Menstrual blood organoids and uses thereof

Abstract

The application discloses a menstrual blood organoid and application thereof, and belongs to the technical field of organoid construction. The menstrual blood organoid is preserved in the China Center for Type Culture Collection, is named as human menstrual blood organoid MFO-Jennifer (Homo sapiens), has a preservation number of CCTCC NO: C2024133, and is preserved on September 25, 2024. The menstrual blood organoid is applied to construction of an embryo implantation model, construction of an individualized drug screening model, and construction of a receptivity test model of in vitro fertilization-embryo transfer. The application establishes a method for inducing the menstrual blood organoid to an implantation window stage, and provides a powerful research tool for studying changes of endometrium during embryo implantation and interaction of a maternal-fetal interface.

Description

TECHNICAL FIELD

[0001] The present application relates to a menstrual blood organoid and its application, and belongs to the field of organoid construction. BACKGROUND

[0002] An organoid is a 3D structure self-assembled by adult stem cells or pluripotent stem cells, which is very similar to in vivo tissues or organs in structure and function, can be continuously and stably subcultured in vitro, and has important potential in the fields of developmental biology, regenerative medicine, precision medicine, etc.

[0003] In 2021, Margherita Y. Turco et al. pioneered a method of constructing endometrial organoids using menstrual blood, but the menstrual blood organoids constructed by them can only simulate the morphological structure, hormone responsiveness and other functions of the in vivo endometrium, and cannot completely simulate the uterine endometrial microenvironment. Moreover, after hormone treatment, the organoids are only induced to the proliferation phase and the secretion phase, and cannot completely simulate the state of the uterine endometrium during embryo implantation, and cannot study the changes of the endometrium during the embryo implantation stage. SUMMARY

[0004] In view of the above prior art, the present application provides a menstrual blood organoid and its application, which belongs to the technical field of organoid construction.

[0005] The present application is realized by the following technical solutions:

[0006] A menstrual blood organoid, which is preserved in the China Center for Type Culture Collection, named Homo sapiens menstrual blood organoid MFO-Jennifer (Homo sapiens), with the preservation number CCTCC NO: C2024133 and the preservation date September 25, 2024.

[0007] Application of the menstrual blood organoid in constructing an embryo implantation model.

[0008] Application of the menstrual blood organoid in constructing an individualized drug screening model.

[0009] Application of the menstrual blood organoid in constructing a receptivity test model for in vitro fertilization-embryo transfer.

[0010] The application optimizes the construction method when constructing menstrual blood organoids, improves the digestion solution formula when digesting the tissue into single cells, adds DNA enzyme to ensure the digestion effect, and retains as many endometrial microenvironment cells as possible. At the same time, after the tissue is digested into single cells, the mixture is passed through a 40 μm cell screen to retain as many stromal cells as possible, and the constructed menstrual blood organoids contain stromal cells, immune cells and the like, which better simulate the in vivo endometrial environment. The application also establishes a method for inducing menstrual blood organoids to the implantation window stage, and the receptivity marker expression of the menstrual blood organoids at the implantation window stage is increased, which is beneficial to the study of the changes of endometrium during embryo implantation and also beneficial to the embryo implantation model to answer early embryo development and the like, thereby providing a powerful research tool for the study of endometrial changes during embryo implantation and the interaction of the maternal-fetal interface. At the same time, the menstrual blood organoid preserved by the application has obvious advantages in cell activity, passage number and receptivity to embryo implantation compared with other menstrual blood organoids constructed by the same method.

