CN116478912A - A medium for inducing directed differentiation of mouse intestinal organoids into enteroendocrine cells and its application - Google Patents

Abstract

The invention provides a culture medium for inducing directional differentiation of a mouse intestinal organoid into an enteroendocrine cell and application thereof, and relates to the technical field of organoid induction culture. According to the invention, mek inhibitor PD0325901, wnt inhibitor IWP-2 and Notch inhibitor DAPT are added into a standard condition (ENR) culture medium of a small intestine organoid of a mouse to induce the small intestine organoid of the mouse to differentiate into mature enteroendocrine cells, so that the construction of an enteroendocrine cell induction model of the mouse is realized.

Description

A medium for inducing the directed differentiation of mouse intestinal organoids into enteroendocrine cells and its application

technical field

The invention belongs to the technical field of organoid induction culture, in particular to a culture medium for inducing the directional differentiation of mouse intestinal organoids into enteroendocrine cells and its application

Background technique

Small intestinal endocrine cells are differentiated from small intestinal stem cells and distributed throughout the intestinal tract to regulate physiological activities such as intestinal secretion, motility, digestion and absorption, blood sugar regulation, and metabolism. It can secrete different types of intestinal hormones, and play a key role in intestinal peristalsis, vasoconstriction, nutrient absorption, epithelial growth, and central biological rhythm regulation through endocrine and paracrine pathways. At the same time, it plays an important role in the research of non-clinical and clinical systems for medicine, nutrition and tissue engineering. In the past, the in vitro research on enteroendocrine cells has always remained at the cell line level, cultured in a single layer, and there are large differences in cell environment, genetic background and functional characteristics with normal enteroendocrine cells.

Studies have shown that Lgr5 ⁺ intestinal stem cells in the intestine have the potential to differentiate into all types of cells in the intestinal epithelium, including Paneth cells, endocrine cells, intestinal epithelial cells, etc. Therefore, extracting Lgr5 ⁺ intestinal stem cells in small intestinal crypts and inducing them can form intestinal organoids that can maintain and proliferate. The organoids contained all intestinal epithelial cell types, possessed a cellular environment similar to that of the gut, and expressed gut hormones in a manner similar to that of the native gut. However, enteroendocrine cells account for only 1% of intestinal organoids. Considering that it is difficult to obtain enteroendocrine cell populations by controlling their specific differentiation direction in current culture methods, it is urgent to develop a stable, efficient and convenient method for constructing mouse intestinal endocrine cell induction models.

Contents of the invention

In view of this, the purpose of the present invention is to provide an induction medium, which can successfully and rapidly differentiate into mature enteroendocrine cells after treating mouse intestinal organoids with the induction medium.

In order to realize the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The invention provides an induction medium, wherein the medium components include intestinal organoid standard condition ENR medium 10-15mL, Mek inhibitor PD03259010.5-2μM, Wnt inhibitor IWP-23-8μM and Notch inhibitor DAPT8-15μM; the ENR medium components are Advanced DMEM medium 10-15ml, Glutamax 100-150μL, Hepes buffer 100-15 0 μL, N-2 supplement 100～150 μL, B-27 supplement 100～150 μL, penicillin 100～150 μL, Noggin 80～120ng/ml, R-spondin1400～600ng/ml, EGF 30～80ng/ml.

The present invention also provides a method for inducing directed differentiation of mouse intestinal organoids into enteroendocrine cells, comprising the following steps: using the induction medium to cultivate mouse small intestinal organoids.

Preferably, the culture conditions are 35-37°C, 5% CO ₂ .

Preferably, when the mouse small intestine organoid differentiation begins, the medium is renewed every 2 days; after 4 days of differentiation, aspirate the culture medium, add PBS buffer to pipette, centrifuge, discard the supernatant, resuspend in Advanced DMEM at 4°C, centrifuge, discard the supernatant, and resuspend in Advanced DMEM and Matrigel.

More preferably, the conditions of the centrifugation are 0-4°C, 280-290g for 2-8 minutes.

Preferably, the mouse small intestinal organoid is obtained by subculturing the mouse small intestinal crypt stem cells in the intestinal organoid standard condition ENR medium for 5-7 days.

