An ovary organ chip, a manufacturing method and application thereof

Abstract

The application relates to an ovary organ chip, a manufacturing method and application thereof. The ovary organ chip is sealed by a four-layer structure from top to bottom, and comprises an upper ovary chip layer, a follicle culture layer, a porous filter membrane layer and a lower ovary chip layer. An upper end of an upper culture medium perfusion chamber of the upper ovary chip layer is communicated with an upper inlet pool through an upper perfusion channel, and a lower end of the upper culture medium perfusion chamber is communicated with an upper outlet pool through the upper perfusion channel. An upper end of a lower culture medium perfusion chamber of the lower ovary chip layer is communicated with a lower inlet pool through a lower perfusion channel, and a lower end of the lower culture medium perfusion chamber is communicated with a lower outlet pool through the lower perfusion channel. The follicle culture layer is provided with a round hole or a cylinder. The ovary organ chip is constructed to simulate the physiological environment and pathological changes of substantial cells in an ovary in vitro, so that researches on hormone synthesis, ovary development and maturation, disease occurrence mechanism, tumor occurrence, development, invasion and metastasis and the like are carried out, and the ovary organ chip is used for researching and developing drugs, individualized treatment of tumors and fertility preservation.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of reproductive medicine, in particular to an ovary organ chip, a manufacturing method and application thereof. BACKGROUND

[0002] With the rapid development of economic level and great progress of medical technology, since the 21st century, the life expectancy of human beings has been significantly prolonged. It is estimated that the total number of the elderly population in 2050 will be 5 times that in the 1950s. China, as the country with the largest population in the world, has entered an aging society. According to the latest census data, the size and growth rate of the elderly population in China are far beyond the international general level, which brings huge social pension burden and health problems caused by organ aging. In addition, organ aging is closely related to tumor, diabetes, cardiovascular disease and obesity, which will jointly lead to the decline of the quality of life and the extension of the adjustment of disability years. Therefore, how to delay organ aging is particularly important.

[0003] The ovary is one of the organs that age earliest in the human body, characterized by a significant decrease in the number and quality of follicles, and eventually menopause. The main function of the ovary is to secrete sex hormones and produce oocytes. When the ovary gradually ages, the levels of estrogen and progesterone secreted by the ovary decrease, while the levels of follicle-stimulating hormone and luteinizing hormone increase. Under the influence of hormones, the risk of osteoporosis, Alzheimer's disease and cardiovascular disease in women significantly increases. Therefore, ovarian aging is also called the "pacemaker" of female body aging. Extensive and in-depth research on the ovary helps to find strategies to delay ovarian aging.

[0004] On the other hand, more and more women choose to delay marriage and childbirth, but ovarian aging accelerates with age, and the number and quality of oocytes decrease sharply, which greatly challenges the fertility of women. At present, more than 15% of couples worldwide are facing infertility problems. Research on the survival and atresia of follicles is expected to provide new solutions for female infertility. In addition, infertility caused by the ovary is also affected by ovarian diseases such as ovarian fibrosis, endometriosis and ovarian cancer. They are affected by excessive deposition of extracellular matrix, infiltration of inflammatory factors and immune cells, or influenced by iatrogenic intervention during disease treatment, which will eventually cause varying degrees of ovarian damage. SUMMARY

[0005] The purpose of the present application is to provide an ovary organ chip, a manufacturing method and application thereof to improve the above problems.

[0006] In order to achieve the above purpose, the embodiments of the present application provide the following technical solutions:

[0007] In one aspect, the embodiments of the present application provide an ovary organ chip, comprising:

[0008] The ovary organ chip is sealed by a four-layer structure from top to bottom in turn, the ovary chip upper layer, the follicle culture layer, the porous filter membrane layer and the ovary chip lower layer, the ovary chip upper layer includes the upper layer inlet pool, the upper layer medium perfusion chamber, the upper layer outlet pool and the upper layer perfusion channel; the upper end of the upper layer medium perfusion chamber is communicated with the upper layer inlet pool through the upper layer perfusion channel, and the lower end is communicated with the upper layer outlet pool through the upper layer perfusion channel; the ovary chip lower layer comprises a lower layer inlet pool, a lower layer medium perfusion chamber, a lower layer outlet pool and a lower layer perfusion channel, the upper end of the lower layer medium perfusion chamber is communicated with the lower layer inlet pool through the lower layer perfusion channel, and the lower end is communicated with the lower layer outlet pool through the lower layer perfusion channel; the perfusion channel of the ovary chip upper layer (1) and the ovary chip lower layer (4) is perpendicular to each other; the follicle culture layer is provided with a round hole or a cylinder.

[0009] Optionally, the materials used in the ovary chip upper layer, the follicle culture layer and the ovary chip lower layer are selected from at least one or a combination of polydimethylsiloxane (PDMS), polystyrene, glass or polycaprolactone (PCL).

