A method for three-dimensional culture of secondary follicles using agarose-calcium alginate composite hydrogel microchamber

Abstract

The present invention belongs to the technical field of follicle in vitro culture, and relates to a method for three-dimensional culture of secondary follicles using agarose-calcium alginate composite hydrogel microcavity. Follicles are dispersed into a sodium alginate solution to obtain a follicle solution, and the follicles are inoculated into an agarose microcavity through the follicle solution; then a calcium salt solution is added to the agarose microcavity to cross-link the sodium alginate, and an agarose-calcium alginate composite hydrogel microcavity loaded with follicles is obtained, and then follicle growth medium is added to the agarose-calcium alginate composite hydrogel microcavity loaded with follicles for follicle culture. The present invention first discovered and disclosed a method for three-dimensional culture of secondary follicles using agarose-calcium alginate composite hydrogel microcavity, and proved through experiments that the agarose-calcium alginate composite hydrogel microcavity has a good three-dimensional culture effect on secondary follicles, so it has good practical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of follicle in vitro culture, and relates to a method for three-dimensionally culturing secondary follicles by utilizing agarose-calcium alginate composite hydrogel microcavity. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Reproductive system damage caused by factors such as chemotherapy and radiotherapy seriously threatens human health and quality of life. As the trend of younger cancer patients increases and the cure rate of cancer increases, how to effectively preserve the fertility of female survivors of childbearing age and prepubertal women is a difficult problem faced in clinical practice. Currently, the commonly used oocyte cryopreservation and embryo cryopreservation processes in clinical practice are cyclical and only applicable to women of childbearing age. There is a lack of effective methods for preserving the fertility of patients of childbearing age who are in urgent need of treatment and prepubertal female cancer patients.

[0004] The purpose of in vitro follicle culture is to culture the preantral follicles in the ovaries into antral follicles in vitro and obtain mature oocytes by inducing ovulation. This process does not require an ovulation induction cycle and can effectively avoid the reinfusion of tumor cells caused by transplantation after ovarian tissue cryopreservation. It is particularly suitable for women of childbearing age and prepubertal female tumor patients who are in urgent need of chemotherapy. Follicle in vitro culture is divided into two-dimensional culture and three-dimensional culture. A large number of studies have shown that compared with traditional two-dimensional culture, the three-dimensional follicle culture system can provide a three-dimensional growth microenvironment for follicles, which is more conducive to maintaining the follicle morphology and supporting the growth and development of follicles in vitro. However, the existing three-dimensional follicle culture systems, such as the commonly used three-dimensional follicle culture system based on calcium alginate hydrogel microspheres, have the disadvantages of low follicle growth efficiency, limited egg recovery rate, poor uniformity and stability of culture effect, which is not conducive to promotion and application. Summary of the invention

[0005] In order to address the deficiencies of the prior art, the purpose of the present invention is to provide a method for three-dimensional culture of secondary follicles using agarose-calcium alginate composite hydrogel microchambers, which can stably and efficiently promote the in vitro growth, development and mature ovulation of follicles, thereby promoting the application of in vitro follicle culture in fertility preservation.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] A method for three-dimensionally culturing secondary follicles using an agarose-calcium alginate composite hydrogel microcavity comprises the following steps: dispersing follicles into a sodium alginate solution to obtain a follicle solution, inoculating the follicles into an agarose microcavity through the follicle solution; then adding a calcium salt solution into the agarose microcavity to cross-link the sodium alginate to obtain an agarose-calcium alginate composite hydrogel microcavity loaded with follicles, and then adding a follicle growth medium into the agarose-calcium alginate composite hydrogel microcavity loaded with follicles to culture the follicles.

