Reagent composition and method for in-vitro maturation three-dimensional culture of human oocytes

Abstract

The invention relates to the technical field of cell culture, and discloses a reagent composition and method for in-vitro maturation three-dimensional culture of human oocytes, and the reagent composition comprises a three-dimensional hydrogel scaffold material formed by compounding sodium alginate, hyaluronic acid and collagen; the invention also relates to a culture medium containing a specific bioactive additive, wherein the additive comprises recombinant human anti-mullerian hormone, recombinant human follicle stimulating hormone and recombinant human epidermal growth factor with specific concentrations. The method comprises the following steps: mixing to-be-mature human oocytes and granular cells, embedding the mixture in the three-dimensional hydrogel scaffold to form cell hydrogel microspheres, and culturing the cell hydrogel microspheres in the culture medium. According to the method, the bionic three-dimensional microenvironment is constructed, and a specific factor combination is utilized to synergistically regulate and control ripening synchronization of nucleoplasm, so that the problem of low maturation quality of oocytes in traditional culture is solved, and the maturation rate, the fertilization rate and the blastocyst formation rate of the oocytes are remarkably improved.

Description

Technical Field

[0001] The present invention relates to the field of cell culture technology, in particular to a reagent composition and method for in vitro maturation three-dimensional culture of human oocytes. Background Art

[0002] In vitro maturation (IVM) of human oocytes refers to the process of culturing immature oocytes obtained from the ovaries in a simulated physiological environment in vitro to allow them to mature. It is an important technology in the field of assisted reproductive technology, especially for patients with polycystic ovary syndrome (PCOS) and women who need to preserve their fertility due to tumors and other reasons.

[0003] However, despite years of development, IVM remains unsatisfactory in clinical application. Currently, widely used in vitro maturation techniques rely primarily on two-dimensional (2D) culture systems, where cells are cultured on a flat surface in a culture dish. This culture method differs significantly from the environment in which oocytes grow in follicles in vivo and cannot effectively mimic the complex three-dimensional spatial structure and extracellular matrix environment of follicles in vivo.

[0004] This non-physiological culture condition blocks the two-way communication between the oocyte and the surrounding granulosa cells, which in turn leads to a core technical problem: the nuclear maturation and cytoplasmic maturation of the oocyte are not synchronized. Although many oocytes cultured under two-dimensional conditions have reached nuclear maturity (i.e., the expulsion of the first polar body) in terms of morphology, key processes such as organelle function, messenger RNA storage, and protein synthesis in their cytoplasm are not fully completed, that is, the cytoplasm is immature. This state of nuclear and cytoplasmic developmental asynchrony is the main reason for the low subsequent fertilization rate, poor embryonic development potential, and low clinical pregnancy rate of in vitro matured oocytes. In addition, the in vitro culture environment also exposes oocytes to higher levels of oxidative stress, further damaging their quality.

[0005] Therefore, how to construct a culture system that is closer to the physiological state to overcome the limitations of traditional two-dimensional culture and promote the synchronous maturation of oocyte nucleus and cytoplasm is a technical problem that needs to be solved urgently in this field. Summary of the Invention

[0006] The technical problem to be solved by the present invention is that the existing human oocyte in vitro maturation technology adopts a two-dimensional culture method, which cannot effectively simulate the physiological microenvironment of the follicle, resulting in asynchrony between the nuclear maturation and cytoplasmic maturation of the oocyte, ultimately affecting its developmental potential.

[0007] To solve the above technical problems, the present invention provides a reagent composition and method for three-dimensional culture of human oocytes for in vitro maturation, which improves the quality and efficiency of in vitro maturation of human oocytes by constructing a bionic three-dimensional microenvironment and optimizing the bioactive factors in the culture system.

[0008] The first aspect of the present invention provides a reagent composition for in vitro maturation and three-dimensional culture of human oocytes.

