Methods for isolating different types of single cells from the ovary

Abstract

The present invention provides a method for separating mammalian early ovarian follicles to obtain a single oocyte and a single granulosa cell thereof. The method of the present invention can separate mammalian early ovarian follicles to obtain active single oocytes and their corresponding granulosa cells. The present invention also provides a kit for obtaining mammalian early ovarian follicle single oocytes and their single granulosa cells.

Description

Technical Field

[0001] The present invention relates to the field of cell biology and in particular to a method for processing early ovarian follicles of mammals to obtain a single oocyte and its granulosa cells. Background Art

[0002] Cells are the basic units of life. Since the cell theory was proposed, humans have been trying to find ways to study cells. In recent years, major breakthroughs in nucleic acid amplification and sequencing technology have been of great significance to the physiological and biochemical research of single cells, making the technical methods for analyzing single cells a hot research direction. Therefore, it is particularly important to accurately capture a single specific type of cell for research and analysis.

[0003] The follicle is the basic structural and functional unit of the female mammalian ovary, mainly composed of the middle oocyte and one or several layers of granulosa cells surrounding it. According to the differences in follicle morphology, structure and function, it can be divided into several stages of follicles, including primordial follicles (primordial follicles), primary follicles, secondary follicles, preantral follicles, antral follicles and mature follicles. The diameter of human follicles can grow from the initial 30-50μm to about 20mm for mature follicles. The primordial follicles of mice are only about 20μm, and the diameter is about 0.5mm after maturity. The primordial follicles and primary follicles with the smallest diameter can be collectively referred to as early follicles. At present, the methods for separating mammalian secondary and later stage follicles are very mature and widely used in basic research and clinical treatment. However, there is no unified and standardized standard for the separation of single early follicles, and the separation methods for the two cells (i.e., oocytes and their corresponding granulosa cells) that make up the primordial follicles and primary follicles are still in the exploratory stage.

[0004] As for the method of separating follicles, the most commonly used method is currently the enzymatic digestion method, which usually uses one or two enzymes to digest the ovarian tissue. An appropriate amount of DNA enzyme (DNase) can be added to the digestion solution to prevent cell adhesion during the digestion process. During the digestion process, the digestion solution can be shaken or blown at intervals to promote cell separation. The digestion is terminated by centrifugation and resuspension to obtain follicles. The shortcomings of the existing follicle separation method are that the separation process often requires a centrifugation and resuspension step, which has the disadvantage of reducing the efficiency of cell recovery, and due to improper control of the action of the digestive enzyme, it is easy to cause certain damage to the cells.

[0005] As for the separation of oocytes and corresponding granulosa cells in a single early follicle, the existing method is to cut and separate the ovarian tissue after fixed slices by laser capture microdissection (LCM). This method can accurately cut and obtain single cells in the ovarian tissue. Although the LCM technology is very accurate to separate single cells, the tissue must be fixed and sliced ​​first, and active cells cannot be obtained. In addition, the cell structure after slicing is often incomplete, which will have a certain impact on subsequent research and analysis.

[0006] Therefore, there is a need in the art for a more efficient method for obtaining a single active oocyte and its corresponding granulosa cell from a mammal. Summary of the invention

[0007] The present invention provides for the first time a method for efficiently and accurately separating early follicle tissue of mammals to obtain active single oocytes and their corresponding granulosa cells.

[0008] The present invention provides a method for obtaining a single oocyte and a single granulosa cell of an early ovarian follicle of a mammal, characterized by comprising the following steps:

[0009] (1) mechanically separating the ovarian tissue of the mammal into small pieces;

[0010] (2) digesting the mammalian ovarian tissue fragments in a first digestion solution at about 37° C. for about 20-60 minutes, for example, about 30 minutes; the first digestion solution contains collagenase I, collagenase II or collagenase IV or a mixture thereof;

[0011] (3) passing the digestion liquid of step (2) through a first pore size cell strainer, wherein the pore size of the first pore size cell strainer is about 40-100 μm (e.g., about 70-80 μm), and then filtering the filtrate again through a second pore size cell strainer, wherein the pore size of the second pore size cell strainer is about 8-12 μm (e.g., about 8 μm); discarding the filtrate;

[0012] (4) washing the precipitate in the second pore size cell filter with culture medium, and then resuspending the precipitate with culture medium;

[0013] (5) Aspirate the single follicle from the resuspension solution;

[0014] (6) After washing the single early follicle, digest it in a second digestion solution for about 3-10 minutes, preferably within 5 minutes, wherein the second digestion solution contains trypsin or

[0015] (7) The digestion solution mixture obtained in step (6) is transferred into the culture medium, and a single follicle cell and a single granulosa cell thereof are separated by blowing.

