KR102096522B1 - Primordial follicle culture medium under hormone-free conditions and in-vitro culture method using the same - Google Patents

Abstract

The present invention comprises the steps of culturing the isolated primordial follicle (primordial follicle) in vitro in a medium containing a composition for primitive follicular in vitro culture, comprising insulin, knockout serum replacement (KO-SR) and basic fibroblast growth factor (bFGF). Including, culturing method for early development of primitive follicles, relates to a method for producing a mature egg using the follicle and a composition used therefor.
Using the initial method of culturing the primitive follicles of the present invention, primitive follicles can be cultured even under non-hormonal or hormone-free conditions, from which eggs can be obtained, resulting in low-hormone patients, early menopause or hormones -It can be useful for in vitro fertilization and infertility studies in elderly patients with only non-reactive follicles.

Description

Primordial follicle in vitro culture medium composition under non-hormonal or hormonal exclusion conditions and in vitro culture method using the same {Primordial follicle culture medium under hormone-free conditions and in-vitro culture method using the same}

The present invention comprises the steps of culturing the isolated primordial follicle (primordial follicle) in vitro in a medium containing a composition for primitive follicular in vitro culture, comprising insulin, knockout serum replacement (KO-SR) and basic fibroblast growth factor (bFGF). Including, culturing method for early development of primitive follicles, relates to a method for producing a mature egg using the follicle and a composition used therefor.

In vitro culture of follicles is an important method for studying factors that control the development of oocytes at different stages and is also an alternative to preserving fertility (Khosravi et al. , 2013; 30 (11): 1397-406; Kim YJ et al. , 2013; 28 (11): 3050-61). However, in vitro culture methods of follicles, which are currently used in basic and clinical studies, mainly use a method of transplanting embryos into the uterus by collecting, fertilizing, and culturing various follicles after administration of superovulation injection. The current assisted reproductive technique based on the induction of hyperovulation is a method in which a number of follicles in response to the hyperovulation inducing hormone are grown and an egg is collected.

However, the above method has a limitation in that it is difficult to apply to a patient with a low hormone group, an early menopause, or an elderly patient aged 40 years or older with only hormone-reactive follicles remaining. Due to social changes, the average age of maternity and pregnancy continues to increase, and the survival rate after chemotherapy is also increasing, so preserving the fertility of surviving patients after chemotherapy is also emerging as a new issue.

The female reproductive system, the ovary, is very sensitive to cytotoxic drugs, and it is well known that drugs such as cyclophosphamide, which is used as an anticancer drug, damages the egg and granulosa cells. It is known that in combination with abdominal radiation therapy and drug therapy, ovarian premature insufficiency is induced, leading to infertility in most patients. Because adolescent women do not start their menstrual cycle or are pre-marriage, most of them cannot preserve eggs or embryos using cryopreservation before chemotherapy using current assisted reproduction, and preservation of reproductive ability before treatment Development of a new adjuvant reproductive technique for the above-mentioned patient group, which is difficult to treat with current adjuvant reproductive technique, is also very necessary because there are limitations that should be performed and at least 2-3 weeks before the start of chemotherapy.

The egg is surrounded by the follicle, a complex unit that surrounds the egg, and undergoes growth and development to reach ovulation, which is achieved by the stimulation of gonadotropin secreted by the pituitary gland. The follicle (composite unit) consists of an egg and a granular membrane cell surrounding the egg and a follicle membrane cell (theca cell), and the granular membrane cell and the follicular membrane cell are each a follicle stimulating hormone (FSH) receptor and an emperor-forming hormone. (Luteinizing Hormone, LH) Receptor is located and is associated with stimulation according to estrogen concentration, and plays a role in controlling the development of complex units and maturation of the egg.

The stage of follicle development is divided into primitive, primary, secondary, and pre-ovulation follicles, and is largely divided into hormone-independent periods and hormone-dependent periods, and the first half of follicular development is hormones. Because it is not affected by, it is impossible to control its development by administering hormones from the outside.

