Culture medium for inducing directional differentiation of mouse intestinal organoids into intestinal endocrine cells and applications therefor

Abstract

To provide a culture medium for inducing directional differentiation of mouse intestinal organoids into intestinal endocrine cells and applications therefor.SOLUTION: A Mek inhibitor PD0325901, a Wnt inhibitor IWP-2 and a Notch inhibitor DAPT are added into a mouse small intestine organoid standard condition (ENR) culture medium, to induce mouse small intestine organoids to be differentiated into mature intestinal endocrine cells, thereby achieving the construction of the mouse intestinal endocrine cell induction model. The induction culture medium is used to induce mouse intestinal organoids to be differentiated into intestinal endocrine cells. The culture medium is replaced every two days following the initiation of differentiation of mouse small intestine organoids. Following 4 days of differentiation, the culture solution is removed by aspiration. A PBS buffer is added and sprayed, followed by centrifugation and removal of a supernatant. Resuspension is conducted in 4°C Advanced DMEM, followed by centrifugation and removal of the supernatant, and then resuspension in Advanced DMEM and Matrigel.SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] The present invention relates to the technical field of inductive culture of organoids, specifically to a medium for inducing directed differentiation of mouse intestinal organoids into enteroendocrine cells and its use. [Background technology]

[0002] Enteroendocrine cells are differentiated from small intestinal stem cells and distributed throughout the intestinal tract, regulating physiological activities such as intestinal secretion, motility, digestion and absorption, blood glucose regulation and metabolism. They can secrete various types of intestinal hormones, and through endocrine and paracrine secretion, they play an important role in intestinal peristalsis, vasoconstriction, nutrient absorption, epithelial growth and central biological rhythm regulation. At the same time, they play an important role in research on non-clinical and clinical systems for medicine, nutrition and tissue projects. In the past, in vitro studies of enteroendocrine cells have always remained at the level of cell lines, cultured in monolayers, and have significant differences in cellular environment, genetic background and functional properties compared with ordinary enteroendocrine cells.

[0003] Research has shown that Lgr5 in the intestinal tract + It has been shown that intestinal stem cells have the potential to differentiate into all types of cells in the intestinal epithelium, including Paneth cells, endocrine cells, and enterocyte cells. + Intestinal stem cells can be extracted and induced to form intestinal organoids that can maintain and grow on their own. The organoids contain all intestinal epithelial cell types, have a similar cellular environment to the intestine, and have a similar expression pattern of intestinal hormones to the natural intestine. However, in intestinal organoids, intestinal endocrine cells account for only 1%. Considering that current culture methods are difficult to obtain intestinal endocrine cell populations by controlling their specific differentiation direction, it is therefore urgent to develop a stable, efficient and convenient method for constructing a mouse intestinal endocrine cell induction model. Summary of the Invention [Problem to be solved by the invention]

[0004] In view of this, the present invention aims to provide an induction medium that allows treating mouse small intestinal organoids for successful and rapid directed differentiation into mature enteroendocrine cells. [Means for solving the problem]

[0005] In order to achieve the above objectives, the present invention provides the following technical solutions:

[0006] The present invention provides an induction medium, and the medium components include 10 to 15 mL of intestinal organoid standard condition ENR medium, 0.5 to 2 μm of Mek inhibitor PD0325901, 3 to 8 μm of Wnt inhibitor IWP-2, and 8 to 15 μm of Notch inhibitor DAPT, and the ENR medium components are 10 to 15 ml of Advanced DMEM medium, 100 to 150 μL of Glutamax, 100 to 150 μL of Hepes buffer, 100 to 150 μL of N-2 supplement, 100 to 150 μL of B-27 supplement, 100 to 150 μL of mycilin, 80 to 120 ng / ml of Noggin, 400 to 600 ng / ml of R-spondin1, and 30 to 80 ng / ml of EGF.

[0007] The present invention also provides a method for inducing mouse intestinal organoids to undergo directed differentiation into enteroendocrine cells, comprising culturing mouse small intestinal organoids using the induction medium.

