Culture method of small intestine tumor primary organoid

Abstract

The invention relates to the technical field of biology, in particular to a culture method of small intestine tumor primary organoids, which comprises the following specific steps: preparing by taking mouse subcutaneous connective tissue cells as feeder cells; mixing the treated feeder cells with the small intestine crypt-like structure of the mouse, and resuspending in matrigel; the method comprises the following steps: inoculating a mixed suspension of feeder cells, mouse small intestine crypt-like structures and matrigel into holes of a 24-hole cell culture plate, and after the matrigel is completely solidified, adding a prepared complete culture medium along the wall; the culture plate is placed in a 37 DEG C carbon dioxide incubator for culture, the culture medium is replaced once every three days, and the growth state of the organoid is monitored. According to the culture method of the small intestine tumor primary organoid, provided by the invention, the survival rate of the small intestine organoid can be remarkably improved, and the construction of the small intestine tumor primary organoid can be completed in a shorter time.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for culturing primary organoids of small intestinal tumors. Background Art

[0002] Organoid culture technology is an in vitro culture method that simulates the structure and function of organs in vivo. It has been widely used in biomedical research in recent years. In 2009, Sato et al. used three-dimensional matrix gel (Matrigel) containing EGF, Noggin, and R-spondin to culture a single mouse intestinal stem cell rich in Lgr5 markers, and successfully formed small intestinal organoids with crypt and villus-like structures, ushering in the era of organoid culture. The development of this technology not only provides a powerful tool for studying intestinal biology, but also provides a new way for the establishment of disease models, drug screening and regenerative medicine research.

[0003] With the deepening of understanding of stem cell niche factors and signaling pathways, subtle changes in various factors in the culture system may produce different culture results. For example, adding various Wnt regulators (CHIR99021, GSK3β inhibitors) and Notch signaling pathway regulators (valproic acid, histone deacetylase inhibitors) to the intestinal culture medium helps to enrich and maintain the number of stem cells, while promoting their differentiation into mature intestinal epithelial cells and goblet cells. The development of complete culture medium enables the intestinal epithelial organoid culture system to reproduce the self-renewal and differentiation capabilities observed in the mature intestine in vivo. In addition to displaying all the known cell types present in the mature intestinal epithelium, another feature of intestinal organoids is that they have crypt-villus structure, epithelial polarization, and functional cavities, which make organoid culture a powerful tool for studying intestinal biology.

[0004] There are commercial complete culture media specifically for mouse intestinal organoid culture on the market, such as the Model Bio Mouse Small Intestinal Organoid Culture Kit, which simplifies the culture process and makes the establishment and culture of organoids more convenient and efficient. These culture media usually contain a variety of growth factors and other additives that can support the growth and differentiation of organoids and reproduce the characteristics of the small intestinal epithelium in vivo. However, the existing traditional culture methods for primary small intestinal tumor organoids have a low success rate, and the activity decreases quickly, and long storage time can easily cause the activity to decrease. Summary of the invention

[0005] In order to overcome the shortcomings of the prior art, the purpose of the present invention is to provide a method for culturing primary organoids of small intestinal tumors, thereby improving the success rate of primary construction of small intestinal tumor organoids while improving the success rate of culturing the same batch of primary organoids of small intestinal tumors.

[0006] The present invention adopts the following technical solutions:

[0007] A method for culturing primary organoids of small intestinal tumors, characterized in that it comprises the following steps:

[0008] S1: Mouse subcutaneous connective tissue cells were prepared as feeder cells;

[0009] S2: The treated feeder cells were mixed with mouse intestinal crypt-like structures and resuspended in Matrigel;

[0010] S3: A mixed suspension of feeder cells, mouse small intestinal crypt-like structures, and Matrigel was inoculated into the wells of a 24-well cell culture plate, 30 μL per well;

[0011] S4: After the matrix gel is completely solidified, slowly add the prepared complete culture medium along the wall, 500 μL per well;

[0012] S5: Place the culture plate in a 37°C CO2 incubator, replace the culture medium every 3 days, and monitor the growth status of the organoids.

[0013] Furthermore, the step S1 specifically includes:

[0014] S101: Inoculate mouse subcutaneous connective tissue cells in a culture dish, add 10% FBS-DMEM complete medium for culture, and stop culturing when the cell density reaches 80-90%;

[0015] S102: irradiating the cells in step S101 with gamma rays at a rate of 150 Gy for 990 seconds;

[0016] S103: Prepare feeder cell suspension: discard the supernatant, wash twice with an appropriate amount of PBS, digest with trypsin for 3 minutes and terminate with complete medium, centrifuge at 1000 rpm for 3 minutes, discard the supernatant, resuspend the cells with complete medium, count and set aside.

