uniform blastomeres 31 hours after insemination, and selecting embryos that have grown to the 8-cell stage or higher 55 hours after insemination (embryo selection steps).

The state of the embryos at each point after insemination can be confirmed by observing the embryos under a microscope during cultivation. Furthermore, selection at each point is carried out by observing them under a microscope and selecting and recovering embryos that meet certain conditions.

This embryo selection process allows for the production of blastocysts that are expected to have a high fertility rate.

The bovine embryo culture method, which includes the above-mentioned embryo selection step, significantly increases the rate of development of blastocysts that meet the four cleavage rate conditions, and significantly decreases the rate of development of blastocysts that do not meet the four conditions. In other words, by culturing embryos using the bovine embryo culture method, which includes the above-mentioned embryo selection step, it is possible to efficiently obtain blastocysts that meet the four cleavage rate conditions, i.e., blastocysts that are expected to have a high conception rate.

According to the bovine embryo culture method including the above-mentioned embryo selection step, the percentage of blastocysts that developed without satisfying the above four conditions (non-selected blastocysts) among the obtained blastocysts is preferably 40% or less, more preferably 20% or less, and even more preferably 10% or less.

In this way, in the bovine embryo culture method, by culturing bovine embryos using the conditioned medium of the present invention and applying the above-mentioned selection process, blastocysts that are expected to have high fertility can be produced extremely efficiently.

The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

Examples of the present invention are described below.

Example 1: Preparation of immortalized bovine oviduct epithelial cells
The present inventors aimed to obtain immortalized bovine oviduct epithelial cells.

To obtain immortalized bovine oviduct epithelial cells capable of producing physiologically active substances for embryonic development, oviduct epithelial cells were first isolated from the oviducts of slaughtered cows and cultured in cell culture medium (D-MEM solution supplemented with 10% fetal bovine serum).

By measuring the telomere length and telomerase activity of bovine oviduct epithelial cells and repeatedly subculturing them over a long period (approximately 10 months or more), we established a cell line with a population doubling level of over 220. Figure 1 shows the growth curve of immortalized bovine oviduct epithelial cells.

Although mammalian cultured cells, with the exception of many tumor cells and some immortalized cells, tend to lose telomerase activity with passage, the bovine oviduct epithelial cell line obtained by the inventors consistently exhibited high telomerase activity. Furthermore, although the telomeres of the bovine oviduct epithelial cell line shortened until the population doubling level reached 120, they subsequently lengthened and restored telomere length.

As described above, the fallopian tube epithelial cell line established by the inventors was shown to be an immortalized cell line based on the population doubling level as well as the characteristics of telomerase activity and telomere length.

Example 2: Preparation of conditioned medium
The immortalized bovine oviduct epithelial cells prepared in Example 1 were used to prepare a conditioned medium for use in culturing bovine embryos.

Immortalized bovine oviduct epithelial cells were cultured in cell culture medium (D-MEM solution supplemented with 10% fetal bovine serum) until confluent in a culture dish. After removing the culture supernatant, the cells were incubated in KSOM medium, which can be used for both cell culture and embryo development culture, at 38.0°C for two days to obtain conditioned medium. The conditioned medium was centrifuged at 2000 x g for 10 minutes to remove contaminating cells and dead cells, and then sterilized by filtration through a 0.22 μm filter to remove impurities, sterilize, and remove extracellular vesicles larger than 0.22 μm before use in subsequent experiments.

The fallopian tube epithelium secretes various factors involved in cell proliferation, and it is thought that conditioned medium prepared using immortalized fallopian tube epithelial cells can also mimic the environment within the fallopian tube and support early development.

Example 3: Analysis of the effect of conditioned medium on the development of in vitro-produced bovine embryos
In vitro-produced bovine embryos were cultured and developed using either the conditioned medium (100%) prepared in Example 2, the KSOM medium (0%) used as a control to prepare the conditioned medium, or a medium (50%) prepared by diluting the conditioned medium with an equal volume of KSOM medium, and the developmental efficiencies were compared.

Bovine immature oocytes were cultured for 20 hours in a known in vitro maturation medium (M199 solution supplemented with 10% fetal bovine serum) and then subjected to in vitro fertilization using a known in vitro fertilization medium (BO solution) and frozen-thawed sperm. From days 0 to 3 after insemination, the oocytes were cultured in either conditioned medium (100%), KSOM medium (0%), or conditioned medium (50%). On day 3 after insemination, half of the medium used from days 0 to 3 after insemination was discarded and replaced with the same amount of KSOM medium. Culture was continued until day 9 after insemination. Culture was carried out at 38.5°C under conditions of 5% oxygen, 5% carbon dioxide, and 90% nitrogen. The concentrations of conditioned medium used up to 3 days after insemination and the blastocyst development rate on day 9 of culture are shown in Table 1 and Figure 2. Figure 2 shows the blastocyst development rate on day 9 of culture for each conditioned medium concentration test group.
The blastocyst development rate was calculated relative to the control (KSOM medium) concentration, and is shown as the mean ± standard error (%).

