Menstrual blood organoid and application thereof

Abstract

The invention 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, and is named as human menstrual blood organoid MFO-Jennifer (Homo sapiens), the preservation number is CCTCC (China Center for Type Culture Collection) NO: C2024133, and the preservation date is September 25, 2024. The application of the menstrual blood organ in construction of an embryo implantation model, the application of the menstrual blood organ in construction of an individualized drug screening model and the application of the menstrual blood organ in construction of an in-vitro fertilization-embryo transplantation receptivity test model. The method for inducing the menstrual blood organoid to the implantation window stage is established, and a powerful research tool is provided for researching endometrial change and interaction of a maternal interface and a fetal interface in the embryo implantation period.

Description

Technical Field

[0001] The present invention relates to a menstrual blood organoid and its application, belonging to the field of organoid construction. Background Art

[0002] An organoid is a 3D structure self-assembled from 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 passaged 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 for constructing endometrial organoids using menstrual blood. Although the constructed menstrual blood organoids can simulate functions such as the morphological structure and hormone responsiveness of the in-vivo endometrium, they cannot fully simulate the endometrial microenvironment. Moreover, after hormone treatment, the organoids are only induced to the proliferative phase and secretory phase, and cannot fully simulate the state of the endometrium during embryo implantation, thus unable to study the endometrial changes during the embryo implantation stage. Summary of the Invention

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

[0005] The present invention is achieved through the following technical solutions: A menstrual blood organoid, which is preserved in the China Center for Type Culture Collection, named Homo sapiens menstrual blood organoid MFO-Jennifer, with the preservation number CCTCC NO: C2024133 and the preservation date September 25, 2024.

[0006] Application of the menstrual blood organoid in constructing an embryo implantation model.

[0007] Application of the menstrual blood organoid in constructing an individualized drug screening model.

[0008] Application of the menstrual blood organoid in constructing a receptivity test model for in vitro fertilization-embryo transfer.

[0009] In constructing menstrual blood organoids, the present invention optimized the construction method, improved the digestion solution formula for digesting tissues into single cells, added DNA enzyme to ensure the digestion effect, and retained as many endometrial microenvironment cells as possible. At the same time, after digesting the tissue into single cells, the mixture was passed through a 40-μm cell sieve to retain stromal cells as much as possible. The constructed menstrual blood organoids contain stromal cells, immune cells, etc., and better simulate the in-vivo endometrial environment. The present invention also established a method for inducing menstrual blood organoids to the implantation window stage. The expression of receptivity markers in menstrual blood organoids at the implantation window stage increased, which is not only conducive to studying the changes in the endometrium during embryo implantation, but also conducive to serving as a model for embryo implantation to answer questions such as early embryo development, providing a powerful research tool for studying the changes in the endometrium during embryo implantation and the interaction at the maternal-fetal interface. At the same time, compared with other menstrual blood organoids constructed by the same method, this strain of menstrual blood organoids preserved by the present invention has obvious advantages in cell activity, passage times, and receptivity to embryo implantation.

[0010] As a culture derived from a non-invasive source, menstrual blood organoids contain stem cells and are similar in structure to the endometrium. They can be used for the injury repair of endometrial disease patients and can be autotransplanted into patients with endometrial diseases such as intrauterine adhesions and thin endometrium to repair the damaged endometrium and restore fertility. Menstrual blood organoids can also be used as a model for embryo implantation to study the key events of early embryo development and explore the biological processes at 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. During assisted reproduction, the success of in vitro fertilization-embryo transfer requires the endometrium after hormone treatment to have synchronous development with the transplanted embryo. At this time, the ability of the endometrium to accept the embryo is called receptivity. Menstrual blood organoids can be used as a model for receptivity testing to individually detect which hormone treatment plan can achieve the best receptivity effect, so as to improve the success rate of assisted reproductive technology and improve the transplantation outcome and pregnancy outcome. The research of the present invention has laid a foundation for the clinical treatment and clinical transformation of endometrial diseases.

[0011] The various terms and phrases used in the present invention have the general meanings well-known to those skilled in the art. Description of the Drawings

[0012] The menstrual blood organoids of the present invention are preserved in the China Center for Type Culture Collection, named Human Menstrual Blood Organoid MFO-Jennifer (Homo sapiens), with the preservation number CCTCC NO: C2024133, the preservation date being September 25, 2024, and the preservation address being: Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Zip Code: 430072.