[0008] Preferably, the culture conditions are 35 to 37° C., 5% CO 2 It is.

[0009] Preferably, the medium is renewed every two days after the initiation of differentiation of mouse small intestinal organoids, and after differentiation for four days, the culture medium is aspirated and removed, an additional PBS buffer is added and sprayed, centrifuged, the supernatant is discarded, resuspended in Advanced DMEM at 4°C, centrifuged, the supernatant is discarded, and resuspended in Advanced DMEM and Matrigel.

[0010] More preferably, the centrifugation conditions are 0 to 4° C. and 280 to 290 g for 2 to 8 minutes.

[0011] Preferably, the mouse small intestinal organoid is obtained by culturing mouse small intestinal crypt cells in standard intestinal organoid condition ENR medium for 5 to 7 days and then subculturing the cells.

[0012] More preferably, the intestinal organoid standard condition ENR medium components are Advanced DMEM medium 10 to 15 ml, Glutamax 100 to 150 μL, Hepes buffer 100 to 150 μL, N-2 supplement 100 to 150 μL, B-27 supplement 100 to 150 μL, mycilin 100 to 150 μL, Noggin 80 to 120 ng / ml, R-spondin1 400 to 600 ng / ml, and EGF 30 to 80 ng / ml.

[0013] More preferably, the passage ratio is 1:3 to 1:5.

[0014] More preferably, the mouse small intestinal crypt dry cells are obtained by digesting mouse small intestinal tissue with EDTA.

[0015] The present invention also provides the use of the induction medium or the method in constructing a mouse enteroendocrine cell organoid model. Effect of the Invention

[0016] Compared with the prior art, the present invention has the following beneficial effects: The induction medium provided by the present invention directs mouse small intestinal organoids to differentiate into enteroendocrine cells. By immunofluorescence detection, the number of enteroendocrine cell markers treated with the induction medium of the present invention increased by 985% compared with the control group, and by qRT-PCR, the expression levels of various cell marker mRNAs in the organoids were detected, and the expression levels of the labeled 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), were all significantly increased. The results show that the induction medium of the present invention can be used to treat mouse small intestinal organoids to direct differentiation into mature enteroendocrine cells, which is advantageous for successfully constructing an induction model of mouse enteroendocrine cells. The model can better simulate the intestinal cell environment than 2D cell models, which can be used to explore the effects and molecular mechanisms of various disease models on enteroendocrine cells and their secretion of intestinal hormones, and can also be used to study intestinal nutrition and health, screening and safety assessment of enteroendocrine diseases. [Brief description of the drawings]

[0017] [Figure 1] Intestinal organoids Standard condition ENR medium was used to culture mouse small intestinal organoids. This shows the culture growth status over 1 to 8 days. [Diagram 2] FIG. 1 shows the growth status of small intestinal organoids on days 0 and 4 under different medium conditions. [Diagram 3] 1 shows the results of immunofluorescence detection of enteroendocrine cell markers in small intestinal organoids under different culture conditions. [Figure 4] This shows the results of cell marker mRNA expression levels in small intestinal organoids under induction medium conditions of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The present invention provides an induction medium, and the medium components preferably include 10 to 15 mL of intestinal organoid standard condition ENR medium, 0.5 to 2 μm of a Mek inhibitor PD0325901, 3 to 8 μm of a Wnt inhibitor IWP-2, and 8 to 15 μm of a Notch inhibitor DAPT. The intestinal organoid standard condition ENR medium components preferably include 10 to 15 ml of Advanced DMEM medium, 100 to 150 μL of Glutamax, 100 to 150 μL of Hepes buffer, 100 to 150 μL of N-2 supplement, 100 to 150 μL of B-27 supplement, 100 to 150 μL of mycilin, 80 to 120 ng / ml of Noggin, 400 to 600 ng / ml of R-spondin1, and 80 to 600 ng / ml of EGF. The concentration is 30 to 80 ng / ml, and more preferably, the medium components include 12 mL of intestinal organoid standard condition ENR medium, 1 μm of Mek inhibitor PD0325901, 5 μm of Wnt inhibitor IWP-2, and 10 μm of Notch inhibitor DAPT, and the intestinal organoid standard condition ENR medium components are more preferably 12 ml of Advanced DMEM medium, 120 μL of Glutamax, 120 μL of Hepes buffer, 120 μL of N-2 supplement, 120 μL of B-27 supplement, 120 μL of mycilin, 100 ng / ml of Noggin, 500 ng / ml of R-spondin1, and 50 ng / ml of EGF. In the present invention, the Mek inhibitor PD0325901, the Wnt inhibitor IWP-2, and the Notch inhibitor DAPT inhibitor are purchased from MedChemExpress, the Advanced DMEM medium, Glutamax, Hepes buffer, N-2 supplement, B-27 supplement, and Mycilin are purchased from Thermo Scientific, and the Noggin, R-spondin1, and EGF are purchased from Propertech.