On the other hand, the follicular development stage in response to hormonal fertility treatment is a hormone-dependent stage in the second half, so during the period of fertility reduction, the follicles in the late stages of development are lost a lot and the menstrual function weakens, and pregnancy is also possible with current fertility treatment using hormones. This is a difficult situation.

Accordingly, the inventors of the present invention, as a result of diligent research efforts to culture the raw follicles in vitro without using hormones, confirmed the conditions for culturing the raw follicles in vitro under non-hormonal or hormone-free conditions and completed the present invention.

One object of the present invention is to separate the isolated primary follicle (primordial follicle) insulin, KO-SR (knockout serum replacement) and bFGF (basic fibroblast growth factor), in vitro in a medium containing a composition for primitive follicular in vitro culture It is to provide a culture method for early development of primitive follicles, comprising culturing.

Another object of the present invention is to obtain a primitive follicle initially developed by the culture method; And obtaining an egg by inducing ovulation in the initially developed primitive follicle.

Another object of the present invention is to provide a composition for primitive follicle in vitro culture, including insulin, knockout serum replacement (KO-SR), basic fibroblast growth factor (bFGF), and combinations thereof.

Hereinafter, the present invention will be described in more detail.

Meanwhile, each description and embodiment disclosed herein may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein are within the scope of the present invention. In addition, it cannot be said that the scope of the present invention is limited by the specific descriptions described below.

In addition, one of ordinary skill in the art can recognize or identify a number of equivalents to certain aspects of the invention described in this application using only routine experimentation. In addition, such equivalents are intended to be included in the present invention.

The present inventors tried to establish a condition capable of culturing primitive follicles in vitro under non-hormonal or hormone-free conditions, while medium containing an appropriate concentration of insulin, knock-out serum replacement (KO-SR) and bFGF In the case of culturing in, it was confirmed that primitive follicles can be initially developed, and ovulation can be induced in the initially developed primitive follicles to obtain oocytes.

When primitive follicles are cultured in vitro without including hormones in the medium, follicles can be cultured in vitro in infertile patients who have lost a large number of follicles in the late stages of development that respond to hormones. It can be said that the significance of the present invention is great in that it is possible to treat infertility in elderly patients over 40 years old, where only non-reactive follicles remain.

In order to solve the above problems, the present invention is one aspect, the isolated primordial follicle (primordial follicle) insulin, KO-SR (knockout serum replacement) and bFGF (basic fibroblast growth factor), for primitive follicle in vitro culture Provided is a culture method for early development of primitive follicles, comprising culturing in vitro in a medium containing the composition.

In the present invention, the term "early follicle development" refers to the process of artificially growing hormonal non-reactive primitive follicles in vitro under controlled conditions. Except for the essential technical idea provided by the present invention, the method for initial development of the hormone non-reactive follicle may be performed without limitation using methods well known in the art.

The early development of primitive follicles of the present invention is in particular hormone-free conditions in which gonadotropins such as follicle stimulating hormone (FSH) and luteinizing hormone (LH) hormones are excluded. It has the advantage that primitive follicles can be cultured in vitro.

In the present invention, the term "primary follicle (primordial follicle)" is the follicle grows from the start of menstruation until ovulation from the start of the ovary to grow into a primitive follicle. Specifically, the primitive follicle means that the follicle is in an undeveloped stage and is in the superficial layer of the ovarian cortex, and means that the flat oocyte epithelial cell of the first layer is surrounded by one oocyte, and is not limited to the above-described developmental state. .

In the present invention, the follicle may be from a mammal. Specifically, human, cow, sheep, pig, horse, rabbit, goat, mouse, hamster and rat, but may be derived from an animal cultured in vitro by the method of the present invention and capable of performing the initial development of primitive-follicle Is included without limitation.