[0010] Optionally, the ovary chip upper layer and the ovary chip lower layer are controlled by using independent micro-pumps respectively, and the flow rate of the micro-pump is 10-1000 μL / h.

[0011] Optionally, the sealing method of the ovary organ chip includes but is not limited to any one of PDMS colloidal adhesion connection, oxygen plasma irreversible sealing or epoxy resin sealing.

[0012] Optionally, the round hole or the cylinder is in an array flux, and the number is 10-200.

[0013] Optionally, the follicle culture layer is provided with a round hole to form a honeycomb-shaped ovary chip, and the round hole and the porous filter membrane layer form a cell culture chamber, the cell culture chamber has different pore size structures, the pore size diameter is 30-700 μm, and the depth is 50-700 μm; the closer to the upper layer inlet pool, the smaller the cell culture chamber diameter and the shallower the depth.

[0014] Optionally, the follicle culture layer is provided with a cylinder to form a grid-shaped ovary chip, and the cylinder and the porous filter membrane layer form a cell culture grid to culture by blocking follicles, the cylinder has different spacing, the spacing range is 30-700 μm, the height is 50-700 μm, and the cylinder diameter is 30-100 μm; the closer to the upper layer inlet pool, the greater the spacing between the cylinders.

[0015] Optionally, the porous filter membrane layer comprises a porous filter membrane, and a composition of the porous filter membrane comprises at least one of polycarbonate membrane, cellulose membrane, nylon membrane, polyvinylidene fluoride membrane, polyethersulfone, cell culture membrane or polytetrafluoroethylene membrane, and a thickness of the porous filter membrane is 10-200 μm, and a height of the upper perfusion channel and the lower perfusion channel is 200-1000 μm.

[0016] In a second aspect, the embodiments of the present application provide a manufacturing method of an ovary organ chip, comprising:

[0017] The ovary organ chip is manufactured by selecting a biological 3D printing method or a sacrificial layer manufacturing casting method.

[0018] In a third aspect, the embodiments of the present application provide an application of the ovary organ chip, which is used to construct an ovary physiological model, an ovary pathological model and an ovary intervention model, and the ovary physiological model comprises an ovary ovulation model and an ovary aging model.

[0019] Optionally, a method for constructing the ovary ovulation model comprises:

[0020] The above ovary organ chip is subjected to high-temperature and humid heat sterilization treatment, and then is subjected to drying treatment;

[0021] The ovary first somatic cells and the ovary second somatic cells are separated from tissues, and the cell types thereof are identified by an immunofluorescence method. The chips are injected, inverted, and the ovary first somatic cells are attached to the porous filter membrane layer to grow. The ovary organ chip is placed in a 37℃ incubator to stand and culture for 2 hours. Then, the DMEM culture medium is slowly injected from the lower inlet pool to flush out the unattached cells.

[0022] The chips are placed upright, the ovary second somatic cells are injected into the chips from the upper inlet pool, the ovary second somatic cells are attached to the porous filter membrane layer to grow. The ovary organ chip is placed in a 37℃ incubator to stand for 2 hours. Then, the M5a culture medium is slowly injected from the upper inlet pool to flush out the unattached cells, and the chips are placed upright overnight. The ovary first somatic cells and the ovary second somatic cells need to be identified by an immunofluorescence method.

[0023] The follicles at all levels are injected into the ovary organ chip from the upper inlet pool (5), 5-15 mIU / mL follicle stimulating hormone is added into the follicle growth culture medium, the follicles are injected into the chip from the upper inlet pool (5), and a microfluidic pump is used to maintain a certain perfusion to avoid the follicles from being attached to the substrate in a 2D state. After 7-14 days of culture, one-time 5-15 mIU / mL human chorionic gonadotropin is given, and after 14-18 hours of culture, the occurrence of ovulation is observed to complete the construction of the ovary ovulation model.

[0024] Optionally, a method for constructing the ovary aging model comprises:

[0025] According to the method of constructing the ovary ovulation model, the first ovarian somatic cells, the second ovarian somatic cells and the ovarian follicles of the same age are added, and all the culture media in the ovary ovulation model are replaced with the universal culture medium, and the universal culture medium is used for culture;

[0026] The first ovarian somatic cells, the second ovarian somatic cells and the ovarian follicles of different ages are used, the universal culture medium is used for culture, the influence of aging on the number and quality of ovarian follicles is observed, and the construction of the ovary aging model is completed.

[0027] Optionally, the universal culture medium comprises a basic culture medium, nutritional factors, recombinant proteins, hormones, antibiotics and growth factors, the basic culture medium comprises but is not limited to DMEM, DMEM / F12, aMEM and M199, the hormones comprise but are not limited to estrogen, progesterone, androgen, human chorionic gonadotropin, follicle stimulating hormone and luteinizing hormone, and the growth factors comprise vascular endothelial growth factor, epidermal growth factor and insulin-like growth factor.

