CN116024158A - Preparation method of 3D model for in-vitro construction of mother-child interface of endometrium organoid - Google Patents
Preparation method of 3D model for in-vitro construction of mother-child interface of endometrium organoid Download PDFInfo
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Abstract
The invention discloses a preparation method of a 3D model for constructing a maternal-fetal interface in vitro of an endometrium organoid, which comprises the following steps: establishing a primary endometrial epithelial organoid and endometrial stromal cell line in vitro; improving the extracellular matrix used to assemble the endometrial gland-like organoids with stromal cells; the constructed endometrial organoid model comprises glandular and stromal cells; culturing an endometrium organoid model by adopting a gas-liquid interface to enable the endometrium organoid model to have a cavity epithelium and gland structure; the endometrial organoid model mimics the menstrual cycle and functional identification of the endometrium. The invention establishes an intima organoid model with lumen epithelium and glandular epithelium for the first time, the tissue structure, cell composition, hormone induction change and gene expression characteristics of organoids are similar to those of the human body, and the dynamics of receptive genes and cilia necessary for embryo implantation in the human body can be reproduced; can be used as a model for researching embryo implantation regulation, endometrial diseases and regeneration mechanism.
Description
Technical Field
The invention belongs to the field of reproductive medicine and biotechnology, and particularly relates to a preparation method of a 3D model for constructing a maternal-fetal interface in vitro of an endometrium organoid.
Background
The fertility of humans is low compared to other mammals. Even for healthy women, there is only 30% of the chance of natural conception per menstrual cycle. Based on human fertility statistics, one couple of every six couples was diagnosed with infertility, and about 25% of these groups of couples were diagnosed with infertility of unknown origin. Although assisted reproductive technology is used clinically to increase fertility, success rates are only between 40-55%, and implantation failure is the rate limiting step of this technology. Because the current understanding of embryo implantation by humans is still unclear, we need to better understand the cross-talk of the human maternal-fetal interface, understand the importance of endometrial receptivity to embryo implantation, and thereby further improve pregnancy outcome and pregnancy rate for infertility couples.
Endometrium acts as the "soil" for embryo implantation, which is the key to successful pregnancy. The intima is a multi-layered structure consisting of a luminal epithelium overlying the surface layer, the implantation site for the embryo to interact with, and an underlying glandular epithelium, the latter providing a nutritional environment for the embryo infiltrated therein. During the female menstrual cycle, the endometrium is in a highly dynamic process under the action of the oestrogen, and each cycle undergoes shedding, regeneration and differentiation. Human endometrial remodeling and regeneration abnormalities can lead to infertility, recurrent abortion, endometrial tumors, inflammation, thin endometrium, and endometriosis. Despite the much understanding of the pathology of these diseases, the molecular and cellular mechanisms associated with these diseases remain unresolved. Thus, profiling the cellular and molecular mechanisms involved in endometrial physiology and pathology helps to gain a better understanding of this dynamic organ and its associated diseases, and helps to develop new therapies. Most of the current research models use immortalized cell lines. After long-term passage, immortalized cell lines may differ in phenotype and genotype from the cells from which they were originally derived, and the nature and heterogeneity of these diseases cannot be summarized, thus impeding advances in science and clinic. In 2017, the establishment of the endometrial gland epithelial organoid model of human beings and mice provides a new idea for in vitro research. In vivo, cells are in complex microenvironments, where complex signaling and cell-to-cell interactions exist, which are critical in establishing, maintaining, and modulating cell phenotype and function. Organoids are obtained directly from endometrial biopsies, are believed to be phenotypically and physiologically closer to tissue cells, and exhibit their physiological functions better using 3D culture modes. Because of the limitations in the conversion of research results obtained from animal models to humans, little is known about the pathogenesis of human endometrial disease. Development of organoids advances the steps of precise treatment in the twenty-first century, and personalized treatment is realized by using organoid models with definite genetic backgrounds. Endometrial organoids will become a promising tool for a wide range of biomedical applications, from disease modeling to personalized medicine, which will accelerate our understanding of molecular and cellular mechanisms involved in endometrial development and disease. Most of the in vitro models of endometrium described so far are limited to single endometrial cells or lack of luminal epithelial structures, and do not truly mimic the three-dimensional structure and function of endometrium in vivo.