[0019] The present invention also provides a method for inducing mouse intestinal organoids to undergo directed differentiation into enteroendocrine cells, comprising culturing mouse small intestinal organoids using the induction medium.

[0020] In the present invention, the culture conditions are 35 to 37°C, 5% CO 2 It is.

[0021] In the present invention, the medium is renewed every two days after the initiation of differentiation of mouse small intestinal organoids. After differentiation for four days, the culture medium is aspirated and removed, PBS buffer is added and sprayed, centrifuged, the supernatant is discarded, and the cells are resuspended in Advanced DMEM at 4°C, centrifuged, the supernatant is discarded, and the cells are resuspended in Advanced DMEM and Matrigel.

[0022] In the present invention, the centrifugation conditions are preferably 0 to 4° C. and 280 to 290 g for 2 to 8 minutes.

[0023] In the present invention, the mouse small intestinal organoids are obtained by culturing mouse small intestinal crypt cells in standard intestinal organoid condition ENR medium for 5 to 7 days and then subculturing them. In a specific embodiment of the present invention, the culture conditions are 37° C. and 5% CO 2 In the present invention, the number of subcultures is preferably 3 to 4, and the state of the organoid can be well maintained by subculture.

[0024] In the present invention, the components of the intestinal organoid standard condition ENR medium are preferably 10 to 15 ml of Advanced DMEM medium, 100 to 150 μL of Glutamax, 100 to 150 μL of Hepes buffer, 100 to 150 μL of N-2 supplement, 100 to 150 μL of B-27 supplement, 100 to 150 μL of mycilin, 80 to 120 ng / ml of Noggin, 400 to 600 ng / ml of R-spondin1, and 30 to 80 ng / ml of EGF, and more preferably 12 ml of the intestinal organoid standard condition ENR medium, 12 ml of Advanced DMEM medium, 120 μL of Glutamax, 120 μL of Hepes buffer, 120 μL of N-2 supplement, 120 μL of B-27 supplement, 120 μL of Glutamax, 120 μL of Hepes buffer, 120 μL of N-2 supplement, 120 μL of B-27 supplement, 120 μL of Glutamax, 120 μL of Hepes buffer, 120 μL of N-2 supplement 120μL, B-27 supplement 120μL, mycilin 120μL, Noggin 100ng / ml, R-spondin1 500ng / ml, EGF 50ng / ml.

[0025] In the present invention, the passaging ratio is preferably 1:3 to 1:5, more preferably 1:4. The passaging ratio of the present invention can be appropriately adjusted depending on the state of the crypts.

[0026] In the present invention, the mouse small intestinal crypt dry cells are obtained by digesting mouse small intestinal tissue with EDTA. After digestion with EDTA, the small intestinal tissue becomes loose, and under the action of external mechanical force, the small intestinal crypts are separated from the tissue, and the external mechanical force can be repeated several times to obtain different fraction mixtures, and the fraction with higher crypt content and less parenchyma cells can be selected and cultured in a cell culture plate. In a specific embodiment of the present invention, the obtained small intestinal tissue slice is resuspended in 25mL 4°C EDTA solution, and placed on a shaking table under conditions of 0-4°C, and cultured at a rotation speed of 20 rpm for 30-60 minutes, the supernatant containing EDTA is removed, and the tissue slice is washed three times with PBS, resuspended in 4°C PBS containing 0.1% BSA, and sprayed up and down three times using a pipette. Allow to stand until most of the intestinal tissue slices sink to the bottom, carefully remove the supernatant, filter using a 70 μm filter, collect the filtrate in an EP tube, mark the filtrate as "fraction 1", and repeat the steps three times to obtain fractions 2 to 4. In a specific embodiment of the present invention, fractions 3 and 4 are preferably used to prepare crypts for subsequent culture.

