Immortalized bovine oviduct epithelial cells and their uses

Abstract

To provide immortalized bovine fallopian tube epithelial cells, and to provide uses thereof.SOLUTION: The conditioned medium of the present invention is obtained by culturing immortalized bovine fallopian tube epithelial cells, which are non-genetically modified cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to immortalized bovine oviduct epithelial cells and uses thereof. [Background technology]

[0002] 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 perspective of assisted reproductive technology or the conservation of endangered animals, there is a demand for more efficient technology to produce embryos with the potential for high fertility rates.

[0003] Since embryos reside in the fallopian tube during fertilization and early embryonic development, a culture method that mimics the environment inside 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).

[0004] Next, a method was developed to develop bovine and porcine embryos using a conditioned medium prepared using oviduct epithelial cells (Patent Document 1, Non-Patent Documents 2 and 3).

[0005] It was subsequently discovered that culturing bovine embryos under low oxygen concentrations (5%) allowed them to develop to the blastocyst stage with an efficiency comparable to that of 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). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Laid-Open No. 2003-93054 [Non-patent literature]

[0007] [Non-Patent Document 1] WH Eyestone et. al., J. Reprod. Fert. 85, 715-720.(1989) [Non-patent document 2] P. Mermillod et. al., Biol. Reprod. 49, 582-587.(1993) [Non-patent document 3] A. Van Langendonckt et. al., An overview. Reprod. Nutr. Dev. 36, 493- 502. (1996) [Non-patent document 4] K. Murata et. al., Tiss. Cult. Res. Commun. 25: 119-127 (2006) [Non-Patent Document 5] 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) [Non-patent document 6] Murata Ken et al., Analysis of Robertsonian chromosome fusions in bovine oviduct epithelial cells, 79th Annual Meeting of the Japanese Society for Tissue Culture [Non-Patent Document 7] K. Murata et. al., In Vitro Cellular & Developmental Biology-Animal 43 7): 235-244 (2007) Summary of the Invention [Problem to be solved by the invention]

[0008] In the co-culture method, isolation of oviduct epithelial cells from bovine oviducts 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 isolated from the oviduct frequently. Oviduct epithelial cells are composed of four types of cells, and each time the cells are isolated, their composition changes, potentially resulting in different properties of the cell population. Furthermore, in the case of the co-culture method, not only is cell culture skills 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 other methods before they can be distributed to users.

[0009] In the method using conditioned medium prepared using fallopian tube epithelial cells, the isolation of fallopian tube epithelial cells from the fallopian tube is laborious, as described above, and the amount of conditioned medium is limited because the number of cells that can be obtained is 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 may alter the composition of the conditioned medium.

[0010] Although the hypoxic method improves embryo development rates, it also produces blastocysts with low fertility as well as those with high fertility, so further technical improvements are needed to improve the efficiency of embryo transfer.

[0011] Furthermore, it has been pointed out that immortalized cells based on genetic recombination techniques often lack differentiation potential, although they exhibit high proliferation potential. [Means for solving the problem]

[0012] 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 immortalized bovine oviduct epithelial cells have a population doubling level of more than 200. <1> The conditioned medium described in . <3> The immortalized bovine oviduct epithelial cell culture comprises a substance that precipitates when the immortalized bovine oviduct epithelial cell culture is ultracentrifuged. <1> or <2> The conditioned medium described in . <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> <1> ~ <3> 2. A method for culturing a mammalian embryo, the method comprising culturing a mammalian embryo using the conditioned medium according to any one of the preceding items. <6> The mammalian embryos are bovine embryos, and the method includes the steps of selecting cleaved embryos 27 hours after insemination, selecting embryos at the 2-cell stage with uniform blastomeres 31 hours after insemination, and selecting embryos that have grown to the 8-cell stage or higher 55 hours after insemination. <5> 2. A method for culturing mammalian embryos according to claim 1. <7> Immortalized bovine oviduct epithelial cells, which are non-genetically modified cells. <8> Population doubling level exceeds 200, <7> Immortalized bovine oviduct epithelial cells described in . <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. [Effects of the Invention]