[0028] Optionally, the method for constructing the ovary pathological model comprises:

[0029] On the basis of the ovary ovulation model, all the culture media in the ovary ovulation model are replaced with the universal culture medium, and various recombinant proteins, growth factors, chemotactic factors, inflammatory factors or tumor cells are added in the universal culture medium, so as to obtain the ovary pathological model, which is used for simulating various pathological ovaries.

[0030] The ovary pathological model comprises but is not limited to a fibrosis model, an ovary cell aging model, an ovarian cancer model and an external environment damage model, and the specific construction method is as follows:

[0031] The fibrosis model: before the universal culture medium is injected, an extracellular matrix liquid is injected into the culture chamber, the ovary organ chip is translated and placed into the incubator for incubation for 30 min, so as to promote the gelation, and then the extracellular matrix liquid is flushed out, and the remaining steps are the same as the construction steps of the ovary ovulation model; wherein the extracellular matrix liquid comprises different concentrations of type I collagen, fibronectin, gelatin, matrix gel Matrigel and other natural or artificially synthesized extracellular matrix aqueous solutions, and the components comprise but are not limited to the above-mentioned substances and combinations thereof; or

[0032] In the universal culture medium, factors such as TGFB, IL6 or IL11 are added, and after standing overnight, each level of ovarian follicle is added. The remaining steps are the same as the construction steps of the ovary ovulation model;

[0033] Ovary cell aging model: Add an aging induction reagent or an anti-aging drug to the universal medium, the former including but not limited to 5% hydrogen peroxide and D-galactose, and the latter including but not limited to mifepristone, dasatinib, quercetin and rapamycin, and the rest of the steps are the same as the construction steps of the ovary ovulation model;

[0034] Ovary cancer model: replace the ovary second somatic cells with ovary cancer cell lines, including but not limited to OVCAR3, OVCAR5, SKOV3, OVHM, ID8, UWB1.289, SVOG, MES-OV and other ovary tumor cell lines, and use the universal medium for culture, and the rest of the steps are the same as the construction steps of the ovary ovulation model;

[0035] External environment damage model: add external environment components to the universal medium, including but not limited to atmospheric particulate matter, microplastics, environmental endocrine disruptors, carbon tetrachloride and the like, and the rest of the steps are the same as the construction steps of the ovary ovulation model;

[0036] The universal medium can be used for mixed culture of multiple different cells to meet the growth and functional needs of different cells. The basic medium of the universal medium includes but is not limited to DMEM, DMEM / F12, aMEM, M199, etc. By adding corresponding nutritional factors, recombinant proteins, hormones and growth factors of the cultured cells, combining them in appropriate proportions, and adding the required antibiotics, a complete culture medium is formed. The corresponding factors of the cultured cells include but are not limited to estrogen, progesterone, androgen, human chorionic gonadotropin, follicle-stimulating hormone, luteinizing hormone, vascular endothelial growth factor, epidermal growth factor, insulin-like growth factor, etc.

[0037] Optionally, the method for constructing an ovary intervention model comprises:

[0038] On the basis of the ovary ovulation model, the ovary first somatic cells and the ovary second somatic cells are replaced with young ovary first somatic cells and ovary second somatic cells, and the follicle is replaced with an old follicle, and the young ovary first somatic cells and ovary second somatic cells are used to observe the reversal effect on the old follicle; or

[0039] On the basis of the ovary ovulation model, the ovary first somatic cells and the ovary second somatic cells are replaced with old ovary first somatic cells and ovary second somatic cells, and the follicle is replaced with a young follicle, and the old ovary first somatic cells and ovary second somatic cells are used to observe the damage effect on the young follicle; or

[0040] On the basis of the ovary physiological model or the ovary pathological model, an intervention measure is added, and the reaction of the ovary is observed; the intervention measure includes adding a small molecule compound, a drug, a nano-targeting peptide, a plant extract and a viral vector.

[0041] The above technical solutions provided by the embodiments of the present application have the following advantages compared with the prior art:

[0042] 1. Complete cell components. The present application uses the first ovarian somatic cells (including granulosa cells and various source stem cells), the second ovarian somatic cells (including theca cells, interstitial cells, vascular endothelial cells and various source stem cells) and various follicles (including primordial follicles, primary follicles, secondary follicles, preantral follicles, antral follicles and preovulatory follicles), which cover all cell components in the real ovary, are arranged under the design of the organ chip, simulate the situation that the ovarian somatic cells provide suitable growth environment for the follicle, and can more simulate the real situation of the in-vivo ovary.

[0043] 2. Comprehensive ovarian function. The present application can realize important functions of the ovary such as hormone synthesis, ovarian development and maturation, and can also explore the ovarian state such as the occurrence mechanism of diseases, the occurrence, development and invasion and metastasis of tumors, and even through intervention measures, can be used for research and development of drugs, individualized treatment of tumors, fertility preservation and the like.