[0017] Furthermore, the mouse small intestinal crypt-like structure in step S2 is obtained by the following steps:

[0018] S201: The mice were killed by dislocating the neck, and the surface was sterilized by spraying alcohol. The small intestinal tissue 3-10 cm near the stomach was removed under sterile conditions. The mesentery and fat outside the intestine were removed with forceps, and the small intestinal tissue was placed in a 4°C precooled DPBS solution containing double antibodies.

[0019] S202: Use surgical scissors to cut the intestine open, with the intestinal cavity facing upwards, scrape off the intestinal villi on the surface of the intestinal cavity with a surgical blade, and place the intestinal tissue in a new culture dish containing DPBS solution for washing. Repeat the washing twice, cut the washed small intestinal tissue into pieces of 2 mm in width, transfer it to a new culture dish, and wash it twice with DPBS solution;

[0020] S203: Transfer the cleaned intestinal segment to a pre-cooled DPBS solution containing 5mmol / L EDTA for digestion, incubate at 4℃ or in crushed ice for 20-30min, and gently blow the intestinal segment with a pipette for 20min. Take the supernatant and observe it under a microscope. Stop digestion when complete crypts appear in the supernatant. If there are no crypts, extend the digestion time.

[0021] S204: After digestion, transfer the tissue fragments to a new dish containing DPBS and wash, repeating twice to remove EDTA;

[0022] S205: Use a 5 mL pipette to blow and resuspend tissue fragments in a culture dish or a 50 mL centrifuge tube containing a pre-cooled 0.1% BSA DPBS solution, and make the tissue pass through the pipette tip repeatedly to generate mechanical shear force to separate the crypts from the basal layer. Take a portion of the suspension for microscopic examination. When a large number of crypt-like structures can be seen, stop blowing and filter the tissue suspension after blowing through a 70 μm filter.

[0023] S206: collect the tissue suspension that passes through the filter, centrifuge at 300 g, 4°C, for 3 min;

[0024] S207: Discard the supernatant, resuspend the tissue pellet with 1 mL of 0.1% BSA in DPBS solution, take 20 μL of the suspension for microscopic examination and crypt counting, aspirate the suspension after counting, centrifuge at 300 g, 4°C, for 3 min, discard the supernatant and place on ice to obtain a suspension of mouse small intestinal crypt-like structures.

[0025] Furthermore, the specific operation of resuspending in matrix gel in step S2 is: the resuspension density is 70-100 crypts and 4500 feeder cells per 10 μL matrix gel suspension, and the cells are placed on ice after resuspension, and the resuspension time does not exceed 30 seconds.

[0026] Furthermore, in step S2, the dilution ratio of the matrix gel is controlled to be above 70%.

[0027] Furthermore, the specific operation of completely solidifying the matrix gel in step S4 is: placing the culture plate after inoculation in a 37° C. carbon dioxide constant temperature incubator and incubating for 30 minutes.

[0028] Furthermore, the treated feeder cells are resuscitated and cultured for 1 day, mixed with the mouse small intestinal crypt-like structures, and resuspended in matrix gel.

[0029] Furthermore, in step S4, the complete culture medium is 10% FBS-DMEM culture medium supplemented with Y27632 and chir99021, the working concentration of Y27632 is 10 μM, and the working concentration of chir99021 is 10 μM.

[0030] Furthermore, the matrix gel is a three-dimensional matrix gel Matrigel containing EGF, Noggin, and R-spondin.

[0031] Furthermore, the 24-well cell culture plate is used in combination with a culture rack.

