WO2018103654A1

WO2018103654A1 - Artificial ovary and preparation and application of same

Info

Publication number: WO2018103654A1
Application number: PCT/CN2017/114700
Authority: WO
Inventors: 张键; 阮长顺; 任培根; 杨雅莉; 孙立峰; 翟欣昀; 吴明明
Original assignee: 深圳先进技术研究院
Priority date: 2016-12-09
Filing date: 2017-12-06
Publication date: 2018-06-14
Also published as: CN108611314A

Abstract

An artificial ovary and a preparation and application of the same. The artificial ovary comprises a biocompatible frame, follicular cells, and a coating mixed liquid, wherein the coating mixed liquid covers the outside of the follicular cells, enters inside voids of the biocompatible frame, and is made from a coated excipient and a biological hormone, and the biocompatible frame is fabricated by 3D printing. The artificial ovary is suitable for composing a research tool for cellular studies of the whole or part of primordial follicles, preantral follicles, and luminal follicles of any species, and is also suitable for a screening model for a factor, a prodrug, and the like related to primordial follicles, preantral follicles, and luminal follicles, and for a prevention or treatment tool for a disease directly or indirectly related to ovarian damage caused by any factor.

Description

Artificial ovary and preparation and application thereof

Technical field

The invention relates to the field of biotechnology, in particular to an artificial ovary prepared by a 3D printing technique.

Background technique

The World Health Organization predicts that infertility will be the third largest disease after cancer and cardiovascular disease. There are 1 infertility in every 6 couples in the world, and China’s infertility rate has climbed from 3% 20 years ago to 12.5-15%.

The National Bureau of Statistics released the main data bulletin of the sixth national census on the same day, showing that the total population of the country is 1,339,724,852. Compared with the fifth national census in 2000, the number increased by 73.9 million in the decade, an increase of 5.84%, with an average annual growth rate of 0.57%, which is 0.5 percentage points lower than the annual average growth rate of 1.07% from 1990 to 2000. The data shows that China's population growth is at a stage of low fertility levels.

In October 2015, the communique of the Fifth Plenary Session of the 18th Central Committee of the Communist Party of China pointed out: Promote balanced population development, adhere to the basic national policy of family planning, improve the population development strategy, and fully implement the policy of a couple of children to have two children. Actively respond to the aging of the population.

However, in recent years, the fast-paced work and lifestyle, the pressure of high consumption, all face the challenges of women of marriage and childbearing age. Therefore, women's health, especially women's reproductive health, is a social problem that cannot be ignored.

(1) Premature ovarian failure (POF) refers to the low estrogen and high gonadotropin state that occurs after menarche until the age of 40, accompanied by amenorrhea, infertility, sexual atrophy and menopausal syndrome. In recent years, the incidence of clinical POF has been increasing year by year. Epidemiological surveys show that the incidence of POF is 1%, and the prevalence of women before 30 years old is about 0.1%. The loss of fertility and low estrogen due to POF. Status has become a factor that can not be ignored influencing female reproductive health and social stability. There are many causes of premature ovarian failure, mainly due to premature depletion of primordial follicles in the ovary or inability of primord follicles to develop. At present, assisted reproductive technologies such as IVF technology have become the key technology for the treatment of infertility. In particular, IVF technology has developed rapidly over the past 20 years since its establishment, enabling thousands of families to successfully own their own offspring. However, this technology is not a panacea and does not solve all the problems of infertility. The key to IVF technology is that mothers must be provided with a high-quality mature egg. If the mother has no egg ovulation or low ovulation function, such as for patients with premature ovarian failure, then this technique can't help.

(2) Polycystic ovary syndrome (PCOS)

Polycystic ovary syndrome is a common endocrine disorder disease in women of childbearing age. The incidence rate is 6-10% of women of childbearing age. It is mainly characterized by menstrual thinning or amenorrhea, infertility, ovarian polycystic changes, obesity, hairy, Hyperandrogenemia and the like. Treatment for patients with PCOS, including surgery, medication, and assisted reproductive technology.

