WO2019029084A1 - 3d打印人造子宫内膜及其制备方法和应用 - Google Patents

3d打印人造子宫内膜及其制备方法和应用 Download PDF

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WO2019029084A1
WO2019029084A1 PCT/CN2017/116596 CN2017116596W WO2019029084A1 WO 2019029084 A1 WO2019029084 A1 WO 2019029084A1 CN 2017116596 W CN2017116596 W CN 2017116596W WO 2019029084 A1 WO2019029084 A1 WO 2019029084A1
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endometrial
endometrium
artificial
mixed culture
epithelial cells
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French (fr)
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张键
阮长顺
赵华山
任培根
杨雅莉
孙立峰
翟欣昀
吴明明
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深圳先进技术研究院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/222—Gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A—HUMAN NECESSITIES
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    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3813—Epithelial cells, e.g. keratinocytes, urothelial cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
    • A61L27/3882—Hollow organs, e.g. bladder, esophagus, urether, uterus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • 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
    • B33Y10/00—Processes of additive manufacturing
    • 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
    • B33Y70/00—Materials specially adapted for additive manufacturing
    • G—PHYSICS
    • G09—EDUCATION; CRYPTOGRAPHY; DISPLAY; ADVERTISING; SEALS
    • G09B—EDUCATIONAL OR DEMONSTRATION APPLIANCES; APPLIANCES FOR TEACHING, OR COMMUNICATING WITH, THE BLIND, DEAF OR MUTE; MODELS; PLANETARIA; GLOBES; MAPS; DIAGRAMS
    • G09B23/00—Models for scientific, medical, or mathematical purposes, e.g. full-sized devices for demonstration purposes
    • G09B23/28—Models for scientific, medical, or mathematical purposes, e.g. full-sized devices for demonstration purposes for medicine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
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    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/22—Materials or treatment for tissue regeneration for reconstruction of hollow organs, e.g. bladder, esophagus, urether, uterus

Definitions

  • the invention relates to the field of biotechnology, in particular to an artificial endometrium prepared by 3D printing technology.
  • the uterus is a part of the female reproductive organs of humans and animals. It is the place where human and animal fetuses or larvae grow and develop. In female mammals, not only plays a role in gestating the fetus and progeny, but also plays an important role in the female animal itself. Physiological function.
  • Endometrium refers to the mucosal layer in the mammalian uterus, composed of epithelial cells, stromal cells and blood vessels.
  • the endometrium can be divided into a superficial functional membrane and a deep basal layer according to its periodic functional changes.
  • the functional layer is thicker, accounting for about 4/5 of the thickness of the intima.
  • the basal layer is thinner and denser, accounting for about 1 /5, in the menstrual cycle, the functional layer can be exfoliated, and the basal layer will not exfoliate.
  • the morphology and function of the endometrium follow the sexual cycle ( The estrus cycle and the menstrual cycle show significant morphological and functional changes. Therefore, the pathological and physiological research on endometrium has important practical significance for ensuring the reproductive health of female animals.
  • Endometrial hyperplasia As a gynecological disease that may occur in women of childbearing age and non-menopausal women, it is more common, and the age of onset tends to be younger. The main symptom is abnormal uterine bleeding. Infertility, etc., have a negative impact on the patient's physical and mental health. In the long run, most patients with endometrial hyperplasia are reversible lesions, or continue to maintain a benign state, and some may even recover naturally with the endometrium of the menstrual period, but a small number of patients from Endometrial hyperplasia eventually develops into endometrial cancer. It is currently recognized that endometrial atypical hyperplasia is a precancerous lesion of endometrial cancer.
  • Endometrial carcinoma It is one of the three most common malignant tumors of the female reproductive system. It occurs in the endometrial epithelium of perimenopausal and postmenopausal women. There are nearly 200,000 new cases every year. And the incidence rate is still rising year by year, seriously endangering women's reproductive health. The cause of the disease is still unclear, and may be related to epigenetics and lifestyle. The incidence rate varies from place to place in the world. In developed countries such as North America and Europe, the incidence rate is the highest in gynecological malignancies. In China, the incidence of endometrial cancer is also increasing year by year, and it is close to or even exceeds the incidence of cervical cancer.
  • endometriosis is more common in puberty and women of childbearing age, the incidence rate is about 15%, mostly manifested as dysmenorrhea, menstrual abnormalities, pelvic adhesions, pain, infertility, etc.
  • endometriosis is morphologically benign, it has clinically similar characteristics of malignant tumors, such as metastasis and invasion. In recent years, the incidence rate has increased significantly, and its pathogenesis has not yet been elucidated. Increasingly high rates of caesarean section, induced abortion, and laparoscopic procedures may also be associated with genetic and immune factors.
  • endometrial polyps endometrial polyps as one of the most common endometrial lesions, is caused by endometrial inflammation caused by local blood vessels and connective tissue hyperplasia, forming a benign protruding from the endometrial surface Nodules can cause abnormal bleeding and infertility in the uterus, which can be malignant. Endometrial polyps are common in women over the age of 35, with an increased incidence with age, and the probability of endometrial polyps in infertile patients is higher. At present, the incidence of endometrial polyps in women in China is increasing, about 24%-25%. The main cause of this disease is inflammatory factors, followed by endocrine disorders, especially the high levels of estrogen, which may cause multiple reproductive cancers. Pathogen infection caused by external damage and irritation is also responsible for the formation of endometrial polyps.
  • IVF-ET in vitro fertilization and embryo transfer
  • Embryo implantation is a complex process that involves adhesion, dissolution, and invasion.
  • the implantation process mainly includes blastocyst hatching, decidualization of the endometrium, and is defined to be completed within a specific period of time, that is, the normal 28-day menstrual cycle on the 20th to 24th (LH peak day 7-11 day). This time is called the implantation window.
  • the Implantation window is precisely regulated by ovarian hormones. After the implantation of the endometrium begins to decapitate, this physiological phenomenon is essential for maintaining pregnancy.
  • the successful implantation of the embryo requires a mature endometrium, the endometrium is immature or not in the implantation window, and even if a high quality embryo is transplanted, the implantation rate is not high.
  • the success of embryo implantation depends to a large extent on the interaction between the embryo and the receptive endometrium, which means that the two sides establish a "dialogue" mechanism in which the embryo can be normally implanted in the endometrium. Endometrial tolerance to embryos is considered to be the key to successful embryo implantation.