[0009] In a specific embodiment, the reagent composition comprises a three-dimensional hydrogel scaffold material, a basal culture medium and a plurality of bioactive additives added thereto.

[0010] The three-dimensional hydrogel scaffold material is composed of a composite of sodium alginate, hyaluronic acid, and collagen. Sodium alginate, as the foundational gelling agent, provides a stable three-dimensional spatial structure; hyaluronic acid, a key component of the follicular extracellular matrix, helps maintain intercellular signaling; and collagen provides cell adhesion sites, supporting the normal attachment and function of granulosa cells. This composite scaffold can construct a three-dimensional structure that mimics the in vivo follicular microenvironment.

[0011] In a preferred embodiment, the mass ratio of the sodium alginate to the collagen is 2.8: 1 to 3.2: 1. This specific ratio balances the mechanical strength and biocompatibility of the scaffold.

[0012] In another preferred embodiment, the final concentration of the sodium alginate by weight volume percentage is 1.0-2.0%, and the final concentration of the hyaluronic acid by weight volume percentage is 4.0-6.0%. This concentration range ensures that the formed microspheres have an appropriate porosity, which is conducive to the exchange of nutrients and metabolic waste.

[0013] The biologically active additive comprises recombinant human anti-mullerian hormone, recombinant human follicle-stimulating hormone and recombinant human epidermal growth factor at specific concentrations.

[0014] The concentration of the recombinant human anti-Müllerian hormone is 8-12 ng / mL. The function of this hormone is to inhibit the premature initiation of nuclear maturation in oocytes, thereby providing a time window for the full maturation of the cytoplasm and promoting synchronous maturation of the nucleus and cytoplasm.

[0015] The concentration of the recombinant human follicle-stimulating hormone is 40-60 mIU / mL. The hormone acts on the co-cultured granulosa cells, inducing their functional activation, and then supports the maturation of oocytes through a paracrine pathway.

[0016] The concentration of the recombinant human epidermal growth factor is 15-25 ng / mL. This factor directly acts on oocytes to promote their cytoplasmic maturation process.

[0017] In a further embodiment, the reagent composition further comprises a cholesterol carrier and an antioxidant system.

[0018] The cholesterol carrier is methyl-β-cyclodextrin, and its concentration in the basal culture medium is 0.08-0.12 mM. Its function is to promote the transport of exogenous cholesterol to the oocyte and replenish the precursor substances required for the oocyte membrane structure and steroid synthesis.

[0019] The antioxidant system contains 4-6 μM sodium selenite, 40-60 μg / mL ascorbic acid, and 10-20 μM melatonin. This system reduces the damage of oxidative stress to oocytes by removing reactive oxygen species generated during in vitro culture.

[0020] In another further embodiment, the reagent composition further comprises 5-15% by volume of the patient's autologous serum. The autologous serum can provide personalized, complex growth factors and nutrients to further optimize the culture microenvironment.

[0021] A second aspect of the present invention provides a three-dimensional culture method for improving the in vitro maturation quality of human oocytes using any of the aforementioned reagent compositions.

[0022] The method comprises the following steps:

[0023] In step a, human oocytes to be matured are mixed with granulosa cells and embedded in a three-dimensional hydrogel scaffold composed of sodium alginate, hyaluronic acid, and collagen to form a cell-hydrogel complex. This co-encapsulation of oocytes and granulosa cells reestablishes physical contact and bidirectional communication between the two cells, essential for normal oocyte maturation.

[0024] In a preferred embodiment, in step a, the ratio of the number of oocytes to the number of granulosa cells is 1:4000 to 1:6000. This cell ratio ensures that there are sufficient granulosa cells to support the maturation of a single oocyte.

[0025] In another preferred embodiment, the embedding in step a specifically includes dripping a hydrogel scaffold solution containing oocytes and granulosa cells into a crosslinking solution to form cell-hydrogel microspheres with a diameter of 180-220 μm, which serve as the cell-hydrogel complex. This specific diameter physically simulates the size of an ovarian follicle and defines the effective distance for substance diffusion.

