CN115418340A - Method for culturing endometrial organoids derived from human uterine lavage fluid - Google Patents

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

Method for culturing endometrial organoids derived from human uterine lavage fluid

technical field

The invention belongs to the field of biotechnology, in particular to a method for culturing endometrial organoids derived from human uterine lavage fluid.

Background technique

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 corresponding organ sources, which can simulate or reproduce a series of physiological and pathological conditions in vivo process with relatively stable phenotypic and genetic characteristics. Organoid culture technology has been successfully applied to many tissues and organs, and has been proved to be a powerful tool to study the normal development process of organs and simulate diseases. Organoids can be used to predict drug effects and open new avenues for personalized regenerative medicine.

It has been demonstrated that endometrial organoids (EEOs) can provide a valuable model for studying maternal-fetal interactions in the first trimester and exploring the pathophysiology of gynecological diseases such as endometriosis and endometrial cancer.

In the past, EEO biopsy samples were mainly obtained through laparotomy or laparoscopic surgery, excision or scraping of endometrial tissue. Although this invasive sampling method can make the biopsy sample reproducible and the success rate high, it also has many defects, mainly including the following points:

(1) The way to obtain normal tissue samples is limited by the doctor's clinical experience and the patient's surgical indications.

(2) The endometrial tissue obtained through surgery is an invasive operation, which will cause harm to patients or donors to a certain extent. At the same time, invasive operations inevitably bring potential risks such as intraoperative trauma, surgical accidents, and postoperative infection.

Another way to obtain it is to extract it from menstrual blood, but it cannot become a universal operation method because of the obvious problems such as strict storage conditions and incomplete sample data. Menstrual blood is a mixture of tissue and blood from the endometrium after the estrogen and progesterone withdrawal. Although it has been reported in the literature that endometrial organoids can be obtained from them, endometrial organoids derived from menstrual blood are limited by the sampling time, and this method is not suitable for menopausal or postmenopausal patients, making it difficult to conduct comprehensive research.

Therefore, it is necessary to design a relatively non-invasive and applicable method for culturing endometrial organoids in various scenarios. We harvest endometrial organoids from exfoliated cells and tissue fragments in uterine fluid through a relatively non-invasive method.

Contents of the invention

In order to solve the above problems, the present invention discloses a method and a method for culturing endometrial organoids derived from human uterine lavage fluid. The present invention adopts a relatively non-invasive method to reduce the injury suffered by the patient in the process of obtaining the endometrium, and the patient does not need anesthesia and has little bleeding during the extraction process of the uterine lavage fluid; for some patients who cannot extract the endometrium, For example, patients with thin endometrium, etc., we can use this method to realize the culture of endometrial organoids in patients; the endometrial organoids cultured in uterine cavity lavage fluid can be used for subsequent use in the relationship between mother-fetal interaction in early pregnancy. Mechanism research or drug screening and treatment of patients.

To achieve the above object, the technical solution of the present invention is:

A method for culturing endometrial organoids derived from human uterine lavage fluid, comprising the steps of:

Step 1, using normal saline to lavage the uterine cavity to obtain uterine cavity lavage fluid;

Step 2, enriching the endometrial cells in the uterine cavity lavage fluid and culturing them to form endometrial organoids;

Step 3: Passaging and amplifying the endometrial organoids;

Step 4. Cryopreserving the endometrial organoids;

Step 5: Resuscitate and use the cryopreserved endometrial organoids.

As a further improvement, said step 1 includes the following steps:

1.1 Prepared items: disposable artificial insemination tube, 5ml syringe, 0.9% normal saline, transplantation bag, iodophor, 15ml sterilized centrifuge tube;

1.2 Wearing gloves, inhale 2±1ml of normal saline into a 5ml syringe;

1.3 The patient takes the lithotomy position, routinely disinfects the drape, slowly sends the artificial insemination tube into the fundus of the uterus, withdraws about 1cm, connects a 5ml syringe with normal saline, slowly pushes the normal saline into the uterine cavity, and rinses the uterine cavity Finally, the uterine lavage fluid was slowly sucked out and collected into a sterilized centrifuge tube to obtain the uterine cavity lavage fluid;