  • tissue engineering technology With the development of tissue engineering technology, more and more researchers have tried to use tissue in vitro methods to inoculate endometrial glandular epithelial cells and stromal cells in liquid collagen scaffolds for co-culture to form three-dimensional endometrial tissue.
  • a composite structure that allows cells to grow sterically and replicate human endometrial tissue by secreting extracellular matrices, allowing deeper in vitro studies of adhesion and implantation of the endometrium and its interaction with the embryo.
  • Process research provides a more direct model that can serve as a new tool for further study of the pathophysiological mechanisms of endometrial diseases, providing a more reliable choice for prevention or treatment options for patients with irregular menstruation or infertility, or as a
  • a new model for in vitro embryo implantation is a more intuitive tool for in vitro fertilization-embryo transfer and related derivation techniques, which is important for the prevention and treatment of clinical reproductive health problems.
  • the present invention provides an artificial endometrium which is made of biocompatible scaffold material, endometrial stromal cells, endometrial epithelial cells, and mixed culture solution by 3D bioprinting. .
  • the biocompatible scaffold material is made into endometrium by 3D bioprinting. a stent, and then injecting a mixed culture medium containing endometrial stromal cells and a mixed culture medium containing endometrial epithelial cells into the endometrial stent;
  • the biocompatible scaffold material is mixed with a mixed culture of endometrial stromal cells and endometrial epithelial cells to make an artificial endometrium by 3D bioprinting; or
  • the biocompatible scaffold material is mixed with a mixed culture medium containing endometrial stromal cells or a mixed culture medium containing endometrial epithelial cells, and sequentially made into an artificial endometrium by 3D bioprinting.
  • the mixed culture liquid is a fluid liquid made of a culture medium and a biologically active substance required for promoting follicular development;
  • the medium is selected from DMEM/F12 or M199DMEM medium;
  • the biologically active substance is selected from one or a combination of one or more of biological hormones or growth factors; more preferably, the biological hormone is selected from the group consisting of estrogen (E2), pregnancy A combination of one or more of hormones (P4);
  • the growth factor is selected from the group consisting of one or more of vascular epidermal growth factor (VEGF), epidermal growth factor (EGF), and insulin-like growth factor (IGFs).
  • VEGF vascular epidermal growth factor
  • EGF epidermal growth factor
  • EGF epidermal growth factor
  • IGFs insulin-like growth factor
  • the biocompatible scaffold material is selected from one or a combination of collagen, sodium alginate, gelatin, agarose, matrigel, hyaluronic acid, chitosan, and dextran.
  • the collagen is selected from the group consisting of fibroblast collagen, more preferably one of type I collagen, type II collagen, type III collagen, type XI collagen, type XXIV collagen, and type XXVII collagen. Or any combination of several.
  • the artificial endometrium is made to have voids by 3D bioprinting, and the diameter of the voids is not more than 1.5 mm, more preferably not more than 1 mm.
  • the size of the void has one or more sizes, preferably from 1 to 4 sizes, and more preferably from 2 to 3 sizes.
  • the endometrial stromal cells are selected from the luteal phase endometrial stromal cells, and the endometrial epithelial cells are selected from the luteal phase endometrial epithelial cells.
  • a selection scheme of a biocompatible stent, a stromal cell/epithelial cell, and a mixed culture solution is used.
  • the material used in the present invention is a biocompatible material whose composition mimics the components contained in the endometrial tissue, which may be selected from the group consisting of type I collagen, sodium alginate, gelatin, agarose, matrigel, hyaluronic acid. One or a combination of one of chitosan and dextran.
  • Type 1 Collagen It is a hydrogel matrix extracted from animals. It has good biocompatibility, rich source, high plasticity, convenient clinical application and no immunogenicity. The formed gel network facilitates the free entry and exit of nutrients, has good hydrophilicity and cytocompatibility, and has been successfully applied to the in vitro culture of human endometrial stromal cells.
  • Alginate It is a natural polysaccharide extracted from brown algae or bacteria. It can be used as a food additive for food. It can also be used as a scaffold material for medical purposes and has good biocompatibility.
  • 3 Gelatin It is one of the most important natural biopolymer materials. It has been widely used in food, medicine and chemical industries, and has homology with collagen. It is a commonly used 3D printing scaffold material.
  • Agarose A linear multi-polymer that is alternately linked by 1,3 linked ⁇ -D-galactose and 1,4 linked 3,6-endoether-L-galactose to form a long-chain basic structure. Generally, it dissolves at 90 degrees or more, and a semi-solid gel can be formed at about 37 degrees. It has significant stability, hysteresis, and water absorption and is widely used in food and medicine.
  • Matrigel It 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 mimics the growth of cells in vivo.
  • 6 hyaluronic acid is a high molecular polysaccharide, 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.
  • Chitosan A sugar obtained from chitin which is widely found in nature. Due to its biocompatibility, safety and degradability, it is widely used in the fields of medicine, food and chemical industry.
  • glucan is a homopolysaccharide composed of glycosidic linkages of glucose unit. It can promote immune response, scavenge free radicals, dissolve cholesterol, and have anti-microbial effects. Widely used in food, medicine, cosmetics and other industries.
  • the stromal cells/epithelial cells used in the present invention are selected from the luteal phase endometrium of a human or an animal. After mechanical separation and digestion, a mixed cell composed of stromal cells and epithelial cells is obtained for the preparation of an artificial endometrium.
  • the mixed culture solution used in the present invention is a fluid liquid made of a medium, serum, a biological hormone, penicillin and streptomycin; wherein the medium is selected from DMEM F12 or M199DMEM;
  • the biological hormone is selected from hormones or growth factors required for promoting the development of stromal cells and epithelial cells, preferably
  • the biohormone is selected from the group consisting of estrogen (E2), progesterone (P4), vascular epidermal growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor (IGFs) and the like.
  • the mixed culture solution is selected from the group consisting of MEM F12 + FBS + streptomycin + penicillin + E2 + P4; preferably MEM F12 + 10% FBS + 1% streptomycin + 1% penicillin + 100 nmol / L E2 + 10 nmol / L P4.
