Bionic endometrial organ construction method and intrauterine adhesion disease model construction method

Abstract

The invention relates to a construction method of a bionic endometrial organ, which comprises the following steps: mixing three primary cells to obtain a mixed cell precipitate, and adding the mixed cell precipitate into a bionic organ culture medium; precipitating and resuspending the mixed cells to obtain a cell suspension; and adding Matrigel, and transferring into a constant-temperature cell culture box for culturing, so as to obtain the bionic endometrial organ. The regeneration promoting effect of the bionic endometrial organ provided by the invention is obviously better than that of transplanted pure stromal cells and pure epithelial organ groups, and the endometrial organ of the bionic cells has a huge application value in the aspect of tissue regeneration. According to the construction method of the intrauterine adhesion disease model, a fibrosis inducing factor TGF beta 1 is added into a bionic endometrial organ, immunofluorescent staining of a fibrosis marker alpha SMA is carried out, and the intrauterine adhesion disease model is obtained. By reproducing the pathological change of the intrauterine adhesion inner membrane, the model can be used as an intrauterine adhesion pathological model for disease pathological research or drug screening research.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a method for constructing a biomimetic endometrial organoid and a method for constructing an intrauterine adhesion disease model. BACKGROUND

[0002] An organoid is a three-dimensional tissue with a certain spatial structure formed by three-dimensional culture in vitro. The organoid is highly similar to the real organ, can reproduce the tissue structure and key functions of the corresponding organ in vivo in vitro, and can be stably subcultured for a long time. The organoid has great application prospects in organ reconstruction, disease model, drug screening, precision medicine and other fields. The construction of endometrial organoids has great significance in promoting endometrial regeneration, endometrial disease model, and endometrial disease treatment. However, the current endometrial organoids are too simple compared with normal in vivo endometrial tissue: since 2017, when researchers first cultured human endometrial epithelial organoids, endometrial epithelial organoids based on mouse endometrium, early pregnancy human decidual tissue and other endometrial tissue sources have been constructed in succession. Recently, endometrial organoids containing two types of endometrial cells have also been constructed. However, in addition to a large number of epithelial cells and stromal cells, endometrial tissue also contains a large number of vascular structures. Therefore, the existing established endometrial organoids are mainly epithelial or epithelial-stromal organoids, and the cell types are single. SUMMARY

[0003] The technical problem to be solved by the present application is to provide a method for constructing a biomimetic endometrial organoid and a method for constructing an intrauterine adhesion disease model to overcome the shortcomings of the prior art.

[0004] The technical solution of the present application to solve the above technical problem is as follows: a method for constructing a biomimetic endometrial organoid, comprising the following steps:

[0005] Step S01: obtaining three types of primary cells, the three types of primary cells including primary endometrial epithelial cells, primary stromal cells and primary umbilical vein vascular endothelial cells;

[0006] Step S02: mixing the three types of primary cells, removing the supernatant after centrifugation, and obtaining a mixed cell precipitate;

[0007] Step S03: adding the mixed cell precipitate to a biomimetic organoid culture medium;

[0008] Step S04: resuspending the mixed cell precipitate in the biomimetic organoid culture medium to obtain a cell suspension, and precooling the cell suspension on ice for standby;

[0009] Step S05: After adjusting the cell concentration in step S04, a low concentration of Matrigel is added to the pre-cooled standby cell suspension, and the Matrigel is fully mixed in the cell suspension;

[0010] Step S06: The mixed cell suspension in step S05 is inoculated into a low-temperature pretreated porous low-adhesion carrier, and is placed in a low-temperature environment, and then is transferred to a constant-temperature cell incubator at physiological temperature for culture, to obtain a biomimetic endometrial organoid.