[0027] The present invention also provides the use of the induction medium or the method in constructing a mouse enteroendocrine cell organoid model.

[0028] The technical solutions provided by the present invention are described in detail below with reference to examples, which cannot be understood as limiting the protection scope of the present invention.

[0029] Example 1 The induction medium contained 12 ml of intestinal organoid standard condition (ENR) medium, 1 μm of Mek inhibitor PD0325901, 5 μm of Wnt inhibitor IWP-2, and 10 μm of Notch inhibitor DAPT, and the intestinal organoid standard condition (ENR) medium components contained 12 ml of Advanced DMEM medium, 120 μL of Glutamax, 120 μL of Hepes buffer, 120 μL of N-2 supplement, 120 μL of B-27 supplement, 120 μL of mycilin, 100 ng / ml of Noggin, 500 ng / ml of R-spondin1, and 50 ng / ml of EGF.

[0030] Example 2 The induction medium contained 10 ml of intestinal organoid standard condition (ENR) medium, 0.5 μm of Mek inhibitor PD0325901, 3 μm of Wnt inhibitor IWP-2, and 8 μm of Notch inhibitor DAPT, and the components of the intestinal organoid standard condition (ENR) medium were 10 ml of Advanced DMEM medium, 100 μL of Glutamax, 100 μL of Hepes buffer, 100 μL of N-2 supplement, 100 μL of B-27 supplement, 100 μL of Hepes buffer, 100 μL of N-2 supplement, 100 μL of B-27 supplement, 100 μL of mycilin, 80 ng / ml of Noggin, 400 ng / ml of R-spondin1, and 30 ng / ml of EGF.

[0031] Example 3 The induction medium contained 15 ml of intestinal organoid standard condition (ENR) medium, 2 μm of Mek inhibitor PD0325901, 8 μm of Wnt inhibitor IWP-2, and 15 μm of Notch inhibitor DAPT, and the components of the intestinal organoid standard condition (ENR) medium were 15 ml of Advanced DMEM medium, 150 μL of Glutamax, 150 μL of Hepes buffer, 150 μL of N-2 supplement, 150 μL of B-27 supplement, 150 μL of mycilin, 120 ng / ml of Noggin, 600 ng / ml of R-spondin1, and 80 ng / ml of EGF.

[0032] Comparative Example 1 The difference from Example 1 was that the Wnt inhibitor IWP-2 and the Notch inhibitor DAPT were not added, and other components and dosages were unchanged.

[0033] Comparative Example 2 The difference from Example 1 was that the Mek inhibitor PD0325901 and the Notch inhibitor DAPT were not added, and other ingredients and dosages were unchanged.

[0034] Comparative Example 3 The difference from Example 1 was that the Mek inhibitor PD0325901 and the Wnt inhibitor IWP-2 were not added, and other ingredients and dosages were unchanged.

[0035] Comparative Example 4 The difference from Example 1 was that the Notch inhibitor DAPT was not added, and other ingredients and dosages were unchanged.

[0036] Comparative Example 5 The difference from Example 1 was that the Wnt inhibitor IWP-2 was not added, and other ingredients and dosages were unchanged.

[0037] Comparative Example 6 The difference from Example 1 was that the Mek inhibitor PD0325901 was not added, and other ingredients and dosages were unchanged.