1.4 Put the uterine lavage fluid on ice, and proceed to step 2 within 1 hour.

As a further improvement, the second step includes the following steps:

2.1 Centrifuge the centrifuge tube containing the uterine lavage solution at 800g centrifugal force for 5min, and discard the supernatant;

2.2 Wash the precipitate with PBS 1-2 times to remove cervical mucus;

2.3 Resuspend the pellet in DMEM/F12, pipette the pellet gently, mechanically separate the gland fragments, centrifuge at 800g for 5min, and discard the supernatant;

Resuspend the pellet in 20% EXM/80% Matrigel, take 50uL of the homogeneous suspension and drop it into a preheated 24-well culture plate for plating. Culture in a %CO ₂ incubator and replace the culture medium every 2-3 days.

As a further improvement, said step three includes the following steps:

3.1 Take out the culture plate from the incubator, add pre-cooled DMEM/F12 without enzymes, growth factors and serum to liquefy the matrix gel, and use a pipette to collect all organoids into 15ml sterile centrifuge tubes;

3.2 Add 1X TrypLE digestion solution (Gibco) to the centrifuge tube to digest the organoids at 37°C for 6-10 minutes, during which pipetting is required repeatedly;

3.3 Centrifuge the digested solution at 800g at 4°C for 5 minutes, discard the supernatant, and add 20% EXM/80% Matrigel to resuspend to obtain a homogeneous suspension;

3.4 Take 50 μL of the homogeneous suspension and drop it into the preheated culture plate for plating. After the Matrigel is solidified, add 500ul of complete culture solution, culture in an incubator at 37°C and 5% CO2, and replace it every 2-3 days Culture medium; passaging according to the ratio of 1:2 to 1:4, and take the organoids at passage 3-6 for cryopreservation or for subsequent experiments.

A further improvement also includes step 4, cryopreserving the passaged endometrial organoids;

4.1 Replace the medium in the culture well with 350 μL of pre-cooled cell recovery solution, pipette the EEO-Matrigel mixture, and place the culture dish on ice for 30 minutes to liquefy the Matrigel matrigel;

4.2 Use a low-adsorption pipette tip to collect the mixture in the petri dish into a low-adsorption 15ml centrifuge tube, manually pipette up and down gently 80 times to separate the endometrial organoids from the matrigel, and centrifuge at 800g for 5 minutes to make Endometrial organoid precipitation;

4.3 Discard the supernatant, add 1-2ml of pre-cooled DMEM/F12 medium to the centrifuge tube, pipette up and down gently 80 times manually to destroy the glandular structure of endometrial organoids, add 4ml of pre-cooled DMEM /F12 medium, blow and mix well, then centrifuge at 800g for 5 minutes;

4.4 Discard the supernatant and place the precipitate on ice for 3 minutes;

4.5 Add 0.8-1mL organoid cryopreservation solution to the centrifuge tube, pipette the organoid to mix well and transfer it to the cell cryopreservation tube, mark the name of the organoid, patient number, date, and operator information on the cell cryopreservation tube , transfer the cryopreservation tube to a programmed freezer box, store overnight at -80°C, and transfer the cell cryopreservation tube to a liquid nitrogen tank for long-term freezing the next day.

As a further improvement, said step five includes the following steps:

5.1 After taking out the frozen endometrial organoids from the -80°C refrigerator or liquid nitrogen tank, quickly place them in a 37°C constant temperature water bath to quickly shake and thaw;

5.2 Transfer the organoids to a 15ml centrifuge tube, add 3ml of DMEM/F12 medium to resuspend the organoids, centrifuge at 800g for 5 minutes at room temperature, and discard the supernatant;

5.3 Add DMEM/F12 medium to the pellet, pipette evenly and centrifuge at 800g at 4°C for 5 minutes, add 20% EXM/80% Matrigel to resuspend;

Take 50 μL of the homogeneous suspension and drop it into a preheated 24-well culture plate for plating. After the Matrigel is solidified, add 500 μl of complete culture solution and culture it in an incubator at 37°C and 5% CO2, replacing 1 medium every 2-3 days. Secondary culture medium.