Thus, to address the above problems, a 3D model preparation method for in vitro construction of a maternal-fetal interface by an endometrial organoid is presented herein.
Disclosure of Invention
In order to solve the technical problems, the invention designs a preparation method of a 3D model for constructing a maternal-fetal interface in vitro by using an endometrial organoid, establishes a microenvironment for communication dialogue between the endometrial gland epithelium organoid and endometrial stromal cells, improves the defect that a single epithelial organoid cannot truly simulate menstrual cycle characteristics, and verifies the consistency of hormone response and in vivo; the invention also combines the characteristics of Matrigel and collagen to obtain the extracellular matrix which is better than that of independently culturing the endometrial gland organoid and endometrial stromal cells; by adopting a special culture mode of a gas-liquid interface, an intima organoid model with both luminal epithelium and glandular epithelium is established.
In order to achieve the technical effects, the invention is realized by the following technical scheme: a method for preparing a 3D model of an endometrial organoid in vitro constructed maternal-fetal interface, comprising the steps of:
s1, in vitro establishing a primary endometrial epithelial organoid and endometrial stromal cell line;
s2, improving extracellular matrix for assembling the endometrial gland-like organoids and stromal cells;
s3, constructing an endometrium organoid model which comprises glands and matrix cells;
s4, culturing an endometrium organoid model by adopting a gas-liquid interface so as to enable the endometrium organoid model to have a cavity epithelium and gland structure;
s5, simulating menstrual cycle and functional identification of the endometrium by using the endometrium organoid model.
Further, the medium of the endometrial epithelial organoids in S1 is called an Expansion medium (ExM), wherein the components include Advanced DMEM/F12, N2 supply, B27supplement minus vitamin A, penicillin/streptomycin, 0.5-2mM N-Acetyl-L-cysteine,0.5-3mM L-glutamine,30-70ng/ml EGF,50-200ng/ml Noggin,50-100ng/ml Rspondin-1, 50-200ng/ml FGF-10, 20-80ng/ml HGF,200-1000nM A83-01,5-20nM nicotinamide and 5-15nM Y27632.
Further, the medium components of the endometrial stromal cells in S1 are 5-20% fetal bovine serum and 0.5-5 mug/ml L-ascorbic acid.
Further, in the step S2, the endometrial gland-like epithelial organoids and the cells of the stromal cells are mixed according to a certain proportion; improving the extracellular matrix of the assembly of the endometrial gland-like epithelial organoids and stromal cells, wherein the extracellular matrix is formed by mixing Matrigel and type I collagen; after the two are assembled, the two are placed in an upper chamber or a culture dish of a Transwell chamber, incubated for more than 1 hour at 37 ℃, and then added with a culture medium for culture after being completely solidified.
Further, constructing an endometrial organ cavity epithelial structure in the step S3, after the assemblies of the endometrial organ epithelial structure and the endometrial organ epithelial structure are solidified in the step S2, collecting the endometrial organ in the step of ExM culture in a low-adsorption centrifuge tube, and removing extracellular matrixes in a repeated gentle blowing and centrifugation mode; the obtained cell pellet was resuspended in ExM medium, then added to the upper chamber of the Transwell chamber, and the same ExM medium was added to the lower chamber.
Further, in the step S4, an endometrium organoid combination is cultivated by adopting a gas-liquid interface, the endometrium organoid model constructed in the step S3 exists in a Transwell small chamber of a 24-pore plate, and the upper chamber and the lower chamber are cultivated by using an ExM culture medium for 0-4 days; removing liquid in the Transwell upper chamber by adopting a special culture mode of a gas-liquid interface, and keeping the upper part of the endometrium organoid model in contact with air and the lower part in contact with a culture medium to obtain nutrition; culturing for 4-20 days by adopting a gas-liquid interface culture mode.
Furthermore, the structure and function of the endometrium organoid model in the step S5 are verified, 5-15nM beta-estradiol (E2) is externally administered for 3-8 days, and the gas-liquid interface culture differentiation is carried out for 4-20 days so as to simulate the proliferation period of menstrual cycle; after 5-15nM beta-estradiol (E2) treatment for 3-8 days, 1 mu M progesterone (P4) and 1 mu M8-bromoadenosine 3',5' -cyclic monophosphate (cAMP) are added for 3-8 days, and the gas-liquid interface culture differentiation is carried out for 4-20 days to simulate the secretion phase of menstrual cycle; by single cell transcriptome analysis and immunofluorescent staining, the tissue structure, cell composition, hormone-induced changes, gene expression profile and in vivo similarity of organoids were confirmed as compared with endometrial tissue at different menstrual periods, and the dynamics of the receptive genes and cilia necessary for embryo implantation in vivo could be reproduced.