[0044] 3. Wide modeling coverage. The present application includes physiological ovary modeling (including ovulation model and ovary aging model), pathological ovary modeling (including fibrosis model, ovarian cell aging, ovarian cancer model and external environment damage model, which simulates diseases such as ovarian fibrosis, endometriosis cyst, ovarian simple cyst and ovarian cancer), and ovary intervention model (small molecule compounds, drugs, nano-targeting peptides, plant extracts and viral vectors).

[0045] 4. High efficiency of ovarian research transformation. At present, the ovary cell culture is mostly in the form of 2D culture, and the current 3D culture system is not mature enough, which makes it difficult for related scientific research results to be clinically transformed, and the impaired ovarian function of patients cannot be effectively delayed or even reversed. The present application adopts the ovary organ chip to construct a new 3D culture system, and can be adjusted to simulate the situation of the ovary in physiological, pathological and intervention states, so as to realize the research on the physiological development of the ovary, the discussion on the pathogenesis of the ovary, and the effect after intervention in various ways, which will fundamentally improve the research method in the field of ovary and improve the clinical transformation efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0046] The accompanying drawings, which are incorporated into and form a part of the specification, illustrate an embodiment consistent with the present application and, together with the description, serve to explain the principles of the application.

[0047] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the accompanying drawings needed to be used in the description of the embodiments or the prior art will be briefly introduced. Obviously, for those of ordinary skill in the art, other drawings can be obtained based on these drawings without any creative effort.

[0048] Figure 1 is an exploded view of the overall structure of the ovarian organ chip when a round hole is provided on the follicle culture layer in the embodiments of the present application.

[0049] Figure 2 is an exploded view of the overall structure of the ovarian organ chip when a cylinder is provided on the follicle culture layer in the embodiments of the present application.

[0050] Figure 3 is a cell type identification diagram of the first ovarian somatic cell and the second ovarian somatic cell.

[0051] The labels in the figure are as follows: 1, upper layer of the ovarian chip; 2, follicle culture layer; 3, porous filter membrane layer; 4, lower layer of the ovarian chip; 5, upper layer inlet pool; 6, upper layer culture medium perfusion chamber; 7, upper layer outlet pool; 8, upper layer perfusion channel; 9, round hole; 10, cylinder; 11, porous filter membrane; 12, lower layer inlet pool; 13, lower layer culture medium perfusion chamber; 14, lower layer outlet pool; 15, lower layer perfusion channel; 16, cell culture chamber. DETAILED DESCRIPTION

[0052] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without any creative effort fall within the scope of protection of the present application.

[0053] Embodiment 1

[0054] As Figure 1 and Figure 2As shown, the present embodiment provides an ovary organ chip, which is sealed by a four-layer structure, from top to bottom in turn is ovary chip upper layer 1, follicle culture layer 2, porous filter membrane layer 3 and ovary chip lower layer 4, the ovary chip upper layer 1 includes upper layer inlet pool 5, upper layer medium perfusion chamber 6, upper layer outlet pool 7 and upper layer perfusion channel 8; the upper end of the upper layer medium perfusion chamber 6 is communicated with the upper layer inlet pool 5 through the upper layer perfusion channel 8, and the lower end is communicated with the upper layer outlet pool 7 through the upper layer perfusion channel 8; the ovary chip lower layer 4 contains lower layer inlet pool 12, lower layer medium perfusion chamber 13, lower layer outlet pool 14 and lower layer perfusion channel 15, the upper end of the lower layer medium perfusion chamber 13 is communicated with the lower layer inlet pool 12 through the lower layer perfusion channel 15, and the lower end is communicated with the lower layer outlet pool 14 through the lower layer perfusion channel 15; the follicle culture layer 2 is provided with a round hole 9 or a cylinder 10.

[0055] In one specific embodiment of the present disclosure, the materials used in the ovary chip upper layer 1, the follicle culture layer 2 and the ovary chip lower layer 4 are selected from at least one or a combination of polydimethylsiloxane PDMS, polystyrene, glass or polycaprolactone PCL.

[0056] In one specific embodiment of the present disclosure, the ovary chip upper layer 1 and the ovary chip lower layer 4 are controlled by using independent micro-pumps respectively, the micro-pump can be used for parameter adjustment by using program, and the flow rate of the micro-pump is 10-1000 μL / h.

[0057] In one specific embodiment of the present disclosure, the sealing method of the ovary organ chip includes but is not limited to any one of PDMS colloidal adhesive connection, oxygen plasma irreversible sealing or epoxy resin sealing.

[0058] In one specific embodiment of the present disclosure, the round hole 9 or the cylinder 10 is in array flux, the number is 10-200, and the array can be coded to in-situ track observation on the development process of follicle and pathophysiological changes.

[0059] In one specific embodiment of the present disclosure, the follicle culture layer 2 is provided with a round hole 9, which constitutes a honeycomb-shaped ovary chip, and the round hole 9 and the porous filter membrane layer 3 form a cell culture chamber 16, the cell culture chamber 16 has different pore size structures, the pore size diameter is 30-700 μm, and the depth is 50-700 μm, the cell culture chamber 16 is smaller in diameter and shallower in depth as it is closer to the upper layer inlet pool 5, the bottom of the cell culture chamber 16 is the porous filter membrane layer (3), the upper layer perfusion channel 8 and the lower layer perfusion channel 15 of the cell culture chamber 16 can be added with fluid at the same time, and the material exchange of nutrients and metabolic products in the upper and lower layers can be realized.