[0013] The immortalized bovine oviductal epithelial cells of the present invention enable the mass production of homogeneous oviductal epithelial cells, and also enable the mass production of a homogeneous conditioned medium containing the immortalized bovine oviductal epithelial cells. Furthermore, the method for culturing mammalian embryos of the present invention enables the efficient production of blastocysts with high fertility. [Brief explanation of the drawings]

[0014] [Figure 1] 1 shows the growth curve of immortalized bovine oviduct epithelial cells according to Example 1 of the present invention. [Figure 2]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. [Figure 3] 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. [Figure 4] 1 shows the proportion of non-selected blastocysts (not satisfying the four conditions for embryo development) in each conditioned medium concentration test group according to Example 4 of the present invention. [Figure 5] 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. [Figure 6] 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. [Figure 7] 1 shows the percentage of non-selected blastocysts (which do not satisfy the four conditions for embryo development) in each medium according to Example 6 of the present invention. [Figure 8] 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. DETAILED DESCRIPTION OF THE INVENTION

[0015] [Definitions of terms, etc.] As used herein, "immortalized cells" refers to cell lines that have vigorous proliferation capacity even when their population doubling level exceeds 200. The "population doubling level" refers to the number of times a cell can divide in a culture environment. A population doubling level of more than 200 means that the number of times a cell can divide in a culture environment exceeds 200. Note that normal fallopian tube epithelial cells can only divide approximately 25 to 50 times.

[0016] As used herein, the term "in vitro produced embryo" refers to an embryo that has been subjected to in vitro maturation and in vitro fertilization.

[0017] As used herein, the term "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. The cellular factors contained in the conditioned medium support cell differentiation, proliferation, and embryonic development when cells or embryos are cultured in the medium.

[0018] As used herein, "insemination" refers to the process of placing sperm and eggs in a single fertilization medium.

[0019] [1. Immortalized bovine oviduct epithelial cells] The present invention provides immortalized bovine oviduct epithelial cells, which are non-genetically modified cells.

[0020] The immortalized bovine oviduct epithelial cells are derived from bovine oviduct epithelial cells isolated from bovine oviducts.

[0021] The types of cattle used include cows (Bos taurus), zebu cattle (Bos indicus), and water buffalo (Bubalus bubalis). They may be any of dairy breeds, meat breeds, dual-purpose breeds, draft breeds, and dual-purpose breeds. The types of cattle are not particularly limited, but examples include black cattle. Examples include Wagyu cattle such as Japanese Hair Cattle, Japanese Brown Cattle, and Japanese Shorthorn Cattle, as well as Holstein, Jersey, and native breeds from each country.

[0022] 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.

[0023] It has been pointed out that immortalized cells produced by 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.

[0024] Furthermore, the immortalized bovine oviduct epithelial cells of the present invention have the ability to produce physiologically active substances for embryonic development.

[0025] Exosomes are an example of physiologically active substances for embryonic development. 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.

[0026] The proliferation potential of cells can be evaluated, for example, using the population doubling level as an index. 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.

[0027] Furthermore, the level of cell proliferation ability can be evaluated using, for example, telomerase activity as an indicator.

[0028] Immortalized bovine oviduct epithelial cells can be preserved semi-permanently if frozen. In the case of frozen storage, it is preferable to use liquid nitrogen or the like.

[0029] 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).

[0030] The bovine oviduct epithelial cells before subculture are isolated and collected from the oviducts of slaughtered bovines, for example. The types of bovines are as described above.

[0031] In one embodiment, the culture of fallopian tube epithelial cells is passaged for, for example, 10 months or more, 12 months or more, or 24 months or more.

[0032] The number of passages of the oviduct epithelial cells until immortalized bovine oviduct epithelial cells are obtained is preferably 70, more preferably 75, and even more preferably 80.