[0008] Agarose is a linear polysaccharide polymer, and its aqueous solution is temperature-sensitive within a certain concentration range, solidifies at low temperatures to form a gel, and has good biocompatibility, chemical stability, easy plasticity, and biodegradability. Therefore, the present invention uses agarose as the outer microcavity scaffold material to support the three-dimensional culture of follicles. Alginate is a natural polysaccharide carbohydrate that can be cross-linked by calcium ions under mild cross-linking conditions. The obtained calcium alginate hydrogel has good biocompatibility and biodegradability. Therefore, the present invention uses calcium alginate hydrogel as the inner filling material to further maintain the three-dimensional morphology of follicles in the microcavity. The agarose-calcium alginate composite hydrogel microcavity prepared by the present invention has an outer agarose microcavity structure and an inner calcium alginate hydrogel filling material with elastic moduli similar to those of the ovarian cortex and medulla, respectively, and can provide a growth microenvironment for follicles that simulates the mechanical properties of the ovary. The composite hydrogel microchamber is convenient for batch operation of three-dimensional culture of follicles, can provide a uniform and stable three-dimensional growth microenvironment for multiple follicles at the same time, and effectively supports the stable encapsulation and growth and development of follicles in low-strength calcium alginate hydrogel. Therefore, the sheet-like agarose-calcium alginate composite hydrogel microchamber selected by the present invention as a three-dimensional culture system for secondary follicles has a significant effect on the regulation of the growth and development of secondary follicles in vitro.

[0009] In some embodiments, the follicle is from a mouse ovary. Specifically, the mouse ovary is a 2-week to 8-week-old mouse ovary. Specifically, the diameter of the follicle is 100 μm to 130 μm.

[0010] In some embodiments, the concentration of the sodium alginate solution is 0.1 wt% to 2 wt%. Specifically, the sodium alginate solution is filtered through a sterilizing filter and then used.

[0011] In some embodiments, agarose is made into agarose microcavities by a template method. Specifically, agarose is dissolved to form an agarose solution, the agarose solution is injected into a template containing a microcolumn array, the agarose is cooled to solidify, and the solidified agarose is demolded to form a sheet agarose microcavity array, and the sheet agarose microcavity array is divided to obtain an agarose microcavity. More specifically, the temperature for cooling to solidify the agarose is 0 ℃~40 ℃, and the time is 30 min~120 min. More specifically, in the microcolumn array, the microcolumn diameter is 100 μm~1000 μm, the microcolumn height is 100 μm~1000 μm, and the microcolumn spacing is 50 μm~500 μm. Since agarose forms an agarose microcavity through a microcolumn, the agarose microcavity is a microcolumn-shaped cavity with a diameter of 100 μm~1000 μm and a depth of 100 μm~1000 μm. More specifically, the material of the micro-pillars in the micro-pillar array is silica gel. More specifically, the preparation process is as follows:

[0012] Dissolve 0.1 g to 1 g of agarose powder in 1 mL to 100 mL of deionized water at 60 ℃ to 100 ℃. After it is completely dissolved, immediately pour 0.1 mL to 1 mL of agarose solution into a silica gel microcolumn array template with a microcolumn diameter of 100 μm to 1000 μm, a microcolumn height of 100 μm to 1000 μm, and a microcolumn spacing of 50 μm to 500 μm purchased from Taizhou Microchip Pharmaceutical Technology Co., Ltd. Cool the agarose solution and the microcolumn template at 0 ℃ to 40 ℃ for 30 min to 120 min, and demold after the agarose is fully solidified to obtain a sheet agarose microcavity array, which is fully soaked and cleaned with deionized water, and the cleaned sheet agarose microcavity is cut into 0.5×0.5 cm 2 ~2×2 cm 2 Small pieces for spare use.

[0013] In some embodiments, before use, the agarose microcavity is first soaked in 70-80% alcohol by volume, sterilized by ultraviolet irradiation, and then washed with phosphate buffered saline (PBS). Specifically, the soaking time is 12 h to 48 h. Specifically, the ultraviolet irradiation sterilization time is 30 min to 180 min.

[0014] In some embodiments, one agarose microchamber is seeded with one follicle.

[0015] The calcium salt solution of the present invention refers to an aqueous solution in which calcium salt is dissolved, wherein the calcium salt is a compound whose cation is calcium ion, and can be calcium chloride, calcium nitrate, etc., and calcium chloride is generally used.

[0016] In some embodiments, the concentration of the calcium salt solution is 10 mM to 100 mM.