[0011] The beneficial effects of the present application are: based on endometrial epithelial cells, endometrial stromal cells, and vascular endothelial cells, the present application constructs a biomimetic endometrial organoid based on three types of cells through 3D culture of various types of cells. The endometrial organoid is a dense spherical structure containing epithelial cells, stromal cells and endothelial cells, and has the polarity of stromal cells wrapping epithelial cells. In terms of endometrial damage model repair and regeneration, the regenerative effect of the biomimetic endometrial organoid is significantly better than that of the stromal cell and epithelial organoid groups, and the biomimetic endometrial organoid can achieve endometrial thickness and embryo implantation number comparable to the normal control group after two weeks of repair, proving the great application value of the biomimetic endometrial organoid in tissue regeneration.

[0012] On the basis of the above technical solution, the present application can also be improved as follows.

[0013] Further, the method for obtaining the three types of primary cells in step S01 includes the following steps:

[0014] Step S11: Collecting human endometrial tissue samples and umbilical cord samples from the clinic, wherein the human endometrial samples are derived from normal endometrial tissue obtained by hysteroscopy biopsy, or normal endometrial tissue obtained by total hysterectomy due to cervical disease; the umbilical cord sample is derived from discarded umbilical cord tissue of full-term pregnancy;

[0015] Step S12: Digesting the clinically collected human endometrial tissue to obtain primary endometrial epithelial cells and primary stromal cells; digesting the endometrium of the umbilical cord tissue to obtain primary umbilical vein endothelial cells.

[0016] Further, the mixing ratio of the three types of primary cells in step S02 is specifically: mixing according to the cell ratio 2:2:1.

[0017] Further, the centrifugation in step S02 is specifically: under the action of a centrifugal force of 500g, centrifuging for 5min.

[0018] Further, step S03 is specifically: adding 5% fetal bovine serum FBS and 1% endothelial cell growth factor ECGS to the endometrial epithelial organoid expansion medium ExM, mixing uniformly to obtain a biomimetic organoid culture medium, and adding the mixed cell precipitate to the biomimetic organoid culture medium.

[0019] Further, step S05 specifically includes the following steps:

[0020] Step S51: thawing Matrigel at a temperature of 4°C;

[0021] Step S52: using a pre-cooled gun head to suck the thawed Matrigel and add it to the pre-cooled cell suspension prepared in step S04;

[0022] Step S53: repeatedly pipetting the Matrigel in the cell suspension to fully mix the Matrigel in the cell suspension.

[0023] Further, the Matrigel in step S52 is added at a volume concentration of 1% or 2%.

[0024] Further, step S06 is specifically:

[0025] The cell suspension mixed in step S05 is immediately inoculated into a 4°C pre-cooled 96-well low-adhesion U-shaped plate at a volume of 200ul / well, and quickly transferred to a 4°C refrigerator for cooling for 0.5-1 hours, and then transferred to a 37°C constant-temperature cell culture box for culture, to obtain a biomimetic endometrial organoid.

[0026] The application also discloses a method for constructing an intrauterine adhesion disease model of a biomimetic endometrial organoid, based on the biomimetic endometrial organoid obtained by the method for constructing a biomimetic endometrial organoid, and comprising the following steps:

[0027] Step H01: adding a fibrosis inducer TGFβ1 on the 7th day of formation of the biomimetic endometrial organoid;

[0028] Step H02: performing immunofluorescence staining of the fibrosis marker αSMA after 5 days of induction by the fibrosis inducer TGFβ1, to obtain an intrauterine adhesion disease model.

[0029] The application has the beneficial effect that the fibrosis-induced endometrial organoid model reproduces the pathological changes of the intrauterine adhesion endometrium, and can be used as an intrauterine adhesion pathological model for disease pathology research or drug screening research. BRIEF DESCRIPTION OF DRAWINGS

[0030] Fig. 1 Characterization chart of the biomimetic endometrial organoid of the application;

[0031] Fig. 2 Application effect diagram of the application of the biomimetic endometrium organoid in endometrium regeneration treatment;

[0032] Fig. 3 Fibrosis characterization diagram of the application of the biomimetic endometrium organoid in intrauterine adhesion disease model. DETAILED DESCRIPTION

[0033] The principles and features of the present application are described below in conjunction with the accompanying drawings, and the examples are only used to explain the present application and are not used to limit the scope of the present application.