[0033] The culture is carried out using a cell culture medium such as D-MEM supplemented with 10% fetal bovine serum, etc. The culture temperature during subculture is preferably maintained at 38.0°C.

[0034] The immortalized bovine oviduct epithelial cells of the present invention have high proliferation ability and high differentiation ability, and therefore it is possible to produce and supply a large amount of oviduct epithelial cells without having to extract epithelial cells again from bovine oviducts.

[0035] Therefore, it is possible to efficiently obtain immortalized bovine oviduct epithelial cells without the troublesome task of repeatedly isolating them from bovine oviducts.

[0036] Non-genetically modified immortalized bovine oviduct epithelial cells can be suitably used as a component of conditioned medium for the developmental culture of mammalian embryos. Furthermore, by using non-genetically modified immortalized bovine oviduct epithelial cells, conditioned medium with uniform properties can be mass-produced, 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.

[0037] [3. Conditioned medium] The present invention provides a conditioned medium obtained by culturing immortalized bovine oviduct epithelial cells, which are non-genetically modified cells.

[0038] Immortalized bovine oviduct epithelial cells include those described in [1. Immortalized bovine oviduct epithelial cells], and provide the active components of the conditioned medium.

[0039] Furthermore, 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 (precipitate) that precipitates when the conditioned medium is ultracentrifuged. 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.

[0040] The 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.

[0041] Other components may also be included, such as various amino acids, vitamins, inorganic salts, antibiotics, and fetal bovine serum.

[0042] 4. Preparation of 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).

[0043] Examples of cell culture media used in the cell culture process include D-MEM solution supplemented with 10% fetal bovine serum, etc. Culture is carried out in a desired culture vessel such as a culture dish.

[0044] The cell culture temperature may be any suitable temperature for cell culture, for example, 38.0°C.

[0045] The cell culture period is preferably the period until the cells become confluent, for example, 3 to 4 days.

[0046] In one embodiment, the method further comprises 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, filtration, or the like.

[0047] Examples of media that can be used for cell culture and embryo development culture in the incubation step include KSOM medium, WM medium, and CZB medium.

[0048] The incubation temperature is, for example, 38.0°C, and the incubation period is, for example, from one day to two days.

[0049] 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 incubated cell culture to light centrifugation, filtration, etc.

[0050] 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 until confluent, and after removing the culture supernatant, the cells are incubated for two days in KSOM medium, which can be used for both cell culture and embryo development. The resulting preparation is designated as conditioned medium.

[0051] In another example of a method for preparing a conditioned medium, the cell culture obtained after incubation using the above-described method 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 thus obtained can also be used as a conditioned medium. For example, the conditioned medium can be ultracentrifuged by the pellet-down method using an ultracentrifuge. The centrifugal force used may be any force suitable for the separation of exosomes. For example, the centrifugal force is preferably 100,000 g or higher. In this example, centrifugation is performed at 24,700 rpm for 2 hours.

[0052] By using the precipitated material 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.

[0053] Furthermore, the sediment obtained by ultracentrifugation of the above-described cell culture has a smaller volume than the conditioned medium itself containing immortalized cells, and therefore 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 hundreds of times and transported. Therefore, it is suitable as an additive for medium preparation.

[0054] Because the fallopian tube epithelium secretes various factors involved in cell proliferation, the conditioned medium prepared using immortalized fallopian tube epithelial cells mimics the environment within the fallopian tube due to the factors secreted by the fallopian tube epithelial cells, and is therefore thought to be able to support early development.

[0055] As described above, the conditioned medium of the present invention does not require the isolation of fallopian tube epithelial cells, which eliminates the need for cell culture and makes preparation extremely easy. Furthermore, the conditioned medium can be stored refrigerated, allowing for easy distribution to users. Furthermore, since the conditioned medium of the present invention is prepared from immortalized cells, it is possible to produce a large amount of homogeneous conditioned medium.