[0011] As a non-invasive source of culture, menstrual blood organoids contain stem cells and are similar to endometrial structures, can be used for injury repair of endometrial disease patients, and can be autologously transplanted into patients with endometrial diseases such as intrauterine adhesion and thin endometrium, thereby repairing damaged endometrium and restoring fertility. Menstrual blood organoids can also be used as a model for embryo implantation to study key events of early embryo development and explore the biological processes of the maternal-fetal interface. Menstrual blood organoids can also be used as a model for high-throughput and individualized drug screening. Menstrual blood organoids can also be used as a model for receptivity testing. In the process of assisted reproduction, the success of in vitro fertilization-embryo transfer requires that the endometrium after hormone treatment and the implanted embryo have synchronous development, at which time the receptivity of the endometrium to the embryo is called receptivity; menstrual blood organoids can be used as a model for receptivity testing to individually detect which hormone treatment scheme can achieve the best effect of receptivity, thereby improving the success rate of assisted reproductive technology, improving the outcome of transplantation and pregnancy. The research of the application lays a foundation for the clinical treatment and clinical transformation of endometrial diseases.

[0012] Various terms and phrases used in the application have the general meanings known to those skilled in the art. BRIEF DESCRIPTION OF DRAWINGS

[0013] The menstrual blood organoids of the application are preserved in the China Center for Type Culture Collection, named Homo sapiens menstrual blood organoids MFO-Jennifer (Homo sapiens), with a preservation number of CCTCC NO: C2024133, a preservation date of September 25, 2024, and a preservation address of Wuhan University, No. 8th of August Road, Wuchang District, Wuhan City, Hubei Province, China, with a postcode of 430072.

[0014] Figure 1 : Comparative photographs of 4 menstrual blood organoids, wherein, from top to bottom, the first row is a photograph at day 8 of P0 generation culture, the second row is a photograph at day 8 of P1 generation culture, the third row is a photograph at day 8 of P3 generation culture, and the fourth row is a photograph at day 7 of P7 generation culture; from left to right, the first column is a photograph of menstrual blood organoid 1, the second column is a photograph of menstrual blood organoid 2, the third column is a photograph of menstrual blood organoid 3, and the fourth column is a photograph of menstrual blood organoid 4.

[0015] Figure 2 : Photograph of menstrual blood organoid 1.

[0016] Figure 3 : Immunofluorescence detection results of epithelial cells and stromal cells, wherein the cell nucleus is labeled with DAPI.

[0017] Figure 4 : Immunofluorescence detection results of epithelial cell marker E-cadherin.

[0018] Figure 5 : Immunofluorescence detection results of stromal cell marker vimentin.

[0019] Figure 6 : Figure 3 , Figure 4 , Figure 5 : Superimposed display.

[0020] Figure 7 : Immunofluorescence detection results of luminal epithelial cells and glandular epithelial cells, wherein the cell nucleus is labeled with DAPI.

[0021] Figure 8 : Immunofluorescence detection results of luminal epithelial marker Wnt-7a.

[0022] Figure 9 : Immunofluorescence detection results of glandular epithelial marker FOXA2.

[0023] Figure 10 : Figure 7 , Figure 8 , Figure 9 : Superimposed display.

[0024] Figure 11 : Bright field photograph of embryo-menstrual blood organoid co-culture system at day 6 after fertilization.

[0025] Figure 12 : Immunofluorescence detection results of OCT4.

[0026] Figure 13 : Immunofluorescence detection results of SOX17.

[0027] Figure 14 : Immunofluorescence detection results of GATA3.

[0028] Figure 15 : Figure 12 , Figure 13 , Figure 14 : Superimposed presentation of the photographs, wherein d.p.f. 9 represents day 9 post-fertilization, the dashed line above represents menstrual blood organoids structure, the white arrow above indicates menstrual blood organ epithelium, and the white arrow below indicates embryonic hypoblast structure.

[0029] Figure 16 : Photographs of menstrual blood organoids co-cultured with embryos for 7, 8, 9 and 11 days, wherein A: photograph at 7 days; B: photograph at 8 days; C: photograph at 9 days; D: photograph at 11 days. The arrow indicates the embryo.

[0030] Figure 17 : Photographs of endometrial organoids 1 co-cultured with embryos for 7, 8, 9 and 11 days, wherein A: photograph at 7 days; B: photograph at 8 days; C: photograph at 9 days; D: photograph at 11 days. The arrow indicates the embryo.