[0017] In some embodiments, the time for cross-linking the sodium alginate is 1 min to 10 min.

[0018] In some embodiments, after cross-linking, phosphate buffered saline (PBS) is used for washing to remove calcium salts that are not involved in the reaction.

[0019] In some embodiments, the follicles are cultured at a temperature of 36-38°C and a volume fraction of 4-6% CO. 2 It is carried out in a humid environment saturated with water vapor.

[0020] In some embodiments, the follicle growth medium is a basic medium containing fetal bovine serum with a mass concentration of 9-11%, insulin-transferrin-selenium with a mass concentration of 0.9-1.1%, follicle stimulating hormone with a concentration of 9-11 mIU / mL, and double antibody with a mass concentration of 0.45-0.55%, and the basic medium is Leibovitz's L-15 medium, McCoy's 5A medium or MEM α medium.

[0021] Specifically, Leibovitz's L-15 medium can be obtained commercially, and its main components are 0.9 g / L D-galactose, sodium pyruvate, amino acids, vitamins, inorganic salts and phenol red, etc.; McCoy's 5A medium can be obtained commercially, and its main components are 3 g / L glucose, bacterial peptone, reduced glutathione, amino acids, vitamins, inorganic salts and phenol red, etc.; MEM α medium can be obtained commercially, and its main components are 1.0 g / L glucose, lipoic acid, sodium pyruvate, amino acids, vitamins, inorganic salts, ribonucleosides, deoxyribonucleosides and phenol red, etc. Fetal bovine serum is a nutrient used for animal cell culture, which is obtained by removing fibrin from the plasma of an eight-month-old fetus after blood is drawn by cardiac puncture; insulin-transferrin-selenium is a basal culture medium supplement (100×), follicle-stimulating hormone is a hormone secreted by the pituitary gland, and the double antibiotic is penicillin-streptomycin solution (100×), with a penicillin content of 10,000 U / ml and a streptomycin content of 10 mg / ml.

[0022] The beneficial effects of the present invention are:

[0023] (1) The present invention first discovered and disclosed a method for culturing secondary follicles to antral follicles using agarose-calcium alginate composite hydrogel microchambers and obtaining mature oocytes by inducing ovulation. The composite hydrogel microchambers provide a three-dimensional growth microenvironment for the growth and development of follicles that is similar to the ovarian structure and mechanical properties and is uniform and stable, integrating the structural plasticity and adjustable mechanical properties of the biomaterial itself, and promoting the application of hydrogel materials in three-dimensional in vitro culture of follicles. Experiments have confirmed that the agarose-calcium alginate composite hydrogel microchambers of the present invention can promote the growth of secondary follicles to the antral follicle stage, and after 8 days of culture, the secondary follicles have a three-dimensional growth microenvironment that is similar to the ovarian structure and mechanical properties and is uniform and stable. Compared with the follicles cultured in calcium alginate hydrogel microspheres, the ovulation rate of follicles cultured in agarose-calcium alginate composite hydrogel microcavities increased from 63.3% to 90.7%, the cumulus-oocyte complex formation rate increased from 63.9% to 91.2%, the oocyte maturation rate increased from 28.8% to 65.7%, and the spindle abnormality rate decreased from 35.2% to 9.9%. This proves that the sheet-like agarose-calcium alginate composite hydrogel microcavities have a good effect in promoting the growth, development, maturation and ovulation of secondary follicles in vitro.

[0024] (2) Compared with the commonly used calcium alginate hydrogel microsphere encapsulated follicle three-dimensional culture method, the method of secondary follicle three-dimensional culture disclosed in the present invention does not need to use excessive calcium chloride solution to encapsulate the follicles one by one in three-dimensional form. It uses agarose microcavity as the outer scaffold material and calcium alginate hydrogel as the inner filling material. Follicles can be inoculated in batches in the microcavity through a simple oral pipette transfer method, and a small amount of calcium chloride solution can be used to uniformly and quickly encapsulate the follicles, thereby promoting the growth, development, maturation and ovulation of the follicles in vitro.