At present, drug treatment of PCOS has replaced surgical treatment as a first-line treatment, and the purpose of treatment is mainly related to the patient's fertility requirements. Such as the use of drugs to reduce hyperandrogenemia, ovulation drug therapy, or insulin-sensitizing drugs (ISD) treatment.

Surgical treatment of PCOS can reduce some granulosa cells in the ovary, reduce the production of androgen in the ovarian stroma, and thus reduce the level of androgen in the circulation, and then reduce the GnRH, causing the serum androgen concentration to further decrease, which also indicates that the ovarian stroma is also affected. Pituitary-ovarian axis regulation. Mainly include bilateral ovarian wedge resection (BOWR), laparoscopic ovarian electrocautery or laser perforation (LOD) and transvaginal hydrolaparoscopy (THL).

There are also PCOS patients who use assisted reproductive technology, especially for PCOS patients who have ovulation but have not been pregnant after 6 months of ovulation induction therapy, or multiple drug ovulation therapy and adjuvant therapy for anovulation and acute pregnancy. Patients can choose the assisted reproductive technology of embryo transfer. In vitro fertilization (IVF) and in vitro maturation (IVM) are included.

However, the above treatment methods have advantages and disadvantages, the key is that the pathogenesis of PCOS is still unclear so far, so the current treatment method is only a symptom.

(three) ovarian cancer (Ovarian Cancer, OAC)

Among female reproductive cancers, some are associated with endocrine. For example, breast cancer, endometrial cancer and Ovarian Cancer (OAC). Among them, ovarian cancer is a malignant tumor that occurs in the ovary, 90% to 95% of which are primary ovarian cancers, and 5% to 10% of the primary cancers in other parts are transferred to the ovaries. Although the incidence of ovarian cancer in China is not as high as in Europe and the United States, eradication surgery and cytotoxic chemotherapy lack the effectiveness of effectively reducing ovarian cancer mortality. Because of the early absence of symptoms in ovarian cancer, even if symptoms are not specific, the role of screening is limited, so early diagnosis is difficult, 60% to 70% of patients have advanced in the treatment, and advanced cases are not effective. Therefore, although the incidence of ovarian cancer is second only to cervical cancer and endometrial cancer, the third place in gynecological malignancies, but the mortality rate is more than the sum of cervical cancer and endometrial cancer, the highest in gynecological cancer, is serious The biggest threat to women's health.

The above three diseases that endanger women cause the occurrence, development and ovulation of normal eggs in women, leading to infertility. In addition, many women also suffer from multiple factors such as endocrine disorders, irregular menstruation and other symptoms, seriously affecting the physical and psychological state. Therefore, the development of 3D printed ovaries with secretory hormones, developmental follicles or ovulation function, implanted into females with ovarian insufficiency, relieve endocrine disorders, irregular menstruation, and even help females to ovulate and give birth to healthy offspring. It is of great social importance to provide new possibilities for the treatment of infertility in humans.

Several types of 3D printed ovarian bioprostheses invented in this application, whether in terms of ovarian structural morphology, stent channel design, cell type and culture protocol, are closer to physiological conditions of the ovary, and can be used as a screening for primordial follicles. A powerful research tool for recruitment, follicular cycle recruitment, follicular development, and ovulation mechanisms can also provide a more reliable treatment for endocrine dysfunction, irregular menstruation, or infertility The choice of prevention or treatment options.

Fully combining biology, cytology and materials science, using 3D printer technology to develop functional 3D printed ovarian bioprostheses with follicles of different developmental stages, or for in vitro and in vivo mechanisms; or implanting them into ovarian insufficiency Females, restore female physiological characteristics, and can help females to produce healthy offspring, provide new tools for revealing the mechanism of infertility, and also apply to menstrual irregularities, infertility, for the relief, prevention or treatment of humans. Wait for new options.