[0031] Figure 18 : Photographs of endometrial organoids 2 co-cultured with embryos for 7, 8, 9 and 11 days, wherein A: photograph at 7 days; B: photograph at 8 days; C: photograph at 9 days; D: photograph at 11 days. The arrow indicates the embryo. DETAILED DESCRIPTION

[0032] The application will be further described in conjunction with the examples below. However, the scope of the application is not limited to the examples below. Those skilled in the art can understand that various changes and modifications can be made to the application without departing from the spirit and scope of the application.

[0033] The instruments, reagents, materials and the like involved in the following examples are all conventional instruments, reagents, materials and the like existing in the prior art if not specifically stated, and can be obtained through regular commercial channels. The experimental methods, detection methods and the like involved in the following examples are all conventional experimental methods, detection methods and the like existing in the prior art if not specifically stated.

[0034] Example 1 Construction of menstrual blood organoids

[0035] The steps are as follows:

[0036] (I) Establishment of menstrual blood organoids

[0037] (1) Washing of blood cells

[0038] The menstrual blood was centrifuged (4°C, 400 g, 10 min) to discard the supernatant blood cells; the pre-cooled DPBS added with 1% antibiotic-antimycotic (Gibco 15240062) was used to wash 4 times, and the supernatant was discarded after each washing, and only the cell components were reserved; after the washing, the cell screen with a pore size of 100 μm was inverted on a culture dish, and the pre-cooled DPBS was used to backwash the screen to wash the tissue fragments on the screen into the culture dish.

[0039] The menstrual blood was collected by using a high-temperature sterilized menstrual cup on the 1st to 2nd day of the heaviest menstrual flow, and the total amount was not less than 7 ml. After the collection, the menstrual blood was placed in a sterile centrifuge tube and stored at 4°C temporarily, and was transported on ice to the laboratory.

[0040] (2) Digesting the tissue fragments into single cells

[0041] The tissue collected in the culture dish was cut into small pieces, and the cut tissue was transferred into the digestion solution and was digested at 37°C under the condition of rotary shaking for 30 min; after the digestion, an equal volume of neutralization medium was added to prevent further digestion, and a mixed solution was obtained.

[0042] The digestion solution was composed of the following components: water; collagenase V, 0.4 mg / mL; dispase II, 1.5 U / mL; deoxyribonuclease I (DNase I), 15 μg / mL.

[0043] During the digestion, the rotation mode of the incubation container was 15 rpm, the shaking mode was 1 rpm, and the mixture was blown and mixed every 10 min to make it more uniform.

[0044] The neutralization medium was DMEM / F12 medium added with 10% FBS and 1% antibiotic-antimycotic.

[0045] (3) Model establishment

[0046] The mixed solution was vortexed fully, and was left to stand for 1 min to precipitate the undigested tissue fragments; the supernatant was passed through a cell screen with a pore size of 40 μm to retain as many cells in the endometrial microenvironment as possible, such as stromal cells and immune cells; the cell screen was inverted on a culture dish, and the pre-cooled DPBS was used to backwash the screen to collect the cell clusters, and a cell suspension was obtained.

[0047] The cell suspension is centrifuged (400 g, 5 min), the cell pellet is resuspended with DMEM / F12 medium, centrifuged again (400 g, 5 min), the cell pellet is resuspended with DMEM / F12 medium, mixed with Matrigel without phenol red, and plated at a volume ratio of DMEM / F12 medium to Matrigel of 1:3, and incubated in an incubator at 37℃, 5% CO2 for more than 30 min to solidify the Matrigel.

[0048] After the Matrigel is solidified, the cell plate is plated, the endometrial organ culture medium is added, and the cell plate is placed in an incubator at 37℃, 5% CO2, and the medium is changed every 2 days, and the cells are subcultured at 8 days.