  • the invention provides a method for preparing an artificial endometrium comprising the following steps:
  • the biocompatible scaffold material is made into a three-dimensional scaffold by 3D bioprinting, and then the cell mixture containing the endometrial stromal cells and epithelial cells is injected into the endometrial stent to make an artificial endometrium; or
  • the biocompatible scaffold material is mixed with the cell mixture of endometrial stromal cells and epithelial cells to make an artificial endometrium by 3D bioprinting;
  • the biocompatible scaffold material is mixed with a mixed culture medium containing endometrial stromal cells or a mixed culture medium containing endometrial epithelial cells, and sequentially made into an artificial endometrium by 3D bioprinting.
  • the biocompatible scaffold material is mixed with the mixed culture medium containing the endometrial stromal cells or the mixed culture medium containing the endometrial epithelial cells, respectively, and the artificial endometrium is made by 3D bio-printing;
  • Printing method The artificial endometrium is made by printing the substrate layer through 3D biofilm and printing the epithelial layer on the substrate layer (as shown in Fig. 1).
  • the matrix layer is 3D printed with a biocompatible scaffold material containing endometrial stromal cells
  • the epithelial layer is 3D printed with a biocompatible scaffold material containing endometrial epithelial cells.
  • the method for preparing the artificial endometrium further comprises the step 5), and the prepared endometrium is cultured in the mixed culture solution.
  • the endometrial stent or the artificial endometrium has a gap to facilitate sufficient contact between the cells and the mixed culture solution, and the diameter of the void is ⁇ 1 mm.
  • the 3D printing size of the biocompatible stent of the present invention is: pore size (R): 50 ⁇ m to 800 ⁇ m; line stacking angle: 0-90°.
  • the void size and the stacking angle in the biocompatible stent prepared by 3D printing can be adjusted according to the cell size, and the size used in the present invention can support the endometrial thinness.
  • the cell in turn, exerts its physiological role.
  • type I rat tail collagen and matrigel are used as a scaffold material, wherein type I collagen is used to encapsulate endometrial stromal cells, and matrigel is used to wrap endometrial epithelial cells; Rear
  • the term "mixed culture solution” refers to a medium and a composition made of a biologically active substance required for promoting cell development, and the medium may be selected from any cell culture medium capable of culturing animal cells, preferably DMEM. /F12 or M199DMEM medium.
  • the invention discloses a 3D printed artificial endometrium prepared for treating endometrial diseases, endometrial hyperplasia, endometrial atypical hyperplasia, endometrial cancer, uterine cancer, endometriosis, Use of endometrial polyps, factors such as infertility caused by endometrium, embryo implantation problems, endometrial damage, hysterectomy-related diseases or conditions, medical devices for direct or indirectly related diseases of endometrial lesions .
  • the present invention discloses the use of a 3D printed artificial endometrium for the preparation of a medical model for the study of embryonic implantation, a directly related disease of endometrial lesions or an indirectly related disease.
  • the artificial endometrium of the present invention can be used as a research and treatment tool for a directly related disease or an indirectly related disease of endometrial lesions caused by any factor;
  • the artificial endometrium of the present invention can be used as a research model for studying physiological problems such as embryo implantation, such as directly observing a series of changes in the positioning, adhesion, and invasion of an embryo during implantation, and observing the epithelial cell decidualization.
  • physiological problems such as embryo implantation, such as directly observing a series of changes in the positioning, adhesion, and invasion of an embryo during implantation, and observing the epithelial cell decidualization.
  • the phenomenon of increased stromal gland and the changes of related regulatory factors and signal pathways in this process are examples of diseases and the embryo implantation, such as directly observing a series of changes in the positioning, adhesion, and invasion of an embryo during implantation, and observing the epithelial cell decidualization.
  • Figure 1 Schematic diagram of 3D printed endometrial process: A, computer-aided design of endometrial structure, print model; B, for printing substrate; C, rat tail collagen/stromal cell mixture; D, matrigel / epithelium Cell mix.
  • Figure 2 3D printed endometrial stent preparation process.
  • FIG. 3 Implantation site formation after repair of damaged uterus: The left side shows the mouse Y-shaped double-horned uterus. The embryonic implantation process still occurs at the wound repair site and forms an implantation site (ie The embryo is implanted in the endometrium. The fluorescence signal is detected after taking part of the uterus tissue. As shown in the right figure, the tissue around the implantation site has a fluorescent signal indicating that the damaged endometrium is repaired, and Implantation of the embryo is acceptable, indicating the restoration of normal function of the endometrium.
  • the composition of the mixed culture solution was: DMEM F12 + 10% FBS + 1% streptomycin + 1% penicillin + 100 nmol / L E2 + 10 nmol / L P4).
  • the mixed culture solution was sterilized by filtration at 0.22 ⁇ m, and then equilibrated in a 37 ° C, 5% CO 2 incubator for 2-4 hours.
  • Example 2 Acquisition of endometrial epithelial cells and stromal cells
  • the mouse endometrial tissue was taken out under aseptic conditions, washed 3 times with PBS (containing 1% cyan, streptomycin) to remove blood clots and mucus on the surface of the tissue, placed in a Petri dish, and the endometrium was cut and added.
  • PBS containing 1% cyan, streptomycin
  • 2 ml of 0.3% type I collagenase mixed, placed in a 37 ° C water bath and shaken for 60 min; repeatedly blown and mixed, filtered through a 100 mesh filter to remove undigested tissue, collect the filtrate; obtain epithelial cells and stromal cells Crude mixture.
  • the stromal cells and epithelial cells in the endometrium were separated by centrifugation: centrifuged at 600 r/min for 10 min to precipitate epithelial cells; the suspension was stromal cells;
  • the stromal cell-rich suspension was centrifuged at 1200 r/min for 10 min, the supernatant was discarded, and the pellet was suspended in 1 ml of the culture solution, and then slowly dropped into a centrifuge tube containing 8 ml of the culture solution, and centrifuged at 400 r/min for 3 min to remove A small amount of cells were pelleted; the supernatant was transferred to another centrifuge tube, centrifuged at 1200 r/min for 10 min, the supernatant was discarded, and the bottom was purified endometrial stromal cells.