Further improvement also includes step 6, detecting the responsiveness of endometrial organoids to sex hormones:

6.1 After the organoids are grouped and subcultured, they are cultured for 3-4 days, and the medium is replaced every other day;

6.2 Add 10nM estrogen for 2 days on the 5th and 6th day, add the following hormones on the 7th and 8th day: add 10nM estrogen to the E2 group, add 10nM estrogen and 1uM progesterone to the EP group; add 10nM estrogen and 1uM progesterone to the EPC group Progesterone and 10uMcAMP;

6.3 After hormone treatment, the organoids were fixed with formaldehyde, and the organoids were stained with PAS after embedding sections in agarose;

6.4 After hormone treatment, separate and extract by TRIzol method;

6.5 After the hormone treatment, the organoids were fixed with glutaraldehyde, and the organoids were dissociated by aspiration with a 1ml syringe needle, and the surface morphology of the organoids was detected by a scanning electron microscope.

Advantages of the present invention:

Beneficial effects brought by the technical solution of the present invention

(1) The non-invasive sampling method is used to reduce the harm to the patient during the process of obtaining the endometrium, and the patient does not need anesthesia and has little bleeding during the extraction of the uterine lavage fluid.

(2) For some patients who cannot obtain endometrium by conventional methods, such as patients with thin endometrium, we can use this method to achieve endometrial organoid culture in patients.

(3) The endometrial organoids cultured in uterine lavage fluid can be subsequently used for research on the mechanism of maternal-fetal interaction in early pregnancy or for drug screening and treatment of patients.

Description of drawings

Fig. 1A is a schematic diagram of the extraction and cultivation of UF-EEO of the present invention.

Figure 1B is an EEO light microscope picture;

Figure 1C is a photomicrograph of UF-EEO;

Figure 1D is the immunofluorescence images of EEO glandular epithelial markers EpCAM, Cytokeratin-7 (CK7), and E-cadherin;

Figure 1E is the immunofluorescence images of UF-EEO glandular epithelial markers EpCAM, Cytokeratin-7 (CK7), and E-cadherin;

Figure 2 is the photomicrograph of UF-EEO after passage;

Figure 3 is the light microscope picture of UF-EEO after freezing and thawing;

Figure 4 is the PAS staining before and after organoid hormone treatment;

Figure 5 is the expression detection of hormone response-related genes before and after organoid hormone treatment;

Figure 6 is a microscopic view of the disassembled endometrial organoid.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

Example

A method for culturing endometrial organoids derived from human uterine lavage fluid, comprising the steps of:

(1) Extraction method of intrauterine lavage fluid

① Prepared items: disposable artificial insemination tube, 5ml syringe, 0.9% normal saline, transplantation bag, iodophor, 15ml sterilized centrifuge tube;

②Wearing gloves, inhale 2±1ml of normal saline into a 5ml syringe;

③The patient takes the lithotomy position, routinely disinfects and spreads the towel, slowly sends the artificial insemination tube into the fundus of the uterus, withdraws about 1cm, connects a 5ml syringe with normal saline, and slowly pushes the normal saline into the uterine cavity to moisten the uterine cavity Finally, the uterine lavage fluid was slowly sucked out and collected into a sterilized centrifuge tube to obtain the uterine cavity lavage fluid;

④ Place the sample on ice, transfer it to the laboratory, and process it within 1 hour.

(2) The extraction method of human endometrial organoids (UF-EEO) derived from intrauterine lavage fluid is shown in Figure 1A:

① Centrifuge the centrifuge tube containing the uterine lavage solution at 800g for 5 minutes, and discard the supernatant;

②Wash the precipitate several times with PBS to remove cervical mucus;

③Resuspend the pellet in DMEM/F12, pipette the pellet gently, mechanically separate the gland fragments, centrifuge at 800g for 5min, and discard the supernatant;

④Resuspend the pellet in DMEM/F12, pipette the pellet gently, mechanically separate the gland fragments, centrifuge at 1000g for 5min, and discard the supernatant;

⑤ Resuspend the precipitate in 20% EXM/80% Matrigel, take 50uL of the homogeneous suspension and drop it into a preheated 24-well culture plate for plating. Culture in a 5% CO ₂ incubator and replace the culture medium every 2 days.