Summary of the invention

In order to solve the above problems, an aspect of the invention provides an artificial ovary comprising a biocompatible stent, a follicular cell, and a coating mixture, wherein the coating mixture is wrapped outside the follicular cell and is embedded in the biological phase The interior of the void of the capacitive support.

In a specific technical solution, the coating liquid is a fluid liquid made of a coating auxiliary;

The coating material is selected from the group consisting of agar, agarose, calcium alginate, hyaluronic acid, matrigel, polyglycol polymer, polyvinyl alcohol polymer, collagen gel, collagen, and extracellular matrix protein. One or more of a hydrogel formed by cross-linking of alginate or calcium;

In a specific technical solution of the present invention, the coating mixture is set to a concentration of 1.5% by sodium alginate and gelatin, respectively, and then tried to mix in different ratios, and the ratio is selected to be 1:1, 4:6, 3:7, 2:8, 1:9. It is possible to prepare a coating mixture having a suitable concentration by conducting experiments in each ratio.

Preferably, the coating mixture further comprises a biological hormone selected from the group consisting of hormones or growth factors required for promoting follicular development. Preferably, the biological hormone is selected from the group consisting of follicle stimulating hormone (FSH), luteinizing hormone (LH), epidermis. Cell growth factor (EGF).

In the technical solution of the present invention, the material of the biocompatible scaffold is selected from the group consisting of collagen I, sodium alginate, gelatin, agarose, Matrigel, hyaluronic acid, chitosan, and dextran. Or any combination of several.

In the technical solution of the present invention, the biocompatible stent has a void having a diameter which is 1 to 2 times, preferably 1-1.5 times, the diameter of the follicular cell.

In the technical solution of the present invention, the follicular cells are selected from the group consisting of primordial follicles, preantral follicles, luminal follicles, tissues containing primordial follicular cells, tissues including preantral follicular cells, and tissues containing luminal follicular cells. Kind or more.

In the solution of the present invention, the artificial ovary has more than one void size for different follicular cells, preferably 1-4 kinds of void size, more preferably 2-4 kinds of void size.

In the technical solution of the present invention, the biocompatible stent is made by 3D printing, and the coating mixture of the follicular cells is poured into the interior of the biocompatible stent; or

The biocompatible scaffold material and the coating mixture of the encapsulated follicular cells are sequentially produced by 3D printing in a layer-by-layer manner.

The material used in the present invention is a biocompatible material whose composition mimics the ovarian tissue contained therein. A component which may be selected from one or a combination of the following materials.

Collagen I is a hydrogel matrix extracted from animals. It has good biocompatibility, rich source, high plasticity, convenient clinical application and no immunogenicity. The gel network formed by it facilitates free entry and exit of nutrients, and has good hydrophilicity and cell compatibility.

Sodium alginate is the most widely used three-dimensional in vitro culture system for follicles. The scaffolding effect is good and can easily simulate natural tissues.

Agarose gel has been successfully applied to preantral follicular culture of humans and hamsters. At the same time, due to the low melting temperature of the low melting point agarose, it is more suitable for the embedding of follicles.

Matrigel is a hydrogel matrix with a pore size of about 20-50 nm. Its main components are laminin III, type IV collagen, heparan sulfate proteoglycan and nestin, similar to the embryonic basement membrane. A microenvironment that simulates cell growth in vivo.

Hyaluronic acid is a high molecular polysaccharide, which is the main component of the extracellular matrix, has vegetative cells, and promotes the physiological role of cell differentiation. However, the mechanical properties are poor, and the mechanical properties can be improved and the application range can be extended by the method of modification modification or the method of compounding other materials.

Another aspect of the invention provides a method of making an artificial ovary comprising the steps of:

1) preparing a biocompatible scaffold material;

2) preparing a coating mixture and coating the follicular cells;

3) obtaining a biocompatible scaffold by 3D printing with a biocompatible scaffold material, and infusing the follicular-containing coating mixture into the internal space of the biocompatible scaffold, or

3) The biocompatible scaffold material and the coating mixture of the encapsulated follicular cells are sequentially produced by 3D printing in a layer-by-layer manner.