[0016] The method of the present invention may further include step (8), using the single oocyte and / or single granulosa cell obtained in step (7) for subsequent single cell experiments, such as cell culture, nucleic acid extraction and analysis, etc. In one aspect of the present invention, the single oocyte and / or single granulosa cell obtained in step (7) can be directly transferred to a culture medium for subsequent single cell experiments.

[0017] In the present invention, the early follicles of mammals refer to primordial follicles and primary follicles. According to the differences in follicle morphology, structure and function, they can be divided into several periods, including primordial follicles (primordial follicles), primary follicles, secondary follicles, preantral follicles, antral follicles and mature follicles. Primordial follicles and primary follicles can be collectively referred to as early follicles. Primordial follicles are often in a dormant state as ovarian reserves, and their structure consists of a central oocyte and a layer of flat granulosa cells wrapped around it. Once activated, the primordial follicles will be transformed into primary follicles consisting of an oocyte and a single layer of cubic granulosa cells. The volume and structure of the primordial follicles and primary follicles of mammals are relatively similar. The diameter of the primordial follicle is generally 20-40μm, which is composed of a layer of flat granulosa cells wrapped by an oocyte of about 20μm in the middle. The diameter of the primary follicle is generally 50-80μm, which is composed of a layer of cubic granulosa cells wrapped by an oocyte of about 40μm in the middle. Statistics show that the volume and structure of primordial follicles and primary follicles of mammals, especially small and medium-sized mammals, are within the above range. Small mammals (generally referring to mammals with a body length of less than 0.5 meters) include mice, rats, guinea pigs, rabbits, cats, dogs, etc. Medium-sized mammals (generally referring to mammals with a body length of less than 2 meters) include humans, monkeys, sheep, pigs, cattle, etc.

[0018] In step (3), the digestion solution of step (2) is passed through a first pore size cell strainer, the pore size of which is selected to be larger than the diameter of the primary follicle or primordial follicle of the mammal to be treated according to the purpose, so that the target follicle can pass through the first pore size cell strainer. The pore size of the first pore size cell strainer is generally about 40-100 μm (e.g., about 70-80 μm).

[0019] In step (1), common medical instruments such as forceps, needles, etc. can be used to mechanically and bluntly separate the ovarian tissue of the mammal. Generally, the ovarian tissue is divided into pieces of about 1×10 6 ~9×10 6 μm 3The mechanical separation operation is usually carried out in a culture dish containing a dissection solution, or it can be directly carried out in the first digestion solution of step (2).

[0020] In one aspect of the present invention, the first digestive solution contains a mixture of collagenase I, collagenase II or collagenase IV. In another aspect of the present invention, the first digestive solution contains a mixture of Liberase and collagenase IV. TM TL Research Grade, Liberase (Cat. No. 05401020001) is a widely used digestive enzyme standard preparation in recent years, which contains a mixture of high-purity collagenase I and collagenase II. Liberase removes impurities such as clostripain and trypsin, and removes >98% of endotoxins and other dead cell components in the raw materials. Liberase is often used to replace traditional Collagenase I and / or Collagenase II preparations.

[0021] In one aspect of the present invention, the ratio of Liberase to Collagenase IV in the first digestion solution is about 1:4. In another aspect of the present invention, the Liberase in the first digestion solution is 0.02-0.2 mg / ml, preferably about 0.05 mg / ml, and the Collagenase IV is 0.05-0.5 mg / ml, preferably about 0.2 mg / ml.

[0022] In the present invention, the first digestion solution may further contain neutral protease and / or metalloprotease, such as thermolysin. In yet another aspect of the present invention, the first digestion solution may further contain DNase.

[0023] In one aspect of the present invention, the second digestion solution contains trypsin, such as trypsin-EDTA solution (0.25%:0.02%) reagent.

[0024] In another aspect of the present invention, the second digestive fluid contains Accutase (such as Cell Dissociation Reagent, ThermoFisher, Catalog No. A1110501). Accutase is a widely used standard preparation of digestive enzyme in the field. It has protease and collagenase activities and does not contain any animal or bacterial components. It is often used as a substitute for pancreatic enzymes. Its digestion method is mild and has low cell damage. In use, the single follicle obtained in step (5) is directly added to the Accutase reagent for digestion.