⑥As shown in Figure 1B-E, there was no difference in the morphology of EEO and UF-EEO under the light microscope, and both expressed glandular epithelial markers EpCAM, Cytokeratin-7 (CK7), and E-cadherin.

(3) Passage of UF-EEO

Human endometrial glandular cells will form a spherical gland-like structure in Matrigel, and it takes 7-10 days to passage, and passage according to the ratio of 1:2 to 1:4 (according to the growth efficiency), 3 The -6 passage organoids were used for subsequent experiments (as shown in Figure 2).

①Take out the 24-well plate from the incubator, add pre-cooled DMEM/F12 (without enzymes, growth factors and serum) to liquefy the matrix gel, and use a pipette to collect as much organoids as possible into a 15ml sterile centrifuge tube;

② Add 1X TrypLE (Gibco) to dissociate the organoids at 37°C for 6-10 minutes, during which repeated pipetting is required;

③Centrifuge at 800g for 5min at 4°C, discard the supernatant, add 20% EXM/80% Matrigel to resuspend to obtain a homogeneous suspension;

④Take 50 μl of homogeneous suspension dropwise into a preheated 24-well culture plate for plating. After the Matrigel is solidified, add 500ul of complete culture solution, culture in an incubator at 37°C and 5% CO2, and replace it every 2-3 days culture medium.

(4) Cryopreservation of UF-EEO

① Replace the medium in the culture well with 350 μL of pre-cooled cell recovery solution, pipette the EEO-Matrigel mixture, and place the culture dish on ice for 30 minutes to liquefy the Matrigel;

②Use a low-adsorption pipette tip to collect the mixture in the petri dish into a low-adsorption 15ml centrifuge tube, manually pipette up and down gently 80 times to separate the endometrial organoids from the matrigel, and centrifuge at 800g for 5 minutes to make Endometrial organoid precipitation;

③ Discard the supernatant, add 1-2ml of pre-cooled DMEM/F12 to the centrifuge tube, pipette up and down gently 80 times manually to destroy the glandular structure of endometrial organoids, add 4ml of pre-cooled DMEM/F12 , centrifuge at 800g for 5 minutes after mixing by pipetting;

④ Discard the supernatant and place the precipitate on ice for 3 minutes;

⑤Add 0.8-1mL organoid freezing solution to the centrifuge tube, pipette the organoid to mix well and transfer to the cell cryopreservation tube, mark the name of the organoid, patient number, date, and operator information on the cell cryopreservation tube , transfer the cryopreservation tube to a programmed freezer box, store overnight at -80°C, and transfer the cell cryopreservation tube to a liquid nitrogen tank for long-term freezing the next day. Organoid cryopreservation solution composition: 10% DMSO + 90% serum.

(5) Recovery of UF-EEO

① After taking out the frozen organoids from the -80°C refrigerator or liquid nitrogen tank, quickly place them in a 37°C constant temperature water bath to quickly shake and dissolve;

②Transfer to a 15ml centrifuge tube, add 3ml of DMEM/F12 to resuspend the organoids, centrifuge at 800g for 5 minutes at room temperature, and discard the supernatant;

③ Add DMEM/F12 medium to the pellet, pipette evenly and centrifuge at 800g at 4 c for 5 minutes, discard the supernatant, and resuspend in 20% EXM/80% Matrigel;

④Take 50 μl of homogeneous suspension dropwise into a preheated 24-well culture plate for plating. After the Matrigel is solidified, add 500 μL of complete culture solution, culture in an incubator at 37°C and 5% CO2, and replace it every 2-3 days 1 culture medium (as shown in Figure 3).

(6) Detection of the response of UF-EEO to hormones

1. UF-EEO and EEO were subcultured for 3 days, and then sequentially treated with estrogen and cAMP for 4 days. The final concentration of estrogen is 10nM, progesterone is 1uM, and cAMP is 10uM. E2 is the estrogen group, treated continuously for 4 days. E2+MPA+cAMP was treated with estrogen for 2 days, and then treated with estrogen+progesterone+cAMP for 2 days.