[0026] Step b: placing the cell-hydrogel complex in a culture medium containing specific concentrations of recombinant human anti-Müllerian hormone, recombinant human follicle-stimulating hormone and recombinant human epidermal growth factor for culture.

[0027] In a preferred embodiment, the culture in step b is performed at 37°C and 5% O2. The purpose of using a low oxygen concentration of 5% for culture is to simulate the physiological hypoxic environment of ovarian tissue in vivo and reduce oxidative damage to cells caused by a high oxygen environment.

[0028] The present invention provides a reagent composition and method for in vitro maturation and three-dimensional culture of human oocytes.

[0029] It has the following beneficial effects:

[0030] 1. The present invention adopts a three-dimensional hydrogel scaffold composed of sodium alginate, hyaluronic acid and collagen, and embeds oocytes and granulosa cells therein, thereby constructing a microenvironment that simulates the in vivo follicle in terms of spatial structure and extracellular matrix components. This three-dimensional structure restores the necessary physical contact and communication path between oocytes and granulosa cells, providing conditions for the maturation of oocytes that are more in line with physiological conditions than traditional two-dimensional planar culture.

[0031] 2. The present invention uses a specific concentration combination of anti-Müllerian hormone, follicle-stimulating hormone and epidermal growth factor in the culture system. Among them, the use of anti-Müllerian hormone can effectively delay the initiation of oocyte nuclear maturation, buying time for the full maturation of the cytoplasm; at the same time, follicle-stimulating hormone and epidermal growth factor synergistically promote the function of granulosa cells and the maturation of oocyte cytoplasm. This combined regulation method solves the problem of asynchronous nuclear and cytoplasmic maturation that is common in the existing technology, and helps to achieve synchronous development of the two.

[0032] 3. The present invention introduces an antioxidant system and a cholesterol carrier into the reagent composition. The antioxidant system can remove the reactive oxygen species generated by environmental changes during in vitro culture and reduce the damage to oocytes caused by oxidative stress. The cholesterol carrier supplements the oocyte's demand for lipids during the maturation process to maintain the structural integrity of the cell membrane, thereby comprehensively improving the intrinsic developmental potential of oocytes after in vitro maturation. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the three-dimensional culture medium structure of the present invention. DETAILED DESCRIPTION

[0034] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the drawings in the present specification. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0035] The sources and specifications of the main raw materials and reagents used in the following examples and comparative examples are as follows. Reagents not specifically specified are all commercially available products of analytical grade or higher.

[0036] Culture media and solutions:

[0037] Basal medium G-1PLUS TM :Vitrolife Sweden AB;

[0038] G-MOPS handling and washing medium TM :Vitrolife Sweden AB;

[0039] G-2PLUS medium for embryo culture to blastocyst stage TM :Vitrolife Sweden AB;

[0040] Phosphate buffered saline (DPBS), calcium- and magnesium-free: Gibco;

[0041] Three-dimensional hydrogel scaffold related reagents:

[0042] Sodium Alginate, medical grade, CAS No.: 9005-38-3;

[0043] Sodium Hyaluronate, medical grade, CAS No. 9067-32-7;

[0044] Type I collagen (Col lagen, Type I), acid-soluble, sourced from bovine Achilles tendon;

[0045] Calcium chloride, anhydrous, CAS number: 10043-52-4;

[0046] Biologically active additives and other chemical reagents:

[0047] Recombinant human anti-Müllerian hormone (AMH);

[0048] Recombinant human follicle-stimulating hormone (FSH);

[0049] Recombinant human epidermal growth factor (Recombinant Human EGF);

[0050] Methyl-β-cyclodextrin, CAS No.: 128446-36-6;

[0051] Sodium Selenite, CAS No.: 10102-18-8;

[0052] L-Ascorbic acid, CAS No.: 50-81-7;

[0053] Melatonin, CAS No.: 73-31-4;

[0054] Hyaluronidase, derived from bovine testicles, CAS number: 9001-54-1.