[0060] In one specific embodiment of the present disclosure, the follicle culture layer 2 is provided with a cylinder 10, which constitutes a grid-shaped ovary chip. The cylinder 10 and the porous filter membrane layer 3 form a cell culture grid, and the cylinder 10 is cultured by blocking the follicle. The cylinders 10 have different spacings, and the spacing ranges from 30 to 700 μm. The height of the cylinder is 50-700 μm, and the diameter of the cylinder is 30-100 μm. The closer to the upper inlet pool 5, the larger the spacing between the cylinders 10.

[0061] In one specific embodiment of the present disclosure, the porous filter membrane layer 3 includes a porous filter membrane 11, and the composition of the porous filter membrane 11 includes at least one of polycarbonate membrane, cellulose membrane, nylon membrane, polyvinylidene fluoride membrane, polyether sulfone, cell culture membrane or polytetrafluoroethylene membrane. The thickness of the porous filter membrane 11 is 10-200 μm, and the height of the upper perfusion channel 8 and the lower perfusion channel 15 is 200-1000 μm.

[0062] The present embodiment can simulate the physiological environment and pathological changes of parenchymal cells in the ovary in vitro, so as to study hormone synthesis, ovary development and maturation, disease mechanism, tumor occurrence, development and invasion and metastasis, and use the same for the development of drugs, individualized treatment of tumors and fertility preservation. The organ chip can also construct an ovarian fibrosis, endometrial cyst, ovarian simple cyst and ovarian cancer model, study the mechanism of these diseases, and explore the strategy of fertility protection while treating the diseases.

[0063] Embodiment 2

[0064] A method for manufacturing an ovary organ chip, comprising:

[0065] The above ovary organ chip is manufactured by a biological 3D printing method or a sacrificial layer manufacturing casting method. When the biological 3D printing method is used, the model of the ovary chip is established in advance, the model is sliced by computer slicing software, and finally the 3D printing path data is obtained. The printing is performed by an extrusion type or light curing printer. When the sacrificial layer manufacturing casting method is used, the mold and the sacrificial layer material are manufactured according to the three-dimensional model.

[0066] Embodiment 3

[0067] An application of an ovary organ chip, which is used to construct an ovulation model of the ovary. The construction method comprises:

[0068] Step one, the above ovary organ chip is subjected to high temperature and humidity sterilization treatment, and the drying temperature is 65-80℃;

[0069] Step two, the first ovarian somatic cells and the second ovarian somatic cells are separated from the tissue, and the cell types are identified by immunofluorescence method, and the cell type identification diagram is as shown in Figure 3 The first ovarian somatic cells are injected from the lower inlet pool into the chip, inverted, and the first ovarian somatic cells are attached to the growth of the multi-well filter membrane layer, the ovarian organ chip is placed in a 37°C incubator for 2 hours, then DMEM medium is slowly injected from the lower inlet pool, and the unattached cells are washed out; wherein the first ovarian somatic cells include granulosa cells and various source stem cells;

[0070] Step three, the chip is upright, the second ovarian somatic cells are injected from the upper inlet pool 5 into the chip, the second ovarian somatic cells are attached to the growth of the multi-well filter membrane layer 3, the ovarian organ chip is placed in a 37°C incubator for 2 hours, then M5a medium is slowly injected from the upper inlet pool 5, and the unattached cells are washed out, and the upright is overnight; wherein the second ovarian somatic cells include theca cells, interstitial cells, vascular endothelial cells and various source stem cells. The first ovarian somatic cells and the second ovarian somatic cells can be of animal origin, including but not limited to monkeys, mice, rats, pigs, cows, sheep and other mammals, and the uterine specimens of animals are obtained from experimental animal centers and slaughterhouses. It can also be a primary cell from a clinical patient specimen, as well as various source cell lines, including but not limited to KGN, COV434, etc. All cells are digested into single cell suspension before entering the ovarian organ chip, and the cell suspension density is 1-8x10 6 cells / mL;