The beneficial effects of the invention are as follows:
the invention utilizes the epithelium and the stroma cells of endometrium sources to construct an organoid model with similar cell composition in vivo, discusses the influence of the stroma cells on the growth proliferation and gene phenotype of gland-like organoids, verifies the consistency of hormone response and in vivo, and improves the defect that the original single gland-like organoids can not simulate menstrual cycle change;
the invention also combines the characteristics of Matrigel and collagen to obtain extracellular matrix which is more suitable for in vitro organoid culture; by combining a gas-liquid interface culture mode, an endomembrane organoid model with a luminal epithelium and an glandular epithelium is established for the first time, and compared with endometrial tissues in different menstrual periods through single cell transcriptome analysis and immunofluorescence staining, the characteristics of tissue structure, cell composition, hormone induction change and gene expression of the organoid are proved to be similar to those of the endomembrane organoid in vivo, and the dynamics of receptive genes and cilia necessary for embryo implantation in vivo can be reproduced; can be used as a model for researching embryo implantation regulation, endometrial diseases and regeneration mechanism.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 in vitro construction of endometrial epithelial organoids and endometrial stromal cell lines;
FIG. 2 improves the extracellular matrix of an assembled endometrial organoid model;
FIG. 3 in vitro endometrium organoid model mimic proliferative phase;
FIG. 4 in vitro endometrium organoid model mimics the secretory phase;
FIG. 5 single cell map analysis of endometrial differences in vivo and in vitro.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
Medium preparation of endometrial gland epithelial organoids and endometrial stromal cells:
(1) Endometrial epithelial organoid medium formulation (abbreviated as ExM) comprising the ingredients Advanced DMEM/F12, N2supplement, B27supplement minus vitamin A, penicillin/streptomycin, 0.5-2mM N-Acetyl-L-cysteine,0.5-3mM L-glutamine,30-70ng/ml EGF,50-200ng/ml Noggin,50-100ng/ml Rspondin-1, 50-200ng/ml FGF-10, 20-80ng/ml HGF,200-1000nM A83-01,5-20nM nicotinamide and 5-15nM Y27632.
(2) The medium for endometrium stroma cell comprises DMEM,5-20% fetal bovine serum and 0.5-5 μg/ml L-ascorbic acid.
Example 2
Establishment and function identification of in vitro endometrial gland-like epithelial organoids:
(1) Tissues were collected in PBS phosphate buffer followed by cell separation treatment within 2-4 h. The cells were cultured at 37℃in a humidified incubator with 5% carbon dioxide. The centrifugation and incubation steps were all performed at room temperature unless otherwise indicated. The obtained endometrial biopsy tissue is crushed for 5-10 minutes by surgical scissors, collected in a 50ml centrifuge tube, added with 5-20ml of separation culture solution (RPMI 1640 medium containing 0.5-5U/ml neutral proteinase/dispase II and 0.2-2mg/ml collagenase V and 5-20% FBS.37 ℃ water bath for 30-60 minutes (whether a large number of glands are observed by a microscope), the supernatant is filtered by one or more cell sieves with 70 mu m, and the filtered cell sieves are washed with 1640 medium for a plurality of times.
(2) The cell sieve was inverted and glands remaining above the cell sieve were backwashed in a petri dish with basal medium, centrifuged to remove supernatant, and the pellet was resuspended in pre-chilled 50-100% matrigel. Mu.l of the suspension per well was transferred to a 48-well plate, placed in an incubator at 37℃for 30 minutes or more, and cultured in a well-based culture medium by adding 200 to 300. Mu.l of ExM.
(3) Subculturing of endometrial gland-like epithelial organoids: matrigel wrapping the endometrial gland-like epithelial organoids is gently scraped by using a gun tip in a low adsorption centrifuge tube, supernatant is removed by centrifugation, 100-500 mu L of Advanced DMEM/F12 is added for resuspension and precipitation, and a pipette is gently blown under 100-500. The supernatant was removed by centrifugation, the tube was prevented from being inverted, the above procedure was repeated, and the cell pellet was resuspended in pre-chilled 50-100% matrigel. Mu.l of the suspension per well was transferred to a 48-well plate, placed in an incubator at 37℃for 30 minutes or more, and cultured in a well-based culture medium by adding 200 to 300. Mu.l of ExM. In the subsequent subculture process, the liquid is routinely changed every 2 days, and the subculture is carried out every 6-10 days.