In the present invention, the term "medium" refers to a medium that can support the growth and survival of cells, including follicular cells in vitro, and includes all mediums used in the art suitable for culturing cells. It can contain. The basic medium used for culture is Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Opti-MEM, BME (Basal Medium Eagle), RPMI1640, F-10, F-12, MEM α (Minimal Essential Medium) , GMEM (Glasgow's Minimal Essential Medium) and Iscove's Modified Dulbecco's Medium, DMEM / F12, KO-DMEM, etc., but are not limited thereto.

In addition, the medium of the present invention may further include growth factors, hormones, polypeptides, and inorganic substances to help the culture and maintenance of follicular cells. More specifically, the growth factors, hormones, polypeptides, and inorganic substances are bovine pituitary extract, epidermal growth factor, insulin, hydrocortisone, transferrin, epinephrine, CaCl ₂ and amphotericin It may be selected from the group consisting of B. In addition, the medium may include antibiotics such as penicillin, streptomycin or gentamicin to prevent bacterial infection.

In a specific embodiment of the present invention, using the culture medium containing insulin, KO-SR (knockout serum replacement), bFGF (basic fibroblast growth factor), the culture follicle is made into a culture dish, and the raw follicles collected in each drop 1 Each was seeded and incubated at 37 ° C. in a 5% CO ₂ incubator for about 2 weeks.

The culture method may be performed for 5 to 25 days, 7 to 17 days, or 10 to 15 days, but is not limited thereto.

The culture medium may include insulin, KO-SR (knockout serum replacement), or basic fibroblast growth factor (bFGF). The concentration of insulin contained in the culture medium may be 5 mg / mL to 50 mg / mL, 10 mg / mL to 30 mg / mL, 15 to 25 mg / mL in the composition, but is not limited thereto, and KO-SR The concentration of may be 5% to 50%, 20% to 40%, 25% to 35% in the composition, but is not limited thereto, and the concentration of bFGF is 0.4 ng / mL to 100 ng / mL in the composition, It may be 6 ng / mL to 80 ng / mL, 8 ng / mL to 50 ng / mL, but is not limited thereto.

The culture medium may include insulin, glial cell-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF), or leukemia inhibitory factor (LIF). The concentration of insulin contained in the culture medium may be 5 mg / mL to 50 mg / mL, 10 mg / mL to 30 mg / mL, 15 to 25 mg / mL in the composition, but is not limited thereto, and the concentration of GDNF May be 5 ng / mL to 100 ng / mL, 10 ng / mL to 80 ng / mL, 20 ng / mL to 50 ng / mL in the composition, but is not limited thereto, and the concentration of bFGF is within the composition. 0.4 ng / mL to 100 ng / mL, 6 ng / mL to 80 ng / mL, 8 ng / mL to 50 ng / mL, but is not limited thereto, and the concentration of LIF is 10 units to 10 ⁸ units, 10 ² units to 10 ⁷ units. It may be 10 ³ units to 10 ⁶ units, but is not limited thereto.

In a specific embodiment of the present invention, insulin, glial cell-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF) are treated to develop primitive follicles when primitive follicles are cultured in vitro and Although egg maturation was not achieved efficiently, initial development of primitive follicles is achieved when primitive follicles are cultured in vitro by treating medium (20SR, B +) containing insulin, KO-SR (knockout serum replacement) and bFGF, 3 After culturing weekly, it was confirmed that the ovulated egg matured according to gonadotropin treatment (FIG. 1). Therefore, when primitive follicles are cultured in vitro in a medium containing insulin, KO-SR (knockout serum replacement) or bFGF (basic fibroblast growth factor), the initial development of primitive follicles, even under non-hormonal or hormone-free conditions This was done, and further confirmed that the egg can be discharged. Moreover, it can be seen that the initial development of primitive follicles by the method of the present invention has an excellent yield of 25%.