[0013] Figure 1 : Comparative photos of 4 menstrual blood organoids. From top to bottom, the first row is the photo at the 8th day of P0 generation culture, the second row is the photo at the 8th day of P1 generation culture, the third row is the photo at the 8th day of P3 generation culture, and the fourth row is the photo at the 7th day of P7 generation culture; from left to right, the first column is the photo of menstrual blood organoid 1, the second column is the photo of menstrual blood organoid 2, the third column is the photo of menstrual blood organoid 3, and the fourth column is the photo of menstrual blood organoid 4.

[0014] Figure 2 : Photo of menstrual blood organoid 1.

[0015] Figure 3 : Immunofluorescence detection results of epithelial and stromal cells, where the nucleus is labeled with DAPI.

[0016] Figure 4 : Immunofluorescence detection results of the epithelial cell marker E-cadherin.

[0017] Figure 5 : Immunofluorescence detection results of the stromal cell marker vimentin.

[0018] Figure 6 : Figure 3 、 Figure 4 、 Figure 5 Superimposed display.

[0019] Figure 7 : Immunofluorescence detection results of luminal epithelial and glandular epithelial cells, where the nucleus is labeled with DAPI.

[0020] Figure 8 : Immunofluorescence detection results of the luminal epithelial marker Wnt-7a.

[0021] Figure 9 : Immunofluorescence detection results of the glandular epithelial marker FOXA2.

[0022] Figure 10 : Figure 7 、 Figure 8 、 Figure 9 Superimposed display.

[0023] Figure 11 : Bright-field photo of the co-culture system of embryo-menstrual blood organoids on the 6th day after fertilization.

[0024] Figure 12 : Immunofluorescence detection results of OCT4.

[0025] Figure 13 : Immunofluorescence detection results of SOX17.

[0026] Figure 14 : Immunofluorescence detection results of GATA3.

[0027] Figure 15 : Figure 12 , Figure 13 , Figure 14 Superimposed display of, where d.p.f. 9 represents the 9th day after fertilization, the structure of menstrual blood organoids is shown above the dotted line, the upper white arrow indicates the epithelium of menstrual blood organoids, and the lower white arrow indicates the structure of the embryonic hypoblast.

[0028] Figure 16 : Photos of the co - culture of menstrual blood organoids and embryos at 7, 8, 9, and 11 days. Among them, A: Photo at 7 days; B: Photo at 8 days; C: Photo at 9 days; D: Photo at 11 days. The arrow indicates the embryo.

[0029] Figure 17 : Photos of the co - culture of endometrial organoids 1 and embryos at 7, 8, 9, and 11 days. Among them, A: Photo at 7 days; B: Photo at 8 days; C: Photo at 9 days; D: Photo at 11 days. The arrow indicates the embryo.

[0030] Figure 18 : Photos of the co - culture of endometrial organoids 2 and embryos at 7, 8, 9, and 11 days. Among them, A: Photo at 7 days; B: Photo at 8 days; C: Photo at 9 days; D: Photo at 11 days. The arrow indicates the embryo. Detailed implementation manners

[0031] The present invention will be further described below in conjunction with embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art can understand that various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention.

[0032] For the instruments, reagents, materials, etc. involved in the following embodiments, unless otherwise specified, they are all conventional instruments, reagents, materials, etc. existing in the prior art and can be obtained through regular commercial channels. For the experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, they are all conventional experimental methods, detection methods, etc. existing in the prior art.

[0033] Example 1 Construction of menstrual blood organoids The steps are as follows: (I) Establishment of menstrual blood organoids (1) Washing of blood cells Collect menstrual blood and centrifuge it (at 4°C, 400 g, for 10 min) to discard the upper layer of blood cells; wash it 4 times with pre-cooled DPBS supplemented with 1% Antibiotic-Antimycotic (Gibco 15240062). After each wash, discard the upper layer of blood sample supernatant and only retain the cell components; after washing, pass the cells through a cell sieve with a pore size of 100 μm. Invert the cell sieve on a culture dish and backwash the sieve with pre-cooled DPBS to backwash tissue debris and the like on the sieve into the culture dish.

[0034] The menstrual blood is obtained in the following way: On the 1st to 2nd day with the largest menstrual volume, collect menstrual blood using a menstrual cup sterilized at high temperature, and the total collected amount is not less than 7 ml. After collection, place the menstrual blood in a sterile centrifuge tube, temporarily store it at 4°C, transport it on ice throughout the process, and quickly transfer it to the laboratory.