[0025] In one aspect of the present invention, the second pore size cell filter uses a cell permeable chamber (Transwell). Transwell is a permeable cup-shaped device with a permeable polycarbonate membrane (polycarbonate membrane) at the bottom of the cup-shaped device, and the material of the rest of the cup-shaped device is the same as that of an ordinary cell culture well plate. The permeable polycarbonate membrane has micropores with a pore size range of 0.4 to 12.0 μm. In this method, a transwell with a pore size of 8 μm is used. When the precipitate in the transwell is resuspended in step (4), the transwell is suspended in the air to perform step (5).

[0026] In the method of the present invention, step (5) can be performed by using a capillary glass tube or other automatic or semi-automatic cell aspiration device or apparatus to aspirate the single follicle in the resuspended solution. After the single follicle is aspirated, the aspirated follicle is washed in a clean culture medium before being transferred to the digestion solution 2 for step (6).

[0027] The present invention also provides a kit for implementing the above-mentioned method of obtaining a single oocyte of an early follicle and a single granulosa cell thereof from a mammal. The kit includes the first digestion solution and the second digestion solution. The components of the first digestion solution and the second digestion solution are as defined above. For example, in the kit, the first digestion solution contains a mixture of collagenase I, collagenase II or collagenase IV. In another aspect of the present invention, the first digestion solution contains a mixture of Liberase and collagenase IV. In another aspect of the present invention, the ratio of Liberase to collagenase IV in the first digestion solution is about 1:4. In the present invention, the first digestion solution may also contain a neutral protease and / or a metalloprotease, such as thermolysin. In another aspect of the present invention, the first digestion solution may also contain DNase. In one aspect of the present invention, the second digestion solution contains trypsin. In another aspect of the present invention, the second digestion solution is Accutase. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The separation of oocytes and granulosa cells in a single early follicle of a mouse is observed under a microscope. Figure 1 A is the free early follicle observed under a microscope; Figure 1 B is the oocyte and granulosa cell after separation observed under a microscope

[0029] Figure 2 Oocytes observed under a microscope. Figure 2 A is a free single oocyte of a primordial follicle separated according to the method of Example 2 observed under a microscope; Figure 2 B is a free single oocyte of a primary follicle separated according to the method of Example 2 observed under a microscope.

[0030] Figure 3 Real-time PCR amplification curve and melting curve of Gapdh gene in a single early oocyte. Figure 3 A is the amplification curve; Figure 3 B is the melting curve.

[0031] Figure 4 Gene expression in single oocytes of mouse primordial and primary follicles and its hierarchical cluster analysis and principal component analysis. Figure 4 A is a heat map based on the differences in gene expression levels in different cells; Figure 4 B is the principal component analysis. DETAILED DESCRIPTION

[0032] The essential contents and beneficial effects of the present invention will be further described below in conjunction with embodiments, which are only used to illustrate the present invention rather than to limit the present invention.

[0033] Example 1 Preparation of test materials and preparation of animals

[0034] Prepare the following test materials:

[0035] Dissection medium: Leibowitz L15 medium: contains 4% fetal bovine serum (FBS) and 100ug / ml of penicillin-streptomycin double antibody.

[0036] Digestion solution 1: αMEM medium: containing 0.05 mg / ml Liberase TMTL Research Grade (Sigma, product number 05401020001, purchase information http: / / www.sigmaaldrich.com / catalog / product / roche / 05401020001?lang=zh®ion=CN) and 0.2 mg / ml collagenase IV (ThermoFisher, 17104-019) and 0.1% DNase.

[0037] Digestive juice 2: Cell Dissociation Reagent (ThermoFisher, product number A1110501, purchase information: https: / / www.thermofisher.com / order / catalog / product / A1110501). Accutase is a widely used digestive enzyme preparation in recent years. It has protease and collagenase activities and does not contain any animal or bacterial components. It is often used as a substitute for pancreatic enzymes. Its digestion method is gentle and has low damage to cells.

[0038] Culture medium:

[0039] αMEM medium: contains 10mIU / mL follicle-stimulating hormone (FSH), 3mg / mL fetal bovine serum (BSA), 1mg / mL bovine serum albumin (Fetuin: Sigma, F3385), 5mg / mL insulin, 5mg / mL transferrin, and 5ng / ml selenium (ITS, Sigma, I3146).

[0040] Experimental Animals:

[0041] One to two-week-old female C57 mice

[0042] Example 2 Isolation of single oocytes from mouse primordial follicles and primary follicles and single granulosa cells from the oocytes

[0043] Experimental steps:

[0044] (1) The mouse ovaries were removed and freed, transferred to a culture dish containing dissection solution and washed three times, and then transferred to a culture dish containing 2 ml of digestion solution.