In the technical solution of the present invention, before the coating of the follicular cells with the coating mixture in the step 2), the step of culturing the follicular cells is further included.

The artificial ovary of the present invention is used as a drug screening and follicle research model.

The artificial ovary of the present invention is a preventive or therapeutic tool for a directly related disease or an indirectly related disease caused by ovarian damage caused by any factor.

In a preferred embodiment, the 3D printing size of the biocompatible stent of the present invention is: pore size (R): 50 μm to 800 μm; line stacking angle: 0° to 180°.

In a preferred embodiment of the present invention, sodium alginate is used as a scaffold material, and various shapes of scaffolds (for example, circular, rectangular, square) are prepared by 3D printing, and the gap length in the 3D printing scaffold is 150 um. Angle: 90°, containing preantral follicles coated with (1.5% (w/v)) sodium alginate gelatin coating. The preantral follicles were cultured in culture medium (a-MEM + 10% FBS + 5.5 mg/ml PNa + ITS + 5 ng / ml EGF + 400 mIU PMSG) before the follicles were coated.

The stent portion of the artificial ovary of the present invention may be provided in a plurality of sizes to accommodate follicles of different sizes and at different stages, thereby enabling the follicles to sequentially grow into dominant follicles. The artificial ovary of the present invention is suitable for any research on primordial follicles, preantral follicles, whole or partial constitutive cells of any species The tool is also suitable for screening models of primordial follicles, preantral follicles, luminal follicle related factors, prodrugs, etc. It is also suitable for the prevention or treatment of directly related diseases or indirect related diseases caused by ovarian damage caused by any factor. tool.

Beneficial effect

The 3D printed ovary of the present invention can be cited as ovarian damage caused by any factor, and as long as there are still some ovarian cortex containing primordial follicles, preantral follicles, and luminal follicles, the preparation method of the artificial ovary of the present invention can be used for preparation. Ovarian bioprosthesis, which stimulates the development of primordial follicles, preantral follicles and luminal follicles in the prosthesis, so that it can enter the mature follicles, and will continue to exercise the function of ovarian secretion of hormones and excretion of eggs, maintaining female physiology. Features to relieve symptoms such as endocrine disorders and irregular menstruation.

DRAWINGS

Figure 1 is a schematic diagram of the preparation process of the 3D printed ovarian bioscaffold, wherein A is a computer aided design of the ovarian structure and a printed model. B is a printed substrate. C is a collagen/matrix gum mixture. D is gelatin.

2 is a schematic flow chart of a 3D printed ovarian biological stent,

Where a) is CAD design, b) is 3D printing, and c) is a schematic representation of a three-dimensional ovarian stent.

Figure 3 is a schematic view showing the preparation process of the follicle/matrix mixture.

1 is primodial follicle, 2 is preantral follicle, 3 is antral follicle, 4 is matrigel, 5 is primordial follicle/matrix mixture, and 6 is preantral follicle/ Matrigel mixture, 7 is a cavity follicle / matrigel mixture.

Figure 4 is a schematic view showing the structure of several different types of ovarian bioprostheses (type I, type II, type III, type IV, type V, and type VI).

Among them, 1 is the original follicle/matrix mixture, 2 is the pre-cavity follicle/matrix mixture, 3 is the cavity follicle/matrix mixture, 4 is the biological stent, and 5 is the type I or IV ovarian bioprosthesis. , 6 is type II or V ovarian bioprosthesis, 7 is type III or type VI ovarian bioprosthesis

Figure 5 is an electron micrograph of the present invention.