[0071] Step four, each level of follicle is injected from the upper inlet pool (5) into the ovarian organ chip, 5-15 mIU / mL follicle stimulating hormone is added to the follicle growth medium, the chip is injected from the upper inlet pool (5), and a microfluidic pump is used to maintain a certain perfusion to avoid follicle settlement and attachment to a 2D state. After 7-14 days of culture, a one-time 5-15 mIU / mL human chorionic gonadotropin is given, and after 14-18 hours of culture, the occurrence of ovulation is observed, and the construction of the ovary ovulation model is completed. Each level of follicle includes primary follicle, primary follicle, secondary follicle, pre-antral follicle, antral follicle and pre-ovulatory follicle; the main body of the follicle growth medium is αMEM medium, and fetal bovine serum, penicillin, streptomycin, recombinant human follicle stimulating hormone and ITS are added, wherein the fetal bovine serum accounts for 10% of the total volume, the penicillin and streptomycin account for 0.1% of the total volume, the concentration of the recombinant human follicle stimulating hormone in the culture medium is 10 mIU / mL, and the ITS is composed of insulin, transferrin and sodium selenite, and the concentrations in the culture medium are insulin 5 μg / mL, transferrin 5 μg / mL and sodium selenite 5 ng / mL, respectively;

[0072] Example 4

[0073] The application of the ovarian organ chip is used for constructing an ovarian aging model, and the construction method comprises the following steps:

[0074] According to the method for constructing the ovarian ovulation model, the ovarian first somatic cells, the ovarian second somatic cells and the follicles of the same age stage are added, all the culture mediums in the ovarian ovulation model are replaced with universal culture mediums, and the universal culture mediums are used for culture; the ovarian first somatic cells, the ovarian second somatic cells and the follicles of different age stages are used for culture, the influence of aging on the quantity and quality of the follicles is observed, and the construction of the ovarian aging model is completed.

[0075] Specifically, according to the method steps for constructing the ovarian ovulation model (all the steps are the same as the steps for constructing the ovarian ovulation model), the difference lies in that, first, the ovarian first somatic cells, the ovarian second somatic cells and the follicles of young age (1-3 months old) are added, and then the ovarian first somatic cells, the ovarian second somatic cells and the follicles of old age (1.5-2 years old) are added; second, all the culture mediums in the method for constructing the ovarian ovulation model are replaced with universal culture mediums for culture. The ovarian first somatic cells, the ovarian second somatic cells and the follicles of different age stages are added to observe the influence of aging on the quantity and quality of the follicles.

[0076] During the construction of the ovarian ovulation model and the ovarian aging model, the function of the ovarian chip can also be detected, and the detection method is as follows:

[0077] During the culture, the culture medium is collected from the cell outlet pool every day, the changes in the levels of estrogen and progesterone are detected to reflect the functions of the upper layer 1 of the ovarian chip, the follicle culture layer 2 and the follicles; the cell activity is detected by using a CCK-8 kit; the cell survival state is detected by using a live and dead staining kit; the formation of blood vessels in the follicle surrounding and the ovarian somatic cells is displayed by using CD31 and other indexes for immunofluorescence; the formation of lymphatic vessels in the follicle surrounding and the ovarian somatic cells is displayed by using LYVE1 and other indexes for immunofluorescence.

[0078] Embodiment 5

[0079] The application of the above ovarian organ chip is used for constructing an ovarian pathological model on the basis of the ovarian ovulation model constructed in Embodiment 3, and the construction method comprises the following steps:

[0080] On the basis of the ovarian ovulation model, all the culture mediums in the ovarian ovulation model are replaced with universal culture mediums, and various recombinant proteins, growth factors, chemotactic factors, inflammatory factors or tumor cells are added in the universal culture mediums to obtain an ovarian pathological model, which is used for simulating various pathological ovaries.

[0081] The ovarian pathological model comprises a fibrosis model, an ovarian cell aging model, an ovarian cancer model and an external environment damage model, and the specific construction method is as follows:

[0082] Fibrosis model: before injecting the universal culture medium, inject extracellular matrix liquid into the culture chamber, translate the ovary organ chip into the incubator for incubation for 30 min to promote the gelation, then flush with the extracellular matrix liquid, and the remaining steps are the same as those in the construction steps of the ovary ovulation model; wherein the extracellular matrix liquid comprises natural or artificially synthesized extracellular matrix aqueous solution of different concentrations of collagen type I, fibronectin, gelatin, matrix gel Matrigel, and the like, and the components include but are not limited to the above-mentioned substances and combinations thereof; or

[0083] Add factors such as TGFB, IL6 or IL11 to the universal culture medium, and after standing overnight, add the follicles at different levels. The remaining steps are the same as those in the construction steps of the ovary ovulation model;

[0084] Ovary cell aging model: add an anti-aging reagent or an anti-aging drug to the universal culture medium, the former includes but is not limited to 5% hydrogen peroxide and D-galactose, and the latter includes but is not limited to mifepristone, dasatinib, quercetin and rapamycin, and the remaining steps are the same as those in the construction steps of the ovary ovulation model;

[0085] Ovarian cancer model: replace the ovary second somatic cells with an ovarian cancer cell line, including but not limited to OVCAR3, OVCAR5, SKOV3, OVHM, ID8, UWB1.289, SVOG, MES-OV and the like, and use the universal culture medium for culture, and the remaining steps are the same as those in the construction steps of the ovary ovulation model;

[0086] External environment damage model: add external environment components to the universal culture medium, including but not limited to atmospheric particulate matter, microplastics, environmental endocrine disruptors, carbon tetrachloride and the like, and the remaining steps are the same as those in the construction steps of the ovary ovulation model;