[0045] The ovarian tissue was mechanically dissected using a sterile needle and divided into approximately 1 × 10 6 -4×10 6 μm 3 of fragments.

[0046] (2) Place the culture dish in a sterile cell culture incubator and digest at 37°C for 30 minutes. During the digestion period, blow the digestion solution every 10 minutes. During the operation, observe the follicle separation in real time under a microscope. When most of the follicles are in a free state and no large pieces of tissue are observed, proceed to the next step.

[0047] (3) The digestion solution was passed through a 70 μm cell strainer ( 70 μm Cell Strainer, Corning, 352350), filtered twice; then the filtrate was added to a Transwell (SPLInsert TM Hanging, SPL Life Sciences, 35206) and filtered again, and the filtrate was discarded;

[0048] (4) Rinse the precipitate in the Transwell three times with culture medium, and finally resuspend the precipitate with 1 ml of culture medium. During the resuspending process, suspend the Transwell above the liquid surface of the culture dish.

[0049] The resuspended culture medium contains follicles and cells with diameters between 8μm-70μm:

[0050] The morphology of early mouse follicles is as follows:

[0051] The primordial follicle (20-40 μm) is composed of an oocyte with a middle diameter of about 20 μm surrounded by a layer of flat granulosa cells.

[0052] The primary follicle (50-70 μm) is composed of an oocyte with a central diameter of about 40 μm surrounded by a layer of cuboidal granulosa cells.

[0053] The present method uses a Transwell with a pore size of 8 μm. When the Transwell is placed in a culture dish, liquid can easily pass through the filter membrane, and when the Transwell is suspended in the air, the liquid can stay on the filter membrane due to surface tension. The present invention utilizes this feature to allow the digestion fluid after the first digestion and filtration to pass through the filter membrane of the Transwell again, and then recover the cells remaining in the Transwell. The advantages of the method of the present invention are: (a) by filtering the culture medium containing digestive enzymes, digestion can be terminated directly without centrifugation; (b) by using a Transwell with a pore size of 8.0 μm, the granular cells and theca cells with smaller diameters, which are the largest in number in the digestion fluid, can be directly discarded through the filter membrane, while the desired early follicles are left on the filter membrane, and then the characteristics of the filter membrane are utilized to resuspend and aspirate the cells with larger diameters on the filter membrane.

[0054] (5) Use the PicoPipet single-cell separation and extraction system (NEPA GENE) to aspirate a single follicle. During the operation, the early follicles and their components are manipulated according to the cell morphology and size: 30μm S glass capillaries are used to manipulate primordial follicles and their corresponding oocytes and granulosa cells, and 50μm S glass capillaries are used to manipulate primary follicles and their corresponding oocytes and granulosa cells. When aspirating early oocytes, the suction force should be controlled at 0.5-1.5V, and the fine-tuning amplitude should be set to 0.05V. When removing cells, the suction force should be increased by 0.5V after the cells reach the equilibrium position before removal.

[0055] (6) The aspirated follicles are then washed in a clean culture medium, transferred to a container containing digestion solution 2 with digestive enzyme activity, and placed in a cell culture incubator at 37°C for digestion for 5-10 minutes.

[0056] The digestive solution 2 in the method of this embodiment is CellDissociation Reagent. Accutase digestive enzyme preparation has mild and effective digestive enzyme activity, which can effectively digest and separate oocytes and granulosa cells in follicles while minimizing damage to cells. At the same time, the inventors found that treating the follicles for a shorter time during the digestion process, for example within 3 to 5 minutes, can minimize damage to cells, so that after the oocytes and their corresponding granulosa cells are separated into free single cells, they can be directly used for subsequent experiments on single cells, such as cell culture, nucleic acid extraction and amplification, etc. Longer digestion time can easily reduce the activity of oocytes and even cause cell degradation.

[0057] Experiments have found that using trypsin in the second digestion solution, such as a trypsin-EDTA solution (0.25%: 0.02%) reagent, can also digest and separate the oocytes and granulosa cells in the follicles. However, after the oocytes and their corresponding granulosa cells are separated into free single cells, they need to be digested and stopped before they can be used in subsequent single cell experiments, such as adding culture fluid or trypsin inhibitors to stop the action of trypsin on the cells, and then washing the cells, or filtering them again through a suitable cell filter.