Figure 6 is a graph showing the results of levels of progesteron secreted by follicles in different proportions.

detailed description

Example 1 Design and preparation of 3D printed ovarian bioscaffold

(1) Combining computer aided design (CAD), using three-dimensional printing technology to construct ovarian scaffolds, in which the shape, composition and internal structure have good designability;

(2) The shape of the three-dimensional ovarian stent can be designed as a circular, rectangular, square or ovarian-like shape, but the internal structure is ensured to communicate with a certain porosity;

(3) The material used for the three-dimensional ovarian stent, the biocompatible material used in the invention can be selected not only for follicular growth but also for supporting, and also convenient for three-dimensional printing. Alternative materials: collagen I, sodium alginate, gelatin, agarose, Matrigel, hyaluronic acid, chitosan, One or a combination of any of the dextran is printed separately.

(4) Internal three-dimensional ovarian structural parameters (see figure): pore size (R): 150 μm (depending on the size of follicles at different developmental stages of the implant); line stacking angle (α): 90° to 180°.

(5) The prepared stent was stored at -80 ° C for use.

Example 2 Acquisition of follicles

(1) Obtainment of preantral follicles

Naturally obtained method: Female mice 12-14 days after birth were sacrificed by cervical dislocation. After disinfecting the skin, the ovaries were aseptically removed from the back and placed in the separation medium (L-15+10% FBS+100 IU). /ml penicillin + 100ug / ml streptomycin). The attached tissue around the ovary was removed under a stereoscopic microscope and washed 3 times in the separation solution. Then, the ovary was injected under the microscope with insulin, and the preantral follicles with a diameter of 100-130 μm in the ovary of the mouse were released.

DES treatment: First, 19 days old mice were implanted with DES subcutaneously. After 3 days, they were sacrificed by cervical dislocation. After disinfecting the skin, the ovaries were aseptically removed from the back and placed in the separation medium (L-15+). 10% FBS + 100 IU / ml penicillin + 100 ug / ml streptomycin). The attached tissue around the ovary was removed under a stereoscopic microscope and washed 3 times in the separation solution. Then, the ovary was injected under the microscope with insulin, and the preantral follicles with a diameter of 100-130 μm in the ovary of the mouse were released.

(2) Obtaining follicles

21 days old female mice were injected intraperitoneally with PMSG. After 48 hours, they were sacrificed by cervical dislocation. After disinfecting the skin, the ovaries were aseptically removed from the back and placed in the separation medium (L-15+10% FBS+100 IU). /ml penicillin + 100ug / ml streptomycin). The attached tissue around the ovary was removed under a stereoscopic microscope and washed 3 times in the separation solution. Then, under the microscope, the ovary was injected by insulin, and the ovarian follicles having a diameter of 200-3000 μm were released.

(3) Acquisition of primordial follicles

Female mice of 3 days old were sacrificed by cervical dislocation. After disinfecting the skin, the ovaries were aseptically removed from the back and placed in the separation medium (L-15+10% FBS+100 IU/ml penicillin+100 ug/ml chain). Mycin). The attached tissue around the ovary was removed under a stereoscopic microscope and washed 3 times in the separation solution. The ovary is then dosed with insulin under the microscope, and the mouse ovary is divided into tissue fragments containing primordial follicles.

Example 3 Preparation of coating mixture

The coating mixture is agar/agarose, calcium alginate and hyaluronic acid, matrigel, synthetic polyethanol or polyvinyl alcohol polymer, collagen gel, collagen and extracellular matrix protein mixed gel, Or a hydrogel formed by the cross-linking of alginate with calcium for dilution. The concentration when sodium alginate and gelatin were compounded was 1.5% (w/v). The sodium alginate salt and gelatin were respectively arranged at a concentration of 1.5%, and then tried to mix in different ratios, and the ratio was selected to be 1:1, 4:6, 3:7, 2:8, 1:9. It is possible to prepare a coating mixture having a suitable concentration by conducting experiments in each ratio. See Figure 6.

Example 4 Coating the mixture to coat the follicle

(1) Preparation of preantral follicle/coating mixture.

