CN115418340A - Method for culturing endometrioid organs from human uterine cavity lavage fluid - Google Patents
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Abstract
The invention discloses a method for culturing an endometrial organoid from human uterine cavity lavage fluid, which comprises the following steps: firstly, irrigating the uterine cavity by using normal saline to obtain uterine cavity irrigating solution; step two, enriching endometrial cells in uterine cavity lavage fluid to culture endometrial organs; step three, carrying out passage amplification on the endometrioid organ; step four, carrying out cryopreservation on the endometrium organoids; and fifthly, recovering the frozen endometrioid organs. The invention adopts a non-invasive sampling mode to reduce the injury of the patient in the process of obtaining the endometrium, and the patient does not need anesthesia and rarely bleeds in the process of obtaining the lavage fluid of the uterine cavity; for some patients who cannot scratch endometrium, the scheme can be used for culturing the endometrioid of the patient; the endometrioid organ cultured in the uterine cavity lavage fluid can be subsequently used for researching the interaction related mechanism of the mother and fetus in the early pregnancy or screening and treating the medicine for patients.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a culture method of an endometrial organoid from human uterine cavity lavage fluid.
Background
Organoids (Organoids): refers to the use of 3D culture technology to culture cells with stem cell potential derived from human or animal tissues, so as to form tissue structures similar to those of corresponding organ sources, and to be capable of simulating or reproducing a series of in vivo physiological and pathological processes in vitro, and having relatively stable phenotypic and genetic characteristics. Organoid culture techniques have been successfully applied to many tissues, organs, and have proven to be a powerful tool for studying the normal developmental processes of organs and for mimicking disease. Organoids can be used to predict drug effects and open up new avenues for individualized regenerative medicine.
It has now been demonstrated that endometrioid (EEO) can provide a valuable model for studying maternal-fetal interactions in early pregnancy and for exploring the pathophysiology of gynaecological disorders such as endometriosis and endometrial cancer.
The EEO biopsy sample is obtained mainly by cutting or scraping the endometrial tissue through an open abdominal operation or a hysteroscopic operation. Although this invasive sampling method can make the biopsy sample have good repeatability and high success rate, there are many drawbacks, including the following:
(1) The access to normal tissue samples is limited by the clinical experience of the physician and the surgical indications of the patient.
(2) Obtaining endometrial tissue by surgery is an invasive procedure that to some extent may cause injury to the patient or donor. Meanwhile, the invasive operation inevitably brings potential risks of postoperative trauma, surgical accidents, postoperative infection and the like.
The other acquisition mode is extracted from menstrual blood, but the other acquisition mode cannot be a universal operation mode due to the obvious problems of severe storage conditions, incomplete sample data and the like. Menstrual blood is a mixture of tissue and blood that is stripped from the endometrium after withdrawal of the estrogen and progestogen. Although the literature reports that endometrial organoids can be obtained from it, the time of sampling endometrial organoids from menstrual blood is limited and the procedure is not suitable for patients with menopause or menopause, making full study difficult.
Therefore, there is a need to devise a culture method for endometrial organoids that is relatively non-invasive and suitable for a variety of settings. We collect exfoliated cells and tissue fragments in the uterine fluid by a relatively non-invasive method and culture the endometrial organoids therefrom.
Disclosure of Invention
In order to solve the problems, the invention discloses a method and a device for culturing an endometrioid organ from human uterine cavity lavage fluid. The invention adopts a relatively non-invasive mode to reduce the damage to the patient in the process of obtaining the endometrium, and the patient does not need anesthesia and rarely bleeds in the process of extracting the lavage fluid in the uterine cavity; for some patients who cannot extract endometrium, such as thin endometrium patients and the like, the culture of the endometrium organoid of the patient can be realized by the scheme; the cultured endometrioid organ in the uterine cavity lavage fluid can be subsequently used for researching the mechanism related to the interaction between the maternal and fetal in the early pregnancy or screening and treating the medicine of a patient.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a method for culturing human endometrial organoids from uterine lavage fluid, comprising the steps of:
firstly, irrigating a uterine cavity by using normal saline to obtain uterine cavity irrigating solution;
step two, enriching endometrial cells in the uterine cavity lavage fluid and culturing to form an endometrial organoid;
step three, carrying out passage amplification on the endometrioid organ;
step four, freezing and storing the endometrioid organ;
and fifthly, recovering and using the frozen endometrioid organ.