2. After hormone treatment, the organoids were embedded in agar and sliced, and the changes in the secretory function of the organoids were detected by PAS staining. The results showed that UF-EEO after hormone sequential treatment showed endometrium-like changes in the secretory phase (Figure 4).

3. Collect organoids after hormone treatment, extract organoid RNA by TRIzol method, and perform real-time fluorescent quantitative PCR to detect expression changes of hormone response-related genes (SPP1, PAEP, 17HSDβ2). The results showed that the expression of endometrial hormone response-related genes significantly increased after hormone sequential treatment (as shown in Figure 5).

4. The organoids after sequential treatment with estrogen and progesterone were fixed with glutaraldehyde, then aspirated with a 1ml syringe needle, the organoids were crushed and embedded, and then the surface morphology of the organoids was detected with a scanning electron microscope. Electron micrographs showed that there were microvilli and pinocytosis on the surface of the organoid (Figure 6).

Although the embodiment of the present invention has been disclosed as above, it is not limited to the use listed in the specification and embodiment, it can be applied to various fields suitable for the present invention, and it can be easily understood by those skilled in the art Further modifications can be effected, so the invention is not limited to the specific details and examples shown and described herein without departing from the general concept defined by the claims and their equivalents.

Claims (7)

1. A culture method for endometrial organoids derived from human uterine lavage fluid, characterized in that, comprising the steps of: Step 1, using normal saline to lavage the uterine cavity to obtain uterine cavity lavage fluid; Step 2, enriching the endometrial cells in the uterine cavity lavage fluid, and culturing them to form endometrial organoids; Step 3: Passaging and amplifying the endometrial organoids; Step 4. Cryopreserving the endometrial organoids; Step 5: Resuscitate and use the cryopreserved endometrial organoids. 2. the sampling method of people's uterine lavage fluid as claimed in claim 1, is characterized in that, described step one comprises the steps: 1.1 Prepared items: disposable artificial insemination tube, 5ml syringe, 0.9% normal saline, transplantation bag, iodophor, 15ml sterilized centrifuge tube; 1.2 Wearing gloves, inhale 2±1ml of normal saline into a 5ml syringe; 1.3 The patient takes the lithotomy position, routinely disinfects the drape, slowly sends the artificial insemination tube into the fundus of the uterus, withdraws about 1cm, connects a 5ml syringe with normal saline, slowly pushes the normal saline into the uterine cavity, and rinses the uterine cavity Finally, the uterine lavage fluid was slowly sucked out and collected into a sterilized centrifuge tube to obtain the uterine cavity lavage fluid; 1.4 Put the uterine lavage fluid on ice, and proceed to step 2 within 1 hour. 3. The method for culturing endometrial organoids derived from human uterine lavage fluid as claimed in claim 2, wherein said step 2 comprises the following steps: 2.1 Centrifuge the centrifuge tube containing the uterine lavage solution at 800g centrifugal force for 5min, and discard the supernatant; 2.2 Wash the precipitate with PBS 1-2 times to remove cervical mucus; 2.3 Resuspend the pellet in DMEM/F12, pipette the pellet gently, mechanically separate the gland fragments, centrifuge at 800g for 5min, and discard the supernatant; 2.4 Resuspend the pellet in 20% EXM/80% Matrigel Matrigel, take 50uL of the homogeneous suspension and drop it into a preheated 24-well culture plate for plating. Culture in a 5% CO ₂ incubator and replace the culture medium every 2-3 days. 4. The method for passage of endometrial organoids derived from human uterine lavage fluid as claimed in claim 3, wherein said step 3 comprises the following steps: 3.1 Take out the culture plate from the incubator, add pre-cooled DMEM/F12 without enzymes, growth factors and serum to liquefy the matrix gel, and use a pipette to collect all organoids into 15ml sterile centrifuge tubes; 3.2 Add 1X TrypLE digestion solution (Gibco) to the centrifuge