More preferably, the components of the intestinal organoid standard condition ENR medium are Advanced DMEM medium 10-15ml, Glutamax 100-150μL, Hepes buffer 100-150μL, N-2supplement 100-150μL, B-27supplement 100-150μL, Penicillin 100-150μL, Noggin 80-12 0ng/ml, R-spondin1400~600ng/ml, EGF 30~80ng/ml.

More preferably, the passage ratio is 1:3-1:5.

More preferably, the mouse small intestinal crypt stem cells are obtained by digesting mouse small intestinal tissue with EDTA.

The present invention also provides an application of the induction medium or the method in constructing a mouse enteroendocrine cell organoid model.

Compared with the prior art, the present invention has the following beneficial effects:

The induction medium provided by the invention differentiates the mouse intestinal organoids into enteroendocrine cells. Through immunofluorescence detection, compared with the control group, the number of enteroendocrine cell markers treated with the induction medium of the present invention increased by 985%; through qRT-PCR detection of the mRNA expression levels of various cell markers in organoids, the expression levels of marker hormone mRNAs of common endocrine cell subtypes in the small intestine such as CCK (I cells), GLP-1 (L cells), Ghrelin (A cells), Secrtin (S cells), GIP (K cells) and Somatostatin (D cells) all increased significantly. The results show that the induction medium of the present invention can successfully differentiate the mouse intestinal endocrine organoids into mature enteroendocrine cells after being treated with the induction medium, which is beneficial to the successful establishment of a mouse intestinal endocrine cell induction model. This model can better simulate the intestinal cell environment than the 2D cell model. It can be used to explore the influence and molecular mechanism of different disease models on enteroendocrine cells and their intestinal hormone secretion. It can also be used to study intestinal nutrition and health, enteroendocrine disease drug screening and safety evaluation.

Description of drawings

Figure 1 is a diagram of the growth state of mouse small intestinal organoids cultured on days 1-8 using intestinal organoid standard condition ENR medium;

Figure 2 is a diagram of the growth status of small intestinal organoids on day 0 and day 4 under different medium conditions;

Figure 3 is the results of immunofluorescence detection of enteroendocrine cell markers of small intestinal organoids under different culture medium conditions;

Fig. 4 is the result of mRNA expression of cell markers in small intestinal organoids under the conditions of the induction medium of the present invention.

Detailed ways

The invention provides an induction medium, wherein the medium components preferably include intestinal organoid standard condition ENR medium 10-15 mL, Mek inhibitor PD03259010.5-2 μM, Wnt inhibitor IWP-23-8 μM and Notch inhibitor DAPT 8-15 μM; the intestinal organoid standard condition ENR medium component is preferably Advanced DMEM medium 10-15 ml, Glutamax 100-150 μL, Hepes buff er 100-150 μL, N-2 supplement 100-150 μL, B-27supplement 100-150 μL, penicillin 100-150 μL, Noggin 80-120 ng/ml, R-spondin 1400-600 ng/ml, EGF 30-80 ng/ml; more preferably, the medium components include intestinal organoid standard condition ENR medium 1 2mL, Mek inhibitor PD03259011μM, Wnt inhibitor IWP-25μM and Notch inhibitor DAPT 10μM; the intestinal organoid standard condition ENR medium components are more preferably Advanced DMEM medium 12ml, Glutamax120μL, Hepes buffer 120μL, N-2supplement 120μL, B-27supplement 120μL, penicillin and streptomycin 1 20 μL, Noggin 100ng/ml, R-spondin 1500ng/ml, EGF 50ng/ml. In the present invention, the Mek inhibitor PD0325901, Wnt inhibitor IWP-2 and Notch inhibitor DAPT inhibitor were purchased from MedChemExpress; the Advanced DMEM medium, Glutamax, Hepes buffer, N-2supplement, B-27supplement and penicillin and streptomycin were purchased from Thermo Scientific; the Noggin, R-spondin1 and EGF were purchased from Propertech.

The present invention also provides a method for inducing directed differentiation of mouse intestinal organoids into enteroendocrine cells, comprising the following steps: using the induction medium to cultivate mouse small intestinal organoids.

In the present invention, the culture conditions are 35-37° C., 5% CO ₂ .

In the present invention, the culture medium was renewed every 2 days when the mouse small intestinal organoids began to differentiate; after 4 days of differentiation, the culture medium was aspirated, PBS buffer was added to pipette, centrifuged, the supernatant was discarded, resuspended in Advanced DMEM at 4°C, centrifuged, discarded the supernatant, and resuspended in Advanced DMEM and Matrigel matrigel.