[0035] (2) Digest the tissue fragments into single cells Cut the tissue collected in the above culture dish into small pieces, transfer the cut tissue into the digestive solution, and digest it at 37°C under rotary oscillation conditions for 30 min; after digestion, add an equal volume of neutralization medium to prevent further digestion to obtain a mixed solution.

[0036] The digestive solution consists of the following components: water; collagenase V, 0.4 mg / mL; dispase II, 1.5 U / mL; deoxyribonuclease I (DNase I), 15 μg / mL.

[0037] During digestion, the incubation container rotates at a mode of 15 rpm and oscillates at a mode of 1 rpm. Pipette the mixture every 10 min to make it more evenly dispersed.

[0038] The neutralization medium is DMEM / F12 medium supplemented with 10% FBS and 1% Antibiotic-Antimycotic.

[0039] (3) Model establishment Vortex the mixed solution thoroughly, let it stand for 1 min to precipitate undigested tissue fragments. The supernatant passes through a cell sieve with a pore size of 40 μm to retain as many cells in the endometrial microenvironment such as stromal cells and immune cells as possible. Invert the cell sieve on a culture dish and backwash the sieve with pre-cooled DPBS to collect cell clusters to obtain a cell suspension.

[0040] The cell suspension was centrifuged (400 g, 5 min). The cell pellet was resuspended in DMEM / F12 medium, centrifuged again (400 g, 5 min), and the cell pellet was resuspended in DMEM / F12 medium. After mixing with Matrigel without phenol red, it was plated. The volume ratio of DMEM / F12 medium to Matrigel was 1:3. It was placed in an incubator and incubated at 37 °C and 5% CO2 for more than 30 min to solidify the Matrigel.

[0041] After the Matrigel solidified, it was plated on a cell culture plate. Endometrial organoid medium was added and it was placed in an incubator and cultured at 37 °C and 5% CO2. The medium was changed every 2 days and subculture was carried out on the 8th day of culture.

[0042] The endometrial organoid medium is DMEM / F12 medium supplemented 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, and 1% antibiotic-antifungal agent.

[0043] (4)Results Using the above method, the present invention collected menstrual blood from 4 volunteers, constructed 4 strains of menstrual blood organoids (temporarily named: menstrual blood organoid 1, menstrual blood organoid 2, menstrual blood organoid 3, menstrual blood organoid 4) and carried out subculture; observed under bright field microscopy and compared the states of these 4 strains of menstrual blood organoids at the same passage number and the same culture days.

[0044] The comparison photos of the 4 strains of menstrual blood organoids are as Figure 1 shown. By comparison, it can be seen that menstrual blood organoid 1 has the strongest proliferation activity and can be stably subcultured to P7 with good cell growth state; when menstrual blood organoid 2 was subcultured to P3, the cell viability was poor and it could not be subcultured continuously; when menstrual blood organoid 3 was subcultured to P1, the cell viability was poor and it could not be subcultured continuously; when menstrual blood organoid 4 was in primary culture, the cell viability was poor and it could not be subcultured.

[0045] Subsequently, volunteer 1 (the volunteer of menstrual blood organoid 1) donated menstrual blood 3 more times. The 3 strains of menstrual blood organoids constructed therefrom could all be stably subcultured to P7 with good cell viability. At the same time, several groups of parallel experiments were also carried out using the menstrual blood of other volunteers, but none of them could be stably subcultured.

[0046] The above results indicate that only the menstrual blood organoids constructed from the menstrual blood of Volunteer 1 can be stably passaged, showing obvious advantages in cell viability and the number of passages. Presumably, the reason is that the menstrual blood of this volunteer is special (compared with ordinary menstrual blood, some unknowable and unpredictable changes may have occurred). Therefore, the menstrual blood organoid 1 of the present invention was deposited in the China Center for Type Culture Collection, named Human Menstrual Blood Organoid MFO_Jennifer (Homosapiens), with the deposit number CCTCC NO: C2024133, the deposit date being September 25, 2024, and the deposit address being: Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Zip Code: 430072.