  • the cells were resuspended by adding the mixed culture medium, inoculated into a culture flask, and cultured in a 37 ° C, 5% CO 2 , 95% saturated humidity incubator for 24 hours, and the growth medium was replaced (to remove unattached stromal cells and blood cells);
  • the epithelial-rich pellet is added to the Hanks solution for rinsing, then centrifuged, and the precipitate is added.
  • DMEM/F12 containing 10% fetal bovine serum and 1% cyan, streptomycin
  • culture medium was inoculated into the culture flask under the same culture conditions as the stromal cells. After 24 hours, the cells were changed, and the growth of the cultured cells was observed every day, and the cell changes were recorded in detail.
  • the shape of the three-dimensional endometrial stent is designed to resemble the shape of the endometrium, and the internal structure is ensured to communicate with a certain pore structure;
  • the materials used for the three-dimensional endometrial stent are selected from gelatin, collagen I, and matrigel.
  • the prepared stent was stored at -80 ° C for use.
  • the crude mixture of epithelial cells and stromal cells was resuspended in mixed culture and the mixture was injected into the pores of a 3D printed endometrial bioscaffold.
  • the prepared 3D printed endometrium was placed in a mixed culture solution for in vitro culture.
  • Endometrial stromal cells subcultured to 2-3 passages were mixed at a final concentration of 2 x 10 6 cells/mL and subcultured with 2 x 10 5 cells/mL to endometrial epithelial cells of 2-3 passages.
  • the mixed cells are mixed with a biocompatible scaffold material to provide a biocompatible scaffold material comprising endometrial epithelial cells and endometrial stromal cells.
  • the prepared 3D printed endometrium was placed in a mixed culture solution for in vitro culture.
  • liquid collagen type I fetal bovine serum (4:1) was mixed with 2 ⁇ DMEM/F12 in equal proportions, and the pH was adjusted to neutral with 0.1 mol/L NaOH to obtain a liquid collagen mixture of type I; subculture was carried out until The 2-3 generation endometrial stromal cells were mixed with a type I liquid collagen mixture to a final concentration of 2 x 10 6 cells/mL.
  • a mixture containing endometrial epithelial cells and stromal cells is printed on the endometrial stromal cell layer by a double-tip, and an epithelial cell layer is printed on the upper part of the stromal cells to prepare a 3D printed endometrium.
  • the prepared 3D printed endometrium was placed in a mixed culture solution for in vitro culture.