[0049] The endometrial organ culture medium is DMEM / F12 medium added with 1X B-27, 1X N-2, 1X ITS-G, 2 mM glutamine supplement, 1.25 mM N-acetyl-L-cysteine, 1 mM nicotinamide, 200 ng / mL human Wnt-3a recombinant protein, 200 ng / mL human R-spondin 1 recombinant protein, 50 ng / mL human EGF recombinant protein, 100 ng / mL human FGF2 recombinant protein, 100 ng / mL human Noggin recombinant protein, 0.5 μM A 83-01, 10 μM SB 202190, 1% antibiotic-antimycotic.

[0050] (4) Results

[0051] Using the above method, menstrual blood of 4 volunteers was collected, and 4 strains of menstrual blood organoids (temporarily named menstrual blood organoid 1, menstrual blood organoid 2, menstrual blood organoid 3, and menstrual blood organoid 4, respectively) were constructed and subcultured; bright field observation was performed under a microscope, and the states of the 4 strains of menstrual blood organoids at the same subculture generation and the same culture time were compared.

[0052] The comparison photos of the 4 strains of menstrual blood organoids are shown in Figure 1 As can be seen from the comparison, the proliferation activity of menstrual blood organoid 1 is the strongest, and it can be stably subcultured to P7 with good cell growth state; menstrual blood organoid 2 has poor cell viability when subcultured to P3, and cannot be subcultured; menstrual blood organoid 3 has poor cell viability when subcultured to P1, and cannot be subcultured; menstrual blood organoid 4 has poor cell viability when primary cultured, and cannot be subcultured.

[0053] The volunteer 1 (the volunteer of menstrual blood organoid 1) donated menstrual blood 3 times again, and the 3 strains of menstrual blood organoids constructed therefrom can be stably subcultured to P7 with good cell viability. At the same time, a number of parallel tests were also performed using menstrual blood of other volunteers, but all of them could not be stably subcultured.

[0054] The above results show that only the menstrual blood organoids constructed from the menstrual blood of volunteer 1 can be stably passaged, and have obvious advantages in cell activity and passage number. It is speculated that the reason is that the menstrual blood of this volunteer is special (compared with ordinary menstrual blood, some unknown and unpredictable changes may have occurred). Therefore, the menstrual blood organoid 1 is preserved in the present application, and is preserved in the China Center for Type Culture Collection, named as human menstrual blood organoid MFO_Jennifer (Homosapiens), with a preservation number of CCTCC NO: C2024133, a preservation date of September 25, 2024, and a preservation address of Wuhan University, Wuhan, Hubei, China, with a postcode of 430072.

[0055] After the menstrual blood organoid 1 is assembled (on the 12th day of P0 generation culture), bright field observation is performed under a microscope, and the photo of the menstrual blood organoid 1 is as shown in Figure 2 It can be seen that the menstrual blood organoid has a cystic structure, and is in the form of a hollow spherical structure surrounded by epithelial cells, and most of the organoids have a diameter of 200 μm.

[0056] (II) Hormone treatment to make the menstrual blood organoid enter the implantation window stage

[0057] After the menstrual blood organoid 1 is assembled in (I), the menstrual blood organoid 1 is cultured in the endometrial organoid culture medium added with 10 nM β-estradiol for 2 days, and then cultured in the endometrial organoid culture medium added with 1 μM medroxyprogesterone acetate, 1 μM dibutyryl CAMP, 1 μg / mL human chorionic gonadotropin, 20 ng / mL human placental prolactin, and 20 ng / mL prolactin for 6 days, so as to induce the menstrual blood organoid 1 to enter a stage beneficial to embryo implantation and implantation, which is called an implantation window stage.

[0058] (III) Immunofluorescence identification of the basic structure of the menstrual blood organoid

[0059] The steps are as follows:

[0060] (1) The menstrual blood organoid 1 cultured for 12 days is fixed in 4% PFA (polyformaldehyde solution) at room temperature for 30 min, washed with PBS for three times, dehydrated in a 20% sucrose solution at 4°C overnight, embedded with OCT, and cut into 10 μm-thick sections by using a freezing microtome.