[0055] Example 1: Preparation of the reagent composition of the present invention and implementation of the three-dimensional culture method

[0056] Preparation of reagent composition:

[0057] Preparation of the three-dimensional hydrogel scaffold stock solution: Dissolve 1.5g of sodium alginate powder and 5.0g of sodium hyaluronate powder in a sufficient amount of calcium-magnesium-free DPBS solution, dilute to 100mL, and stir magnetically overnight at 4°C to prepare a 1.5% (w / v) sodium alginate stock solution and a 5.0% (w / v) sodium hyaluronate stock solution. The two stock solutions were mixed with a medical-grade type I collagen solution (acid-soluble) under sterile ice bath conditions, and the volumes of each component were adjusted so that the mass ratio of sodium alginate to type I collagen in the final mixed stock solution was 3:1. The mixed stock solution was sterilized by filtering through a 0.22μm filter membrane to obtain the three-dimensional hydrogel scaffold stock solution to be used.

[0058] The final IVM medium was prepared in 10 mL of G-1PLUS TM Aseptically add the following components to the basal medium to achieve the following final concentrations:

[0059] Recombinant human anti-Müllerian hormone (AMH): 10 ng / mL;

[0060] Recombinant human follicle-stimulating hormone (FSH): 50 mIU / mL;

[0061] Recombinant human epidermal growth factor (EGF): 20 ng / mL;

[0062] Methyl-β-cyclodextrin: 0.1 mM;

[0063] Sodium selenite: 5 μM;

[0064] L-ascorbic acid: 50 μg / mL;

[0065] Melatonin: 15 μM;

[0066] Patient's autologous serum: 10% (v / v);

[0067] The above culture medium was gently mixed, filtered through a 0.22 μm filter membrane, and placed in a 37° C., 5% CO 2 incubator for pre-equilibration for at least 4 hours to obtain the final IVM culture medium.

[0068] Implementation of the three-dimensional culture method:

[0069] Cell preparation:

[0070] Human oocyte-cumulus cell complexes were obtained from patients with informed consent. Cumulus cells were mechanically removed, and naked oocytes in the germinal vesicle stage (GV) were selected. Granulosa cells were also isolated and purified from the patient's follicular fluid.

[0071] Construction of cell hydrogel microspheres GV stage oocytes and suspended granulosa cells were mixed at a cell number ratio of 1:5000, collected by centrifugation, and resuspended in the three-dimensional hydrogel scaffold stock solution prepared according to the above steps. Using a micropipette, the scaffold stock solution containing cells was squeezed out in the form of droplets and dripped into a sterile calcium chloride solution with a concentration of 100mM. After standing for cross-linking for 3 minutes, cell hydrogel microspheres with an average diameter of 200μm were formed. Subsequently, G-1PLUS TM The microspheres were washed three times with culture medium to remove residual calcium chloride.

[0072] nourish:

[0073] Transfer the washed cell hydrogel microspheres to a culture dish and add 0.5 mL of the final IVM medium prepared in step 1.1.2 to each dish. Incubate the culture dish in a three-gas incubator at 37°C, 5% O2, 5% CO2, and 95% humidity for 24 hours.

[0074] The above IVM culture medium can refer to the attached Figure 1 , Figure 1 This is a schematic diagram of the three-dimensional culture medium structure of the present invention, wherein A is a mixture of basal culture medium and autologous serum, B is GV and M1 stage oocytes, C is a sodium alginate + hyaluronic acid + collagen composite hydrogel scaffold, and D is the patient's autologous granulosa cells.