(4) Cryopreservation of endometrial gland-like epithelial organoids: gentle scraping of the coating endometrial gland-like epithelium using a gun tipOrganoids were pelleted by centrifugation of the supernatant in a low adsorption centrifuge tube, and 100-500. Mu.L of Advanced DMEM/F12 was added to resuspend the pellet, and the pipette gently blown down at 100-500. Centrifuging to remove supernatant, preventing reversal of centrifuge tube, repeating the above operation, and using precooled Recovery TM Cell culture cryopreservation media resuspended cell pellet and the cell suspension was added to the cryopreservation tube, 500 μl/tube, containing 20-100 glandular organoids per tube. Then the frozen tube containing the cell suspension is put into a program cooling box, and is transferred into liquid nitrogen for long-term storage after being cooled to minus 80 ℃ for overnight.
(5) The primary endometrial gland epithelial organoids were obtained according to step (2), after two generations of subculture according to step (3), the experiment was set up to three groups, the first group was collected from the ExM-cultured gland organoids, the second group was collected from the gland organoids treated with 5-15nM beta-estradiol (E2) added under ExM culture conditions, and the third group was collected from the gland organoids treated with 5-15nM beta-estradiol (E2), 0.5-2 μm progesterone (P4) and 0.5-2 μm 8-bromoadenosine 3',5' -cyclic monophosphate (cAMP) added under ExM culture conditions. The detection of the epithelial markers and hormone response markers was confirmed by immunofluorescence imaging. As shown in FIGS. 1a-c, we established that endometrial epithelial organoids specifically expressed the epithelial markers CK7 and E-cadherein and were able to be subcultured in vitro for long periods of time. Figures 1e-i show that an endometrial epithelial organoid established in vitro is responsive to the action of an oestrogen. Can induce differentiation to produce ciliated cells under the action of estrogen, and promote proliferation of epithelial organoids under the action of hormone.
Example 3
Establishment and functional identification of in vitro endometrium matrix cell line:
(1) After filtration of the suspension according to step (1) of example 2, the cells in the filtrate were collected, cultured for several days using endometrial stromal cell medium, and after cell attachment growth, a primary endometrial stromal cell line was obtained.
(2) Subculturing of endometrial stromal cells: endometrium stroma cells are digested into single cells by 50% TrypLE, the supernatant is removed by centrifugation, cell pellet is resuspended in endometrium stroma cell culture medium, and inoculated into 6cm petri dish at cell density of 1X 10-2.5X10-4 cells/cm 2. In the subsequent subculture process, the liquid is routinely changed every 2 days, and the subculture is carried out every 6-10 days.
(3) Cryopreservation of endometrial stromal cells: endometrium stroma cells are resolved into single cells by adopting 50% TrypLE, after supernatant is removed by centrifugation, cell sediment is resuspended in chilled liquid which is suspended in precooling, and the cell suspension is added into a frozen tube with the concentration of 500 mu l/tube, and each tube contains 5X 10-6 cells. Then the frozen tube containing the cell suspension is put into a program cooling box, and is transferred into liquid nitrogen for long-term storage after being cooled to minus 80 ℃ for overnight.
(4) After two generations of culture, deciduation treatment was performed with 0.5-2. Mu. M P4 and 0.5-2. Mu.M cAMP, and the expression level of the deciduation marker was confirmed by immunofluorescent staining for characterization of endometrial stromal cells and real-time fluorescent quantitative PCR. As shown in FIGS. 1j-l, vimentin and the stromal cell specific markers COL6A3, FN1 and LUM were expressed in the 2D culture mode of the primary endometrial stromal cells obtained. FIGS. 1j, m show that, after the matrix cells were subjected to the progestogen deciduation treatment, the matrix cells changed from shuttle morphology to polygonal shape, and the significant increase in the expression levels of PRL and IGFBP1 was confirmed by fluorescent quantitative PCR. FIGS. 1n and p show the tendency that the proliferation ability of the decidua-treated stromal cells was decreased, and the expression of PGR, connexin43 was decreased and the expression of FOXO1 was increased.