[0047] After the assembly of the menstrual blood organoid 1 (on the 12th day of P0 culture), under bright-field observation with a microscope, the photo of the menstrual blood organoid 1 is as Figure 2 shown. It can be seen that the menstrual blood organoid presents a cystic structure, showing a hollow sphere surrounded by epithelial cells, and most of the organoids reach a diameter of 200 μm.

[0048] (II) Hormone treatment to make the menstrual blood organoid enter the implantation window stage After the assembly of the menstrual blood organoid 1 in (I), it is cultured in the endometrial organoid medium supplemented with 10 nM β-estradiol for 2 days, and then cultured in the endometrial organoid medium supplemented with 1 μM medroxyprogesterone acetate, 1 μM dibutyryl CAMP, 1 μg / mL human chorionic gonadotropin, 20 ng / mL human placental lactogen precursor, and 20 ng / mL prolactin for 6 days to induce the menstrual blood organoid 1 to enter the stage favorable for embryo implantation and implantation, which is called the implantation window stage.

[0049] (III) Immunofluorescence identification of the basic structure of the menstrual blood organoid The steps are as follows: (1) Fix the menstrual blood organoid 1 cultured for 12 days in 4% PFA (paraformaldehyde solution) at room temperature for 30 min, wash it three times with PBS, dehydrate it overnight at 4°C in 20% sucrose solution, embed the organoid with OCT, and cut it into 10-μm-thick sections with a cryostat.

[0050] (2) Restore the frozen sections to room temperature, fix them with 4% PFA for 5 min, and permeate them with PBS containing 0.3% TritonX-100 for 20 min. Perform antigen retrieval with sodium citrate at 95°C for 20 min. After restoring to room temperature, the sections are blocked in the immunostaining blocking buffer (Beyotime, P0260) for 20 min.

[0051] (3) Incubate the sections with primary antibodies (mouse anti-E-cadherin antibody, rabbit anti-Vimentin antibody, rabbit anti-FOXA2 antibody, mouse anti-Wnt-7a antibody) overnight at 4°C, and then wash three times with PBS containing 0.1% TritonX-100. Incubate with secondary antibodies (donkey anti-mouse, donkey anti-rabbit secondary antibodies) for 2 hours at room temperature, and wash three times with PBS containing 0.1% TritonX-100. Incubate with DAPI (Beyotime, C1002) for 15 min, and mount with an anti-fluorescence quencher.

[0052] (4) Collect images using a confocal laser scanning microscope (Andor Dragonfly 200), and process them with Imaris x64 9.0.1.

[0053] Images for identifying epithelial cells and stromal cells in menstrual blood organoids are shown as Figures 3 - 6 shown, among which, the immunofluorescence detection results of epithelial and stromal cells are shown as Figure 3 shown, the immunofluorescence detection results of the epithelial cell marker E-cadherin are shown as Figure 4 shown, the immunofluorescence detection results of the stromal cell marker Vimentin are shown as Figure 5 shown, Figure 3 , Figure 4 , Figure 5 The overlay display of Figure 6 is shown as Figures 3 - 6 shown. It can be seen from

[0054] that menstrual blood organoids express E-cadherin and Vimentin, indicating that menstrual blood organoids contain endometrial epithelial cells and stromal cells. Figures 7 - 10 shown, among which, the immunofluorescence detection results of luminal epithelial and glandular epithelial cells are shown as Figure 7 shown, the immunofluorescence detection results of the luminal epithelial marker Wnt-7a are shown as Figure 8 shown, the immunofluorescence detection results of the glandular epithelial marker FOXA2 are shown as Figure 9 shown, Figure 7 , Figure 8 , Figure 9 The overlay display of Figure 10 is shown as Figures 7 - 10 shown. It can be seen from

[0055] Example 2 Menstrual Blood Organoids as a Model for Embryo Implantation Example 1: A menstrual blood organoid induced to the implantation window stage was constructed. This menstrual blood organoid was co-cultured with embryos to study the key events in the development of early embryos during the peri-implantation period, using endometrial organoids induced from endometrial samples obtained by hysterectomy for benign uterine diseases (hereinafter simply referred to as endometrial organoids) as controls (the construction method refers to the literature PMC5410172). There were 2 controls, namely endometrial organoid 1 and endometrial organoid 2 respectively.