The preantral follicles were washed 1-2 times in the separation solution and seeded into the mixed culture medium in a 96-well plate. (The composition of the mixed culture solution was: a-MEM + 10% FBS + 5.5 mg / ml PNa + ITS + 5 ng / ml EGF + 100 mIUrFSH). The mixed culture was first equilibrated in a 37 ° C, 5% CO ₂ incubator for 2-4 hours. The individual pre-cavity follicles are then wrapped with the sterilized coating mixture. )

(2) The cavity follicles are mixed with the matrigel: a cavity follicle/matrix mixture.

The luminal follicles were washed 1-2 times in the separation solution, and then seeded into the mixed culture solution in a 96-well plate.

(Note: Mixed culture solution: a-MEM + 10% FBS + 5.5 mg / ml PNa + ITS + 5 ng / ml EGF + 100 mIUrFSH. The mixed culture solution was first equilibrated in a 37 ° C, 5% CO ₂ incubator for 2-4 hours. The individual cavity follicles are then wrapped with a sterilized coating mixture.)

(3) The original follicle is mixed with Matrigel to form: primordial follicle/matrix mixture.

The ovarian cortical tissue fragments containing the primordial follicles were washed 1-2 times in the separation solution, and then seeded into the mixed culture solution in a 96-well plate.

(Note: Mixed culture solution: a-MEM + 10% FBS + 5.5 mg / ml PNa + ITS + 5 ng / ml EGF + 100 mIUrFSH. The mixed culture solution was first equilibrated in a 37 ° C, 5% CO ₂ incubator for 2-4 hours. Then, the sterilized coating mixture is used to wrap the tissue fragments containing the original follicles.)

Example 5 Preparation and Functional Determination of Type I 3D Printed Ovarian Bioprosthesis

(1) Storage of spare ovarian bioscaffold pretreatment: The ovarian bioscaffold stored at -80 ° C was taken out, placed at room temperature for 20 minutes, and then incubated in physiological saline for 30 minutes.

(2) Injecting the original follicle/matrix mixture into the ovarian bioscaffold: adding the sterilized CaCl ₂ (50 mM)-NaCl (150 mM) mixture to the ovarian bioscaffold, and then using the loading gun to contain the primordial follicle The coating mixture was suspended into different parts of the stent, and an ovarian bioscaffold with primordial follicles was formed 2-5 minutes to form a type I 3D printed ovarian bioprosthesis.

(3) Type I 3D printing ovarian bioprosthesis and function measurement:

Type I ovarian bioprosthesis (containing 20 primordial follicular matrigel solutions) of length (5 mm) x width (2.5 mm) x thickness (2.5 mm) using 100 μM bpV (a PTEN inhibitor, Calbiochem) and/or 500 μg /mL 740Y-P (a PI3K agonist, Tocris) was treated for 24 hours; the control group was incubated in the culture medium without any inhibitor for 24 hours. After 24 hours, the ovarian bioprosthesis of the experimental group and the control group were subjected to tissue section Foxo3 staining, and the positive signal was stained with activated primordial follicles. The results showed that more than 50% of the primordial follicles in the bpV and/or 740Y-P treated ovarian prosthesis showed a Foxo3 positive signal. The above experimental results indicate that the primordial follicle is in a normal state in the ovarian bioprosthesis.

Example 6 Preparation and Functional Determination of Type II 3D Printed Ovarian Bioprosthesis

(1) Preservation of spare ovarian bioscaffold pretreatment: storage of spare ovarian bioscaffolds at -80 °C The mixture was taken out, left at room temperature for 20 minutes, and then incubated in physiological saline for 30 minutes.

(2) Inject the pre-cavity follicle/matrix mixture into the ovarian bioscaffold: add the sterilized CaCl ₂ (50 mM)-NaCl (150 mM) mixture to the scaffold, and then use a sample gun to contain a single pre-cavity follicle. The mixture was suspended into different parts of the stent, and an ovarian bioscaffold with preantral follicles was formed 2-5 minutes to form a type II 3D printed ovarian bioprosthesis.

(3) Preparation of ovariectomized mice: 8-10 week old (C57BL6/6JxCBA/Ca) female mice were subjected to ovarian ablation and rested for 4 weeks after surgery.