Example 4
In vitro assembling endometrium organoid and stroma cell, constructing endometrium organoid model cavity epithelial structure:
(1) Endometrial adenoid organoids and endometrial stromal cells were passaged as in example 1 and example 2, and stromal cells and glandular cells were collected, respectively, as in example 1 and example 2, and endometrium adenoid organoids and endometrial stromal cells were cultured in ExM as in example 1 and example 2.
(2) The endometrial organoids were resuspended in 100-500. Mu.L of Advanced DMEM/F12 following step (3) of example 2, after the supernatant was removed by centrifugation, and kept on ice with a pipette gently blown 100-500. The method comprises the steps of sucking old culture medium from endometrium stromal cells, adopting 50% TrypLE to digest the endometrium stromal cells into single cells, centrifuging to remove supernatant, and mixing 3X 10-5-8X 10-5 cells into pre-cooled endometrium gland-like organoids. After centrifugation to remove the supernatant, it was washed according to 1:1: (0.5-2) (v/v) mixing gland-like organoids resuspended in precooling (Matrigel: type I collagen: advanced DMEM/F12) with matrix, plated in 24-well plate Tanswell upper chamber at 20-50 μl per well, placed at 37deg.C for more than 30 min, and subjected to the next procedure after clotting. As shown in FIG. 2, the novel extracellular matrix (MAC for short) developed in vitro, which is improved by combining the characteristics of Matrigel and type I collagen, was found to be similar in hardness to tissues by measuring Young's modulus. Morphology and proliferation capacity of epithelial organoids were determined by immunofluorescent staining, and it was found that in the modified MAC, columnar epithelium production and stromal cell growth were favored.
(3) The glandular organoids cultured in ExM medium in 1-2 wells were further taken in a low adsorption centrifuge tube, cell pellets of glandular organoids were obtained by passaging centrifugation in step (3) of example 2, cell pellets were resuspended in 50-200. Mu.L of ExM medium, and added to the Transwell upper chamber of the coagulated cell mix gel in step (2), which was added 200-500. Mu.L of ExM medium to the corresponding lower chamber.
Example 5
Culturing an endometrial organoid assembly using a gas-liquid interface:
(1) If the proliferation period of menstrual cycle is simulated in vitro, after the endometrium organoid model constructed in example 3 is cultured for 0-3 days as above, 5-15nM beta-estradiol (E2) is added to the ExM culture medium for 3-8 days, the liquid in the upper chamber of Transwell is removed, the lower chamber is provided with 200-500 μl of ExM culture medium, and the culture is carried out at an air-liquid interface for 4-20 days after the new addition of 5-15nM beta-estradiol (E2). Figures 3a-c show that the proliferative hormone treatment of the menstrual cycle was simulated in vitro and used as a control in a common culture for the same time. The endometrial organoids simulate menstrual cycle proliferation in vitro, and compared with organoids cultured under liquid all the time, the gas-liquid level is more favorable for the formation of columnar cavity epithelium. Figures 3d, e show that ciliated cells occurred more similarly to in vivo by 15 days of differentiation. Fig. 3f shows that endometrial cells are estrogen-regulated, wherein estrogen receptor (ESR) and progestogen receptor (PGR) exhibit high expression in an in vitro organoid model, conforming to in vivo characteristics.
(2) If the secretory phase of menstrual cycle is simulated in vitro, the endometrium organoid model constructed in example 3 is cultured for 0-3 days as above, 5-15nM beta-estradiol (E2) is added to the ExM medium to act on the endometrium organoid model for 3-8 days, 0.5-2 mu M progesterone (P4) and 0.5-2 mu M8-bromoadenosine 3',5' -cyclic monophosphate (cAMP) are added together to act on the endometrium organoid model for 3-8 days, then the liquid in the upper chamber of Transwell is removed, 200-500 mu L of medium is kept in the lower chamber, and the medium is changed every 2 days, and the gas-liquid interface is cultured for 4-20 days. FIGS. 4a-c show that the secretory phase hormone treatment of the menstrual cycle is simulated in vitro and used as a control in a common culture for the same time. The in-vitro endometrium organoid model simulates the secretion period, and compared with the common liquid culture mode, the gas-liquid flat culture mode is also beneficial to the formation of columnar cavity epithelium. Figures 4d, e show that the progestogen antagonizes the effect of estrogen, reducing the proportion of ciliated cells to facilitate adherent implantation of embryos. As shown in fig. 4f, PGR and ESR expressed by epithelial cells are reduced, but PGR in stromal cells are continuously expressed, and the dynamic trend of estrogen receptors with menstrual cycle is similar to that in vivo. During the secretory phase, endometrial epithelial organoids also specifically express PAEP.