[0032] The present invention has at least the following beneficial effects:

[0033] 1. Improve the survival rate: By using mouse subcutaneous connective tissue cells as feeder cells and combining them with matrix gel to culture small intestinal tumor primary organoids, the survival rate of small intestinal organoids can be significantly improved; 2. Improve uniformity: Using organoid proliferation culture racks for culture to ensure that the growth conditions of organoids in each well are consistent, improving the production uniformity of the same batch of organoids. This helps to obtain consistent experimental results in large-scale culture and reduces experimental variability;

[0034] 3. Shortened culture cycle: The culture method for small intestinal tumor primary organoids provided by the present invention optimizes the culture conditions and adds feeder cells. Compared with the traditional method, it shortens the proliferation culture cycle of small intestinal organoids and can complete the construction of small intestinal tumor primary organoids in a shorter time. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 A flow chart of a method for culturing primary organoids of small intestinal tumors provided by the present invention;

[0036] Figure 2 A schematic diagram of the use of a primary organoid culture of small intestinal tumor provided by the present invention;

[0037] Figure 3 A comparison of feeder cell recovery methods for organoid culture;

[0038] Figure 4 This is a comparison of organoid culture on day 0 using different culture methods;

[0039] Figure 5 This is a comparison of organoid culture on the 6th day using different culture methods. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] Embodiment 1

[0042] Combination Figure 1 and Figure 2 As shown, the present invention discloses a method for culturing primary organoids of small intestinal tumors, and the specific operation steps are as follows:

[0043] S1: Preparation of mouse subcutaneous connective tissue cells as feeder cells:

[0044] S101: Inoculate mouse subcutaneous connective tissue cells in a culture dish, add 10% FBS-DMEM complete medium for culture, and stop culturing when the cell density reaches 90%;

[0045] S102: irradiating the cells in step S101 with gamma rays at a rate of 150 Gy for 990 seconds;

[0046] S103: Prepare feeder cell suspension: discard the supernatant, wash twice with an appropriate amount of PBS, digest with trypsin for 3 minutes and terminate with complete medium, centrifuge at 1000 rpm for 3 minutes, discard the supernatant, resuspend the cells with complete medium, count and set aside;

[0047] S2: Mix the treated feeder cells with the mouse intestinal crypt-like structures and resuspend them in Matrigel:

[0048] S201: The mice were killed by dislocating the neck, and the surface was sterilized by spraying alcohol. The small intestinal tissue 3-10 cm near the stomach was removed under sterile conditions. The mesentery and fat outside the intestine were removed with forceps, and the small intestinal tissue was placed in a 4°C precooled DPBS solution containing double antibodies.

[0049] S202: Use surgical scissors to cut the intestine open, with the intestinal cavity facing upwards, scrape off the intestinal villi on the surface of the intestinal cavity with a surgical blade, and place the intestinal tissue in a new culture dish containing DPBS solution for washing. Repeat the washing twice, cut the washed small intestinal tissue into pieces of 2 mm in width, transfer it to a new culture dish, and wash it twice with DPBS solution;

[0050] S203: Transfer the cleaned intestinal segment to a pre-cooled DPBS solution containing 5 mmol / L EDTA for digestion, incubate at 4°C or in crushed ice for 30 min, and gently blow the intestinal segment with a pipette for 20 min. Take the supernatant and observe it under a microscope. Stop digestion when complete crypts appear in the supernatant. If there are no crypts, extend the digestion time.

[0051] S204: After digestion, transfer the tissue fragments to a new dish containing DPBS and wash, repeating twice to remove EDTA;

[0052] S205: Use a 5 mL pipette to blow and resuspend tissue fragments in a culture dish or a 50 mL centrifuge tube containing a pre-cooled 0.1% BSA DPBS solution, and make the tissue pass through the pipette tip repeatedly to generate mechanical shear force to separate the crypts from the basal layer. Take a portion of the suspension for microscopic examination. When a large number of crypt-like structures can be seen, stop blowing and filter the tissue suspension after blowing through a 70 μm filter.

[0053] S206: collect the tissue suspension that passes through the filter, centrifuge at 300 g, 4°C, for 3 min;

[0054] S207: discard the supernatant, resuspend the tissue pellet with 1 mL of 0.1% BSA DPBS solution, take 20 μL of the suspension for microscopic examination and crypt counting, aspirate the suspension after counting, centrifuge at 300 g, 4°C, for 3 min, discard the supernatant and place on ice to obtain a suspension of mouse small intestinal crypt-like structures;

[0055] S208: Resuspend the tissue pellet and feeder cells with an appropriate amount of matrix gel. The recommended resuspension density is 90 crypts and 4500 feeder cells per 10 μL of matrix gel suspension. Place on ice after resuspension. The resuspension time should not exceed 30 seconds to avoid premature coagulation of the matrix gel. The matrix gel dilution ratio should be above 70% to ensure the structural stability of the matrix gel during the culture process. The matrix gel is a three-dimensional matrix gel Matrigel containing EGF, Noggin, and R-spondin.