[0034] As shown in Figs. 1-3 Example 1, a method for constructing a biomimetic endometrium organoid, comprising the following steps:

[0035] Step S01: Obtain three kinds of primary cells, including primary endometrial epithelial cells, primary stromal cells and primary umbilical vein endothelial cells;

[0036] Step S02: Mix the three kinds of primary cells, remove the supernatant after centrifugation, and obtain a mixed cell precipitate;

[0037] Step S03: Add the mixed cell precipitate to the biomimetic organoid culture medium;

[0038] Step S04: Resuspend the mixed cell precipitate in the biomimetic organoid culture medium to obtain a cell suspension. In a specific implementation, the cell concentration in the cell suspension is adjusted to 5x10 4 / ml, the cell concentration can be appropriately increased to obtain a larger volume of organoid, but the highest concentration does not exceed 25x10 4 / ml, and the cell suspension is pre-cooled on ice for standby;

[0039] Step S05: After adjusting the cell concentration in step S04, add a low concentration of Matrigel to the pre-cooled standby cell suspension, and mix the Matrigel thoroughly in the cell suspension;

[0040] Step S06: Seed the mixed cell suspension in step S05 into a low-temperature pretreated multi-well low-adhesion carrier, stand in a low-temperature environment to prevent Matrigel from solidifying, and then transfer to a constant-temperature cell culture incubator under physiological temperature conditions for culture, to obtain a biomimetic endometrium organoid.

[0041] The assembly state of the organoids can be observed under a microscope after 24 hours; in a specific implementation, the culture medium is replaced every two days, and it is observed under a light microscope that the organoids can self-assemble into a dense spherical structure on the fourth day, and the density is higher on the seventh day. Immunofluorescence staining results show that the organoids contain epithelial cells (Krt8 positive and E-cadherin positive), stromal cells (Vimentin positive), and vascular endothelial cells (CD31 positive), and the cells exhibit a polar distribution of stromal cells wrapping epithelial cells. Fig. 1

[0042] The present application is based on 3D culture of various combinations of endometrial epithelial cells, endometrial stromal cells, and vascular endothelial cells, and constructs a biomimetic endometrial organoid based on the three cell types. The endometrial organoid is a dense spherical structure containing epithelial cells, stromal cells, and endothelial cells, and has a polarity of stromal cells wrapping epithelial cells. The regenerative effect of transplanting the biomimetic endometrial organoid is significantly better than that of transplanting pure stromal cells and pure epithelial organoids. After two weeks of repair, the endometrial organoid transplanted with the biomimetic cells can achieve an endometrial thickness and embryo implantation number comparable to the normal control group, demonstrating the great application value of the biomimetic endometrial organoid in tissue regeneration. Meanwhile, after treatment with a fibrosis-inducing factor (TGFβ1), the expression of the fibrosis marker alpha-SMA in the endometrial organoid significantly increases, indicating that the fibrosis-induced endometrial organoid model can reproduce the pathological changes of the endometrium in adhesions, and has potential application value in disease pathology research or drug screening as an adhesions pathological model.

[0043] Embodiment 2, which is a further improvement based on Embodiment 1, is as follows:

[0044] The method for obtaining the three types of primary cells in step S01 includes the following steps

[0045] Step S11: Collecting human endometrial tissue samples and umbilical cord samples from clinics, wherein the human endometrial samples are derived from normal endometrial tissue obtained by hysteroscopic biopsy or normal endometrial tissue obtained by total hysterectomy due to cervical diseases; the umbilical cord sample is derived from discarded umbilical cord tissue of full-term pregnancy;

[0046] Step S12: Digesting the clinically collected human endometrial tissue to obtain primary endometrial epithelial cells and primary stromal cells; digesting the endometrium of the umbilical cord tissue to obtain primary umbilical vein vascular endothelial cells.