[0087] Example 6

[0088] The above ovary organ chip is applied, on the basis of the above-mentioned ovary physiological and pathological models, to construct an ovary intervention model, and the construction method comprises:

[0089] On the basis of the ovary ovulation model, replace the ovary first somatic cells and the ovary second somatic cells with young ovary first somatic cells and ovary second somatic cells, replace the follicles with old follicles, and use the young ovary first somatic cells and the ovary second somatic cells to observe the reversal effect on the old follicles; or

[0090] On the basis of the ovary ovulation model, the ovary first somatic cells and the ovary second somatic cells are replaced by old ovary first somatic cells and ovary second somatic cells, and the follicle is replaced by a young follicle, and the damage to the young follicle is observed by using the old ovary first somatic cells and ovary second somatic cells; or

[0091] On the basis of the ovary physiological model and the ovary pathological model, an intervention measure is added, and the reaction of the ovary is observed; the intervention measure includes adding small molecule compounds, drugs, nano-targeting peptides, plant extracts, and viral vectors.

[0092] By using the ovary organ chip in the embodiment, the construction of the ovary physiological model can be performed, and by using the ovary physiological model, the research on delaying ovary aging can be performed to correspond to the early ovarian aging and menopause and the like; the construction of the ovary pathological model can be performed, and by using the ovary pathological model, the research on the ovary fibrosis, endometrial cyst, ovary simple cyst, and ovarian cancer and the like can be performed; the construction of the ovary intervention model can be performed, and on the basis of the ovary physiological and pathological model, various drugs, small molecule compounds, plant extracts and the like are added to research the treatment of various diseases. Meanwhile, when different models are constructed, human specimens can also be applied to make high-quality research, and by this way, the clinical conversion level can be significantly improved.

[0093] The above only describes specific embodiments of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features applied herein.

Claims

1. An ovarian organ chip, characterized by, The ovarian organ chip is sealed by a four-layer structure from top to bottom in turn, which is an ovarian chip upper layer (1), a follicle culture layer (2), a porous filter membrane layer (3) and an ovarian chip lower layer (4), the ovarian chip upper layer (1) comprises an upper layer inlet pool (5), an upper layer medium perfusion chamber (6), an upper layer outlet pool (7) and an upper layer perfusion channel (8); the upper end of the upper layer medium perfusion chamber (6) is communicated with the upper layer inlet pool (5) through the upper layer perfusion channel (8), and the lower end is communicated with the upper layer outlet pool (7) through the upper layer perfusion channel (8); the ovarian chip lower layer (4) comprises a lower layer inlet pool (12), a lower layer medium perfusion chamber (13), a lower layer outlet pool (14) and a lower layer perfusion channel (15), the upper end of the lower layer medium perfusion chamber (13) is communicated with the lower layer inlet pool (12) through the lower layer perfusion channel (15), and the lower end is communicated with the lower layer outlet pool (14) through the lower layer perfusion channel (15); the upper layer inlet pool (5) and the upper layer outlet pool (7) are linearly communicated with the upper layer perfusion channel (8), and the lower layer inlet pool (12) and the lower layer outlet pool (14) are linearly communicated with the lower layer perfusion channel (15); the perfusion channels of the ovarian chip upper layer (1) and the ovarian chip lower layer (4) are perpendicular to each other; the follicle culture layer (2) is provided with a round hole (9) or a cylinder (10); the round hole (9) or the cylinder (10) is in an array flux, and the number is 10-200; The follicle culture layer (2) is provided with a round hole (9), which constitutes a honeycomb-shaped ovarian chip, and the round hole (9) and the porous filter membrane layer (3) form a cell culture chamber (16), the cell culture chamber (16) has different pore size structures, the pore size diameter is 30-700 μm, and the depth is 50-700 μm; the closer to the upper layer inlet pool (5), the smaller the diameter and the shallower the depth of the cell culture chamber (16); or The follicle culture layer (2) is provided with a cylinder (10), which constitutes a grid-shaped ovarian chip, and the cylinder (10) and the porous filter membrane layer (3) form a cell culture grid, and the follicle is cultured by blocking, the cylinder (10) has different spacing, the spacing range is 30-700 μm, the height is 50-700 μm, and the cylinder diameter is 30-100 μm; the closer to the upper layer inlet pool (5), the greater the spacing between the cylinders (10).

2. The ovarian organ chip according to claim 1, characterized in that, The materials used in the ovarian chip upper layer (1), the follicle culture layer (2) and the ovarian chip lower layer (4) include but are not limited to at least one or a combination of polydimethylsiloxane PDMS, polystyrene, glass and polycaprolactone PCL.

3. The ovarian organ chip according to claim 1, wherein, Independent micropumps are used to control the ovarian chip upper layer (1) and the ovarian chip lower layer (4) respectively, and the flow rate of the micropump is 10-1000 μL / h.