[0025] (3) Agarose and calcium alginate hydrogels are recognized as excellent biomedical materials with low price and easy production. At the same time, since the method of three-dimensional culture of follicles is simple and fast, the method of the present invention can be used to culture secondary follicles in vitro in a batch and uniform manner, so as to promote the application of biocompatible and biodegradable hydrogel materials in follicle culture in vitro and fertility preservation.

[0026] In summary, the application of the agarose-calcium alginate composite hydrogel microchamber disclosed in the present invention in the three-dimensional culture of secondary follicles provides a relatively ideal method for fertility preservation technology based on in vitro culture of follicles, and has important application prospects; at the same time, agarose and calcium alginate hydrogels used as components of the three-dimensional culture system of follicles are widely available, low in price, easy to produce, suitable for mass production and application, and have broad development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0028] Figure 1 This is a flow chart of the preparation of agarose-calcium alginate composite hydrogel microcavity and follicle culture in an embodiment of the present invention;

[0029] Figure 2 Graph showing elastic modulus test results of agarose hydrogel and calcium alginate hydrogel in the embodiments of the present invention;

[0030] Figure 3 This is a scanning electron microscope (SEM) image of agarose-calcium alginate composite hydrogel microcavity after freeze drying in an embodiment of the present invention;

[0031] Figure 4 This is a statistical result diagram of the growth diameter of follicles cultured in a sheet-like agarose-calcium alginate composite hydrogel microcavity from 0 to 8 days in an embodiment of the present invention;

[0032] Figure 5 This is a bright field image of the follicles cultured in the sheet-shaped agarose-calcium alginate composite hydrogel microcavity after 0 to 8 days in the embodiment of the present invention;

[0033] Figure 6 It is a statistical result diagram of the ovulation rate after the follicles are cultured in the sheet-like agarose-calcium alginate composite hydrogel microcavity and the calcium alginate hydrogel microsphere in the embodiment of the present invention;

[0034] Figure 7 The statistical results of the formation rate of cumulus-oocyte complex obtained by ovulation after ovarian follicles were cultured in sheet-shaped agarose-calcium alginate composite hydrogel microcavity and calcium alginate hydrogel microspheres in the embodiment of the present invention;

[0035] Figure 8 This is a bright field image of the cumulus-oocyte complex obtained after ovulation of the follicle cultured in the sheet-shaped agarose-calcium alginate composite hydrogel microcavity in the embodiment of the present invention;

[0036] Fig. 9 The statistical results of the proportion of oocytes at different stages obtained after ovulation of follicles cultured in sheet-shaped agarose-calcium alginate composite hydrogel microcavities and calcium alginate hydrogel microspheres in the embodiments of the present invention;

[0037] Fig.10 This is a bright field image of an oocyte obtained after ovulation of an ovarian follicle cultured in a sheet-like agarose-calcium alginate composite hydrogel microcavity in an embodiment of the present invention;

[0038] Fig.11The statistical results of the abnormality rate of spindles of oocytes obtained after ovulation of follicles cultured in sheet-shaped agarose-calcium alginate composite hydrogel microcavities and calcium alginate hydrogel microspheres in the embodiments of the present invention;

[0039] Fig.12 This is a result of immunofluorescence staining of the spindle of a mature oocyte obtained by ovulation after culturing an ovarian follicle in a sheet-like agarose-calcium alginate composite hydrogel microcavity in an embodiment of the present invention;

[0040] Fig.13 This is a statistical result diagram of the proportion of embryos at various stages obtained after in vitro fertilization of oocytes obtained after ovulation of ovarian follicles cultured in agarose-calcium alginate composite hydrogel microcavities in an embodiment of the present invention;

[0041] Fig.14 This is a bright field image of a blastocyst obtained after in vitro fertilization of an oocyte obtained after ovulation of an ovarian follicle cultured in a sheet-shaped agarose-calcium alginate composite hydrogel microcavity in an embodiment of the present invention. DETAILED DESCRIPTION

[0042] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0043] The follicle growth medium used in the following examples is: MEM α medium + 10% fetal bovine serum + 1% insulin-transferrin-selenium + 10 mIU / mL follicle stimulating hormone + 0.5% double antibody.