tube to digest the organoids at 37°C for 6-10 minutes, during which pipetting is required repeatedly; 3.3 Centrifuge the digested solution at 800g at 4°C for 5 minutes, discard the supernatant, and add 20% EXM/80% Matrigel to resuspend to obtain a homogeneous suspension; 3.4 Take 50 μL of the homogeneous suspension and drop it into the preheated culture plate for plating. After the Matrigel is solidified, add 500ul of complete culture solution, culture in an incubator at 37°C and 5% CO2, and replace it every 2-3 days Culture medium; passaging according to the ratio of 1:2 to 1:4, and take the organoids at passage 3-6 for cryopreservation or for subsequent experiments. 5. The method for culturing endometrial organoids derived from human uterine lavage fluid as claimed in claim 4, further comprising step 4, cryopreserving the passaged endometrial organoids; 4.1 Replace the medium in the culture well with 350 μL of pre-cooled cell recovery solution, pipette the EEO-Matrigel mixture, and place the culture dish on ice for 30 minutes to liquefy the Matrigel matrigel; 4.2 Use a low-adsorption pipette tip to collect the mixture in the petri dish into a low-adsorption 15ml centrifuge tube, manually pipette up and down gently 80 times to separate the endometrial organoids from the matrigel, and centrifuge at 800g for 5 minutes to make Endometrial organoid precipitation; 4.3 Discard the supernatant, add 1-2ml of pre-cooled DMEM/F12 medium to the centrifuge tube, pipette up and down gently 80 times manually to destroy the glandular structure of endometrial organoids, add 4ml of pre-cooled DMEM /F12 medium, blow and mix well, then centrifuge at 800g for 5 minutes; 4.4 Discard the supernatant and place the precipitate on ice for 3 minutes; 4.5 Add 0.8-1mL organoid cryopreservation solution to the centrifuge tube, pipette the organoid to mix well and transfer it to the cell cryopreservation tube, mark the name of the organoid, patient number, date, and operator information on the cell cryopreservation tube , transfer the cryopreservation tube to a programmed freezer box, store overnight at -80°C, and transfer the cell cryopreservation tube to a liquid nitrogen tank for long-term freezing the next day. 6. The method for culturing endometrial organoids derived from human uterine lavage fluid as claimed in claim 5, wherein said step five comprises the following steps: 5.1 After taking out the frozen endometrial organoids from the -80°C refrigerator or liquid nitrogen tank, quickly place them in a 37°C constant temperature water bath to quickly shake and thaw; 5.2 Transfer the organoids to a 15ml centrifuge tube, add 3ml of DMEM/F12 medium to resuspend the organoids, centrifuge at 800g for 5 minutes at room temperature, and discard the supernatant; 5.3 Add DMEM/F12 medium to the pellet, pipette evenly and centrifuge at 800g at 4°C for 5 minutes, add 20% EXM/80% Matrigel to resuspend; 5.4 Take 50 μL of homogeneous suspension and drop it into a preheated 24-well culture plate for plating. After the Matrigel is solidified, add 500ul of complete culture solution, culture in an incubator at 37°C and 5% CO2, and replace it every 2-3 days 1 culture medium. 7. The method for culturing endometrial organoids derived from human uterine lavage fluid as claimed in claim 6, further comprising step 6: detecting the reactivity of endometrial organoids to sex hormones: 6.1 After the organoids are grouped and subcultured, they are cultured for 3-4 days, and the medium is replaced every other day; 6.2 Add 10nM estrogen for 2 days on the 5th and 6th day, add the following hormones on the 7th and 8th day: add 10nM estrogen to the E2 group, add 10nM estrogen and 1uM progesterone to the EP group; add 10nM estrogen and 1uM progesterone to the EPC group Progesterone and 10uM cAMP; 6.3 After hormone treatment, the organoids were fixed with formaldehyde, and the organoids were stained with PAS after embedding sections in agarose; 6.4 After hormone treatment, separate and extract by TRIzol method; 6.5 After the hormone treatment, the organoids were fixed with glutaraldehyde, and the organoids were dissociated by aspiration with a 1ml syringe needle, and the surface morphology of the organoids was detected by a scanning electron microscope.

Images