[0056] S3: A mixed suspension of feeder cells, mouse small intestinal crypt-like structures, and matrix gel was inoculated into the wells of a 24-well cell culture plate on a scaffold, 30 μL per well, and a 24-well cell culture plate was used in combination with a culture rack;

[0057] S4: Place the inoculated culture plate in a 37°C carbon dioxide constant temperature incubator and incubate for about 30 minutes. After the matrix gel is completely solidified, slowly add the prepared complete culture medium along the wall, 500 μL per well, to avoid destroying the solidified structure. The complete culture medium is 10% FBS-DMEM culture medium with Y27632 and chir99021 added. The working concentration of Y27632 is 10 μM, and the working concentration of chir99021 is 10 μM.

[0058] S5: Place the culture plate in a 37°C CO2 incubator, replace the culture medium every 3 days, and monitor the growth status of the organoids.

[0059] Embodiment 2

[0060] In order to study the effect of feeder cell recovery method on organoid culture, this example adopts the culture method of small intestinal tumor primary organoids in Example 1 to conduct experiments.

[0061] The experiment included two groups, one group of feeder cells was used directly after recovery, and the other group of feeder cells was used after recovery and culture for 1 day. The specific operation steps are as described in Example 1 and will not be elaborated here. The difference is:

[0062] The treated feeder cells are first cryopreserved and then revived.

[0063] The specific operation of feeder cell recovery is as follows: take out the frozen feeder cells from liquid nitrogen or -80℃ refrigerator, quickly put them into a 37℃ water bath to thaw, transfer the thawed cell suspension to a sterile centrifuge tube, centrifuge at 1000rpm for 3 minutes, discard the supernatant, and resuspend the cells with complete culture medium.

[0064] The revived feeder cells were divided into two groups, one group was used directly, and the other group was used after culturing for 1 day.

[0065] In addition, four different culture media were prepared: 1. MI serum-free mouse intestinal organoid culture medium; 2. basal culture medium; 3. 10% FBS-DMEM culture medium; 4. 10% FBS-DMEM culture medium (supplemented with Y27632 and chir99021);

[0066] In the specific experiment, each group used four different culture media to culture primary organoids of small intestinal tumors. On the third day, the growth of organoids in each well under the four culture media of each group was observed and recorded under a microscope, including morphology, quantity and distribution.

[0067] The experimental results are as follows Figure 3 As shown, the results show that:

[0068] Between the two groups: the morphological distribution of organoids was more uniform and the number of organoids was greater when the feeder cells were used after 1 day of recovery compared with those used directly after recovery;

[0069] Between each group: the number of organoids in 10% FBS-DMEM medium (supplemented with Y27632 and chir99021) was greater and the growth condition was better.

[0070] Therefore, it can be concluded that:

[0071] The feeder cells after one day of resuscitation culture have a positive effect on the growth of organoids and can effectively improve the survival rate of primary organoid culture of small intestinal tumors; and when the complete culture medium is 10% FBS-DMEM culture medium (with added Y27632 and chir99021), it can increase the number of organoids and promote the growth of organoids.

[0072] Embodiment 3

[0073] In order to study the effect of feeder cell density on organoid culture, this example uses the culture method of small intestinal tumor primary organoids in Example 1 to conduct experiments.

[0074] The experiment included six groups. The feeder cell density of the first group was 0; the feeder cell density of the second group was 1.5×10 3 The feeder cell density of the third group was 3×10 3 The feeder cell density of the fourth group was 4.5×10 3 The feeder cell density of the fifth group was 6×10 3 The feeder cell density of the sixth group was 7.5×10 3 The specific operation steps are as described in Example 1 and will not be elaborated here.

[0075] The final experimental results show that: with 4.5×10 3 The number of organoids that eventually grew was higher when 4.5×10 3 Mixing individual feeder cells with mouse small intestinal crypt-like structures and resuspending them in matrix gel can effectively improve the survival rate of mouse small intestinal organoid culture.

[0076] Embodiment 4

[0077] In order to study the effects of different culture methods (no feeder cells, culture in gel, culture outside gel, and culture on scaffold) on the culture effect of primary small intestinal tumor organoids, the culture method of primary small intestinal tumor organoids in Example 1 was used to conduct experiments.