[0038] Example 4 A method to induce directed differentiation of mouse small intestinal organoids into enteroendocrine cells is described. After the mouse was sacrificed by dislocating the cervical vertebrae, a small intestine of about 20 cm length was taken from the mouse near the stomach, and the membrane, blood vessels, and fat of the outer part of the intestine were removed using tweezers. The intestine was then dissected longitudinally, and washed clean with a pipette containing 1% mycilin and pre-cooled at 4°C in sterile PBS buffer (step 1); (2) shearing the treated small intestine segments with ophthalmic scissors into intestinal segments of approximately 2-5 mm, transferring them into a 50 ml sterile EP tube, pre-wetting a 10 ml pipette with PBS and purging the intestinal segments up and down three times, then allowing the segments to settle by gravity, aspirating the supernatant, adding 15 ml PBS, and repeating the above steps 15 to 20 times until the supernatant is clear; (3) removing the supernatant and resuspending the tissue segments in 25 mL of 4°C EDTA solution and incubating them at 4°C on a shaking table with a rotation speed of 20 rpm for 30 minutes; The supernatant containing EDTA was removed, and the tissue slices were washed three times with PBS. The tissue slices were resuspended in 10 mL of 4°C PBS containing 0.1% BSA and sprayed up and down three times using a pipette. The tissue slices were left to stand until most of the intestinal tissue slices had sunk to the bottom, and the supernatant was carefully removed and filtered using a 70 μm filter. The filtrate was collected in a 50 mL EP tube, and the filtrate was marked as "fraction 1". Step (4) was repeated three times to obtain fractions 2 to 4. All fractions were centrifuged at 290g for 5 min at 4°C, the supernatant was carefully discarded, and resuspended in 10 mL of PBS buffer containing 0.1% BSA. The suspension from each tube was transferred to a 15 mL EP tube, centrifuged at 200g for 5 min at 4°C, and resuspended in 10 mL of 4°C Advanced DMEM (step (5)); Take 10 μL from every fraction and check the mass of the fraction using an inverted microscope. In general, fractions 3 and 4 are more suitable for crypts for subsequent culture (step 6), and Calculate the number of crypts in the 10μL sample, and calculate the number of crypts per milliliter in the fraction. Extract the fraction of the required number of crypts * 200 crypts per milliliter. Centrifuge at 200g for 5 minutes at 4℃. Resuspend in the required number of crypts * 25μL in intestinal organoid standard condition (ENR) medium. Add the required number of crypts * 25μL Matrigel. Resuspend the pellet by aspirating and blowing up and down, being careful to avoid creating air bubbles. Here, the components of the intestinal organoid standard condition (ENR) medium are Advanced DMEM medium 15ml, Glutamax 150 μL, Hepes buffer 150 μL, N-2 supplement 150 μL, B-27 supplement 150 μL, Glutamax 150 μL, Hepes buffer 150 μL, N-2 supplement 150 μL, B-27 supplement 150 μL, Mycilin 150 μL, Noggin Step (7) was 120ng / ml, R-spondin1 600ng / ml, and EGF 80ng / ml. Add the crypts to a pre-warmed 24-well plate, the samples should form a solidified droplet in the center of each hole, place the culture plate at 37 °C for 10 min until the Matrigel is completely solidified, gently add 500 μL Intestinal Organoid Standard Condition (ENR) medium to each hole along the hole sidewall using a pipette, add sterile PBS to the holes that are not inoculated with other droplets, and place in an incubator at 37 °C and 5% CO. 2 Step (8): The cells were cultured in 100 ml of ... After three passages, the crypts were spread onto a plate, 500 μL of the induction medium of Example 1 was added until the matrigel was completely solidified, and the plate was placed in an incubator at 37° C. and 5% CO 2 (9) The medium was renewed every two days after differentiation began. After differentiation for 4 days, the culture medium in the cell culture plate is aspirated, 1 mL of PBS buffer pre-cooled at 4 ° C is added to each hole, and the intestinal organoids are broken down by spraying 10 times, centrifuged at 290 g for 5 minutes at 4 ° C, the supernatant is discarded, resuspended in 10 mL of 4 ° C Advanced DMEM, centrifuged at 200 g for 5 minutes at 4 ° C, the supernatant is discarded, and resuspended in the required number of holes * 25 μL Advanced DMEM and Matrigel (step (10); The crypts were uniformly spread, 500 μL of Advanced DMEM was added until the Matrigel was completely solidified, and then the experimental treatment was waited for (11).