[0056] The donated human blastocysts frozen on the fifth day after fertilization were thawed using a vitrification kit (Kitazato, VT602). The blastocysts were directly transferred from liquid nitrogen to 1 ml of pre-warmed embryo thawing solution with a pipette. After 2 minutes, the blastocysts were transferred to the following solutions: dilution solution (room temperature, 3 minutes), washing solution (room temperature, 5 minutes). Subsequently, the embryos were placed in a small amount of embryo medium, and the zona pellucida was mechanically dissected with a glass needle. The embryos were cultured in G-2 PLUS medium (Vitrolife, 10232), coated with oil (Vitrolife, 10029), and incubated at 37 °C, 5% O2 and 6% CO2 for at least 2 hours until the embryos recovered.

[0057] The above recovered embryos were implanted on the organoids, ensuring that the polar trophoblast cells were against the side of the organoids, and cultured at 37 °C, 6% CO2. After culturing for two days using HIVC1 medium (human embryo in vitro medium 1), it was replaced with HIVC2 medium (human embryo in vitro medium 2), and the experiment was terminated on the 14th day after fertilization.

[0058] The bright-field photograph of the embryo-menstrual blood organoid co-culture system on the 6th day after fertilization is as Figure 11 shown. It can be seen that the embryo on the 6th day after fertilization attached to the menstrual blood organoid with the inner cell mass side, the embryo morphology expanded, and there were no obvious signs of cell death during the development process.

[0059] On the 9th day after fertilization, immunofluorescence identification was performed on the embryo-menstrual blood organoid co-culture system to identify the lineage development of the embryos. SOX17 was used to label the glandular epithelium of the endometrial organoids and the hypoblast structure of the embryos, OCT4 was used to label the epiblast structure of the embryos, and GATA3 was used to label the trophoblast cells of the embryos.

[0060] The immunofluorescence identification results on the 9th day after fertilization are as Figures 12 - 15 shown, among which, the immunofluorescence detection results of OCT4 are as Figure 12 shown, the immunofluorescence detection results of SOX17 are as Figure 13 shown, the immunofluorescence detection results of GATA3 are as Figure 14 shown, Figure 12 、 Figure 13 、 Figure 14The superimposed display is as follows Figure 15 as shown. As can be seen from Figures 12 - 15 Figure Figures 12 - 15 , in the embryo-menstrual blood organoid co-culture system on the 9th day after fertilization, the glandular epithelium of the endometrial organoids and the markers of the embryonic hypoblast SOX17, the embryonic epiblast marker OCT4, and the trophoblast cell marker GATA3 were all expressed, indicating that after co-culture with menstrual blood organoids, the embryo presented a bilaminar disc structure and achieved the differentiation of three lineages (epiblast, hypoblast, trophoblast). Under the premise of ethical permission, menstrual blood organoids can support the in vitro development of embryos to 14 days and achieve the normal development and differentiation of embryos.

[0061] Photographs of the co-culture of menstrual blood organoids and embryos at 7, 8, 9, and 11 days are as follows Figure 16 shown. Photographs of the co-culture of endometrial organoid 1 and embryos at 7, 8, 9, and 11 days are as follows Figure 17 shown. Photographs of the co-culture of endometrial organoid 2 and embryos at 7, 8, 9, and 11 days are as follows Figure 18 shown. It can be seen that the morphology of the embryos in the co-culture of menstrual blood organoids expanded, and the trophoblast developed normally during the development process, while the trophoblast of some embryos in the co-culture of endometrial organoids developed slower.

[0062] In summary, it can be seen that the menstrual blood organoids constructed and preserved in the present invention have obvious advantages in the receptivity to embryo implantation compared with the endometrial organoids induced from endometrial samples obtained by hysterectomy due to benign uterine diseases, are more conducive to the development and growth of embryos during the peri-implantation period, and are more suitable for studying the physiological changes of embryos and endometrium during the embryo implantation stage.

[0063] The above embodiments are provided to those skilled in the art to fully disclose and describe how to implement and use the claimed embodiments, rather than to limit the scope of the disclosure herein. Modifications that are 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: Deposited with the China Center for Type Culture Collection, named human menstrual blood organoid MFO-Jennifer (Homo sapiens), deposit number CCTCC NO: C2024133, deposit date September 25, 2024.

2. Use of the menstrual blood organoid according to claim 1 in constructing an embryo implantation model.

3. Use of the menstrual blood organoid according to claim 1 in constructing an individualized drug screening model.

4. Use of the menstrual blood organoid according to claim 1 in constructing a receptivity test model for in vitro fertilization-embryo transfer.

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