[0044] Example 1

[0045] A method for three-dimensional culture of secondary follicles using agarose-calcium alginate composite hydrogel microcavity, such as Figure 1 As shown, the steps are as follows:

[0046] (1) 0.1 g of agarose powder was dissolved in 1 mL of 60 °C deionized water. After it was completely dissolved, 0.1 mL of agarose solution was immediately poured into a silica gel microcolumn array template with a microcolumn diameter of 100 μm, a microcolumn height of 100 μm, and a microcolumn spacing of 50 μm purchased from Taizhou Microchip Pharmaceutical Technology Co., Ltd. The agarose solution and the microcolumn template were cooled at 0 °C for 30 min. After the agarose was fully solidified, it was demolded to obtain a sheet agarose microcavity array. It was fully soaked and cleaned with deionized water. The cleaned sheet agarose microcavity was divided into 0.5×0.5 cm 2 Prepare a 0.1 wt% sodium alginate solution and filter it with a 0.22 μm sterilizing filter for later use.

[0047] In order to detect the elastic modulus of agarose hydrogel, the agarose hydrogel was tested with an electronic universal testing machine; in order to detect the elastic modulus of calcium alginate hydrogel, the sodium alginate aqueous solution was cross-linked with 10 mM calcium chloride solution for 1 min, and the obtained calcium alginate hydrogel was tested with an electronic universal testing machine. The elastic modulus obtained is as follows Figure 2 As shown, it shows that the mechanical properties of agarose hydrogel and calcium alginate hydrogel can simulate the cortex with higher hardness and the medulla with lower hardness in the ovary, respectively.

[0048] In order to detect the morphology of agarose-calcium alginate composite hydrogel microcavities, 0.1 wt% sodium alginate solution was added to the agarose microcavities and cross-linked with 10 mM calcium chloride solution for 1 min. The obtained agarose-calcium alginate composite hydrogel microcavities were freeze-dried and observed using a scanning electron microscope (SEM). The morphology of the composite microcavities is shown in Figure 2. Figure 3 As shown, it is explained that the composite hydrogel microcavity is a microcavity array structure with agarose microcavity as the outer layer scaffold and calcium alginate hydrogel as the inner layer filling.

[0049] (2) Place the agarose microcavity sheet in a well plate, add 75% alcohol and soak it in it for 12 h. At the same time, irradiate it with ultraviolet light for 30 min in a sterile operating table for sterilization. Wash off the alcohol on the agarose microcavity sheet with PBS and set aside.

[0050] (3) Five follicles with a diameter of 100 μm to 130 μm isolated from the ovaries of 2-week-old mice were dispersed in a 0.1 wt% sodium alginate solution and placed in a sheet agarose microcavity array in a well plate with a single follicle per microcavity. The sodium alginate was then cross-linked with a 10 mM calcium chloride solution for 1 min and washed with PBS to obtain an agarose-calcium alginate composite hydrogel microcavity loaded with follicles. 0.5 mL of follicle growth medium was added to the well plate and the follicles were cultured in the sheet agarose-calcium alginate composite hydrogel microcavity. The follicle growth was observed and recorded under a microscope every 2 days, and the follicle growth medium was replaced once. The growth diameters of the follicles from day 0 to day 8 of culture were counted, and the results were as follows: Figure 4 As shown, the results showed that the agarose-calcium alginate composite hydrogel microchamber could effectively support follicle growth.

[0051] Example 2

[0052] A method for three-dimensional culture of secondary follicles using agarose-calcium alginate composite hydrogel microcavity, the steps are as follows:

[0053] (1) Dissolve 0.55 g of agarose powder in 50.5 mL of 80°C deionized water. After it is completely dissolved, immediately pour 0.55 mL of agarose solution into a silica gel microcolumn array template with a microcolumn diameter of 550 μm, a microcolumn height of 550 μm, and a microcolumn spacing of 275 μm purchased from Taizhou Microchip Pharmaceutical Technology Co., Ltd. Cool the agarose solution and the microcolumn template at 20°C for 75 min. After the agarose is fully solidified, demold the template to obtain a sheet agarose microcavity array. Soak and clean it with deionized water. Divide the cleaned sheet agarose microcavity into 1.25×1.25 cm 2 Prepare a 1.05 wt% sodium alginate solution and filter it with a 0.22 μm sterilizing filter for later use.