[0047] Embodiment 3, which is a further improvement based on Embodiment 1, is as follows:​

[0048] The specific mixing ratio of the three primary cells in step S02 is 2:2:1. Under this mixing ratio, the organoids exhibit better spheroidization.

[0049] Example 4 is a further improvement based on Example 1, and its details are as follows:

[0050] The centrifugation in step S02 specifically involves centrifuging for 5 minutes under a centrifugal force of 500g.

[0051] Example 5 is a further improvement based on Example 1, and its details are as follows:

[0052] Step S03 specifically involves adding 5% fetal bovine serum (FBS) and 1% endothelial growth factor (ECGS) to the endometrial epithelial organoid amplification medium ExM, mixing them thoroughly to obtain the biomimetic organoid culture medium, and then adding the mixed cell precipitate to the biomimetic organoid culture medium.

[0053] Example 6 is a further improvement on Example 1, and its details are as follows:

[0054] Step S05 specifically includes the following steps:

[0055] Step S51: Melt Matrigel at a temperature of 4°C;

[0056] Step S52: Use a pre-cooled pipette tip to draw up the melted Matrigel and add it to the pre-cooled cell suspension prepared in step S04;

[0057] Step S53: Use a pipette to repeatedly pipette and mix Matrigel thoroughly in the cell suspension.

[0058] In practice, step S05 is performed entirely on ice to prevent Matrigel from gelling during the process.

[0059] Example 7 is a further improvement based on Example 6, and its details are as follows:

[0060] In step S52, Matrigel is added at a volume concentration (v / v) of 1% or 2%. This concentration yields the best organoid spheroidization effect.

[0061] Example 8 is a further improvement based on Example 1, and its details are as follows:

[0062] Step S06 is as follows:

[0063] The cell suspension mixed in step S05 is immediately inoculated into a 4℃ pre-cooled 96-well low-adhesion U-shaped plate at a volume of 200ul / well, and quickly transferred to a 4℃ refrigerator for cooling for 0.5-1 hour, and then transferred to a 37℃ constant-temperature cell incubator for culture, to obtain the biomimetic endometrial organoids.

[0064] An endometrial injury model is constructed, and the biomimetic endometrial organoids loaded with Matrigel are implanted into the endometrial injury site, and the repair effect is observed after two weeks of repair. The control groups include a normal control group (Sham), a simple injury group (Injury), a stromal cell group implanted after injury (Stromal), an epithelial organoid group implanted after injury (Epithelial), and an endometrial organoid group of the application implanted after injury (Organoids). The results show that the endometrial repair effect of the endometrial organoid group of the application is the best, and the endometrial thickness and the number of embryo pregnancies are comparable to those of the normal control group. Fig. 2 ).

[0065] Example 9, a method for constructing an intrauterine adhesion disease model of a biomimetic endometrial organoid, based on the method for constructing a biomimetic endometrial organoid of any one of examples 1 to 8, the biomimetic endometrial organoid obtained by the method comprises the following steps:

[0066] Step H01: adding a fibrosis inducer TGFβ1 on the 7th day of the formation of the biomimetic endometrial organoid;

[0067] Step H02: performing immunofluorescence staining of the fibrosis marker αSMA after 5 days of induction by the fibrosis inducer TGFβ1, to obtain an intrauterine adhesion disease model.

[0068] The results show that the expression of the fibrosis marker αSMA of the organoids in the fibrosis inducer treatment group is significantly higher than that in the control group without fibrosis inducer treatment ( Fig. 3 ), indicating that the fibrosis-induced endometrial organoid model can reproduce the pathological changes of the intrauterine adhesion endometrium, and can be used as an intrauterine adhesion pathological model for disease pathology research or drug screening research.

[0069] Although the embodiments of the application have been shown and described above, it should be understood that the above embodiments are exemplary and should not be construed as limiting the application, and those of ordinary skill in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the application.