Example 6
Endometrial organoid assembly model identification:
(1) We used different extracellular matrices to encapsulate endometrial organoids model, and treated with estrogen for 3-8 days. Complete endometrial organoid models were carefully acquired from Transwell superrooms. Sample hardness determination was performed by using a nanoindenter. To ensure the accuracy of the statistical analysis, three biological replicates were performed.
(2) The in vitro endometrial organoid model mimics the proliferative and secretory phases of the menstrual cycle. The Transwell lower chamber medium was gently aspirated. Adding 4% paraformaldehyde for fixation for 1-3 hours, dehydrating 20% sucrose for 30-60 minutes, embedding with OCT, cutting frozen sections of 5-15 μm by a frozen microtome after OCT freezing, and collecting on an adhesive glass slide. The slides were washed with PBS to clear OCT. Each piece was permeabilized with 100-200. Mu.l of 3% BSA containing 0.3-0.5% TritonX-100 for blocking at room temperature for 3-4h or overnight at 4℃and the primary antibody was incubated overnight at 4 ℃. After the next day of PBS washing, secondary antibodies (1:500) and DAPI (1:1000) of the corresponding species were added and labeled for 2 hours at room temperature. The cells were washed 3 times for 10 minutes in PBS. Mu.l of 20% glycerol was added dropwise, blocked with a cover slip, and then imaged by analysis using an Leica SP8 confocal microscope and Leica X software.
(3) The model of the endometrium organoid simulates the proliferation and secretion of the menstrual cycle, after a period of time of estrogenic action, the model is carefully removed from the Transwell chamber, placed in a 0.1-1mg/ml type I collagenase, water-bath at 37℃for 30-60 minutes, and gently shaken every ten minutes to dissociate the gel mixture. After centrifugation of the supernatant, the pellet was gently blown by addition of Advanced DMEM/F12 medium. The supernatant was centrifuged again, 50% TrypLE was added and the pellet digested in a 37℃water bath for 30-60 minutes. Gently beating the cell mass to precipitate quickly, and completely dissociating the cell mass into single cells. The pellet was harvested by centrifugation and suspended in 0.1% bovine serum albumin to prevent single cell adhesion. Cell viability was assessed by cell counting with trypan blue staining. FIG. 5 shows the results of single cell transcriptome sequencing, comparing single cell data of an in vitro constructed endometrial organoid model with data of in vivo epithelial cells and stromal cells, and subdividing in vivo and in vitro cell data into ciliated cells, non-ciliated cells and stromal cells. It was confirmed that the cellular composition of organoids, gene markers of subpopulations and functions were similar to those in vivo.
It can be concluded that: the endometrial gland-like organoids and the stromal cells are co-cultured, so that the composition of endometrial cells is perfected. By improving extracellular matrix and combining the characteristics of matrigel and collagen, the hardness is enhanced on the basis of being beneficial to the growth of epithelial organoids, so that stromal cells have better adhesion sites for proliferation and differentiation. Differentiation is carried out by utilizing a special culture mode of a gas-liquid interface, and a tissue structure which is more similar to that in vivo is constructed in vitro, and the tissue structure has a luminal epithelial-like structure and an glandular epithelial-like structure. Immunofluorescent staining and single cell transcriptome sequencing analysis revealed that the in vitro constructed endometrium organoid model can form tissue structure characteristics in vivo, and can simulate the characteristics of menstrual cycle gene dynamic change in vitro. Can be used as a novel model for researching embryo implantation regulation, endometrial disease occurrence mechanism, endometrial periodic regeneration mechanism and drug screening.