[0039] Test Example 1 According to the steps of Example 4, during the induction culture of step (9), the induction culture medium of Example 1 was replaced with the induction culture medium of Comparative Example 1 to Comparative Example 6, respectively, to perform induction culture on the small intestinal organoids, and a total of eight sets of mouse small intestinal organoids cultured in the intestinal organoid standard condition (ENR) medium alone were used as controls. The morphology and growth state of the organoids under different culture conditions were observed.

[0040] Figure 1 shows the culture growth state of mouse small intestinal organoids obtained by culturing in intestinal organoid standard condition (ENR) medium for 1 to 8 days. The extracted mouse intestinal crypt endothelial cells were cultured and differentiated in Matrigel to form balloon-shaped mid-jejunal organoids, whose central cavity contained villi-like epithelial cells. After 2 to 4 days of culture, the crypt endothelial cells constantly proliferated and differentiated into epithelial cells, increasing the volume of the intestinal organoids. After 4 to 6 days of culture, the organoids folded outward from the epithelial cells to form new bud-like structural domains, and the interior contained intestinal crypt endothelial cells, which, when separated, could form new intestinal organoids. With increasing culture time, dead epithelial cells fell into the central cavity, forming a black shadow under the microscope on the 8th day, and contained a large amount of cell apoptosis factors.

[0041] Figure 2 shows the growth status of small intestinal organoids on day 0 and day 4 under different culture medium conditions (treated with MEK, Wnt, Wnt, Notch inhibitors alone or after co-treatment), and it is clear that after 4 days, the growth status of the iMek / iWnt / iNotch (3i) group was good, while there were many organoid deaths in the iNotch, iMek, and iMek / iNotch groups. The experiment showed that only by suppressing the Wnt, Notch, and Mek pathways in combination, it is possible to prevent the proliferation of intestinal crypt stem cells and avoid the excessive increase of the whole organoids to initiate the apoptosis process.

[0042] Test Example 2 ChgA detection: The Matrigel was removed by centrifugation, and the intestinal organoids under different culture conditions obtained in Test Example 1 were transferred to slides coated with lysine, and fixed overnight in a refrigerator at 4 ° C. using 4% paraformaldehyde. Paraformaldehyde was adsorbed, washed three times with PBS, and the cell membrane was crushed with 0.5% Triton X-100 at room temperature for 10 minutes, and washed three times with PBS. Then, the slides were closed with 3% BSA for 30 minutes at room temperature, and the slides were washed three times, and then the primary antibody (Chromogranin A (ChgA) antibody, abcam) and the secondary antibody (goat anti-rabbit IgG H & L, abcam) were cultured, mounted, observed, and photographed. Here, DPAI marked the position of the cell nucleus, and ChgA marked the enteroendocrine cells.

[0043] According to the results in Figure 3, the number of enteroendocrine cell marker ChgA in each organoid in each set detected by immunofluorescence (ChgA in green, DPAI in blue) was shown. The results showed that the number of enteroendocrine cell markers co-processed by iMek / iWnt / iNotch(3i) was increased by 985% compared to the ENR set, and the difference was significant.

[0044] Test Example 3 Gene detection: After culturing mouse enteroendocrine organoids in the induction medium of Example 1 for 4 days or mouse small intestinal organoids cultured in ENR medium, the medium in the well plate was aspirated, 400 μL Trizol was added directly to each hole to dissolve, sprayed, and left at room temperature for 5 minutes, and RNA extraction and reverse transcription were performed. qRT-PCR was used to detect the expression levels of various cell marker mRNAs in the organoids, the maturation marker genes of enteroendocrine cells, and the expression levels of hormone mRNAs labeled with common endocrine cell subtypes in the small intestine.