In a further improvement, the first step comprises the following steps:
1.1 preparing the article: a disposable artificial insemination tube, a 5ml syringe, 0.9% normal saline, a transplant package, iodophor and a 15ml sterilized centrifuge tube;
1.2 wearing gloves, sucking 2 plus or minus 1ml of physiological saline into a 5ml syringe;
1.3 taking the lithotomy position of a patient, carrying out conventional disinfection and drape, slowly feeding an artificial insemination tube into the uterine bottom, withdrawing for about 1cm, externally connecting a 5ml syringe filled with physiological saline, slowly pushing the physiological saline into the uterine cavity, rinsing the uterine cavity, slowly sucking the uterine cavity lavage fluid out, and collecting the lavage fluid into a sterilized centrifuge tube to obtain the uterine cavity lavage fluid;
1.4 the uterine cavity lavage fluid is placed on ice, and the second step is carried out within 1 hour.
In a further improvement, the second step includes the following steps:
2.1 centrifuging the centrifuge tube filled with the uterine cavity lavage fluid for 5min under the centrifugal force of 800g, and discarding the supernatant;
2.2 washing the precipitate with PBS for 1-2 times to remove cervical mucus;
2.3 resuspending the precipitate in DMEM/F12, slightly blowing and beating the precipitate, mechanically separating gland fragments, centrifuging at 800g for 5min, and removing supernatant;
resuspending the pellet in 20% EXM/80% matrigel, plating 50uL of the homogeneous suspension into a preheated 24-well culture plate, adding complete culture medium after the matrigel has solidified, and 5% CO at 37 ℃% 2 Culturing in incubator, and changing culture solution every 2-3 days for 1 time.
In a further improvement, the third step comprises the following steps:
3.1 taking out the culture plate from the incubator, adding precooled DMEM/F12 without enzyme, growth factors and serum to liquefy the matrix gel, and collecting all organoids to a 15ml sterile centrifuge tube by using a pipette gun;
3.2 adding 1X trypLE digestive juice (Gibco) into the centrifuge tube to digest the organoid at 37 ℃ for 6-10 minutes, and repeatedly beating the organoid;
3.3 centrifuging the digested solution at 4 ℃ at 800g for 5min, discarding the supernatant, adding 20% EXM/80% of the matrigel resuspension to obtain a homogeneous suspension;
3.4 taking 50 μ L of the homogeneous suspension, dripping into a preheated culture plate for plating, adding 500ul of complete culture solution after matrigel is solidified, culturing in an incubator with 5% of CO2 at 37 ℃, and replacing the culture solution for 1 time every 2-3 days; passages were performed at the ratio of 1.
The further improvement comprises the fourth step of carrying out cryopreservation on the surrogate endometrioid;
4.1 replacing the culture medium in the culture well with 350. Mu.L of pre-cooled cell recovery solution, blowing an EEO-Matrigel mixture, and placing the culture dish on ice for 30 minutes to liquefy Matrigel;
4.2 collecting the mixture in the culture dish into a low-adsorption 15ml centrifuge tube by using a low-adsorption gun head, manually and gently pipetting up and down for 80 times to separate the endometrioid from the matrigel, and centrifuging at 800g for 5 minutes to precipitate the endometrioid;
4.3, discarding the supernatant, adding 1-2ml of pre-cooled DMEM/F12 culture medium into the centrifuge tube, manually and gently transferring the liquid up and down for 80 times to destroy the glandular cavity structure of the endometrioid organ, adding 4ml of pre-cooled DMEM/F12 culture medium, blowing, beating and uniformly mixing, and centrifuging at 800g for 5 minutes;
4.4 discarding the supernatant and putting the precipitate on ice for 3 minutes;
4.5 adding 0.8-1mL of organoid cryopreservation liquid into the centrifuge tube, uniformly blowing and beating the organoids, transferring the organoids into the cell cryopreservation tube, marking the names of the organoids, the serial numbers of patients, the dates and the operator information on the cell cryopreservation tube, transferring the cryopreservation tube to a programmed cryopreservation box, standing overnight at-80 ℃, and transferring the cell cryopreservation tube to a liquid nitrogen tank for long-term cryopreservation the next day.