(4) Transplantation type II 3D printing ovarian bioprosthesis and functional measurement:

Experimental group: An ovarian bioprosthesis of length (5 mm) x width (2.5 mm) x thickness (2.5 mm) was implanted (containing 50 pre-cavity eggs to the subcutaneous back of the ovariectomized female rats).

Control group: A bioscaffold (containing a Matrigel solution) of a length (5 mm) x width (2.5 mm) x thickness (2.5 mm) was transplanted subcutaneously into the ovarian resection of the neck.

Three days after transplantation, mice were intraperitoneally injected with 1 IU of FSH every 48 hours until 4 weeks. One week later, the estrus cycle was measured every day, and blood was taken from the eye to detect serum estrogen and progesterone levels. 36 hours prior to recovery of the transplanted ovarian bioprosthesis, 20 IU of human chorionic gonadotropin (hCG) was injected into the spleen of each mouse. The transplanted ovarian bioprosthesis is then removed for morphological analysis and ovarian count.

The results showed that compared with the control group, after 4 weeks, the experimental group began to have a regular physiological cycle, and the levels of estrogen and progesterone increased significantly. In the experimental group, the ovarian bioprosthesis had dominant follicles and fresh after ovulation. The corpus luteum suggests that the preantral follicles in the type II ovarian bioprosthesis can develop, secrete hormones, maintain the female physiological cycle, and be able to ovulate.

Example 7 Preparation and Functional Determination of Type III 3D Printed Ovarian Bioprosthesis

(2) Injecting a vesicular follicle/matrix mixture into the ovarian bioscaffold: adding a sterilized mixture of CaCl ₂ (50 mM)-NaCl (150 mM) to the scaffold, and then using a sample gun to contain a single luminal follicle The coating was suspended into different parts of the stent and formed an ovarian bioscaffold with luminal follicles after 2-5 minutes.

(4) Transplantation of type III 3D printed ovarian bioprosthesis and functional measurement:

Experimental group: An ovarian bioprosthesis (containing 50 luminal follicles) of length (5 mm) x width (2.5 mm) x thickness (2.5 mm) was transplanted subcutaneously into the neck of the ovariectomized female.

Control group: A biological scaffold (length of matrix coating) containing a length (5 mm) x width (2.5 mm) x thickness (2.5 mm) was transplanted subcutaneously into the neck of the ovariectomized female.

Three days after transplantation, mice were intraperitoneally injected with 1 IU of FSH every 48 hours until 4 weeks. One week later, the estrus cycle was measured every day, and blood was taken from the eye to detect serum estrogen and progesterone levels. Recovering transplanted ovarian biopsy Thirty hours before the body, 20 IU of human chorionic gonadotropin (hCG) was injected into the spleen of each mouse. The transplanted ovarian bioprosthesis is then removed for morphological analysis and ovarian count.

The results showed that compared with the control group, after 2 weeks, the experimental group began to have a regular physiological cycle, and the levels of estrogen and progesterone increased significantly. In the experimental group, the ovarian bioprosthesis had dominant follicles and fresh after ovulation. The corpus luteum suggests that the preantral follicles in the type III ovarian bioprosthesis can develop, secrete hormones, maintain the female physiological cycle, and be able to ovulate.

Example 8 Preparation of Type IV 3D Printed Ovarian Bioprosthesis

The ovarian scaffold print material and the primordial follicle/coating mixture were prepared, and the preparation of the ovarian scaffold print material was the same as in Example 1, and the preparation of the original follicle/coating mixture was the same as in Example 4. The ovarian scaffold material and the primordial follicle/coating mixture were printed on a 3D printer with a double gun head. After printing a layer of scaffold material, a layer of primordial follicle/coating mixture is printed, thus completing the primordial follicular ovarian bioprosthesis, a type IV 3D printed ovarian bioprosthesis.