In the present invention, the centrifugation conditions are preferably 0-4°C, 280-290 g for 2-8 minutes.

In the present invention, the mouse small intestinal organoid is obtained by subculturing the mouse small intestinal crypt stem cells in the intestinal organoid standard condition ENR medium for 5-7 days. In a specific embodiment of the present invention, the culture condition is to culture at 37° C. and 5% CO ₂ . In the present invention, the number of passages is preferably 3 to 4 times, and the passage culture can keep the state of the organoid in good condition.

In the present invention, the components of the intestinal organoid standard condition ENR medium are preferably 10-15 ml of Advanced DMEM medium, 100-150 μL of Glutamax, 100-150 μL of Hepes buffer, 100-150 μL of N-2supplement, 100-150 μL of B-27supplement, 100-150 μL of penicillin and streptomycin, 80-12 μL of Noggin 0 ng/ml, R-spondin 1400-600 ng/ml, EGF 30-80 ng/ml; more preferably, the intestinal organoid standard condition ENR medium components are Advanced DMEM medium 12ml, Glutamax 120 μL, Hepes buffer 120 μL, N-2supplement 120 μL, B-27supplement 120 μL, penicillin and streptomycin 120 μL, No ggin 100ng/ml, R-spondin 1500ng/ml, EGF 50ng/ml.

In the present invention, the passage ratio is preferably 1:3-1:5; more preferably 1:4. The passage ratio of the present invention can be appropriately adjusted according to the state of the crypt.

In the present invention, the mouse small intestine crypt stem cells are obtained by digesting mouse small intestine tissue with EDTA. After EDTA digestion, the small intestinal tissue will become loose, and the small intestinal crypts will be separated from the tissue under the action of external mechanical force, which can be repeated several times to obtain the mixture of different fractionated fractions through the action of external mechanical force, or select fractionated fractions with higher crypt content and fewer miscellaneous cells for seed plate culture. In a specific embodiment of the present invention, the obtained small intestine tissue fragments were resuspended in 25 mL of EDTA solution at 4°C, and placed on a shaker at 0 to 4°C and incubated at 20 rpm for 30 to 60 minutes; the supernatant containing EDTA was removed, washed three times with PBS, and the tissue fragments were resuspended in 4°C PBS containing 0.1% BSA, and pipetted up and down three times. After standing until most of the intestinal tissue fragments settled to the bottom, carefully remove the supernatant and filter with a 70 μm filter, collect the filtrate in an EP tube, and mark the filtrate as "fraction 1"; repeat the steps three times to obtain fractions 2-4. In a particular embodiment of the invention, it is preferred to use fractions 3 and 4 for subsequent cultured crypts.

The present invention also provides an application of the induction medium or the method in constructing a mouse enteroendocrine cell organoid model.

The technical solutions provided by the present invention will be described in detail below in conjunction with the examples, but they should not be interpreted as limiting the protection scope of the present invention.

Example 1

An induction medium comprising the following components: intestinal organoid standard condition (ENR) medium 12ml, Mek inhibitor PD03259011μM, Wnt inhibitor IWP-25μM and Notch inhibitor DAPT 10μM; the intestinal organoid standard condition (ENR) medium component is Advanced DMEM medium 12ml, Glutamax 120μL, Hepes buffer 120μL, N-2supplement120μM L, B-27supplement 120μL, Penicillin 120μL, Noggin 100ng/ml, R-spondin1500ng/ml, EGF 50ng/ml.

Example 2

An induction medium comprising the following components: intestinal organoid standard condition (ENR) medium 10ml, Mek inhibitor PD03259010.5 μM, Wnt inhibitor IWP-23 μM and Notch inhibitor DAPT 8 μM; the intestinal organoid standard condition (ENR) medium component is Advanced DMEM medium 10ml, Glutamax 100 μL, Hepes buffer 100 μL, N-2supplement100 μL, B-27supplement 100μL, Penicillin 100μL, Noggin 80ng/ml, R-spondin1400ng/ml, EGF 30ng/ml.