Table 1 clearly shows that when embryos 0 to 3 days after insemination are cultured using conditioned medium (100%), the blastocyst development rate is significantly higher than when cultured using 0% or 50% conditioned medium. This demonstrates that blastocysts can be obtained with high efficiency when the conditioned medium of the present invention is used.

Example 4: Analysis of the effect of conditioned medium on the production of embryos with appropriate cleavage rates
Bovine IVF embryos were cultured in vitro using either the same conditioned medium (100%) used in Example 3, the KSOM medium (0%) from which the conditioned medium was prepared, or a medium (50%) prepared by diluting the conditioned medium with an equal volume of KSOM medium. Cleavage embryos were selected 27 hours after insemination. Embryos with 2-cell stage and uniform blastomeres were then selected 31 hours later. Embryos developing to the 8-cell stage or greater were further selected 55 hours later. On day 3 after insemination, half of the medium used between days 0 and 3 was discarded and replaced with the same volume of KSOM medium. Culture was continued until day 9 after insemination. Culture was carried out at 38.5°C in an atmosphere of 5% oxygen, 5% carbon dioxide, and 90% nitrogen. The development rates of embryos that met the selection criteria at 27, 31, and 55 hours after insemination, and the development rates of blastocysts obtained through such cleavage rates on day 9, were then investigated for each concentration of conditioned medium. The results are shown in Table 2 and Figure 3. The development rates (%) are shown as mean ± standard error. Figure 3 shows the percentage of embryos that met the selection criteria at each time point after insemination for each conditioned medium concentration test group.

Table 2 and Figure 3 show that use of the conditioned medium increases the percentage of embryos that developed at an appropriate cleavage rate 27 hours, 31 hours, and 55 hours after insemination, and that the percentage of blastocysts obtained after 9 days that experienced such a cleavage rate is significantly higher. This demonstrates that by using the conditioned medium of the present invention and selecting embryos that meet the four cleavage rate conditions, blastocysts that are expected to have a high conception rate can be obtained with high efficiency.

Table 3 and Figure 4 show the percentage of blastocysts produced from each concentration of conditioned medium that developed without meeting the four conditions (non-selected blastocysts). The development rate (%) is shown as the mean ± standard error. Figure 4 shows the percentage of non-selected blastocysts (those that did not meet the four conditions for embryo development described above) for each conditioned medium concentration test group.

Table 3 shows that when this conditioned medium is used, the percentage of blastocysts obtained through developmental culture that develop into blastocysts without undergoing an appropriate cleavage rate (i.e., cleavage occurs either too quickly or too slowly) is low.

Furthermore, Table 4 and Figure 5 show the results of a comparison of the effectiveness of each concentration of conditioned medium in generating blastocysts that meet the four conditions and those that do not. The development rate (%) is shown as the mean ± standard error. Figure 5 shows the development rate of blastocysts that meet the four conditions for embryo development and the development rate of blastocysts that do not meet the four conditions for embryo development for each conditioned medium concentration test group.

Table 4 shows that the use of conditioned medium significantly increases the rate of development of blastocysts that meet the four cleavage rate conditions, while also decreasing the rate of development of blastocysts that do not meet the four conditions. The results in Tables 2 to 4 and Figures 2 to 5 show that when using the conditioned medium of the present invention in bovine embryo culture and selecting embryos that meet the four cleavage rate conditions, blastocysts that are expected to have a high conception rate can be obtained with high efficiency.

Example 5: Separation of sediment by ultracentrifugation of conditioned medium
Thirty milliliters of the same conditioned medium used in Examples 2-4 was placed in a centrifuge tube and subjected to ultracentrifugation at 24,700 rpm at 4°C for 2 hours according to the pellet-down method. After ultracentrifugation, extracellular vesicles containing exosomes and impurities should settle to the bottom of the tube, and the supernatant should contain significantly fewer exosomes. A portion of this supernatant was saved, while the majority was removed by decantation. The sediment was then resuspended in 5 mL of phosphate buffered saline (PBS) and vortexed to separate the impurities from the exosomes. An additional 25 mL of PBS was added and ultracentrifugation was performed a second time. The supernatant was again decanted to obtain the sediment. The sediment was resuspended in 1.5 mL of KSOM medium. This resulted in a 20-fold concentration of the sediment components in the conditioned medium.

Example 6: Analysis of the effect of ultracentrifugation sedimentation of conditioned medium on the production of embryos with appropriate cleavage rates.
Bovine in vitro fertilized embryos were cultured in vitro using one of four media: (1) the same conditioned medium used in Example 4; (2) a control medium prepared by heating the KSOM medium, the base medium for the conditioned medium, in a _CO2 incubator for 48 hours; (3) the supernatant obtained by ultracentrifuging the conditioned medium to significantly reduce exosomes and other components; and (4) a medium prepared by adding a 20-fold dilution of the sediment to KSOM medium. As in Example 4, cleaved embryos were selected 27 hours after insemination. Subsequently, embryos with two-cell stage and uniform blastomeres were selected 31 hours later. Further selection was made after 55 hours for embryos that had developed to the 8-cell stage or greater. On day 3 after insemination, half of the medium used between days 0 and 3 after insemination was discarded, and the same amount of KSOM medium was replenished. Culture was continued until day 8 after insemination. Culture was carried out under conditions of 38.5°C, 5% oxygen, 5% carbon dioxide, and 90% nitrogen. The development rates of embryos suitable for selection were then investigated for the four media 27, 31, and 55 hours after insemination, as well as the development rates of blastocysts obtained through such cleavage rates on day 8. The results are shown in Table 5 and Figure 6. The development rates (%) are shown as mean ± standard error. Figure 6 shows the percentage of embryos that met the selection criteria for each medium at each time point after insemination.