[0054] (2) Place the agarose microcavity sheet in a well plate, add 75% alcohol and soak it in it for 30 h. At the same time, irradiate it with ultraviolet light for 105 min in a sterile operating table for sterilization. Wash off the alcohol on the agarose microcavity sheet with PBS and set aside.

[0055] (3) Fifteen follicles with a diameter of 100 μm to 130 μm isolated from the ovaries of 5-week-old mice were dispersed in a 1.05wt% sodium alginate solution and placed in a sheet-shaped agarose microcavity array in a well plate with a single follicle per microcavity. The sodium alginate was then cross-linked with a 55mM calcium chloride solution for 5.5 min and washed with PBS to obtain agarose-calcium alginate composite hydrogel microcavities loaded with follicles. 1.5 mL of follicle growth medium was added to the well plate and the follicles were cultured in the sheet-shaped agarose-calcium alginate composite hydrogel microcavities. The follicle growth was observed and recorded under a microscope every 2 days, and the follicle growth medium was replaced once.

[0056] Secondary follicles cultured in calcium alginate hydrogel microspheres were used as controls, and the steps were as follows: 15 follicles isolated from the ovaries of 5-week-old mice were dispersed in 100 μL of 0.5 wt% sodium alginate solution, and 2.5 μL of sodium alginate solution containing a single follicle was dropped into 55 mM calcium chloride solution by the drop method for cross-linking for 5.5 min. The follicles encapsulated in calcium alginate hydrogel microspheres were transferred to a well plate containing 1.5 mL of follicle growth medium for culture, and the follicle growth was observed and recorded under a microscope every 2 days, and the follicle growth medium was replaced once.

[0057] After culturing for 0 to 8 days, the morphology of the follicles in the microcavities of the agarose-calcium alginate composite hydrogel sheets is as follows: Figure 5As shown. The follicles cultured in the sheet-like agarose-calcium alginate composite hydrogel microcavity and calcium alginate hydrogel microspheres were induced to ovulate, and the oocytes were collected after 16 hours to detect the ovulation rate and oocyte quality of the follicles in the agarose-calcium alginate composite hydrogel microcavity and calcium alginate hydrogel microspheres. The ovulation rate results are shown in Figure 6 As shown in Figure 2, the formation rate of the cumulus-oocyte complex is Figure 7 As shown, the bright field image of the cumulus-oocyte complex is shown Figure 8 As shown, the ratio of oocytes at each stage is Fig. 9 As shown, the bright field image of the oocyte obtained is as follows Fig.10 As shown, the spindle abnormality rate is Fig.11 The results of spindle immunofluorescence staining are shown in Figure 2. Fig.12 As shown. The results showed that compared with follicles cultured in calcium alginate hydrogel microspheres, the ovulation rate of follicles cultured in sheet agarose-calcium alginate composite hydrogel microcavities increased from 63.3% to 90.7%, the cumulus-oocyte complex formation rate increased from 63.9% to 91.2%, the oocyte maturation rate increased from 28.8% to 65.7%, and the spindle abnormality rate decreased from 35.2% to 9.9%. It is proved that sheet agarose-calcium alginate composite hydrogel microcavities can promote follicle maturation and ovulation and improve the quality of obtained oocytes compared with traditional calcium alginate hydrogel microspheres.

[0058] Example 3

[0059] A method for three-dimensional culture of secondary follicles using agarose-calcium alginate composite hydrogel microcavity, the steps are as follows:

[0060] (1) Dissolve 1 g of agarose powder in 100 mL of 100°C deionized water. After it is completely dissolved, immediately pour 1 mL of agarose solution into a silica gel microcolumn array template with a microcolumn diameter of 1000 μm, a microcolumn height of 1000 μm, and a microcolumn spacing of 500 μm purchased from Taizhou Microchip Pharmaceutical Technology Co., Ltd. Cool the agarose solution and the microcolumn template at 40°C for 120 min. After the agarose is fully solidified, demold the template to obtain a sheet agarose microcavity array. Soak and clean it with deionized water. Divide the cleaned sheet agarose microcavity into 2×2 cm 2 Prepare a 2 wt% sodium alginate solution and filter it with a 0.22 μm sterilizing filter for later use.