Example 3

An induction medium comprising the following components: intestinal organoid standard condition (ENR) medium 15ml, Mek inhibitor PD03259012 μM, Wnt inhibitor IWP-28 μM and Notch inhibitor DAPT 15 μM; the intestinal organoid standard condition (ENR) medium component is Advanced DMEM medium 15ml, Glutamax 150 μL, Hepes buffer 150 μL, N-2supplement150 μM L, B-27supplement 150μL, Penicillin 150μL, Noggin 120ng/ml, R-spondin1600ng/ml, EGF 80ng/ml.

Comparative example 1

The difference from Example 1 is that the Wnt inhibitor IWP-2 and the Notch inhibitor DAPT are not added, and other components and dosages remain unchanged.

Comparative example 2

The difference from Example 1 is that Mek inhibitor PD0325901 and Notch inhibitor DAPT are not added, and other ingredients and dosages remain unchanged.

Comparative example 3

The difference from Example 1 is that Mek inhibitor PD0325901 and Wnt inhibitor IWP-2 are not added, and other components and dosages remain unchanged.

Comparative example 4

The difference from Example 1 is that the Notch inhibitor DAPT is not added, and the other ingredients and dosage remain unchanged.

Comparative example 5

The difference from Example 1 is that the Wnt inhibitor IWP-2 is not added, and other components and dosages remain unchanged.

Comparative example 6

The difference from Example 1 is that Mek inhibitor PD0325901 is not added, and other ingredients and dosages remain unchanged.

Example 4

A method for inducing directed differentiation of mouse small intestinal organoids into enteroendocrine cells, comprising the following steps:

(1) After the mice were killed by devertebralization, a small intestine with a length of about 20 cm was taken near the stomach of the mouse, and the membrane, blood vessels and fat outside the intestine were removed with forceps. After being cut longitudinally, it was blown and rinsed with a pipette gun of sterile PBS buffer solution containing 1% penicillin and streptomycin pre-cooled at 4°C until clean;

(2) Use ophthalmic scissors to cut the treated small intestine segment into about 2-5mm intestinal segment, transfer it to a 50ml sterile EP tube, pre-wet a 10mL pipette with PBS, and use it to gently flush the intestinal segment up and down three times, then let the segment settle by gravity, suck out the supernatant, and add 15mL of PBS, repeat the above steps 15 to 20 times until the supernatant is clear;

(3) Remove the supernatant, resuspend the tissue fragments in 25 mL of EDTA solution at 4°C, and incubate on a shaker at 20 rpm for 30 minutes at 4°C;

(4) The supernatant containing EDTA was removed and washed three times with PBS, and the tissue fragment was resuspended in 10 mL of 4° C. PBS containing 0.1% BSA, and pipetted up and down three times. Let stand until most of the intestinal tissue fragments sink to the bottom, carefully remove the supernatant and filter with a 70 μm filter, collect the filtrate in a 50 mL EP tube, and mark the filtrate as "fraction 1"; repeat the steps three times to obtain fractions 2-4.

(5) Centrifuge all fractions at 290g for 5 minutes at 4°C, discard the supernatant carefully, resuspend in 10mL of PBS buffer containing 0.1% BSA, transfer the suspension of each test tube to a 15mL EP tube, centrifuge at 200g for 5 minutes at 4°C, and resuspend in 10mL of Advanced DMEM at 4°C;

(6) Take 10 μL from all fractions, and use an inverted microscope to check the quality of the fractions. Normally, fractions 3 and 4 are more suitable for subsequent cultured crypts;

(7) Count the number of crypts in the 10 μL sample, so as to calculate the number of crypts per ml in the fraction; extract the required number of wells * 200 crypts per ml, centrifuge at 200 g for 5 minutes at 4°C, resuspend in the required number of wells * 25 μL of intestinal organoid standard condition (ENR) medium, and add the required number of wells * 25 μL Matrigel Matrigel, pipette up and down to resuspend the precipitate, pay attention to avoid air bubbles; among them, intestinal organoid standard condition (ENR) medium The components are Advanced DMEM medium 15ml, Glutamax 150μL, Hepes buffer 150μL, N-2supplement 150μL, B-27supplement 150μL, Penicillin 150μL, Noggin 120ng/ml, R-spondin1600ng/ml, EGF 80ng/ml;