[0056] 5. Mammalian Embryo Culture Method The present invention also provides a method for culturing a mammalian embryo, which comprises a step (embryo culturing step) of culturing a mammalian (mammalian) embryo using the above-mentioned conditioned medium.

[0057] The species and breed of mammalian embryos are not limited. In the case of bovine embryos, they may be the same or different species or breed as the bovine from which the immortalized bovine oviduct epithelial cells used to prepare the conditioned medium were derived. The embryos may be, for example, 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.

[0058] When using in vitro fertilized embryos as the embryos to be subjected to the embryo culture step, for example, in vitro fertilization can be performed using known in vitro fertilization techniques. For example, in vitro fertilized embryos can be obtained by culturing immature oocytes in a known in vitro maturation culture medium such as M199 solution, followed by in vitro fertilization using a known in vitro fertilization medium such as BO solution and frozen-thawed sperm. Preferably, the embryos are those immediately after insemination.

[0059] The types of mammalian embryos include human embryos, livestock embryos (pig, horse, goat, sheep, cow, etc.), pet embryos (cat, dog, hamster, rabbit, guinea pig, etc.), and laboratory animal embryos (rodents such as mouse and rat, monkey, etc.), with bovine embryos being particularly preferred. In one example, the embryo is a mammalian embryo other than a human embryo. In another example, the embryo is an embryo of an endangered animal.

[0060] The embryo culture temperature in the embryo culture step may be any temperature suitable for embryo culture, for example, 38.5°C.

[0061] Other conditions, such as the culture medium, culture time, carbon dioxide concentration, and oxygen concentration, can also be appropriately selected so as to provide culture conditions suitable for culturing embryos in vitro.

[0062] 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. Developmental culture under 5% hypoxia can further increase the efficiency of blastocyst development.

[0063] The embryo culture in the embryo culture step is preferably carried out in a medium containing conditioned medium at a concentration of 80% or more per medium volume, more preferably at a concentration of 90% or more per medium volume, and most preferably at 100% conditioned medium per medium volume.

[0064] The embryo culture period using the conditioned medium is, for example, 4 days from day 0 to day 3 after insemination.

[0065] Then, on the third day after insemination, half of the medium used during the first three days after insemination is discarded, and the same amount of embryo development culture medium, such as KSOM medium, is replenished, and the embryos are further cultured under the same temperature and environmental conditions. Culture is preferably continued until the seventh day after insemination, more preferably until the eighth day after insemination, and even more preferably until the ninth day after insemination.

[0066] When the mammalian embryo culture method of the present invention is used for the above-mentioned livestock embryos, it can be used for a wide range of embryo production in the fields of livestock farming, reproductive engineering, etc. Furthermore, when human embryos are used, it can be suitably used for human assisted reproductive technology.

[0067] 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, and 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.

[0068] Embryos that develop when the following four conditions (1) to (4) regarding cleavage rate are satisfied 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 satisfying these four conditions, that is, blastocysts that have developed at an inappropriate cleavage rate, either too fast or too slow, are expected to have a low rate of conception when transferred to the bovine uterus.

[0069] (1) Embryo cleaving 27 hours after insemination (2) 31 hours after insemination, at the 2-cell stage, and (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 rate.

[0070] Therefore, one embodiment of the method for culturing bovine embryos 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 step).

[0071] The state of the embryos at each time point after insemination can be confirmed by observing the embryos under a microscope during culture. Selection at each time point is carried out by selecting and recovering embryos that meet the conditions while observing them under a microscope.

[0072] This embryo selection process makes it possible to obtain blastocysts that are expected to have a high fertility rate.

[0073] According to the bovine embryo culture method including the above-mentioned embryo selection step, the incidence of blastocysts that satisfy the four conditions for cleavage rate is significantly increased, and the incidence of blastocysts that do not satisfy the four conditions is significantly decreased. In other words, by culturing embryos using the bovine embryo culture method including the above-mentioned embryo selection step, blastocysts that satisfy the four conditions for cleavage rate, i.e., blastocysts that are expected to have a high conception rate, can be obtained with high efficiency.

