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WO2018103654A1 - Ovaire artificiel et préparation et application associées - Google Patents

Ovaire artificiel et préparation et application associées Download PDF

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Publication number
WO2018103654A1
WO2018103654A1 PCT/CN2017/114700 CN2017114700W WO2018103654A1 WO 2018103654 A1 WO2018103654 A1 WO 2018103654A1 CN 2017114700 W CN2017114700 W CN 2017114700W WO 2018103654 A1 WO2018103654 A1 WO 2018103654A1
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Prior art keywords
ovarian
follicles
follicular
coating
coating mixture
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Chinese (zh)
Inventor
张键
阮长顺
任培根
杨雅莉
孙立峰
翟欣昀
吴明明
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Shenzhen Institute of Advanced Technology of CAS
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Shenzhen Institute of Advanced Technology of CAS
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
    • C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y80/00—Products made by additive manufacturing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C64/00—Additive manufacturing, i.e. manufacturing of three-dimensional [3D] objects by additive deposition, additive agglomeration or additive layering, e.g. by 3D printing, stereolithography or selective laser sintering
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    • C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
    • G—PHYSICS
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    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
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    • C12N2501/30—Hormones
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    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
    • G—PHYSICS
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    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the invention relates to the field of biotechnology, in particular to an artificial ovary prepared by a 3D printing technique.
  • 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%.
  • Premature ovarian failure 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.
  • POF Premature ovarian failure
  • 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.
  • assisted reproductive technologies such as IVF technology have become the key technology for the treatment of infertility.
  • IVF technology has developed rapidly over the past 20 years since its establishment, enabling thousands of families to successfully own their own offspring.
  • 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.
  • PCOS Polycystic ovary syndrome
  • 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.
  • PCOS has replaced surgical treatment as a first-line treatment, and the purpose of treatment is mainly related to the patient's fertility requirements.
  • ISD insulin-sensitizing drugs
  • 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).
  • PCOS patients who use assisted reproductive technology 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.
  • IVF In vitro fertilization
  • IVM in vitro maturation
  • 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.
  • OAC Ovarian Cancer
  • 3D printed ovarian bioprostheses invented in this application 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.
  • 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.
  • 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.
  • 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.
  • the coating mixture further comprises a biological hormone selected from the group consisting of hormones or growth factors required for promoting follicular development.
  • the biological hormone is selected from the group consisting of follicle stimulating hormone (FSH), luteinizing hormone (LH), epidermis. Cell growth factor (EGF).
  • 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.
  • 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.
  • 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.
  • primordial follicles preantral follicles
  • luminal follicles tissues containing primordial follicular cells
  • tissues including preantral follicular cells tissues including preantral follicular cells
  • tissues containing luminal 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.
  • kinds or more are selected from the group consisting of primordial follicles, preantral follicles, luminal follicles, tissues containing primordial follicular cells, tissues including pre
  • 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.
  • 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.
  • 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:
  • 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 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.
  • the 3D printing size of the biocompatible stent of the present invention is: pore size (R): 50 ⁇ m to 800 ⁇ m; line stacking angle: 0° to 180°.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 2 is a schematic flow chart of a 3D printed ovarian biological stent
  • a) is CAD design
  • b) is 3D printing
  • 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 primodial follicle
  • 2 preantral follicle
  • 3 is antral follicle
  • 4 is matrigel
  • 5 is primordial follicle/matrix mixture
  • 6 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).
  • 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
  • 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.
  • 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;
  • 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.
  • the prepared stent was stored at -80 ° C for use.
  • 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 ⁇ m in the ovary of the mouse were released.
  • the separation medium L-15+10% FBS+100 IU.
  • 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 ⁇ m in the ovary of the mouse were released.
  • L-15+ 10% FBS + 100 IU / ml penicillin + 100 ug / ml streptomycin
  • 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.
  • the separation medium L-15+10% FBS+100 IU/ml penicillin+100 ug/ml chain
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ M bpV (a PTEN inhibitor, Calbiochem) and/or 500 ⁇ g /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.
  • bpV a PTEN inhibitor, Calbiochem
  • 500 ⁇ g /mL 740Y-P a PI3K agonist, Tocris
  • mice 8-10 week old (C57BL6/6JxCBA/Ca) female mice were subjected to ovarian ablation and rested for 4 weeks after surgery.
  • 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.
  • 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.
  • hCG human chorionic gonadotropin
  • mice 8-10 week old (C57BL6/6JxCBA/Ca) female mice were subjected to ovarian ablation and rested for 4 weeks after surgery.
  • 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.
  • 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.
  • hCG human chorionic gonadotropin
  • 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.
  • 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.

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Abstract

L'invention concerne un ovaire artificiel et une préparation et une application associées. L'ovaire artificiel comprend un cadre biocompatible, des cellules folliculaires et un liquide mixte de revêtement, le liquide mixte de revêtement recouvrant l'extérieur des cellules folliculaires, pénétrant à l'intérieur des vides du cadre biocompatible et étant constitué d'un excipient revêtu et d'une hormone biologique et le cadre biocompatible est fabriqué par impression 3D. L'ovaire artificiel est approprié pour composer un outil de recherche pour des études cellulaires de l'ensemble ou d'une partie de follicules primordiaux, de follicules préantraux et de follicules luminaux d'une quelconque espèce et est également approprié pour un modèle de criblage pour un facteur, un promédicament et analogues associés aux follicules primordiaux, aux follicules préantraux et aux follicules luminaux et pour un outil de prévention ou de traitement d'une maladie liée directement ou indirectement à des lésions ovariennes provoquées par un quelconque facteur.
PCT/CN2017/114700 2016-12-09 2017-12-06 Ovaire artificiel et préparation et application associées Ceased WO2018103654A1 (fr)

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