In a further improvement, the step five comprises the following steps:
5.1 taking out the frozen endometrium organoid from a refrigerator or a liquid nitrogen tank at minus 80 ℃, and quickly placing the endometrium organoid in a constant temperature water bath kettle at 37 ℃ for quick vibration and thawing;
5.2 transferring the organoid to a 15ml centrifuge tube, adding 3ml of DMEM/F12 culture medium to resuspend the organoid, centrifuging at 800g at room temperature for 5 minutes, and removing the supernatant;
5.3 adding DMEM/F12 medium to the pellet, pipetting evenly and centrifuging at 4 ℃ at 800g for 5 minutes, adding 20% EXM/80% by weight matrix gel resuspension;
50 μ L of the homogeneous suspension was dropped into a preheated 24-well plate for plating, and after the matrigel was solidified, 500. Mu.l of complete culture solution was added, and the mixture was cultured in an incubator at 37 ℃ and 5% CO2, with the culture solution being changed every 2 to 3 days for 1 time.
In a further improvement, the method also comprises a sixth step of detecting the responsiveness of the endometrioid organ to the sex hormone:
6.1 culturing the organoids after passage for 3-4 days after grouping, and replacing the culture medium for 1 time every other day;
6.2 days 5 and 6 treatments with 10nM estrogen were added for 2 days, and days 7 and 8 with the following hormones: group E2 added 10nM estrogen, group EP added 10nM estrogen and 1uM progestin; EPC group with 10nM estrogen and 1uM progestin and 10uM cAMP;
6.3 after the hormone treatment is finished, fixing the organoid by formaldehyde, embedding the section by agarose and then carrying out PAS dyeing on the organoid;
6.4 after the hormone treatment is finished, separating and extracting by a TRIzol method;
6.5 after the hormone treatment is finished, fixing the organoid by glutaraldehyde, dissociating the organoid by suction with a 1ml syringe needle, and detecting the surface morphology of the organoid by using a scanning electron microscope.
The invention has the advantages that:
the technical scheme of the invention brings beneficial effects
(1) The injury of the patient in the process of obtaining the endometrium is reduced by adopting a non-invasive sampling mode, and the patient does not need anesthesia and rarely bleeds in the process of extracting the lavage fluid from the uterine cavity.
(2) For some patients who cannot obtain endometrium by the conventional method, such as thin endometrium patients, etc., the culture of endometrioid organs of the patients can be realized by the scheme.
(3) The cultured endometrioid organ in the uterine cavity lavage fluid can be subsequently used for researching the mechanism related to the interaction between the maternal and fetal in the early pregnancy or screening and treating the medicine of a patient.
Drawings
FIG. 1A is a schematic diagram of UF-EEO extraction and culture according to the present invention.
FIG. 1B is a drawing of an EEO mirror;
FIG. 1C is a photograph of a UF-EEO mirror;
FIG. 1D is an immunofluorescence map of the EEO glandular epithelial marker EpCAM, cytokeratin-7 (CK 7), E-cadherin;
FIG. 1E is an immunofluorescence map of the UF-EEO glandular epithelial marker, epCAM, cytokeratin-7 (CK 7), E-cadherin;
FIG. 2 is a photograph of UF-EEO light mirror after passage;
FIG. 3 is a photograph of a microscope showing UF-EEO after cryo-resuscitation;
FIG. 4 shows PAS staining before and after organoid hormone treatment;
FIG. 5 shows the measurement of the expression of genes involved in hormone response before and after organoid hormone treatment;
figure 6 is a disassembled microscopic view of an endometrial organoid.
Detailed Description
The invention is further explained by the following embodiments in conjunction with the drawings.
Examples
A method for culturing human uterine cavity lavage fluid-derived endometrioid organs, comprising the following steps:
(1) Uterine cavity lavage liquid extraction method
(1) Preparing an article: a disposable artificial insemination tube, a 5ml syringe, 0.9% normal saline, a transplant package, iodophor and a 15ml sterilized centrifuge tube;
(2) wearing gloves, sucking 2 plus or minus 1ml of normal saline into a 5ml syringe;
(3) taking a lithotomy position, conventionally disinfecting and paving a drape, slowly feeding an artificial insemination tube into the uterine bottom, withdrawing for about 1cm, externally connecting a 5ml syringe filled with physiological saline, slowly pushing the physiological saline into the uterine cavity, rinsing the uterine cavity, slowly sucking the lavage fluid of the uterine cavity out, and collecting the lavage fluid of the uterine cavity to a sterilized centrifuge tube to obtain the lavage fluid of the uterine cavity;
(4) the samples were placed on ice, transferred to the laboratory and processed within 1 hour.