  • mice or rats were intraperitoneally injected with Avertin anesthesia for general anesthesia. Then, the uterus tissue was separated by surgery, and the intact endometrial tissue and the uterine wall of the mesangial half of the 1/4-1/3 mouse were cut. Then, the 3D printed endometrium of the present invention containing the fluorescent protein-labeled epithelial cells and stromal cells to be transplanted is used to repair the damaged uterus, and then the abdominal muscle layer and the skin tissue are respectively sutured, and the recipient animal is restored. After 2-3 months, the cage is caged with the male, and finally the embryo is implanted in the repaired uterus. As shown in Figure 3, the left side shows the mouse in the Y-shaped double-horned uterus.
  • the fluorescence signal is detected after taking this part of the uterus tissue, as shown in the right image, around the implantation site.
  • the tissue has a fluorescent signal indicating that the damaged endometrium is repaired and accepts embryo implantation, indicating the restoration of normal function of the endometrium.

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Abstract

一种人造子宫内膜及其制备和应用，其中人造子宫内膜包括生物相容性支架、子宫内膜基质细胞／上皮细胞以及混合培养液，通过3D生物打印技术，将生物相容性支架与子宫内膜基质细胞／上皮细胞制成具有生物活性的人造子宫内膜。该人造子宫内膜可作为针对人造子宫内膜相关疾病的病理生理机制研究工具，也可作为针对调控和影响胚胎着床过程的相关因子的筛选模型，还可作为任何因素引起的子宫内膜病变的直接或间接相关疾病的预防或治疗研究工具。

Description

3D打印人造子宫内膜及其制备方法和应用 技术领域
本发明涉及生物技术领域，具体涉及3D打印技术制备的人造子宫内膜。
背景技术
子宫是人和动物的雌性生殖器官的一部分，是人和动物胎儿或幼体发育生长的场所，在雌性哺乳动物体内不但起着孕育胎儿和繁衍后代的作用，而且对雌性动物的本身也发挥着重要的生理功能。
子宫内膜，是指哺乳类子宫内的粘膜层，由上皮细胞、基质细胞和血管组成。子宫内膜按其周期性的功能变化特点可分为浅表的功能膜和深部的基底层，功能层较厚，约占内膜厚度的4/5，基底层较薄较致密，约占1/5，在月经周期中，功能层可剥脱，而基底层不会剥脱，这个过程中，在卵巢激素(雌激素和孕激素)的调控下，子宫内膜的形态和功能随着性周期(发情周期、月经周期)呈现明显的形态和功能变化。因此，针对子宫内膜方面的病理和生理研究对于保障雌性动物的生殖健康有着重要的现实意义。
(一)子宫内膜常见疾病问题：
1、子宫内膜增生(Endometrial hyperplasia)：作为一种在育龄期和未绝经期妇女中均可能发生的妇科疾病，较为常见，且发病年龄趋于年轻化，其主要的症状为子宫异常出血、不孕等，对患者的身心健康均有不良影响。从长远角度来看，大部分患者的子宫内膜增生是可逆性病变，或持续保持在良性的状态，有的甚至可能随着月经期子宫内膜的脱落而自然恢复，但也有少部分患者从子宫内膜增生最终发展为子宫内膜癌。目前公认子宫内膜不典型增生是子宫内膜癌的癌前病变。
2、子宫内膜癌(Endometrial carcinoma)：是最常见的女性生殖系统三大恶性肿瘤之一，多发于围绝经期和绝经后女性的子宫内膜上皮，每年有接近20万的新发病例，且发病率还在逐年升高，严重危害女性生殖健康。其发病原因迄今尚不明确，可能与表观遗传和生活方式相关，发病率在世界各地有差异，在北美和欧洲等发达国家其发生率高居妇科恶性肿瘤的首位。在我国，子宫内膜癌的发病率也呈逐年升高趋势，目前已接近甚至超过宫颈癌的发病率。
3、子宫内膜异位症(Endometriosis)：子宫内膜异位症多发育青春期和育龄女性，发病率在15％左右，多表现为痛经、月经异常、盆腔粘连、疼痛、不孕等，具体指子宫内膜组织出现在子宫体以外的部位，绝大多数位于盆腔脏器和腹膜，子宫内膜异位生长、浸润、反复出血，进一步形成结节和包块，是引起疼痛和不育的主要原因。子宫内膜异位症虽然在形态学上呈良性，但在临床上具有类似恶性肿瘤的特点，如转移、侵袭等，且近年来发病率呈明显上升趋势，其发病原因至今尚未阐明，可能与越来越高的剖腹产率、人工流产和宫腹腔镜操作相关，也可能与遗传和免疫等因素相关。
4、子宫内膜息肉(Endometrial polyps)：子宫内膜息肉作为最常见的子宫内膜病变之一，是由子宫内膜炎症反应导致局部血管和结缔组织增生，形成突出于子宫内膜表面的良性结节，可引起子宫异常出血与不孕，可恶变。子宫内膜息肉常见于35岁以上妇女，随年龄增长发病率增高，且不孕症患者患子宫内膜息肉的概率更高。目前我国妇女子宫内膜息肉发病率不断提高，约为24％-25％，其发病原因主要是炎症因素，其次是内分泌紊乱，尤其是雌激素水平过高，可能引发多种生殖癌症，另外，由外来损伤和刺激导致的病原体感染也是诱发子宫内膜息肉形成的原因。
以上子宫内膜疾病均能导致女性不孕，同时伴随月经不调、出血、疼痛等症状，严重影响女性生理和心理健康。而目前相关的研究或治疗手段还是相对不足，因此，开发具有生物活性的3D打印子宫内膜，不仅有助于体外或体内探索子宫内膜相关病变的生理病理机制，而且也为人类的生殖健康研究提供了新的研究或治疗工具。
(二)对于子宫内膜正常生理方面，主要围绕胚胎着床机制的探究