[0078] The experiment included six groups:

[0079] Group A (no feeder cells): Use an appropriate amount of matrix gel to resuspend the mouse small intestinal crypt-like structure, and the resuspension density is 100 crypts per 10 μL of matrix gel suspension; inoculate the mixed suspension into the wells of a 24-well cell culture plate, 30 μL per well; place the culture plate in a 37°C carbon dioxide constant temperature incubator and incubate for 30 minutes to allow the matrix gel to solidify. After the matrix gel is completely solidified, slowly add the prepared complete culture medium along the wall, 500 μL per well.

[0080] Groups B and C (in-gel culture): Use an appropriate amount of matrix gel to resuspend the mouse small intestinal crypt-like structure and feeder cells, and the resuspension density is 100 crypts and 4500 feeder cells per 10 μL of matrix gel suspension; inoculate the mixed suspension into the wells of a 24-well cell culture plate, 30 μL per well; place the culture plate in a 37°C carbon dioxide constant temperature incubator and incubate for 30 minutes to allow the matrix gel to solidify. After the matrix gel is completely solidified, slowly add the prepared complete culture medium along the wall, 500 μL per well.

[0081] Group D (scaffold culture): Place the organoid proliferation culture rack on a 24-well cell culture plate, ensure that the culture rack is tightly combined with the culture plate, and resuspend the mouse small intestinal crypt-like structure and feeder cells with an appropriate amount of matrix gel. The resuspension density is 100 crypts and 4500 feeder cells per 10 μL matrix gel suspension; inoculate the mixed suspension into the wells of the 24-well cell culture plate, 30 μL per well; place the culture plate in a 37°C carbon dioxide constant temperature incubator and incubate for 30 minutes to allow the matrix gel to solidify. After the matrix gel is completely solidified, slowly add the prepared complete culture medium along the wall, 500 μL per well.

[0082] Group E (culture outside the matrix gel): resuspend the mouse small intestinal crypt-like structure and feeder cells with an appropriate amount of complete culture medium, and the resuspension density is 100 crypts and 4500 feeder cells per 10 μL of matrix gel suspension; inoculate the mixed suspension into the wells of a 24-well cell culture plate, 30 μL per well; place the culture plate in a 37°C carbon dioxide constant temperature incubator and incubate for 30 minutes to allow the matrix gel to solidify. After the matrix gel is completely solidified, slowly add the prepared complete culture medium along the wall, 500 μL per well.

[0083] Group F (scaffold culture + extra-gel culture): Place the organoid proliferation culture rack on a 24-well cell culture plate to ensure that the culture rack is tightly combined with the culture plate, and resuspend the mouse small intestinal crypt-like structure and feeder cells with an appropriate amount of complete culture medium. The resuspension density is 100 crypts and 4500 feeder cells per 10 μL of matrix gel suspension; inoculate the mixed suspension into the wells of the 24-well cell culture plate, 30 μL per well; place the culture plate in a 37°C carbon dioxide constant temperature incubator and incubate for 30 minutes to allow the matrix gel to solidify. After the matrix gel is completely solidified, slowly add the prepared complete culture medium along the wall, 500 μL per well.

[0084] Four culture media were prepared for each of the six groups, two of which had complete culture media of MI: serum-free mouse intestinal organoid culture media, and the other two had complete culture media of 10DYC: 10% FBS-DMEM culture media (supplemented with Y27632 and chir99021).

[0085] On days 0 and 6, the growth of organoids in each well of each culture medium in each group was observed and recorded using a microscope, including morphology, quantity, and distribution. The experimental results on day 0 are shown in Figure 2. Figure 4 The experimental results on the 6th day are shown in Figure 5 As shown,

[0086] Combination Figure 4 and Figure 5 By comparison, we can conclude that:

[0087] Comparison between group A and group B / C and between group B / C and group E showed that culture of feeder cells in gel was helpful to improve the survival rate of mouse small intestinal organoid culture;

[0088] Comparison between group B / C and group D, and between group E and group F indicated that scaffold culture was helpful to improve the survival rate of mouse small intestinal organoids;

[0089] In summary, both feeder cell in-gel culture and scaffold culture methods can help improve the survival rate of mouse small intestinal organoids, and the combined use of the two culture methods can significantly improve the survival rate of mouse small intestinal organoids.