[0061] (2) Place the agarose microcavity sheet in a well plate, add 75% alcohol and soak it in it for 48 h. At the same time, irradiate it with ultraviolet light for 180 min in a sterile operating table for sterilization. Wash off the alcohol on the agarose microcavity sheet with PBS and set aside.

[0062] (3) 25 follicles with a diameter of 100 μm to 130 μm isolated from the ovaries of 8-week-old mice were dispersed in 2 wt% sodium alginate solution and placed in a sheet agarose microcavity array in a well plate with a single follicle per microcavity. The sodium alginate was then cross-linked with 100 mM calcium chloride solution for 10 min and washed with PBS to obtain agarose-calcium alginate composite hydrogel microcavities loaded with follicles. 2.5 mL of follicle growth medium was added to the well plate and the follicles were cultured in the sheet agarose-calcium alginate composite hydrogel microcavities. The follicle growth was observed and recorded under a microscope every 2 days, and the follicle growth medium was replaced once. At the same time, the same number of follicles were cultured in calcium alginate hydrogel microspheres as a control group. After 14 days of culture, the follicles cultured in the sheet agarose-calcium alginate composite hydrogel microcavity and the calcium alginate hydrogel microspheres were induced to ovulate, and the oocytes were collected after 16 hours to compare the ovulation rate and oocyte quality of the follicles in the agarose-calcium alginate composite hydrogel microcavity and the calcium alginate hydrogel microspheres. The mature oocytes obtained after the follicles were cultured in the agarose-calcium alginate composite hydrogel microcavity were subjected to in vitro fertilization, and the statistical results of the proportion of embryos at each stage were shown in Figure 2. Fig.13 The morphology of the blastocysts obtained is shown in Fig.14 The results showed that 41.7% of the mature oocytes obtained after ovulation induction in the follicles cultured in the sheet-like agarose-calcium alginate composite hydrogel microcavity were able to develop to the blastocyst stage after in vitro fertilization, proving that the obtained oocytes had good embryonic development potential after in vitro fertilization.

[0063] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for three-dimensional culture of secondary follicles using agarose-calcium alginate composite hydrogel microcavity, characterized in that: The follicles are dispersed in a sodium alginate solution to obtain a follicle solution, and the follicles are inoculated into an agarose microcavity through the follicle solution; then a calcium salt solution is added to the agarose microcavity to cross-link the sodium alginate to obtain an agarose-calcium alginate composite hydrogel microcavity loaded with follicles, and then a follicle growth medium is added to the agarose-calcium alginate composite hydrogel microcavity loaded with follicles to culture the follicles; The follicles are from mouse ovaries; The concentration of sodium alginate solution is 0.1 wt%~2 wt%; Agarose was made into agarose microcavities using the template method; Before use, the agarose microcavity was first soaked in 70-80% alcohol by volume, sterilized by ultraviolet irradiation, and then cleaned with phosphate buffer. One follicle was seeded in one agarose microchamber; The concentration of calcium salt solution is 10 mM~100 mM; The time for cross-linking sodium alginate is 1 min to 10 min; After cross-linking, the samples were washed with phosphate buffered saline; Follicle culture is carried out in a humid environment saturated with water vapor and containing 4-6% CO2 at a temperature of 36-38°C.

2. The method according to claim 1, characterized in that: The follicle growth medium is a basic medium containing fetal bovine serum with a mass concentration of 9-11%, insulin-transferrin-selenium with a mass concentration of 0.9-1.1%, follicle stimulating hormone with a concentration of 9-11 mIU / mL, and double antibody with a mass concentration of 0.45-0.55%, and the basic medium is Leibovitz's L-15 medium, McCoy's 5A medium or MEM α medium.

Images