[0045] According to the results in Figure 4, the mRNA expression levels of various cell markers in organoids were detected by qRT-PCR. After 4 days of Wnt / Notch / Mek co-inhibition, the mRNA expression levels of intestinal epithelial cells, goblet cells, and endocrine cells all increased significantly, increasing by 13.2 times, 8.4 times, and 28.8 times, respectively, while the mRNA expression levels of Paneth cells and intestinal ductal cells decreased significantly (Figure 4A). The maturation marker genes of intestinal endocrine cells were detected by qRT-PCR. After 4 days of Wnt / Notch / Mek co-inhibition, the expression level of isl1 gene was significantly increased more than that of neurog3 gene (Figure 4B). We detected the expression levels of the mRNAs of the common endocrine cell subtypes in the intestine, including CCK (I cells), GLP-1 (L cells), Ghrelin (A cells), Secrtin (S cells), GIP (K cells) and Somatostatin (D cells), all of which were significantly increased after 4 days of co-inhibition of the Wnt / Notch / Mek pathway, with significant differences compared to the ENR group (Figure 4C). Therefore, we can conclude that co-treatment of mouse small intestinal organoids with MEK, Wnt and Notch inhibitors (3i) can enable directed differentiation into mature enteroendocrine cells.

[0046] Example 8 The difference from Example 7 was that the passaging ratio in step (8) was 1:5, and the remaining steps were unchanged.

[0047] Example 9 The difference from Example 7 was that the passaging ratio in step (8) was 1:3, and the remaining steps were unchanged.

[0048] It should be noted that the above is only a preferred embodiment of the present invention, and those skilled in the art can make some improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also fall within the protection scope of the present invention.

Claims

1. The medium components include 10 to 15 mL of ENR medium, which is a standard condition for intestinal organoids, 0.5 to 2 μm of Mek inhibitor PD0325901, 3 to 8 μm of Wnt inhibitor IWP-2, and 8 to 15 μm of Notch inhibitor DAPT. The ENR medium components include 10 to 15 ml of Advanced DMEM medium, 100 to 150 μL of Glutamax, 100 to 150 μL of Hepes buffer, 100 to 150 μL of N-2 supplement, 100 to 150 μL of B-27 supplement, 100 to 150 μL of mycilin, 80 to 120 ng / ml of Noggin, 400 to 600 ng / ml of R-spondin1, and 800 to 600 ng / ml of EGF. An induction medium comprising 30 to 80 ng / ml of the gene.

2. A method for inducing mouse intestinal organoids to differentiate directional into enteroendocrine cells, comprising culturing mouse small intestinal organoids using the induction medium described in claim 1.

3. The culture conditions were 35 to 37° C. and 5% CO ２ 3. The method according to claim 2, wherein

4. The method according to claim 2, characterized in that the medium is renewed every two days after the initiation of differentiation of mouse small intestinal organoids, and after differentiation for four days, the culture medium is removed by aspirating, PBS buffer is added and sprayed, centrifuged, the supernatant is discarded, and the organoids are resuspended in Advanced DMEM at 4°C, centrifuged, the supernatant is discarded, and the organoids are resuspended in Advanced DMEM and Matrigel.

5. The method according to claim 4, wherein the centrifugation conditions are 0 to 4° C. and 280 to 290 g for 2 to 8 min.

6. The method according to claim 2, characterized in that the mouse small intestinal organoid is obtained by culturing mouse small intestinal crypt dry cells in standard intestinal organoid condition ENR medium for 5 to 7 days and then subculturing.

7. The intestinal organoid standard condition ENR medium components are Advanced DMEM medium 10-15 ml, Glutamax 100-150 μL, Hepes buffer 100-150 μL, N-2 supplement 100-150 μL, B-27 supplement 100-150 μL, mycilin 100-150 μL, Noggin 80-120 ng / ml, R-spondin1 400-600 ng / ml, EGF 30-80 ng / ml. The method according to claim 6.

8. The method according to claim 6, wherein the passage ratio is 1:3 to 1:

5.

9. The method according to claim 6, wherein the mouse small intestinal crypt cells are obtained by digesting mouse small intestinal tissue with EDTA.

10. Use 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.

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