[0061] (2) The frozen section was restored to room temperature, fixed with 4% PFA for 5 min, and permeated with PBS containing 0.3% Triton X-100 for 20 min. Antigen repair was performed with sodium citrate at 95°C for 20 min. After being restored to room temperature, the section was blocked in immunostaining blocking buffer (Beyotime, P0260) for 20 min.

[0062] (3) The section was incubated with the primary antibody (mouse anti-E-cadherin antibody, rabbit anti-Vimentin antibody, rabbit anti-FOXA2 antibody, mouse anti-Wnt-7a antibody) at 4°C overnight, and then washed with PBS containing 0.1% Triton X-100 for 3 times. The secondary antibody (donkey anti-mouse, donkey anti-rabbit) was incubated at room temperature for 2 hours, washed with PBS containing 0.1% Triton X-100 for 3 times. DAPI (Beyotime, C1002) was incubated for 15 min, and the section was mounted with an anti-fluorescence quencher.

[0063] (4) Images were collected by a confocal laser scanning microscope (Andor Dragonfly 200), and processed by Imaris x649.0.1.

[0064] The images of identifying epithelial cells and stromal cells in menstrual blood organoids are shown in FIG. 1, wherein the immunofluorescence detection results of epithelial cells and stromal cells are shown in FIG. 2, the immunofluorescence detection results of epithelial cell marker E-cadherin are shown in FIG. 3, the immunofluorescence detection results of stromal cell marker Vimentin are shown in FIG. 4, and the superimposed display of FIGS. 3 and 4 is shown in FIG. 5. Figures 3 to 6 Figure 3 Figure 4 Figure 5 Figure 3 Figure 4 Figure 5 Figure 6 Figures 3 to 6 As shown in FIG. 5, the menstrual blood organoids expressed E-cadherin and Vimentin, indicating that the menstrual blood organoids contained endometrial epithelial cells and stromal cells.

[0065] The images of identifying luminal epithelial cells and glandular epithelial cells in menstrual blood organoids are shown in FIG. 6, wherein the immunofluorescence detection results of luminal epithelial cells and glandular epithelial cells are shown in FIG. 7, the immunofluorescence detection results of luminal epithelial cell marker Wnt-7a are shown in FIG. 8, the immunofluorescence detection results of glandular epithelial cell marker FOXA2 are shown in FIG. 9, and the superimposed display of FIGS. 8 and 9 is shown in FIG. 10. Figures 7 to 10 Figure 7 Figure 8 Figure 9 Figure 7 Figure 8 Figure 9 Figure 10 Figures 7 to 10 ​​​​​​​​​​​​​​​​It can be seen that the menstrual blood organoid expresses the luminal epithelial marker Wnt-7a and the glandular epithelial marker FOXA2, indicating that the menstrual blood organoid contains two types of epithelial cells of the endometrium.

[0066] Example 2 Menstrual blood organoid as a model of embryo implantation

[0067] Example 1 constructed a menstrual blood organoid induced to the implantation window stage. The menstrual blood organoid was co-cultured with embryos to study the key events of early embryo development during the peri-implantation period, and endometrial organoids induced from endometrial samples obtained from uterine surgery for uterine benign disease (hereinafter referred to as endometrial organoids) were used as controls (for construction method, refer to literature PMC5410172). There were 2 controls, namely endometrial organoid 1 and endometrial organoid 2.

[0068] The donated frozen human blastocyst on the fifth day after fertilization was thawed using a vitrification kit (Kitazato, VT602), and the blastocyst was directly transferred from liquid nitrogen to 1 ml of preheated embryo thawing solution using a pipette. After 2 minutes, the blastocyst was transferred to the following solutions: dilution solution (room temperature, 3 minutes), and washing solution (room temperature, 5 minutes). Subsequently, the embryo was placed in a small amount of embryo culture medium and mechanically dissected with a glass needle to remove the zona pellucida. The embryo was cultured in G-2 PLUS medium (Vitrolife, 10232) and coated with oil (Vitrolife, 10029), and incubated at 37°C, 5% O2 and 6% CO2 for at least 2 hours until the embryo recovered.