(2) The extraction method of human endometrioid (UF-EEO) from uterine cavity lavage fluid is shown in figure 1A:
(1) centrifuging the centrifuge tube filled with uterine cavity lavage fluid under 800g centrifugal force for 5min, and removing supernatant;
(2) washing the precipitate with PBS for several times to remove cervical mucus;
(3) resuspending the precipitate in DMEM/F12, slightly blowing and beating the precipitate, mechanically separating gland fragments, centrifuging at 800g for 5min, and removing supernatant;
(4) resuspending the precipitate in DMEM/F12, slightly blowing and beating the precipitate, mechanically separating gland fragments, centrifuging at 1000g for 5min, and removing supernatant;
(5) resuspending the pellet in 20-80% matrix gel, adding 50uL of the homogeneous suspension dropwise to a preheated 24-well culture plate for plating, adding complete culture solution after the matrix gel is solidified, and adding 5% CO at 37 deg.C 2 The culture was carried out in an incubator, and the culture medium was changed 1 time every 2 days.
(6) As shown in FIGS. 1B-E, EEO and UF-EEO were not morphologically different under light microscopy, and both also expressed the glandular epithelial markers EpCAM, cytokeratin-7 (CK 7), E-cadherin.
(3) Passaging UF-EEO
Human endometrial glandular cells will form globular acinar-like structures in matrigel, and will be passaged for 7-10 days, with the ratio of 1.
(1) Taking out a 24-pore plate from the incubator, adding precooled DMEM/F12 (without enzyme, growth factors and serum) to liquefy the matrix gel, and collecting all organoids to 15ml sterile centrifuge tubes as much as possible by using a pipette;
(2) adding 1X TrypLE (Gibco) to dissociate the organoids at 37 deg.C for 6-10 min, repeatedly beating;
(3) centrifuging at 800g for 5min at 4 deg.C, discarding the supernatant, adding 20% EXM/80% matrigel, and resuspending to obtain a homogeneous suspension;
(4) 50 μ l of the homogeneous suspension was dropped into a preheated 24-well plate for plating, and after the matrigel was solidified, 500. Mu.l of complete culture solution was added, and the mixture was cultured in an incubator at 37 ℃ and 5% by volume in CO2, and the culture solution was changed every 2 to 3 days.
(4) Cryopreservation of UF-EEO
(1) Replacing the culture medium in the culture well with 350 μ L of pre-cooled cell recovery solution, blowing an EEO-Matrigel mixture, and placing the culture dish on ice for 30 minutes to liquefy Matrigel;
(2) collecting the mixture in the culture dish into a low-adsorption 15ml centrifuge tube by using a low-adsorption gun head, manually and gently pipetting up and down 80 times to separate the endometrioid organs from the matrigel, and centrifuging at 800g for 5 minutes to precipitate the endometrioid organs;
(3) discarding the supernatant, adding 1-2ml of pre-cooled DMEM/F12 into the centrifuge tube, manually and gently transferring the liquid up and down for 80 times to destroy the glandular cavity structure of the endometrioid organ, adding 4ml of pre-cooled DMEM/F12, blowing, uniformly mixing, and centrifuging at 800g for 5 minutes;
(4) the supernatant was discarded and the precipitate was placed on ice for 3 minutes;
(5) adding 0.8-1mL of organoid cryopreservation liquid into a centrifuge tube, blowing and uniformly mixing the organoids, transferring the organoids into a cell cryopreservation tube, marking the names of the organoids, the serial numbers of patients, the dates and operator information on the cell cryopreservation tube, transferring the cryopreservation tube to a programmed cryopreservation box, standing overnight at-80 ℃, and transferring the cell cryopreservation tube to a liquid nitrogen tank for long-term cryopreservation the next day. Organoid cryopreservation solution composition: 10% DMSO +90% serum.
(5) Resuscitation of UF-EEO
(1) Taking out the frozen organoids from a refrigerator at minus 80 ℃ or a liquid nitrogen tank, and quickly placing the organoids in a constant-temperature water bath kettle at 37 ℃ for quick oscillation and dissolution;
(2) transferring to a 15ml centrifuge tube, adding 3ml DMEM/F12 for resuspending organoids, centrifuging at 800g for 5 minutes at room temperature, and discarding the supernatant;
(3) adding DMEM/F12 medium to the pellet, pipetting evenly and centrifuging at 800g for 5 minutes at 4 c, discarding the supernatant, 20% EXM/80% by weight matrix gel resuspension;
(4) 50 μ l of the homogeneous suspension was dropped into a preheated 24-well plate for plating, and after the matrigel was solidified, 500uL of complete medium was added, and the mixture was cultured in an incubator containing 5% of CO2 at 37 ℃ with 1-time replacement of the medium every 2 to 3 days (see FIG. 3).