[0074] According to the method for culturing bovine embryos that includes the above-mentioned embryo selection step, the proportion 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.

[0075] 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 step, blastocysts that are expected to have high fertility can be produced extremely efficiently.

[0076] 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. [Example]

[0077] An embodiment of the present invention will be described below.

[0078] Example 1: Preparation of immortalized bovine oviduct epithelial cells The present inventors aimed to obtain immortalized bovine oviduct epithelial cells.

[0079] To obtain immortalized bovine oviduct epithelial cells that retain the ability to produce physiologically active substances for embryonic development, we first isolated oviduct epithelial cells from the oviducts of slaughtered cows and cultured them in cell culture medium (D-MEM solution supplemented with 10% fetal bovine serum).

[0080] By measuring telomere length and telomerase activity in bovine oviduct epithelial cells and repeatedly subculturing them for 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.

[0081] Although mammalian cultured cells, except for many tumor cells and some immortalized cells, tend to lose telomerase activity with passage, the bovine oviduct epithelial cell line we obtained consistently exhibited high telomerase activity. Furthermore, the telomeres of the bovine oviduct epithelial cell line were shortened until the population doubling level reached 120, but then elongated and restored telomere length.

[0082] As described above, the fallopian tube epithelial cell line established by the present 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.

[0083] 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.

[0084] 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 2 days to obtain conditioned medium. The conditioned medium was centrifuged at 2000 × g for 10 minutes to remove contaminating cells and their carcasses, 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.

[0085] The fallopian tube epithelium secretes various factors involved in cell proliferation, and it was thought that conditioned medium prepared using immortalized fallopian tube epithelial cells could also mimic the environment within the fallopian tube and support early development.

[0086] 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 efficiency was compared.

[0087] Immature bovine 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 during days 0 to 3 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.

[0088] [Table 1]

[0089] It is clear from Table 1 that when embryos 0 to 3 days after insemination were cultured using conditioned medium (100%), the blastocyst development rate was significantly higher than when the medium was 0% or 50%. This demonstrates that blastocysts can be obtained with high efficiency when the conditioned medium of the present invention is used.

[0090] 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 prepared by diluting the conditioned medium with an equal volume of KSOM medium (50%). Cleavage embryos were selected 27 hours after insemination. Embryos with uniform cleavage were selected 31 hours later. Embryos with development to the 8-cell stage or greater were 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 performed at 38.5°C under conditions of 5% oxygen, 5% carbon dioxide, and 90% nitrogen. The development rates of embryos suitable for selection were then examined 27, 31, and 55 hours after insemination, as well as the development rates of blastocysts obtained through such cleavage rates on day 9. The results are shown in Table 2 and Figure 3. The development rate (%) is shown as the 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.

[0091] [Table 2]

[0092] 3, it can be seen 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 shows that by using the conditioned medium of the present invention and selecting embryos that meet the four conditions for cleavage rate, blastocysts that are expected to have a high conception rate can be obtained with high efficiency.

[0093] Table 3 and Figure 4 show the percentage of blastocysts produced from each concentration of conditioned medium that developed without satisfying 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 satisfy the four conditions for embryo development described above) in each conditioned medium concentration test group.

[0094] [Table 3]

[0095] Table 3 shows that when the conditioned medium is used, the percentage of blastocysts obtained by developmental culture that develop into blastocysts without undergoing an appropriate cleavage rate (i.e., cleavage rates that are either too fast or too slow) is low.

[0096] Furthermore, the effects of each concentration of conditioned medium on the development of blastocysts that met the four conditions and those that did not are shown in Table 4 and Figure 5. The development rate (%) is shown as the mean ± standard error. Figure 5 shows the development rate of blastocysts that met the four conditions for embryo development and the development rate of blastocysts that did not meet the four conditions for embryo development for each conditioned medium concentration test group.