[0069] The recovered embryo was implanted on the organoid, ensuring that the polar trophoblast cells were attached to the side of the organoid, and cultured at 37°C, 6% CO2. After two days of culture using HIVC1 medium (human embryo in vitro culture medium 1), HIVC2 medium (human embryo in vitro culture medium 2) was used for replacement, and the experiment was terminated on the 14th day after fertilization.

[0070] Bright field photographs of the embryo-menstrual blood organoid co-culture system on the 6th day after fertilization are shown in Figure 11 It can be seen that the embryo on the 6th day after fertilization is attached to the menstrual blood organoid with the inner cell mass side, and the embryo expands in shape without obvious signs of cell death during development.

[0071] On the 9th day after fertilization, the embryo-menstrual blood organoid co-culture system was identified by immunofluorescence to identify the lineage development of the embryo. SOX17 was used to mark the glandular epithelium of the endometrial organoid and the hypoblast structure of the embryo, OCT4 was used to mark the epiblast structure of the embryo, and GATA3 was used to mark the trophoblast cells of the embryo.

[0072] The results of immunofluorescence identification on the 9th day after fertilization are shown in Figures 12 to 15As shown, the immunofluorescence detection results of OCT4 are as follows: Figure 12 As shown, the immunofluorescence detection results of SOX17 are as follows: Figure 13 As shown, the immunofluorescence detection results of GATA3 are as follows: Figure 14 As shown, Figure 12 , Figure 13 , Figure 14 Overlay display such as Figure 15 As shown. By Figures 12 to 15 It was observed that in the embryo-menstrual blood organoid co-culture system on day 9 post-fertilization, endometrial organoid glandular epithelium and embryonic hypodermal markers SOX17, OCT4, and trophoblast cell marker GATA3 were expressed, indicating that after co-culture with menstrual blood organoids, the embryo developed a double germinal disc structure and achieved differentiation of the three lineages (epidermis, hypodermis, and trophoblast). Under ethically permissible conditions, menstrual blood organoids can support embryonic development in vitro up to day 14 and achieve normal embryonic development and differentiation.

[0073] Photos of menstrual blood organoids co-cultured with embryos on days 7, 8, 9, and 11, as shown below. Figure 16 As shown. Photos of endometrial organoid 1 co-cultured with embryos on days 7, 8, 9, and 11 are shown. Figure 17 As shown. Photos of endometrial organoids 2 co-cultured with embryos on days 7, 8, 9, and 11 are shown. Figure 18 As shown, embryos in menstrual blood organoid co-culture exhibit expanded morphology and normal trophoblast development during development, while some embryos in endometrial organoid co-culture show slower trophoblast development.

[0074] In summary, the menstrual blood organoids constructed and preserved in this invention have a significant advantage in terms of embryo implantation receptivity compared to endometrial organoids induced from endometrial samples obtained by hysterectomy for benign uterine diseases. They are more conducive to embryonic development and growth around the implantation period and are more suitable for studying the physiological changes of the embryo and endometrium during the implantation stage.

[0075] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A menstrual blood organoid, characterized in that: The menstrual blood organoids are preserved in China Center for Type Culture Collection, named as Menstrual blood organoids MFO-Jennifer Homo sapiens, with the preservation number of CCTCC NO: C2024133, and the preservation date of September 25, 2024.

2. The menstrual blood organoids of claim 1 are applied to construct an embryo implantation model.

3. The menstrual blood organoids of claim 1 are applied to construct an individualized drug screening model.

4. The menstrual blood organoids of claim 1 are applied to construct a receptivity test model for in vitro fertilization-embryo transfer.

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