随着辅助生殖技术的迅速发展，体外受精-胚胎移植(In vitro fertilization and embryo transfer，IVF-ET)及其相关衍生技术成为目前治疗不孕症的重要手段。胚胎着床是成功妊娠的起始和关键环节，大规模流行病学调查显示，近年来IVF-ET的着床率一直徘徊在20％-30％，因此，胚胎着床过程受到生殖医学界的广泛关注和研究。
胚胎着床是一个复杂的过程，包括粘着、溶解、侵入3个阶段。着床过程主要包括胚泡的孵出、子宫内膜的蜕膜化，并被限定在一个特定的时期内完成，即正常28天月经周期的第20-24日(LH峰日第7-11日)。这段时间被称为着床窗口 期(Implantation window)，是由卵巢激素精确调节的。着床后的子宫内膜开始蜕膜化，这一生理现象对维持妊娠至关重要。胚胎的顺利着床需要发育成熟的子宫内膜，内膜不成熟或不处于着床窗口期，即使移植优质的胚胎，着床率也不高。胚胎着床的成功与否在很大程度上还取决于胚胎与可容受性子宫内膜间的相互作用，即双方建立“对话”机制，胚胎才可以在子宫内膜中正常着床。子宫内膜对胚胎的容受性被认为是胚胎着床成功的关键。
关于子宫内膜对胚胎着床的影响，最初通常采用对不同形态分型的子宫内膜进行体外培养，观察其形态特点以及对不同处理条件的反应，尽管相关研究已经取得了较大突破，但是二维培养条件不能完全再现在体子宫内膜的形态、结构和功能，存在一定局限性。
随着组织工程技术的发展，越来越多的研究者尝试采用体外构建组织的方法，将子宫内膜腺上皮细胞和基质细胞依次接种在液态胶原支架材料进行共培养，形成三维子宫内膜组织样复合结构，这种方法使细胞立体生长并通过分泌细胞外基质而复制出人子宫内膜组织，能够更深入地在体外研究子宫内膜及其与胚胎之间的黏附和植入情况。
综上所述，无论是病理还是正常生理情况下，良好的子宫内膜体外或体内模型，对于人类子宫内膜相关问题的研究有着重要的现实意义。因此，本项目在子宫内膜三维培养的基础上，结合组织工程技术和目前先进的3D打印技术，体外打印具有生物学功能的3D子宫内膜，这将为子宫相关疾病研究以及体外胚胎着床过程研究提供了更为直接的模型，既可以作为深入研究子宫内膜疾病病理生理机制的新工具，为月经不调或不孕不育患者提供更可靠的预防或治疗方案的选择，也可以作为体外研究胚胎着床过程的新模型，为体外受精-胚胎移植及相关衍生技术提供更直观的研究工具，这对临床生殖健康问题的预防和治疗均有重要意义。
发明内容
为了解决上述提到的问题，本发明提供了一种人造子宫内膜，其由生物相容性支架材料、子宫内膜基质细胞、子宫内膜上皮细胞以及混合培养液通过3D生物打印方式制成。
在本发明的方案中，将生物相容性支架材料通过3D生物打印制成子宫内膜 支架，再将含子宫内膜基质细胞的混合培养液和含子宫内膜上皮细胞的混合培养液分别注入子宫内膜支架上；或者
生物相容性支架材料与子宫内膜基质细胞和子宫内膜上皮细胞的混合培养液进行混合，通过3D生物打印制成人造子宫内膜；或者
生物相容性支架材料分别与含子宫内膜基质细胞的混合培养液或含子宫内膜上皮细胞的混合培养液混合，分别依次通过3D生物打印制成人造子宫内膜。(1)相关培养液、支架材料、细胞选择在本发明的技术方案中，混合培养液是由培养基和促进卵泡发育所需的生物活性物质制成的流动性液体；
优选地，培养基选自DMEM/F12或M199DMEM培养基；生物活性物质选自生物激素或生长因子中的一种或多种的组合；更优选地，生物激素选自雌激素(E2)、孕激素(P4)中的一种或多种的组合；生长因子选自血管表皮生长因子(VEGF)、表皮生长因子(EGF)、胰岛素样生长因子(IGFs)中的一种或多种的组合。
在本发明的技术方案中，生物相容性支架材料选用胶原蛋白、海藻酸钠、明胶、琼脂糖、基质胶、透明质酸、壳聚糖、葡聚糖中的一种或者几种任意组合；优选地，所述胶原蛋白选自成纤维胶原，更优选为I型胶原蛋白、II型胶原蛋白、III型胶原蛋白、XI型胶原蛋白、XXIV型胶原蛋白、XXVII型胶原蛋白中的一种或者几种任意组合。
在本发明的技术方案中，通过3D生物打印制成人造子宫内膜上具有空隙，空隙的直径不超过1.5mm，更优选不超过1mm。
在本发明的技术方案中，所述空隙的尺寸具有1种以上的尺寸，优选具有1-4种尺寸，更优选具有2-3种尺寸。
在本发明的技术方案中，所述子宫内膜基质细胞选自黄体期的子宫内膜基质细胞，子宫内膜上皮细胞选自黄体期子宫内膜上皮细胞。
在本发明的技术方案中生物相容性支架、基质细胞/上皮细胞、混合培养液的选择方案。
1)支架材料选择：
本发明所用的材料采用生物相容性材料，其组成成份模拟子宫内膜组织中包含的组分，其可以选自Ⅰ型胶原蛋白、海藻酸钠、明胶、琼脂糖、基质胶、透明质酸、壳聚糖、葡聚糖中的一种或者几种任意组合。
①Ⅰ型胶原蛋白：是从动物体内提取出来的一种水凝胶基质，其生物相容性好、来源丰富、可塑性高、临床应用方便、无免疫原性等特点。其形成的凝胶网络利于营养物质的自由进出，具有良好的亲水性和细胞相容性，已成功应用于人子宫内膜基质细胞的体外培养中。
②海藻酸钠：是从褐藻或细菌中提取出的天然多糖，可作为食用的食品添加剂，也可作为支架材料用于医学用途，具备良好的生物相容性。
③明胶：是非常重要的天然生物高分子材料之一，已被广泛应用于食品、医药及化工产业，与胶原具有同源性，是目前常用的3D打印支架材料。
④琼脂糖：一种线性多聚合物，由1,3连结的β-D-半乳糖和1,4连结的3,6-内醚-L-半乳糖交替链接形成长链基本结构。一般90度以上溶解，37度左右可形成半固体凝胶。有着显著的稳定性、滞后性、以及吸水性，被广泛使用于食品医药等领域。
⑤基质胶：是一种孔径大约在20～50nm的水凝胶基质，其主要成分为层粘连蛋白Ⅲ、Ⅳ型胶原蛋白、硫酸乙酰肝素蛋白聚糖及巢蛋白，与胚胎基底膜类似，可良好地模拟体内细胞生长的微环境。
⑥透明质酸：是一种高分子多糖，是细胞外基质的主要成分，有营养细胞、促进细胞分化的生理作用。但是力学性能较差，可通过改性修饰的方法，或复合其它材料的方法，改善其力学性能，扩展应用范围。
⑦壳聚糖：从广泛存在于自然界的几丁质中获得的一种糖类。由于其生物相容性、安全性、可降解性，被广泛用于医药、食品、化工领域。
⑧葡聚糖：是葡萄糖单元仪糖苷键链接组成的同型多糖。能够促进免疫反应，清除游离基，溶解胆固醇，抗微生物作用。广泛用于食品、医药、化妆品等行业。
2)、基质细胞/上皮细胞选择：
本发明所用基质细胞/上皮细胞，选自人或动物的黄体期子宫内膜。经过机械分离，消化处理后，获得基质细胞和上皮细胞组成的混合细胞用于人造子宫内膜的制备。
3)、混合培养液选择：