[0075] Example 2:

[0076] Preparation of reagent composition:

[0077] Preparation of the 3D hydrogel scaffold stock solution: Dissolve 1.0g of sodium alginate powder and 4.0g of sodium hyaluronate powder in a sufficient amount of calcium- and magnesium-free DPBS solution, and dilute to 100mL to prepare a 1.0% (w / v) sodium alginate stock solution and a 4.0% (w / v) sodium hyaluronate stock solution. Mix the two stock solutions with a medical-grade type I collagen solution, adjusting the volumes of the components so that the mass ratio of sodium alginate to type I collagen in the final mixed solution is 2.8:1. Filter and sterilize the mixed solution to obtain the 3D hydrogel scaffold stock solution to be used.

[0078] The final IVM medium was prepared in G-1PLUS TM Aseptically add the following components to the basal medium to achieve the following final concentrations:

[0079] Recombinant human anti-Müllerian hormone (AMH): 8 ng / mL;

[0080] Recombinant human follicle-stimulating hormone (FSH): 40 mIU / mL;

[0081] Recombinant human epidermal growth factor (EGF): 15 ng / mL;

[0082] Methyl-β-cyclodextrin: 0.08 mM;

[0083] Sodium selenite: 4 μM;

[0084] L-ascorbic acid: 40 μg / mL;

[0085] Melatonin: 10 μM;

[0086] Patient's autologous serum: 5% (v / v);

[0087] The culture medium was mixed, filtered, and pre-equilibrated.

[0088] Implementation of the three-dimensional culture method:

[0089] GV-stage oocytes and granulosa cells were mixed at a cell ratio of 1:4000 and resuspended in a three-dimensional hydrogel GLISH gel scaffold prepared as described above. The cell-containing scaffold solution was then added dropwise to a calcium chloride solution for cross-linking, forming cell-hydrogel microspheres with an average diameter of 180 μm. The washed microspheres were placed in the final IVM medium prepared as described above and incubated at 37°C, 5% O₂, 5% CO₂ for 24 hours.

[0090] Example 3:

[0091] Preparation of reagent composition:

[0092] Preparation of the 3D hydrogel scaffold stock solution: Dissolve 2.0g of sodium alginate powder and 6.0g of sodium hyaluronate powder in a sufficient amount of calcium- and magnesium-free DPBS solution, and dilute to 100mL to prepare a 2.0% (w / v) sodium alginate stock solution and a 6.0% (w / v) sodium hyaluronate stock solution. Mix the two stock solutions with a medical-grade type I collagen solution, adjusting the volumes of the components so that the mass ratio of sodium alginate to type I collagen in the final mixed stock solution is 3.2:1. Filter and sterilize the mixed stock solution to obtain the ready-to-use 3D hydrogel scaffold stock solution.

[0093] The final IVM medium was prepared in G-1PLUS TM Aseptically add the following components to the basal medium to achieve the following final concentrations:

[0094] Recombinant human anti-Müllerian hormone (AMH): 12 ng / mL;

[0095] Recombinant human follicle-stimulating hormone (FSH): 60 mIU / mL;

[0096] Recombinant human epidermal growth factor (EGF): 25 ng / mL;

[0097] Methyl-β-cyclodextrin: 0.12 mM;

[0098] Sodium selenite: 6 μM;

[0099] L-ascorbic acid: 60 μg / mL;

[0100] Melatonin: 20 μM;

[0101] Patient's autologous serum: 15% (v / v);

[0102] The culture medium was mixed, filtered, and pre-equilibrated.

[0103] Implementation of the three-dimensional culture method:

[0104] GV-stage oocytes and granulosa cells were mixed at a cell ratio of 1:6000 and resuspended in the 3D hydrogel scaffold solution prepared in step 3.1.1. The cell-containing scaffold solution was then added dropwise to a calcium chloride solution for cross-linking, forming cell-containing hydrogel microspheres with an average diameter of 220 μm. The washed microspheres were placed in the final IVM medium prepared in step 3.1.2 and cultured at 37°C, 5% O₂, 5% CO₂ for 24 hours.