Claims (7)
1. A method for preparing a 3D model of an endometrial organoid in vitro constructed maternal-fetal interface, comprising the steps of:
s1, in vitro establishing a primary endometrial epithelial organoid and endometrial stromal cell line;
s2, improving extracellular matrix for assembling the endometrial gland-like organoids and stromal cells;
s3, constructing an endometrium organoid model which comprises glands and matrix cells;
s4, culturing an endometrium organoid model by adopting a gas-liquid interface so as to enable the endometrium organoid model to have a cavity epithelium and gland structure;
s5, simulating menstrual cycle and functional identification of the endometrium by using the endometrium organoid model.
2. The method for preparing the 3D model for constructing the maternal-fetal interface in vitro by using the endometrium organoid according to claim 1, wherein the method comprises the following steps of: the medium of the endometrial epithelium organoid in S1 is called an amplification medium (ExM), wherein the components comprise advanced DMEM/F12, N2supplement, B27supplementminus vitaminA, penicillin/streptomycin, 0.5-2mMN-Acetyl-L-cysteine,0.5-3mM L-glutamine,30-70ng/ml EGF,50-200ng/ml noggin,50-100ng/ml Rspondin-1, 50-200ng/ml FGF-10, 20-80ng/ml HGF,200-1000nM A83-01,5-20nM nicotinamide and 5-15nMY27632.
3. The method for preparing the 3D model for constructing the maternal-fetal interface in vitro by using the endometrium organoid according to claim 1, wherein the method comprises the following steps of: the medium components of the endometrial stromal cells in S1 are 5-20% fetal bovine serum and 0.5-5 mug/mlL-ascobic acid.
4. The method for preparing the 3D model for constructing the maternal-fetal interface in vitro by using the endometrium organoid according to claim 1, wherein the method comprises the following steps of: in the step S2, the endometrial gland-like epithelial organoids and the cells of the stromal cells are mixed according to a certain proportion; improving the extracellular matrix of the assembly of the endometrial gland-like epithelial organoids and stromal cells, wherein the extracellular matrix is formed by mixing Matrigel and type I collagen; after the two are assembled, the two are placed in an upper chamber or a culture dish of a Transwell chamber, incubated for more than 1 hour at 37 ℃, and then added with a culture medium for culture after being completely solidified.
5. The method for preparing the 3D model for constructing the maternal-fetal interface in vitro by using the endometrium organoid according to claim 1, wherein the method comprises the following steps of: constructing an endometrial organ cavity epithelial structure in the step S3, after the assemblies of the two steps S2 are solidified, collecting the endometrial organ in the ExM culture in a low-adsorption centrifuge tube, and removing extracellular matrixes in a repeated gentle blowing and centrifugation mode; the obtained cell pellet was resuspended in ExM medium, then added to the upper chamber of the Transwell chamber, and the same ExM medium was added to the lower chamber.
6. The method for preparing the 3D model for constructing the maternal-fetal interface in vitro by using the endometrium organoid according to claim 1, wherein the method comprises the following steps of: the endometrial organoid combination is cultured by adopting a gas-liquid interface in the S4, the endometrial organoid model constructed in the S3 is stored in a Transwell small chamber of a 24-pore plate, and the upper chamber and the lower chamber are both cultured by an ExM culture medium for 0-4 days; removing liquid in the Transwell upper chamber by adopting a special culture mode of a gas-liquid interface, and keeping the upper part of the endometrium organoid model in contact with air and the lower part in contact with a culture medium to obtain nutrition; culturing for 4-20 days by adopting a gas-liquid interface culture mode.
7. The method for preparing the 3D model for constructing the maternal-fetal interface in vitro by using the endometrium organoid according to claim 1, wherein the method comprises the following steps of: in S5, the structure and function of the endometrium organoid model are verified, 5-15nM beta-estradiol (E2) is given in vitro for 3-8 days, and the gas-liquid interface culture differentiation is carried out for 4-20 days to simulate the proliferation period of menstrual cycle; after 5-15nM beta-estradiol (E2) treatment for 3-8 days, 1 mu M progesterone (P4) and 1 mu M8-bromoadenosine 3',5' -cyclic monophosphate (cAMP) are added for 3-8 days, and the gas-liquid interface culture differentiation is carried out for 4-20 days to simulate the secretion phase of menstrual cycle; by single cell transcriptome analysis and immunofluorescent staining, the tissue structure, cell composition, hormone-induced changes, gene expression profile and in vivo similarity of organoids were confirmed as compared with endometrial tissue at different menstrual periods, and the dynamics of the receptive genes and cilia necessary for embryo implantation in vivo could be reproduced.
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