Another aspect of the present invention comprises the steps of obtaining a primitive follicle initially developed by the culture method; And obtaining an egg by inducing ovulation in the initially developed primitive follicle.

The ovulation induction refers to a process of inducing the egg to be released from the mature follicle. The material used to induce ovulation may be human chorionic growth hormone (rhCG), human epidermal growth factor (hEGF), human menopausal gonadotropin (hMG), follicle stimulating hormone (FSH), luteinizing hormone (LH), but It is not limited.

In a specific embodiment of the present invention, in order to induce ovulation, recombinant human chorionic growth hormone was treated to induce ovulation.

The method of the present invention surprisingly has the characteristic of confirming that it is possible to secure a mature egg from the early development of primitive follicles under non-hormonal or hormonal exclusion conditions and treatment with gonadotropin.

Another aspect of the present invention provides a composition for primitive follicle in vitro culture comprising insulin, knockout serum replacement (KO-SR), basic fibroblast growth factor (bFGF), and combinations thereof. The primitive follicle, insulin, KO-SR and bFGF are as described above.

Using the composition for in vitro cultivation of the primordial follicle of the present invention, primitive follicles can also be cultured in a non-hormonal or hormone-free medium, from which an egg can be obtained, resulting in low-hormone patients , Early menopause or hormone-reactive follicles can be useful for in vitro fertilization and infertility studies in elderly patients.

Using the initial method of culturing the primitive follicles of the present invention, primitive follicles can be cultured even in non-hormonal or hormone-free conditions, from which ovum can be obtained, resulting in low-hormone patients, early menopause or hormones -It can be useful for in vitro fertilization and infertility studies in elderly patients with only non-reactive follicles.

1 is an in vitro culture of the isolated raw follicles in medium containing insulin, glial cell-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF) (B +, G +, L +) The development of primitive follicles and the efficiency of egg maturation were low, but when cultured in vitro in medium (SR, B +) containing insulin, KO-SR (knockout serum replacement) and bFGF, initial development of primitive follicles was achieved and cultured for 3 weeks This diagram shows that the ovulated egg matures after treatment with gonadotropin.
2 is an analysis of the characteristics of follicles developed from primitive follicles, and as a result of culturing primitive follicles under culture condition 2) of Example 2, it is a diagram confirming whether normal expression of FSH receptors (receptors) is observed in mature follicles.

Hereinafter, the present invention will be described in more detail by examples. However, the following examples are only for illustrating the present invention and the scope of the present invention is not limited to them.

Example 1. Isolation of primordial follicles from mice

Female C57BL / 6 mice were used to isolate primitive follicles for use in the experiment. At this time, the mice were given a contrast period of 12 hours, and water and food were freely consumed.

Specifically, the ovaries were isolated from a 14-day-old mouse, placed in HBSS (Hank's balanced salt solution) containing 5% bovine serum, and then mechanically separated from the early primitive follicles using a 30 gauge syringe needle. Raw follicles were cultured by adding each to the culture drop.

Example 2. In vitro culture of isolated primitive follicles

The isolated primitive follicles are 1) 10 mg / mL insulin, 5% bovine serum, 40 ng / ml, glial cell-derived neurotrophic factor (GDNF), 4 ng / ml basic fibroblast growth factor (bFGF), 10 ⁶ units LIF (MEM alpha medium) containing 2 (Leukemia inhibitory factor) or 2) 10 mg / mL insulin, 20% KO-SR (knockout serum replacement), cultured with MEM alpha medium with 4 ng / ml bFGF added It was replaced with a new culture medium every two days.

The medium was incubated with 20 drops of 20 μL in one culture dish, and primitive follicles were cultured for each drop. Incubation was carried out for about 2 weeks in a 37 ^o C, 5% CO ₂ incubator. The follicle morphology was observed using a phase contrast microscope (Eclips T; Olympus, Tokyo, Japan).