本发明所用的混合培养液由培养基、血清、生物激素、青霉素和链霉素制成的流动性液体；其中，培养基选自DMEM F12或M199DMEM；
生物激素选自促进基质细胞和上皮细胞发育所需的激素或生长因子，优选 地，生物激素选自雌激素(E2)、孕激素(P4)、血管表皮生长因子(VEGF)、表皮生长因子(EGF)、胰岛素样生长因子(IGFs)等。
优选地，混合培养液选自为MEM F12+FBS+链霉素+青霉素+E2+P4；优选为MEM F12+10％FBS+1％链霉素+1％青霉素+100nmol/L E2+10nmol/L P4。(2)、3D打印人造子宫内膜的制备过程
本发明提供了人造子宫内膜的制备方法，其包括以下步骤：
1)制备生物相容性支架材料，
2)制备混合培养液；
3)分离子宫内膜基质细胞和上皮细胞，并以混合培养液分别进行培养；
4)先将生物相容性支架材料，通过3D生物打印制成三维支架，再将含子宫内膜基质细胞和上皮细胞的细胞混合物液注入子宫内膜支架内制成人造子宫内膜；或者
4)生物相容性支架材料与子宫内膜基质细胞和上皮细胞的细胞混合物混合，通过3D生物打印制成人造子宫内膜；或者
4)生物相容性支架材料分别与含子宫内膜基质细胞的混合培养液或以及含子宫内膜上皮细胞的混合培养液混合，依次通过3D生物打印制成人造子宫内膜。
其中，生物相容性支架材料分别与含子宫内膜基质细胞的混合培养液或含子宫内膜上皮细胞的混合培养液混合，分别通过3D生物打印制成人造子宫内膜；进一步采用依次逐层打印方式：通过3D生物打印基质层，并在基质层上打印上皮层，制成人造子宫内膜(如图1所示)。其中基质层以含子宫内膜基质细胞的生物相容性支架材料进行3D打印，上皮层以含子宫内膜上皮细胞的生物相容性支架材料进行3D打印。
在本发明的技术方案中，人造子宫内膜的制备方法还包括步骤5)，将制备得到的子宫内膜在混合培养液中进行培养。
在本发明的技术方案中，所述子宫内膜支架上或者人造子宫内膜上具有空隙，以利于细胞与混合培养液充分接触，空隙的直径<1mm。在一个优选的技术方案中，本发明生物相容性支架的3D打印尺寸为：孔隙尺寸(R)：50μm～800μm；线条堆积夹角:0-90°。
在本发明的技术方案中，采用3D打印制成的生物相容性支架中空隙尺寸和堆积夹角可以根据细胞尺寸而调整，本发明所用的尺寸均能够负载住子宫内膜细 胞，进而发挥其生理作用。
本发明的一个优选的实施方案中以Ⅰ型鼠尾胶原和基质胶作为支架材料，其中Ⅰ型胶原蛋白用以包裹子宫内膜基质细胞，基质胶用以包裹子宫内膜上皮细胞；将其混合后
3D打印子宫内膜的应用：修复受损的子宫内膜组织
(1)、对正常小鼠或大鼠腹腔全身麻醉；
(2)、建立子宫损伤模型：手术分出子宫组织，切去1/4-1/3的小鼠对系膜侧一半的完整子宫内膜组织及子宫壁；
(3)、通过移植手段修复受损子宫组织：用待移植的含有荧光蛋白标记的上皮细胞和基质细胞的3D打印子宫内膜，去修补受损的子宫部位，分别缝合腹壁肌层和皮肤组织，受体动物恢复2-3个月；
(4)、子宫内膜修复效果验证：与雄鼠合笼，检测被修补的子宫部位是否有胚胎植入，以及将来是否有健康动物出生。
在本发明的技术方案中，术语“混合培养液”指培养基和促进细胞发育所需的生物活性物质制成的组合物，培养基可以选择能够培养动物细胞的任何细胞培养基，优选为DMEM/F12或M199DMEM培养基。
本发明公开了一种3D打印人造子宫内膜在制备用于治疗子宫内膜相关疾病、子宫内膜增生、子宫内膜不典型增生、子宫内膜癌、子宫癌、子宫内膜异位症、子宫内膜息肉、因子宫内膜导致的不孕症、胚胎着床障碍、子宫内膜损伤、子宫切除相关疾病或病症，子宫内膜病变的直接相关或间接相关的病症的医疗器械中的用途。
本发明公开了一种3D打印人造子宫内膜在制备用于研究胚胎着床、子宫内膜病变的直接相关疾病或间接相关疾病的医学模型中的用途。
有益效果
1)本发明的人造子宫内膜可作为任何因素引起的子宫内膜病变的直接相关疾病或间接相关疾病的研究和治疗工具；
2)本发明的人造子宫内膜可作为研究胚胎着床等生理问题的研究模型，比如直接观察胚胎在着床过程中的定位、粘附、侵入的一系列变化细节，观察上皮细胞蜕膜化、基质层腺体增多等现象，以及研究此过程中相关调控因子及信号通路的变化情况。
附图说明
图1：3D打印子宫内膜流程示意图：A、为计算机辅助设计子宫内膜结构、打印模型；B、为打印基板；C、为鼠尾胶原/基质细胞混合液；D、为基质胶/上皮细胞混合液。
图2：3D打印子宫内膜支架制备过程。
图3：损伤子宫部位修复后植入位点形成示意：左侧图所示为小鼠Y字型双角子宫中，受创伤修复部位仍有胚胎植入过程发生并形成植入位点(即胚胎植入子宫内膜的部位)，取这部分子宫组织切片后检测荧光信号，可见右侧图所示，在植入位点周围的组织有荧光信号，表明受损子宫内膜被修复，并可接受胚胎的植入，从而表明子宫内膜正常功能的恢复。
具体实施方式
实施例1：混合培养液的配置
混合培养液成分为：DMEM F12+10％FBS+1％链霉素+1％青霉素+100nmol/L E2+10nmol/L P4)。混合培养液0.22μm过滤灭菌后在37℃，5％CO2的培养箱内平衡2-4小时。
实施例2：子宫内膜上皮细胞和基质细胞的获得
无菌条件下取出小鼠子宫内膜组织，用PBS(含1％青、链霉素)洗3次，除去组织表面的血块和粘液，置入培养皿中，将子宫内膜剪碎后加入0.3％I型胶原酶2ml，混匀，置于37℃水浴中振荡消化60min；反复吹打混匀后经100目滤网过滤，以除去未消化尽的组织，收集滤液；得到上皮细胞和基质细胞粗混合物。
用离心法分离子宫内膜中的基质细胞和上皮细胞：以600r/min离心10min，沉淀为上皮细胞；悬液里为基质细胞；
将富含基质细胞的悬液以1200r/min离心10min，弃上清液，将沉淀用1ml培养液混悬后，缓慢滴至含8ml培养液的离心管中，400r/min离心3min，以去除少量成团的细胞；将上清液移入另一离心管中，1200r/min离心10min，弃上清，底部为纯化的子宫内膜基质细胞。加入混合培养液培养液重悬细胞，接种于培养瓶中，置于37℃，5％CO2，95％饱和湿度培养箱中培养24h，换生长培养液(以除去未贴壁的基质细胞和血细胞)；
将富含上皮细胞的沉淀中加入Hanks液漂洗，再离心，沉淀物中加入 DMEM/F12(含10％胎牛血清和1％青、链霉素)培养液，接种于培养瓶中，培养条件同基质细胞。24h后换液，每天观察培养细胞的生长情况，详细记录细胞变化。
实施例3：3D打印子宫内膜的制备
3D打印子宫内膜生物支架的设计及制备