[0105] Comparative Example 1-2:

[0106] Comparative Example 1:

[0107] Compared to Example 1, the difference was that, instead of using a three-dimensional hydrogel scaffold for cell embedding, the oocyte and granulosa cell mixture was placed directly into microdroplets in a culture dish for two-dimensional co-culture. All other conditions, including the composition and concentration of the culture medium, the cell ratio, the culture environment, and the duration, were the same as in Example 1.

[0108] Comparative Example 2:

[0109] Compared with Example 1, the difference is that recombinant human anti-Müllerian hormone (AMH), recombinant human follicle-stimulating hormone (FSH), and recombinant human epidermal growth factor (EGF) are not added to the final IVM culture medium. All other steps, materials, and culture conditions are the same as those in Example 1.

[0110] Test example: Detection of oocyte maturation rate and subsequent developmental potential

[0111] Test method:

[0112] The oocytes cultured for 24 hours using the methods of Example 1, Comparative Example 1 and Comparative Example 2 were removed for subsequent evaluation of developmental potential.

[0113] Maturity rate (MII rate) evaluation:

[0114] Each group of oocytes was briefly treated with hyaluronidase to ensure that no residual cells were attached. The number of oocytes that successfully expelled their first polar body was then observed and recorded under a microscope. These cells were identified as mature MII stage oocytes. The MII rate was calculated using the following formula:

[0115] MII rate (%) = (number of MII stage oocytes / total number of initial GV stage oocytes) × 100%.

[0116] Fertilization rate evaluation:

[0117] MII-stage oocytes obtained from each group underwent intracytoplasmic sperm injection (ICSI). 16-18 hours after injection, the number of normally fertilized zygotes was observed under a microscope and recorded. Normal fertilization was indicated by the presence of two distinct pronuclei (2PN) within the zygote. The fertilization rate was calculated using the following formula:

[0118] Fertilization rate (%) = (number of normally fertilized 2PN zygotes / number of injected MII stage oocytes) × 100%.

[0119] Evaluation of blastocyst formation rate:

[0120] The normally fertilized 2PN zygotes in each group were transferred to G-1PLUS TMContinue to culture in embryo culture medium. On day 5 after ICSI, observe and record the number of embryos that develop to the blastocyst stage. The blastocyst formation rate is calculated using the following formula:

[0121] Blastocyst formation rate (%) = (number of blastocysts formed / number of 2PN zygotes fertilized normally) × 100%.

[0122] Test results:

[0123] The test results of Example 1, Comparative Example 1 and Comparative Example 2 were statistically analyzed, and the specific data are recorded in Table 1 below.

[0124] Table 1. Oocyte developmental potential test results of each group

[0125]

[0126] Result analysis:

[0127] As can be seen from the data in Table 1, the method of Example 1 shows a significant improvement in the oocyte MII rate (72.2% vs 55.1%), fertilization rate (75.0% vs 62.8%), and final blastocyst formation rate (46.2% vs 25.9%) compared to Comparative Example 1 (traditional two-dimensional method). This confirms that the three-dimensional hydrogel scaffold used in the present invention, by constructing a physical microenvironment that simulates the in vivo follicle, can effectively promote the interaction between oocytes and granulosa cells, thereby obtaining higher quality mature oocytes than two-dimensional planar culture.

[0128] In comparison with Comparative Example 2, although the MII rate (68.0%) of Comparative Example 2 (using only a three-dimensional scaffold) was higher than that of Comparative Example 1, its fertilization rate (54.9%) and blastocyst formation rate (17.9%) were significantly lower than those of Example 1. This result shows that the specific combination of bioactive additives contained in the reagent composition of the present invention is the key to obtaining oocytes with high developmental potential. The composition delays nuclear maturation by anti-Müllerian hormone, while promoting cytoplasmic maturation by follicle-stimulating hormone and epidermal growth factor, achieving synchronous nuclear and cytoplasmic maturation, thereby effectively improving the fertilization ability of oocytes and subsequent embryonic development ability.