[0058] Experiments have found that when other digestive enzymes, such as collagenase, are used in digestive solution 2, it is not possible to effectively digest and separate oocytes and granulosa cells in follicles within the short time required to maintain cell activity (e.g., within half an hour, especially within 3-10 minutes).

[0059] (7) After digestion is complete, take out the container of digestion solution 2 and blow it under a microscope (for example, using a capillary or a pipette, etc.) until the oocytes and their corresponding granulosa cells are separated into free single cells.

[0060] (8) Transferring a single oocyte and / or a single granulosa cell thereof directly into culture medium for subsequent single cell experiments.

[0061] Example 3 Identification of single oocytes of mouse primordial follicles and primary follicles and single granulosa cells of the oocytes

[0062] According to the cell morphology and size of the single cells obtained in Example 2, it was determined that a single oocyte of a mouse primordial follicle and a primary follicle and a single granulosa cell of the oocyte were obtained.

[0063] Figure 1 The separation of oocytes and granulosa cells in a single early follicle of a mouse is observed under a microscope. Figure 1 A is the free early follicle observed under a microscope; Figure 1 B shows the oocytes and granulosa cells after separation observed under a microscope.

[0064] like Figure 2 As shown, single oocytes from mouse primordial and primary follicles were obtained. Figure 2 A is a free single oocyte of a primordial follicle separated according to the method of Example 2 observed under a microscope; Figure 2 B is a free single oocyte of a primary follicle separated according to the method of Example 2 observed under a microscope.

[0065] Example 4 Gene expression of single oocytes of mouse primordial follicles and primary follicles and hierarchical cluster analysis and principal component analysis

[0066] 1. Gene expression analysis was performed on single oocytes of mouse primordial follicles and primary follicles obtained in Example 2.

[0067] Experimental reagents: Single Cell Lysis Kit (ThermoFisher, 4458235); SuperScript VILOcDNA Synthesis Kit (ThermoFisher, 1754-050); Platinum Taq DNA Polymerase (ThermoFisher, 10966-034); PowerUp SYBR Green Master Mix (ThermoFisher, A25777).

[0068] Experimental steps:

[0069] (1) The oocytes of the seven primordial follicles and the oocytes of the seven primary follicles separated in Example 2 were transferred to a PCR tube containing 10 μl of cell lysis solution (Single Cell Lysis Kit) and lysed at room temperature for 5 minutes according to the product instructions (after lysis, they can be stored at -20°C).

[0070] (2) Add 1 μl of stop solution to each PCR tube and incubate at room temperature for 2 minutes.

[0071] (3) Perform reverse transcription of a single cell according to the instructions of SuperScript VILO cDNA Synthesis Kit. After reverse transcription, add 50 μl of denuclease-free water to each tube to dilute the obtained cDNA.

[0072] (4) Pre-amplification was performed using the following specific primers. According to the instructions of Platinum Taq DNA Polymerase, the reaction system was as follows:

[0073]

[0074]

[0075] PCR pre-amplification conditions are as follows:

[0076]

[0077] (5) The amplified product can be used for single-cell qPCR. The reaction system is as follows:

[0078]

[0079] qPCR was performed according to the SYBR instructions.

[0080] The experimental results can be Figure 3 For example. Figure 3 The real-time PCR amplification curve and melting curve of the Gapdh gene in a single early oocyte are shown. The experimental results show that a single oocyte can obtain good qPCR results.

[0081] 2. Hierarchical cluster analysis and component analysis were performed on individual oocytes of the mouse primordial follicles and primary follicles obtained in Example 2.

[0082] The qPCR analysis data obtained from the single-cell qPCR analysis of oocytes from 7 primordial follicles and 7 primary follicles were subjected to hierarchical cluster analysis and component analysis.

[0083] The CT values ​​obtained by qPCR were not standardized with the internal reference, but directly processed, and the effective threshold was set, false positive values ​​were deleted, missing values ​​were processed, and finally converted to a log value with a base of 2 for analysis. The processed data were imported into Qlucore Omics software for analysis and plotting. Figure 4 shown. Figure 4 A is a heat map based on the difference in gene expression in different cells. It can be seen that the oocytes of the two periods have their own highly expressed genes. For example, ZP1 is highly expressed in oocytes derived from primordial follicles compared with oocytes derived from primary follicles. In addition, it can be observed that there is no significant difference in the expression of the housekeeping gene Gapdh in the two cells. After cluster analysis, it can be observed that the Gapdh status of primordial follicles and primary follicles is significantly different, which can be used to distinguish primordial follicles from primary follicles.