Claims

1. A method for constructing a biomimetic endometrial organoid, characterized in that, Includes the following steps: Step S01: Obtain three types of primary cells, including primary endometrial epithelial cells, primary stromal cells, and primary umbilical vein endothelial cells; Step S02: Mix the three types of primary cells, centrifuge and remove the supernatant to obtain a mixed cell precipitate; Step S03: Add the mixed cell precipitate to the biomimetic organoid culture medium; Step S04: Resuspend the mixed cell precipitate in the biomimetic organoid culture medium to obtain a cell suspension, and pre-cool the cell suspension on ice for later use; Step S05: After adjusting the cell concentration in step S04, add a low concentration of Matrigel to the pre-cooled cell suspension and mix Matrigel thoroughly in the cell suspension. Step S06: The cell suspension mixed in step S05 is seeded into a porous low-adhesion carrier that has been pretreated at low temperature, placed in a low-temperature environment, and then transferred to a constant temperature cell culture incubator under physiological temperature conditions to obtain a biomimetic endometrial organoid.

2. The method for constructing a biomimetic endometrial organoid according to claim 1, characterized in that, The three methods for obtaining the primary cells in step S01 include the following steps: Step S11: Collect human endometrial tissue samples and umbilical cord samples from the clinic. The human endometrial samples are obtained from normal endometrial tissue obtained by hysteroscopic biopsy or normal endometrial tissue from hysterectomy due to cervical disease. The umbilical cord sample was obtained from discarded umbilical cord tissue from a full-term pregnancy; Step S12: The clinically collected human endometrial tissue is digested to obtain primary endometrial epithelial cells and primary stromal cells; the umbilical cord tissue endometrium is digested to obtain primary umbilical vein endothelial cells.

3. The method for constructing a biomimetic endometrial organoid according to claim 1, characterized in that, The specific mixing ratio of the three primary cells in step S02 is as follows: a cell ratio of 2:2:

1.

4. The method for constructing a biomimetic endometrial organoid according to claim 1, characterized in that, The centrifugation in step S02 specifically involves centrifuging for 5 minutes under a centrifugal force of 500g.

5. The method for constructing a biomimetic endometrial organoid according to claim 1, characterized in that, Specifically, step S03 involves adding 5% fetal bovine serum (FBS) and 1% endothelial growth factor (ECGS) to the endometrial epithelial organoid amplification culture medium ExM, mixing them thoroughly to obtain the biomimetic organoid culture medium, and then adding the mixed cell precipitate to the biomimetic organoid culture medium.

6. The method for constructing a biomimetic endometrial organoid according to claim 1, characterized in that, Step S05 specifically includes the following steps: Step S51: Melt Matrigel at a temperature of 4°C; Step S52: Use a pre-cooled pipette tip to draw up the melted Matrigel and add it to the pre-cooled cell suspension prepared in step S04; Step S53: Use a pipette to repeatedly pipette and mix Matrigel thoroughly in the cell suspension.

7. The method for constructing a biomimetic endometrial organoid according to claim 6, characterized in that, In step S52, Matrigel is added at a volume concentration of 1% or 2%.

8. The method for constructing a biomimetic endometrial organoid according to claim 1, characterized in that, Step S06 specifically involves: The cell suspension, after being mixed in step S05, was immediately seeded at a volume of 200 μL / well into 96-well low-adhesion U-shaped plates pre-cooled at 4°C, and then quickly transferred to a 4°C refrigerator to cool for 0.5-1 hour, and then transferred to a 37°C constant temperature cell culture incubator to obtain biomimetic endometrial organoids.

9. A method for constructing a biomimetic endometrial organoid model of intrauterine adhesions, characterized in that, The biomimetic endometrial organoid obtained by the construction method of any one of claims 1 to 8 includes the following steps: Step H01: Add fibrosis-inducing factor TGFβ1 on day 7 of the formation of the biomimetic endometrial organoids; Step H02: Five days after induction with the fibrosis-inducing factor TGFβ1, immunofluorescence staining of the fibrosis marker αSMA was performed to obtain the intrauterine adhesion disease model.

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