[0129] In summary, this invention addresses the existing issue of low oocyte maturation quality by combining a biomimetic three-dimensional physical structure with precise biochemical signaling regulation. Data clearly demonstrate that the synergistic effect of the physical microenvironment and key bioactive factors enhances the overall developmental potential of oocytes, ultimately manifesting in a substantial improvement in the core functional indicator of blastocyst formation rate.

[0130] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A reagent composition for in vitro maturation and three-dimensional culture of human oocytes, characterized in that: It includes: A three-dimensional hydrogel scaffold material, wherein the scaffold material is composited with sodium alginate, hyaluronic acid and collagen; A basal culture medium; a biologically active additive added to the basal culture medium, comprising: Recombinant human anti-Müllerian hormone at a concentration of 8-12 ng / mL; Recombinant human follicle-stimulating hormone at a concentration of 40-60 mIU / mL; Recombinant human epidermal growth factor at a concentration of 15-25 ng / mL.

2. A reagent composition for in vitro maturation and three-dimensional culture of human oocytes according to claim 1, characterized in that: In the three-dimensional hydrogel scaffold material, the mass ratio of the sodium alginate to the collagen is 2.8:1 to 3.2:

1.

3. A reagent composition for in vitro maturation and three-dimensional culture of human oocytes according to claim 1, characterized in that: In the three-dimensional hydrogel scaffold material, the final concentration of sodium alginate by weight volume percentage is 1.0-2.0%, and the final concentration of hyaluronic acid by weight volume percentage is 4.0-6.0%.

4. A reagent composition for in vitro maturation and three-dimensional culture of human oocytes according to claim 3, characterized in that: The reagent composition further comprises a cholesterol carrier and an antioxidant system, wherein: The cholesterol carrier is methyl-β-cyclodextrin, and its concentration in the basal medium is 0.08-0.12 mM; The antioxidant system comprises sodium selenite at a concentration of 4-6 μM, ascorbic acid at a concentration of 40-60 μg / mL, and melatonin at a concentration of 10-20 μM.

5. The reagent composition for in vitro maturation and three-dimensional culture of human oocytes according to claim 1, characterized in that: The reagent composition further comprises the patient's autologous serum, wherein the volume percentage concentration of the patient's autologous serum is 5-15%.

6. A three-dimensional culture method for in vitro maturation of human oocytes, characterized in that: The method uses the reagent composition for in vitro maturation and three-dimensional culture of human oocytes according to any one of claims 1 to 5, and comprises the following steps: a. Mixing human oocytes to be matured with granulosa cells and embedding them in a three-dimensional hydrogel scaffold composed of sodium alginate, hyaluronic acid and collagen to form a cell-hydrogel complex; b. placing the cell-hydrogel complex in a culture medium containing recombinant human anti-Müllerian hormone, recombinant human follicle-stimulating hormone and recombinant human epidermal growth factor for culturing.

7. A three-dimensional culture method for in vitro maturation of human oocytes according to claim 6, characterized in that: In step a, the embedding specifically includes: dropping a hydrogel scaffold stock solution containing oocytes and granulosa cells into a cross-linking solution to form cell hydrogel microspheres with a diameter of 180-220 μm as the cell hydrogel complex.

8. The method for three-dimensional culture of human oocytes for in vitro maturation according to claim 6, characterized in that: In step a, the ratio of the number of the oocytes to the number of the granulosa cells is 1:4000 to 1:6000.

9. The method for three-dimensional culture of human oocytes for in vitro maturation according to claim 6, characterized in that: In step b, the culture is carried out at 37° C. and 5% O 2 .

10. The method for three-dimensional culture of human oocytes for in vitro maturation according to claim 6, characterized in that: In step b, in the culture medium, the concentration of recombinant human anti-Müllerian hormone is 8-12 ng / mL, the concentration of recombinant human follicle-stimulating hormone is 40-60 mIU / mL, and the concentration of recombinant human epidermal growth factor is 15-25 ng / mL.

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