[0084] The sample composition was analyzed using the expression levels of different genes. Figure 4 As shown in B. Similar to the results of cluster analysis, it can be seen that the oocytes of primordial follicles and primary follicles are significantly different, which can be used to distinguish primordial follicles from primary follicles.

[0085] The above results illustrate that the method for separating single cells of different tissue types in ovarian tissue of the present invention can correctly separate two different early oocytes, i.e. primordial follicles (primordial follicles) and primary follicles, and obtain active single cells thereof. The gene expression of oocytes that have lost cell activity is significantly different from the gene expression of cells with activity. The method of the present invention enables researchers in this field to further study single cells. For example, by selecting genes with specific expression of some periods, oocytes of two different developmental periods can be studied to understand their different characteristics.

[0086] The method of the present invention operates at the level of a single follicle to separate the oocyte and the corresponding granulosa cells contained therein. The method of the present invention reduces the number of operations on the cells, thereby better protecting the integrity of the cells, and being able to obtain a larger number of early follicles from the follicles. With the development and maturity of single-cell technology, experiments at the level of single cells are receiving more and more attention. The separation of oocytes and their corresponding granulosa cells from living early follicles in ovarian tissue has always been a difficult problem. The method of the present invention successfully solves this problem. It provides a platform foundation for subsequent cell activity studies, gene expression analysis, single-cell sequencing, and even proteomics studies at the level of single early oocytes and corresponding granulosa cells.

[0087] The above is an explanation of the present invention and cannot be regarded as a limitation of the present invention. Unless otherwise noted, the practice of the present invention will use the conventional techniques of organic chemistry, polymer chemistry, biotechnology, etc., and it is obvious that the present invention can also be realized in other ways except that it is particularly described in the above description and embodiments. Other aspects and improvements within the scope of the present invention will be apparent to those skilled in the art to which the present invention belongs. According to the teachings of the present invention, many changes and variations are feasible, and therefore they are within the scope of the present invention.

[0088] Unless otherwise specified, the temperature unit "degree" appearing in this document refers to Celsius, that is, ℃.

Claims

1. A method for obtaining a single oocyte and a single granulosa cell of an early ovarian follicle of a mammal, characterized in that The following steps are involved: (1) Blunt dissection of mammalian ovarian tissue; (2) digesting the mammalian ovarian tissue fragments in a first digestion solution at 37° C. for 20-60 minutes, wherein the first digestion solution contains collagenase I, collagenase II or collagenase IV or a mixture thereof; (3) passing the digestion fluid mixture obtained in step (2) through a first pore size cell filter, wherein the pore size of the first pore size cell filter is 40-100 μm, and then passing the filtrate through a second pore size cell filter, wherein the pore size of the second pore size cell filter is 8-12 μm; (4) washing the precipitate in the second pore size cell filter with culture medium, and then resuspending the precipitate with culture medium; (5) Aspirate a single early follicle from the resuspension solution; (6) washing the aspirated single early follicle, and then digesting it in a second digestion solution for 3-10 minutes, wherein the second digestion solution contains trypsin or accutase; (7) transferring the digestion solution mixture obtained in step (6) into a culture medium, and obtaining a single oocyte and its corresponding single granulosa cell by separation, The early follicle is a primordial follicle or a primary follicle.

2. The method of claim 1, wherein the pore size of the first pore size cell filter in step (3) is 70-80 μm.

3. The method of claim 1, wherein the pore size of the second pore size cell filter in step (3) is 8 μm.

4. The method of claim 1, further comprising the steps of: (8) Using a single oocyte and / or a single granulosa cell thereof for single cell experiments.

5. The method of claim 1, wherein the first digestion solution contains a mixture of Liberase and collagenase IV.

6. The method of claim 5, wherein the ratio of Liberase to collagenase IV in the first digestion solution is 1:

4.

7. The method of claim 1, wherein the first digestion solution further comprises a neutral protease and / or a metalloprotease.

8. The method of claim 7, wherein the first digestion solution further contains DNase.

9. The method of claim 1, wherein the second digestion solution contains Accutase.

10. The method of claim 1, wherein in step (6), digestion in the second digestion solution is performed for 3-5 minutes.

11. The method of claim 1, wherein the second pore size cell strainer is a cell permeable chamber.

12. The method of claim 1, wherein the mammal is a small to medium sized mammal.

13. The method of claim 12, wherein the mammal is a monkey, sheep, pig, cow, mouse, rat, guinea pig, rabbit, cat or dog.

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