(8) Add the crypts to the preheated 24-well plate. The sample should form solidified droplets in the center of each well. Leave the culture plate at 37°C for 10 minutes until the Matrigel is completely solidified, and use a pipette gun to gently add 500 μL of intestinal organoid standard condition (ENR) medium to each well along the side wall of the well. Add sterile PBS to other wells that have not been inoculated with droplets, and put them in an incubator at 37°C and 5% CO ₂ for cultivation every 3 days. Change the medium once, usually 5 to 7 days later for subculture, the subculture ratio is 1:4;

(9) After three passages, the crypts were plated, and 500 μL of the induction medium of Example 1 was added after the Matrigel was completely solidified, and placed in an incubator at 37° C. and 5% CO ₂ for cultivation, and the medium was renewed every 2 days at the beginning of differentiation;

(10) After 4 days of differentiation, aspirate the culture medium in the cell culture plate, add 1mL of 4°C pre-cooled PBS buffer to each well, pipette 10 times to break the intestinal organoid, centrifuge at 4°C for 5 minutes at 290g, discard the supernatant, resuspend in 10mL of 4°C Advanced DMEM, centrifuge at 4°C for 5 minutes, discard the supernatant, and resuspend in the required number of wells * 25μL Advanced DMEM and Matrigel;

(11) Spread the crypts evenly, and add 500 μL Advanced DMEM after the matrigel is completely solidified, and then wait for the experimental treatment.

Test example 1

According to the steps of Example 4, during the induction culture of step (9), the induction medium of Example 1 was replaced with the induction medium of Comparative Example 1 to Comparative Example 6 to induce culture of small intestinal organoids, and mouse small intestinal organoids cultured in standard intestinal organoid standard conditions (ENR) medium alone were used as controls, a total of 8 groups. Observe the morphology and growth status of organoids under different culture conditions.

According to Figure 1, it can be seen that the mouse small intestinal organoids were cultured in standard conditions (ENR) culture medium for 1-8 days, and the extracted mouse intestinal crypt stem cells were cultured and differentiated in matrigel to form balloon-shaped hollow intestinal organoids, and the central cavity contained villi-like epithelial cells. After 2-4 days of culture, crypt stem cells continued to proliferate and differentiate into epithelial cells, increasing the volume of intestinal organoids. After 4-6 days in culture, new bud-like domains folded outward from the organoid epithelium and contained intestinal crypt stem cells inside, which were isolated to form new intestinal organoids. As the culture time increased, the dead epithelial cells fell into the central cavity, forming black shadows under the microscope on day 8 and containing a large number of apoptotic factors.

According to Figure 2, it can be seen that the growth status of small intestinal organoids on the 0th day and the 4th day under the conditions of different media (single MEK, Wnt, Notch inhibitor treatment or combined treatment), obviously, after 4 days, the iMek/iWnt/iNotch (3i) group grew well, while a large number of organoids died in the iNotch, iMek, and iMek/iNotch groups. Experiments have shown that only the combined inhibition of Wnt, Notch and Mek pathways can prevent the proliferation of intestinal crypt stem cells and prevent the excessive growth of the entire organoid from initiating the apoptosis program.

Test example 2

ChgA detection: Matrigel matrigel was removed by centrifugation, the intestinal organoids obtained in Experiment 1 under different culture conditions were transferred to lysine-coated glass slides, and fixed overnight in a 4°C refrigerator with 4% paraformaldehyde. Absorb paraformaldehyde, wash 3 times with PBS, rupture the membrane with 0.5% Triton X-100 at room temperature for 10 minutes, and wash 3 times with PBS. Then, they were blocked with 3% BSA at room temperature for 30 minutes, and the slides were washed three times, incubated with primary antibody (Chromogranin A (ChgA) antibody, abcam) and secondary antibody (goat anti-rabbit IgG H&L, abcam), mounted, observed and photographed. Among them, DPAI marks the position of nucleus, and ChgA marks enteroendocrine cells.

According to the results in Figure 3, the number of enteroendocrine cell marker ChgA in each organoid in each group was detected by immunofluorescence (ChgA is green, DPAI is blue). The results showed that compared with the ENR group, the number of enteroendocrine cell markers treated with iMek/iWnt/iNotch (3i) increased by 985%, and the difference was significant.