(6) Detection of UF-EEO response to hormones
1. UF-EEO and EEO were subcultured separately for 3 days and then treated sequentially with oestrogest and cAMP for 4 days. The final concentration of estrogen is 10nM, progestogen is 1uM, cAMP is 10uM. E2 is estrogenic and treatment continued for 4 days. E2+ MPA + cAMP for 2 days of estrogen treatment followed by estrogen + progestin + cAMP for 2 days.
2. After the hormone treatment, the organoids were embedded in agar and sectioned, and the change in secretory function of the organoids was detected by PAS staining. Results it can be seen that UF-EEO after sequential hormone treatment can see endometrioid changes during the secretory phase (see FIG. 4).
3. After hormone treatment, organoid is collected, organoid RNA is extracted by a TRIzol method, and real-time fluorescence quantitative PCR is carried out to detect the expression change of hormone reaction related genes (SPP 1, PAEP, 17HSD beta 2). The results showed that the expression of the endometriotic response-associated genes was significantly increased after sequential hormone treatment (see fig. 5).
4. Fixing organoids after the female progestogen is sequentially treated with glutaraldehyde, then sucking with a 1ml syringe needle, smashing and embedding the organoids, and then detecting the surface morphology of the organoids by using a scanning electron microscope. The electron micrograph shows that the organoid has microvilli and pinocytosis on its surface (see FIG. 6).
While embodiments of the invention have been disclosed above, it is not limited to the applications set forth in the specification and the embodiments, which are fully applicable to various fields of endeavor for which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.
Claims (7)
1. A method for culturing an endometrial organoid from human uterine lavage fluid, comprising the steps of:
firstly, irrigating a uterine cavity by using normal saline to obtain uterine cavity irrigating solution;
step two, enriching endometrial cells in uterine cavity lavage fluid, and culturing to form an endometrial organoid;
step three, carrying out passage amplification on the endometrioid organ;
step four, carrying out cryopreservation on the endometrium organoids;
and fifthly, recovering and using the frozen endometrioid organ.
2. The method of claim 1, wherein said first step comprises the steps of:
1.1 preparing the article: a disposable artificial insemination tube, a 5ml syringe, 0.9% normal saline, a transplant package, iodophor and a 15ml sterilization centrifuge tube;
1.2 wearing gloves, sucking 2 plus or minus 1ml of physiological saline into a 5ml syringe;
1.3 taking the lithotomy position of a patient, carrying out conventional disinfection and drape, slowly feeding an artificial insemination tube into the uterine bottom, withdrawing for about 1cm, externally connecting a 5ml syringe filled with physiological saline, slowly pushing the physiological saline into the uterine cavity, rinsing the uterine cavity, slowly sucking the uterine cavity lavage fluid out, and collecting the lavage fluid into a sterilized centrifuge tube to obtain the uterine cavity lavage fluid;
1.4 the uterine cavity lavage fluid is placed on ice, and the second step is carried out within 1 hour.
3. The method of claim 2, wherein step two comprises the steps of:
2.1 centrifuging the centrifuge tube filled with the uterine cavity lavage fluid for 5min under the centrifugal force of 800g, and discarding the supernatant;
2.2 washing the precipitate with PBS for 1-2 times to remove cervical mucus;
2.3 resuspending the precipitate in DMEM/F12, gently blowing and beating the precipitate, mechanically separating gland fragments, centrifuging at 800g for 5min, and discarding the supernatant;
2.4 resuspending the pellet in 20% EXM/80% matrigel, plating 50uL of the homogeneous suspension by dropping it onto a preheated 24-well plate, adding complete medium after the matrigel has solidified, and 5% CO at 37 ℃% 2 Culturing in incubator, and changing culture solution every 2-3 days for 1 time.