4. The ovarian organ chip of claim 1, wherein, The sealing mode of the ovarian organ chip includes but is not limited to any one of PDMS colloidal adhesion connection, oxygen plasma irreversible sealing or epoxy resin sealing.

5. The ovarian organ chip of claim 1, wherein, The porous filter membrane layer (3) comprises a porous filter membrane (11), the composition of the porous filter membrane (11) comprises at least one of polycarbonate membrane, cellulose membrane, nylon membrane, polyvinylidene fluoride membrane, polyether sulfone, cell culture membrane or polytetrafluoroethylene membrane, the thickness of the porous filter membrane (11) is 10-200 μm, and the height of the upper perfusion channel (8) and the lower perfusion channel (15) is 200-1000 μm.

6. The method of claim 1-5, wherein, Comprise: The ovarian organ chip is prepared by selecting a biological 3D printing method or a sacrificial layer manufacturing casting method.

7. Use of an ovarian organ chip according to any one of claims 1 to 5, wherein The ovarian organ chip is used for constructing an ovarian physiological model, an ovarian pathological model and an ovarian intervention model.

8. Use of an ovarian organ chip according to claim 7, characterized in that, The method for constructing the ovarian ovulation model comprises: The ovarian organ chip in any one of claims 1-5 is subjected to high-temperature moist heat sterilization treatment, and then subjected to drying treatment; The ovarian first somatic cells and the ovarian second somatic cells are separated from tissues, the ovarian first somatic cells are injected into the chip from the lower inlet pool (12), the chip is inverted, the ovarian first somatic cells are attached to the porous filter membrane layer (3) and grow, the ovarian organ chip is placed in a 37 DEG C incubator and is cultured for 2 hours, then DMEM culture medium is slowly injected from the lower inlet pool (12), and unattached cells are washed out; The chip is placed in an upright position, the ovarian second somatic cells are injected into the chip from the upper inlet pool (5), the ovarian second somatic cells are attached to the porous filter membrane layer (3) and grow, the ovarian organ chip is placed in a 37 DEG C incubator and is cultured for 2 hours, then M5a culture medium is slowly injected from the upper inlet pool (5), and unattached cells are washed out, and the chip is placed in an upright position and is cultured overnight; The ovarian first somatic cells and the ovarian second somatic cells are separated from tissues, the ovarian first somatic cells are injected into the chip from the lower inlet pool (12), the chip is inverted, the ovarian first somatic cells are attached to the porous filter membrane layer (3) and grow, the ovarian organ chip is placed in a 37 DEG C incubator and is cultured for 2 hours, then DMEM culture medium is slowly injected from the lower inlet pool (12), and unattached cells are washed out; 9. Use of an ovarian organ chip according to claim 8, characterized in that, The method for constructing the ovarian aging model comprises: According to the method for constructing the ovarian ovulation model, the ovarian first somatic cells, the ovarian second somatic cells and the ovarian follicles of the same age are added, all the culture media in the ovarian ovulation model are replaced with universal culture medium, and the universal culture medium is used for culture; The universal culture medium comprises a basic culture medium, a nutritional factor, a recombinant protein, a hormone, an antibiotic and a growth factor, the basic culture medium comprises but is not limited to DMEM, DMEM / F12, alpha MEM and M199, the hormone comprises but is not limited to estrogen, progestogen, androgen, human chorionic gonadotropin, follicle stimulating hormone and luteinizing hormone, and the growth factor comprises vascular endothelial growth factor, epidermal growth factor and insulin-like growth factor.

10. Use of an ovarian organ chip according to claim 9, characterized in that, ​ 11. Use of an ovarian organ chip according to claim 8, characterized in that, A method for constructing an ovary pathological model, comprising: On the basis of the ovary ovulation model, all of the culture medium in the ovary ovulation model is replaced with a universal culture medium, and various recombinant proteins, growth factors, chemotactic factors, inflammatory factors or tumor cells are added to the universal culture medium to obtain an ovary pathological model for simulating various pathological ovaries.

12. Use of an ovarian organ chip according to claim 8, characterized in that, A method for constructing an ovary intervention model, comprising: On the basis of the ovary ovulation model, the ovary first somatic cells and the ovary second somatic cells are replaced with young ovary first somatic cells and ovary second somatic cells, and the follicle is replaced with an old follicle, and the reverse effect on the old follicle is observed by using the young ovary first somatic cells and the ovary second somatic cells; or On the basis of the ovary ovulation model, the ovary first somatic cells and the ovary second somatic cells are replaced with old ovary first somatic cells and ovary second somatic cells, and the follicle is replaced with a young follicle, and the damage effect on the young follicle is observed by using the old ovary first somatic cells and the ovary second somatic cells; or On the basis of the ovary physiological model or the ovary pathological model, an intervention measure is added, and the reaction of the ovary is observed; the intervention measure comprises adding a small molecule compound, a drug, a nano-targeting peptide, a plant extract and a viral vector.

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