Test example 3

Gene detection: the mouse small intestinal organoids cultured in the induction medium of Example 1 for 4 days or the mouse small intestinal organoids cultured in the ENR medium were sucked off the culture medium in the well plate, and 400 μL Trizol was directly added to each well for lysis, followed by pipetting and then standing at room temperature for 5 minutes for RNA extraction and reverse transcription. qRT-PCR was used to detect the mRNA expression levels of various cell markers in organoids, the mature marker genes of enteroendocrine cells, and the mRNA expression levels of marker hormones of common endocrine cell subtypes in the small intestine.

According to the results in Figure 4, the mRNA expression levels of various cell markers in organoids were detected by qRT-PCR, and it was found that after 4 days of joint inhibition of the Wnt/Notch/Mek pathway, the mRNA expression levels of intestinal epithelial cells, goblet cells, and endocrine cells increased significantly by 13.2 times, 8.4 times, and 28.8 times, respectively, while the mRNA expression levels of Paneth cells and intestinal stem cells decreased significantly (Figure 4A). After inhibiting the Wnt/Notch/Mek pathway for 4 days, the expression level of isl1 gene was significantly increased compared with the expression level of neurog3 gene (Figure 4B); the mRNA expression levels of marker hormones of common endocrine cell subtypes in the small intestine were detected by qRT-PCR. In (D cells) and other common endocrine cell subtypes of the small intestine significantly increased the mRNA expression of marker hormones, which were significantly different from those in the ENR group (Figure 4C). Therefore, it can be concluded that MEK, Wnt, and Notch inhibitors (3i) combined treatment of mouse small intestinal organoids can successfully differentiate them into mature enteroendocrine cells.

Example 8

The difference from Example 7 is that the subculture ratio in step (8) is 1:5, and the rest of the steps remain unchanged.

Example 9

The difference from Example 7 is that the subculture ratio in step (8) is 1:3, and the rest of the steps remain unchanged.

The above is only a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications should also be considered as the protection scope of the present invention.

Claims (10)

1. An induction medium, characterized in that the medium components include intestinal organoid standard condition ENR medium 10～15mL, Mek inhibitor PD03259010.5～2μM, Wnt inhibitor IWP-23～8μM and Notch inhibitor DAPT8～15μM; the ENR medium components are AdvancedDMEM medium 10～15ml, Glutamax100～150μL, Hepesbuffer100～15 0μL, N-2supplement100～150μL, B-27supplement100～150μL, Penicillin 100～150μL, Noggin80～120ng/ml, R-spondin1400～600ng/ml, EGF 30～80ng/ml. 2. A method for inducing directed differentiation of mouse intestinal organoids into enteroendocrine cells, comprising the steps of: using the induction medium according to claim 1 to cultivate mouse small intestinal organoids. 3. The method according to claim 2, characterized in that the cultivation conditions are 35-37°C, 5% CO ₂ . 4. The method according to claim 2, wherein the culture medium was renewed every 2 days when the mouse small intestine organoids differentiated; after 4 days of differentiation, the culture medium was sucked off, and PBS buffer was added to pipette, centrifuged, and the supernatant was discarded, resuspended in AdvancedDMEM at 4°C, centrifuged, discarded the supernatant, and resuspended in AdvancedDMEM and Matrigel. 5. The method according to claim 4, characterized in that the conditions of the centrifugation are 0-4°C, 280-290 g for 2-8 minutes. 6 . The method according to claim 2 , wherein the mouse small intestinal organoid is obtained by subculturing the mouse small intestinal crypt stem cells in standard intestinal organoid condition ENR medium for 5-7 days. 7. The method according to claim 6, wherein the components of the intestinal organoid standard condition ENR medium are 10-15 ml of AdvancedDMEM medium, 100-150 μL of Glutamax, 100-150 μL of Hepesbuffer, 100-150 μL of N-2supplement, 100-150 μL of B-27supplement, 100-150 μL of penicillin and streptomycin, Noggin8 0-120ng/ml, R-spondin1400-600ng/ml, EGF30-80ng/ml. 8. The method according to claim 6, wherein the passage ratio is 1:3-1:5. 9 . The method according to claim 6 , wherein the mouse small intestinal crypt stem cells are obtained by digesting mouse small intestinal tissue with EDTA. 10. The application of the induction medium according to claim 1 or the method according to any one of claims 2 to 9 in constructing a mouse enteroendocrine cell organoid model.