[0097] [Table 4]

[0098] Table 4 shows that the use of the conditioned medium significantly increases the incidence of blastocysts that meet the four cleavage rate conditions, while decreasing the incidence 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.

[0099] 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 other contaminants 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 contaminants 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 then resuspended in 1.5 mL of KSOM medium. This resulted in a 20-fold concentration of the sediment component in the conditioned medium.

[0100] Example 6: Analysis of the effect of ultracentrifugation sedimentation of conditioned medium on the production of embryos with appropriate cleavage rates. Bovine IVF 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, cleavage embryos were selected 27 hours after insemination. Then, 31 hours later, embryos with two-cell stage and uniform blastomeres were selected. 55 hours later, embryos developing to the 8-cell stage or greater were selected. On day 3 after insemination, half of the medium used between days 0 and 3 after insemination was discarded, and the same volume of KSOM medium was replenished. Culture was continued until day 8 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 were then investigated for the four media at 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 media at each time point after insemination.

[0101] [Table 5]

[0102] Table 5 and Figure 6 show that the use of this conditioned medium resulted in a higher percentage of embryos that developed at an appropriate cleavage rate 27, 31, and 55 hours after insemination compared to the control and supernatant from which exosomes and other substances had been removed by ultracentrifugation, and the percentage of blastocysts obtained after 8 days at such a cleavage rate was significantly higher. Furthermore, with the KSOM+ sediment, results similar to those of the conditioned medium were obtained, although not significantly different. This indicates that by using the sediment extracted from the conditioned medium of the present invention and selecting embryos that meet the four cleavage rate conditions, blastocysts with a high probability of conception can be obtained with high efficiency.

[0103] 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.

[0104] [Table 6]

[0105] Table 6 shows that when the conditioned medium and KSOM + sediment were used, the percentage of blastocysts obtained by developmental culture that developed into blastocysts without an appropriate cleavage rate (i.e., cleavage rates that were either too fast or too slow) was low. On the other hand, when the supernatant from ultracentrifugation of conditioned medium was used, the percentage of blastocysts that developed into blastocysts without 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 inevitably contained in the conditioned medium.

[0106] Furthermore, the results of comparing the effects of each medium on the development of blastocysts that meet the four conditions and those that do not are shown in Table 7 and Figure 8. 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.

[0107] [Table 7]

[0108] Table 7 shows that the use of conditioned medium or KSOM+sediment increased the rate of blastocyst development that met the four cleavage rate criteria.

[0109] The results in Tables 5 to 7 and Figures 6 to 8 show that when bovine embryos are cultured using conditioned medium and KSOM + sediment and embryos that meet the four cleavage rate conditions are selected, blastocysts expected to have a high conception rate can be obtained with high efficiency. These findings demonstrate that, among the substances contained in the 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. [Industrial Applicability]

[0110] The present invention can be widely used in the fields of livestock farming, reproductive engineering, assisted reproductive medicine, etc., including the production of mammals such as cattle and the improved propagation of mammals such as cattle.

Claims

1. A conditioned medium containing a culture of immortalized bovine oviduct epithelial cells, which are non-genetically modified cells, and the culture contains exosomes as an active ingredient.

2. A conditioned medium containing a substance that precipitates when a culture of immortalized bovine oviduct epithelial cells, which are non-genetically modified cells, is ultracentrifuged, and the substance contains exosomes as an active ingredient.

3. 3. The conditioned medium of claim 1 or 2, wherein the immortalized bovine oviduct epithelial cells have a population doubling level of more than 200.

4. 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 A method for preparing a conditioned medium, comprising the step of incubating 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 culturing the mammalian embryo using the conditioned medium according to any one of claims 1 to 3.

6. 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.

7. Immortalized bovine oviduct epithelial cells, which are non-genetically modified cells and have a population doubling level of more than 200.

8. 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.

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