After culturing as described above, mouse ovaries were separated and primitive follicles were separated therefrom by physical methods. The isolated follicles are seeded one by one on a culture dish dropper containing each type of culture medium, cultured for 3 weeks, and the maturity of the ovulated egg 48 hours after administration of human chorionic gonadotropin (hCG) (M II). The initial development of primitive follicles was confirmed under both culture conditions of 1) and 2), but the overall yield was excellent in 2) culture conditions and mature eggs were obtained under conditions of 2). Accordingly, it was confirmed that the development and maturation of primitive follicles can be promoted under the culture conditions of 2), that is, non-hormonal or hormonal exclusion conditions (FIG. 1).

Example. 3 Analysis of follicles developed from primitive follicles

In order to analyze the characteristics of the follicles developed in culture condition 2) of Example 2 from the primitive follicles, the culture medium of the developed follicles is removed, washed 3 times with PBS, and then 4% paraformaldehyde is used. It was fixed at room temperature for 20 minutes. After washing with PBS again, 3% bovine serum albumin and 0.3% triton x-100 solution were reacted at 4 ° C for 12 hours or more. Then, after diluting the FSH receptor-specific primary antibody (goat anti-mouse FSH Rc) at a ratio of 1: 100 and reacting at 4 ° C for 12 hours or more, PBS solution containing 0.05% tween 20 ( PBST) and washed 3 times for 10 minutes. Thereafter, the secondary antibody IgG mouse anti-goat 488 specific to the primary antibody was treated at a ratio of 1: 200 to react for 1 hour at room temperature, and washed 3 times with PBS for 10 minutes. For staining the nucleus, DAPI was treated at a concentration of 1 ug / ml at room temperature for 20 minutes, and then observed using a fluorescence microscope. As a result, it was observed that the FSH receptor is normally expressed in the mature follicle, from which it was confirmed that development and maturation can be promoted to normal follicles from primitive follicles cultured by the culture conditions and methods of the present invention.

From the above description, those skilled in the art to which the present invention pertains will appreciate that the present invention may be implemented in other specific forms without changing its technical spirit or essential features. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the following claims rather than the detailed description and equivalent concepts thereof.

Claims (13)

Incubating the isolated primordial follicle (primordial follicle) in a medium containing a composition for primitive follicular in vitro culture, including insulin, knockout serum replacement (KO-SR), basic fibroblast growth factor (bFGF), and combinations thereof. Including, primitive follicle culture method for early development.
Comprising the step of culturing the isolated primordial follicle (primordial follicle) in vitro in a medium comprising a composition for primitive follicular in vitro culture consisting of insulin, knockout serum replacement (KO-SR), and basic fibroblast growth factor (bFGF) , Raw follicle culture method for early development.
The method of claim 1, wherein the culturing method does not use gonadotropin.
The method of claim 1, wherein the culture method for early development of primitive follicles is performed for 5 to 25 days.
Obtaining the initially developed primitive follicle by the culture method of any one of claims 1 to 4; And
A method of producing mature eggs, comprising the steps of inducing ovulation to the initially developed primitive follicles to obtain eggs.
The method of claim 5, wherein the induction of ovulation is performed by treating gonadotropin in an initially developed primitive follicle.
A composition for in vitro culture of primordial follicles, including insulin, KO-SR (knockout serum replacement), basic fibroblast growth factor (bFGF), and combinations thereof.
A composition for primitive follicle in vitro culture consisting of insulin, KO-SR (knockout serum replacement), and basic fibroblast growth factor (bFGF).
The composition for primitive follicular in vitro culture according to claim 7 or 8, wherein the concentration of insulin is 5 mg / mL to 50 mg / mL in the composition.
9. The composition for primitive follicular in vitro culture according to claim 7 or 8, wherein the concentration of KO-SR is 5% to 50% in the composition.
The composition for primitive follicular in vitro culture according to claim 7 or 8, wherein the concentration of bFGF is 0.4 ng / mL to 100 ng / mL in the composition.
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