4. The method for passaging a human uterine cavity lavage derived endometrial organoid of claim 3, wherein said step three comprises the steps of:
3.1 taking out the culture plate from the incubator, adding precooled DMEM/F12 without enzyme, growth factors and serum to liquefy the matrix gel, and collecting all organoids to a 15ml sterile centrifuge tube by using a pipette gun;
3.2 adding 1X trypLE digestive fluid (Gibco) into the centrifuge tube to digest the organoids at 37 ℃ for 6-10 minutes, and repeatedly blowing and beating the organoids in the period;
3.3 centrifuging the digested solution at 4 ℃ at 800g for 5min, discarding the supernatant, adding 20% EXM/80% of the matrigel resuspension to obtain a homogeneous suspension;
3.4 taking 50 μ L of the homogeneous suspension, dripping into a preheated culture plate for plating, adding 500ul of complete culture solution after matrigel is solidified, culturing in an incubator with 5% of CO2 at 37 ℃, and replacing the culture solution for 1 time every 2-3 days; passages were performed at the ratio of 1.
5. The method of claim 4, further comprising the steps of cryopreserving the surrogate endometrial organoids;
4.1 replacing the culture medium in the culture well with 350. Mu.L of pre-cooled cell recovery solution, blowing an EEO-Matrigel mixture, and placing the culture dish on ice for 30 minutes to liquefy Matrigel;
4.2 collecting the mixture in the culture dish into a low-adsorption 15ml centrifuge tube by using a low-adsorption gun head, manually and slightly pipetting up and down for 80 times to separate the endometrioid organ from the matrigel, and centrifuging for 5 minutes at 800g to precipitate the endometrioid organ;
4.3, discarding the supernatant, adding 1-2ml of pre-cooled DMEM/F12 culture medium into the centrifuge tube, manually and gently transferring the liquid up and down for 80 times to destroy the glandular cavity structure of the endometrioid organ, adding 4ml of pre-cooled DMEM/F12 culture medium, blowing, beating and uniformly mixing, and centrifuging at 800g for 5 minutes;
4.4 discard the supernatant and place the precipitate on ice for 3 minutes;
4.5 adding 0.8-1mL of organoid cryopreservation liquid into the centrifuge tube, blowing and uniformly mixing the organoids, transferring the organoids into the cell cryopreservation tube, marking the names of the organoids, the serial numbers of patients, the dates and the operator information on the cell cryopreservation tube, transferring the cryopreservation tube to a programmed cryopreservation box, standing overnight at-80 ℃, and transferring the cell cryopreservation tube into a liquid nitrogen tank for long-term cryopreservation the next day.
6. The method of culturing human uterine cavity lavage fluid-derived endometrial organoids as claimed in claim 5, wherein said step five comprises the steps of:
5.1 taking out the frozen endometrium organoid from a refrigerator or a liquid nitrogen tank at minus 80 ℃, and quickly placing the endometrium organoid in a constant-temperature water bath kettle at 37 ℃ for quick shaking and thawing;
5.2 transferring the organoid to a 15ml centrifuge tube, adding 3ml of DMEM/F12 culture medium to resuspend the organoid, centrifuging at 800g for 5 minutes at room temperature, and discarding the supernatant;
5.3 adding DMEM/F12 medium to the pellet, pipetting homogeneously and centrifuging at 4 ℃ at 800g for 5 minutes, adding 20% EXM/80% of the matrigel resuspension;
5.4 taking 50u L homogeneous suspension dropped into the preheated 24-hole culture plate for plating, after the matrigel solidification, adding complete culture solution 500ul, at 37 degrees C, 5% CO2 in the incubator culture, every 2-3 days change 1 times culture solution.
7. The method of culturing human uterine cavity lavage fluid-derived endometrial organoids according to claim 6, further comprising the steps of: detection of endometrial organoids reactivity to sex hormones:
6.1 culturing after passage for 3-4 days after organ organoids are grouped, and replacing the culture medium for 1 time every other day;
6.2 treatment with 10nM estrogen on days 5 and 6 for 2 days, the following hormones were added on days 7 and 8: group E2 with 10nM estrogen and group EP with 10nM estrogen and 1uM progestin; EPC group with 10nM estrogen and 1uM progestin and 10uM cAMP;
6.3 after the hormone treatment is finished, fixing the organoid by formaldehyde, embedding the section by agarose, and then carrying out PAS dyeing on the organoid;
6.4 after the hormone treatment is finished, separating and extracting by a TRIzol method;
6.5 after the hormone treatment is finished, fixing the organoid by glutaraldehyde, dissociating the organoid by suction with a 1ml syringe needle, and detecting the surface morphology of the organoid by using a scanning electron microscope.
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