Example 9 Preparation and Functional Determination of V-type 3D Printed Ovarian Bioprosthesis

An ovarian stent print material and a pre-cavity follicle/coating mixture were prepared. The preparation of the ovarian stent print material was the same as in Example 1, and the preparation of the pre-cavity follicle/coating mixture was the same as in Example 4. The ovarian scaffold material and the pre-cavity follicle/coating mixture were printed on a 3D printer with a double gun head. After printing a layer of scaffold material, a layer of pre-cavity follicle/coating mixture is printed, thus completing the pre-cavity follicular ovary bioprosthesis, ie, a V-type 3D printed ovarian bioprosthesis.

Example 10 VI-type 3D printing ovarian bioprosthesis preparation and function determination

The ovarian stent print material and the pre-cavity follicle/coating mixture were prepared. The preparation of the ovarian stent print material was the same as in Example 1, and the preparation of the cavity follicle/coating mixture was the same as in Example 4. The ovarian scaffold material and the luminal follicle/coating mixture were printed on a 3D printer with a double gun head. After printing a layer of scaffold material, a layer of cavity follicle/coating mixture is printed, and the ovarian bioprosthesis with a follicular ovary is completed in a cycle, that is, a type VI 3D printed ovarian bioprosthesis.

Claims

An artificial ovary comprising a biocompatible scaffold, a follicular cell, and a coating mixture, wherein the coating mixture is wrapped outside the follicular cell and is embedded inside the void of the biocompatible scaffold.
The artificial ovary according to claim 1, wherein the coating liquid is a fluid liquid made of a coating auxiliary;

The coating material is selected from the group consisting of agar, agarose, calcium alginate, hyaluronic acid, matrigel, polyglycol polymer, polyvinyl alcohol polymer, collagen gel, collagen, and extracellular matrix protein. One or more of a hydrogel formed by cross-linking of alginate or calcium;

Preferably, the coating mixture further comprises a biological hormone selected from the group consisting of hormones or growth factors required for promoting follicular development. Preferably, the biological hormone is selected from the group consisting of follicle stimulating hormone (FSH) and luteinizing hormone (LH). Epidermal growth factor (EGF).
The artificial ovary according to claim 1, wherein the material of the biocompatible scaffold is selected from the group consisting of collagen I, sodium alginate, gelatin, agarose, Matrigel, hyaluronic acid, chitosan, and dextran. One or several combinations.
The artificial ovary according to claim 1, wherein said biocompatible stent has a void having a diameter which is 1 to 2 times, preferably 1 to 1.5 times, the diameter of the follicular cell.
The artificial ovary according to claim 1, wherein the follicular cells are selected from the group consisting of primordial follicles, preantral follicles, luminal follicles, tissues comprising primordial follicular cells, tissues comprising preantral follicular cells, and tissues comprising luminal follicular cells. One or more.
The artificial ovary according to claim 4, wherein the artificial ovary has one or more void sizes for different follicular cells, preferably 1-4 kinds of void sizes, more preferably 2-4 kinds of void sizes.
The artificial ovary according to any one of claims 1 to 6, wherein the biocompatible scaffold is made by 3D printing, and the coating mixture encapsulating the follicular cells is perfused inside the biocompatible scaffold; or

The biocompatible scaffold material and the coating mixture of the encapsulated follicular cells are sequentially produced by 3D printing in a layer-by-layer manner.
A method of producing an artificial ovary according to any one of claims 1 to 7, comprising the steps of:

1) preparing a biocompatible scaffold material;

2) preparing a coating mixture and coating the follicular cells;

3) obtaining a biocompatible scaffold by 3D printing with a biocompatible scaffold material, and infusing the follicular-containing coating mixture into the internal space of the biocompatible scaffold, or

3) The biocompatible scaffold material and the coating mixture of the encapsulated follicular cells are sequentially produced by 3D printing in a layer-by-layer manner.
The preparation method according to claim 8, wherein the step of culturing the follicular cells before coating the follicular cells with the coating mixture in the step 2).
The artificial ovary of the present invention is used as a drug screening and follicle research model.