Table 5 and Figure 6 show that the use of this conditioned medium increases the percentage of embryos that developed at an appropriate cleavage rate 27 hours, 31 hours, and 55 hours after insemination, compared to the control and supernatant from which exosomes and other substances had been removed by ultracentrifugation, and that the percentage of blastocysts obtained after 8 days that had experienced such a cleavage rate was significantly higher. Furthermore, with KSOM + sediment, results similar to those of the conditioned medium were obtained, although not significantly different. This shows that by using sediment extracted from the conditioned medium of the present invention and selecting embryos that meet the four cleavage rate conditions, blastocysts with an expected high conception rate can be obtained with high efficiency.

Table 6 and Figure 7 show the percentage of blastocysts that developed without meeting the four conditions (non-selected blastocysts) among those produced from the control, conditioned medium, the supernatant after ultracentrifugation of conditioned medium, and KSOM + sediment. The development rate (%) is shown as the mean ± standard error. Figure 7 shows the percentage of non-selected blastocysts (those that did not meet the four conditions for embryo development described above) in each medium.

Table 6 shows that when the conditioned medium was used and when KSOM + sediment was used, the percentage of blastocysts obtained by developmental culture that developed into blastocysts without undergoing an appropriate cleavage rate (i.e., cleavage rates that were either too fast or too slow) was lower. On the other hand, when the supernatant after ultracentrifugation of the conditioned medium was used, the percentage of blastocysts that developed into blastocysts without undergoing an appropriate cleavage rate was the highest. This is thought to be due to the significant reduction in exosomes caused by ultracentrifugation and the effects of negative components such as ammonia that are unavoidably contained in the conditioned medium.

Furthermore, Table 7 and Figure 8 show the results of a comparison of the effectiveness of each medium in generating blastocysts that meet the four conditions and those that do not. The development rate (%) is shown as the mean ± standard error. Figure 8 shows the development rate of blastocysts that meet the four conditions for embryo development and the development rate of blastocysts that do not meet the four conditions for embryo development in each medium.

Table 7 shows that the use of conditioned medium or KSOM + sediment increases the rate of blastocyst development that meets the four cleavage rate criteria.

The results in Tables 5-7 and Figures 6-8 show that when culturing bovine embryos, using conditioned medium and KSOM + sediment and selecting embryos that meet the four cleavage rate conditions, blastocysts that are expected to have a high conception rate can be obtained with high efficiency. These findings demonstrate that, of the substances contained in conditioned medium containing immortalized bovine oviduct epithelial cells, the substances that precipitate when the conditioned medium is ultracentrifuged play an important role in producing highly fertile embryos.

The present invention can be widely used in the fields of livestock farming, reproductive engineering, and assisted reproductive medicine, including the production of mammals such as cattle and the improved breeding of mammals such as cattle.

Claims (8)

A conditioned medium containing a culture of immortalized bovine oviduct epithelial cells, which are non-genetically modified cells, and which contains exosomes as an active ingredient. A conditioned medium containing a substance that precipitates when cultures of immortalized bovine oviduct epithelial cells (non-genetically modified cells) are ultracentrifuged, and this substance contains exosomes as an active ingredient. The conditioned medium of claim 1 or 2, wherein the immortalized bovine oviduct epithelial cells have a population doubling level of more than 200. A method for preparing a conditioned medium, comprising the steps of: culturing immortalized bovine oviduct epithelial cells, which are non-genetically modified cells and have a population doubling level of more than 200, in a cell culture medium; and incubating the cells in a medium that can be used for both cell culture and embryo development culture. A method for culturing mammalian embryos, comprising culturing mammalian embryos using the conditioned medium described in any one of claims 1 to 3. the mammalian embryo is a bovine embryo;
6. The method for culturing mammalian embryos according to claim 5, comprising the steps of selecting embryos that have cleaved 27 hours after insemination, selecting embryos that have reached the 2-cell stage and have uniform blastomeres 31 hours after insemination, and selecting embryos that have grown to the 8-cell stage or higher 55 hours after insemination. Non-genetically modified immortalized bovine oviduct epithelial cells with a population doubling level of over 200. A method for producing immortalized bovine oviduct epithelial cells, which are non-genetically modified cells, comprising:
The method comprises subculturing bovine oviduct epithelial cells collected from bovine oviducts for 10 months or more ;
A method for producing immortalized bovine oviduct epithelial cells , comprising measuring telomere length and telomerase activity during the culture period of the subculturing step, and isolating cells with a population doubling level of more than 200 .