(1)结合计算机辅助设计(Computer aided design,CAD)，采用三维打印技术构建子宫内膜支架，其中形状、组成成分及内部结构都具有良好可设计性；
(2)三维子宫内膜支架的形状设计为类似子宫内膜的形状，并保证内部结构联通，具有一定孔隙结构；
(3)所用于三维子宫内膜支架的材料，选择明胶、胶原Ⅰ、基质胶配伍打印。
(4)所构建三维子宫内膜内部结构参数：孔隙尺寸(R)：＝0.2mm；线条堆积夹角：90o(见图2)。
(5)将制备好的支架置于-80℃储存备用。
制备子宫内膜
将上皮细胞和基质细胞粗混合物用混合培养液进行重悬混合，并将混合物注射至3D打印子宫内膜生物支架的孔隙内。
将制备得到的3D打印的子宫内膜放入混合培养液中进行体外培养。
发明人还尝试了不同的孔隙尺寸和堆积夹角，发现在R＝50μm～800μm；线条堆积夹角:1-90°范围内，将混合物注射至3D打印子宫内膜生物支架的孔隙时均能使混合物附着于支架上。
实施例4：3D打印子宫内膜的制备
细胞与3D打印材料的混合
将传代培养至2-3代的子宫内膜基质细胞以终浓度2×106细胞/mL并与2×105细胞/mL传代培养至2-3代的子宫内膜上皮细胞混合。将混合细胞与生物相容性支架材料相混合，得到包含子宫内膜上皮细胞和子宫内膜基质细胞的生物相容性支架材料。
制备子宫内膜
通过3D打印方式，对包含子宫内膜上皮细胞和子宫内膜基质细胞的生物相容性支架材料进行打印，构建具有孔隙的、类似子宫内膜的形状的3D打印子宫 内膜。
将制备得到的3D打印的子宫内膜放入混合培养液中进行体外培养。
实施例5：3D打印子宫内膜的制备
细胞与3D打印材料的混合
将0.5ml的Ⅰ型液态胶原:胎牛血清(4:1)与2×DMEM/F12等比例混合，用0.1mol/L NaOH调pH为中性，得到Ⅰ型液态胶原混合物；将传代培养至2-3代的子宫内膜基质细胞与Ⅰ型液态胶原混合物混合，终浓度达到2×106细胞/mL。
将200ul基质胶与2×105传代培养至2-3代的子宫内膜上皮细胞混合。
制备子宫内膜
将含有子宫内膜上皮细胞以及含有基质细胞的混合物，通过双枪头先打印子宫内膜基质细胞层，再在基质细胞上部再打印上皮细胞层，制成3D打印子宫内膜。
将制备得到的3D打印的子宫内膜放入混合培养液中进行体外培养。
实施例6：宫内膜损伤移植修复实验
对正常小鼠或大鼠腹腔注射Avertin麻药，进行全身麻醉，然后，手术分出子宫组织，切去1/4-1/3的小鼠对系膜侧一半的完整子宫内膜组织及子宫壁，之后将待移植的含有荧光蛋白标记的上皮细胞和基质细胞的本发明的3D打印子宫内膜，去修补受损的子宫部位，然后，分别缝合腹壁肌层和皮肤组织，待受体动物恢复2-3个月后，与雄鼠合笼，最终检测被修补的子宫部位是否有胚胎植入，如图3示意，左侧图所示为小鼠Y字型双角子宫中，受创伤修复部位仍有胚胎植入过程发生并形成植入位点(即胚胎植入子宫内膜的部位)，取这部分子宫组织切片后检测荧光信号，可见右侧图所示，在植入位点周围的组织有荧光信号，表明受损子宫内膜被修复，并可接受胚胎的植入，从而表明子宫内膜正常功能的恢复。

Claims (10)

  1. 一种人造子宫内膜，其由生物相容性支架材料、子宫内膜基质细胞和子宫内膜上皮细胞通过3D生物打印方式制成。
  2. 根据权利要求1所述的人造子宫内膜，通过3D生物打印方式为：
    将生物相容性支架材料通过3D生物打印制成子宫内膜支架，再将含子宫内膜基质细胞和子宫内膜上皮细胞的混合培养液注入子宫内膜支架上；或者
    生物相容性支架材料与含子宫内膜基质细胞和子宫内膜上皮细胞的混合培养液进行混合，通过3D生物打印制成人造子宫内膜；或者
    生物相容性支架材料分别与含子宫内膜基质细胞的混合培养液以及含子宫内膜上皮细胞的混合培养液混合，分别依次通过3D生物打印制成人造子宫内膜。
  3. 根据权利要求1-2任一项所述的人造子宫内膜，其中，混合培养液是由培养基和促进卵泡发育所需的生物活性物质制成的流动性液体；
    优选地，生物活性物质选自生物激素或生长因子中的一种或多种；更优选地，生物激素选自雌激素(E2)、孕激素(P4)中的一种或多种；生长因子选自血管表皮生长因子(VEGF)、表皮生长因子(EGF)、胰岛素样生长因子(IGFs)中的一种或多种。
  4. 根据权利要求1-3任一项所述的人造子宫内膜，生物相容性支架材料选用胶原蛋白、海藻酸钠、明胶、琼脂糖、基质胶、透明质酸、壳聚糖、葡聚糖中的一种或者几种任意组合；优选地，所述胶原蛋白选自成纤维胶原，更优选为I型胶原蛋白、II型胶原蛋白、III型胶原蛋白、XI型胶原蛋白、XXIV型胶原蛋白、XXVII型胶原蛋白中的一种或者几种任意组合。
  5. 根据权利要求1所述的人造子宫内膜，通过3D生物打印制成人造子宫内膜上具有空隙，空隙的直径不超过1.5mm，更优选不超过1mm。
  6. 根据权利要求1-4任一项所述的人造子宫内膜，所述子宫内膜基质细胞选自黄体期的子宫内膜基质细胞，子宫内膜上皮细胞选自黄体期子宫内膜上皮细胞。
  7. 根据权利要求1-6任一项所述的人造子宫内膜的制备方法，其包括以下步骤：
    1)制备生物相容性支架材料，
    2)制备混合培养液；
    3)分离子宫内膜基质细胞和上皮细胞，并以混合培养液分别进行培养；
    4)将生物相容性支架材料通过3D生物打印制成子宫内膜支架，再将含子宫内膜基质细胞的混合培养液和含子宫内膜上皮细胞的混合培养液注入子宫内膜支架上；或者
    4)生物相容性支架材料与含子宫内膜基质细胞和子宫内膜上皮细胞的混合培养液进行混合，通过3D生物打印制成人造子宫内膜；或者
    4)生物相容性支架材料分别与含子宫内膜基质细胞的混合培养液或含子宫内膜上皮细胞的混合培养液混合，分别依次通过3D生物打印制成人造子宫内膜。
  8. 根据权利要求7所述的制备方法，其中，人造子宫内膜的制备方法还包括步骤5)，将制备得到的子宫内膜在混合培养液中进行培养。
  9. 根据权利要求1-6任一项所述人造子宫内膜在制备用于治疗子宫内膜相关疾病、子宫内膜增生、子宫内膜不典型增生、子宫内膜癌、子宫癌、子宫内膜异位症、子宫内膜息肉、因子宫内膜导致的不孕症、胚胎着床障碍、子宫内膜损伤、子宫切除相关疾病或病症，子宫内膜病变的直接相关或间接相关的病症的医疗器械中的用途。
  10. 根据权利要求1-6任一项所述的人造子宫内膜在制备用于研究胚胎着床、子宫内膜病变的直接相关疾病或间接相关疾病的医学模型中的用途。
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