[0090] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for culturing primary organoids of small intestinal tumors, characterized in that: The following steps are involved: S1: Mouse subcutaneous connective tissue cells were prepared as feeder cells; S2: The treated feeder cells were mixed with mouse intestinal crypt-like structures and resuspended in Matrigel; S3: A mixed suspension of feeder cells, mouse small intestinal crypt-like structures, and Matrigel was inoculated into the wells of a 24-well cell culture plate, 30 μL per well; S4: After the matrix gel is completely solidified, add the prepared complete culture medium along the wall, 500 μL per well; S5: Place the culture plate in a 37°C CO2 incubator, replace the culture medium every 3 days, and monitor the growth status of the organoids.

2. The method for culturing primary small intestinal tumor organoids according to claim 1, characterized in that: The step S1 specifically includes: S101: Inoculate mouse subcutaneous connective tissue cells in a culture dish, add 10% FBS-DMEM complete medium for culture, and stop culturing when the cell density reaches 80-90%; S102: irradiating the cells in step S101 with gamma rays at a rate of 150 Gy for 990 seconds; S103: Prepare feeder cell suspension: discard the supernatant, wash twice with an appropriate amount of PBS, digest with trypsin for 3 minutes and terminate with complete medium, centrifuge at 1000 rpm for 3 minutes, discard the supernatant, resuspend the cells with complete medium, count and set aside.

3. The method for culturing primary small intestinal tumor organoids according to claim 1, characterized in that: In step S2, the mouse small intestinal crypt-like structure is obtained by the following steps: S201: The mice were killed by dislocating the neck, and the surface was sterilized by spraying alcohol. The small intestinal tissue 3-10 cm near the stomach was removed under sterile conditions. The mesentery and fat outside the intestine were removed with forceps, and the small intestinal tissue was placed in a 4°C precooled DPBS solution containing double antibodies. S202: Use surgical scissors to cut the intestine open, with the intestinal cavity facing upwards, scrape off the intestinal villi on the surface of the intestinal cavity with a surgical blade, and place the intestinal tissue in a new culture dish containing DPBS solution for washing. Repeat the washing twice, cut the washed small intestinal tissue into pieces of 2 mm in width, transfer it to a new culture dish, and wash it twice with DPBS solution; S203: Transfer the cleaned intestinal segment to a pre-cooled DPBS solution containing 5mmol / L EDTA for digestion, incubate at 4℃ or in crushed ice for 20-30min, and gently blow the intestinal segment with a pipette for 20min. Take the supernatant and observe it under a microscope. Stop digestion when complete crypts appear in the supernatant. If there are no crypts, extend the digestion time. S204: After digestion, transfer the tissue fragments to a new dish containing DPBS and wash, repeating twice to remove EDTA; S205: Use a 5 mL pipette to blow and resuspend tissue fragments in a culture dish or a 50 mL centrifuge tube containing a pre-cooled 0.1% BSA DPBS solution, and make the tissue pass through the pipette tip repeatedly to generate mechanical shear force to separate the crypts from the basal layer. Take a portion of the suspension for microscopic examination. When a large number of crypt-like structures can be seen, stop blowing and filter the tissue suspension after blowing through a 70 μm filter. S206: collect the tissue suspension that passes through the filter, centrifuge at 300 g, 4°C, for 3 min; S207: Discard the supernatant, resuspend the tissue pellet with 1 mL of 0.1% BSA in DPBS solution, take 20 μL of the suspension for microscopic examination and crypt counting, aspirate the suspension after counting, centrifuge at 300 g, 4°C, for 3 min, discard the supernatant and place on ice to obtain a suspension of mouse small intestinal crypt-like structures.

4. The method for culturing primary small intestinal tumor organoids according to claim 3, characterized in that: The specific operation of resuspending in matrix gel in step S2 is: the resuspending density is 70-100 crypts and 4500 feeder cells per 10 μL matrix gel suspension, and the cells are placed on ice after resuspending, and the resuspending time does not exceed 30 seconds.

5. The method for culturing primary small intestinal tumor organoids according to claim 4, characterized in that: In step S2, the dilution ratio of the matrix gel is controlled to be above 70%.

6. The method for culturing primary small intestinal tumor organoids according to claim 5, characterized in that: The specific operation of completely solidifying the matrix gel in step S4 is: placing the culture plate after inoculation in a 37° C. carbon dioxide constant temperature incubator and incubating for 30 minutes.

7. A method for culturing primary small intestinal tumor organoids according to any one of claims 1 to 6, characterized in that: After the treated feeder cells were revived and cultured for 1 day, they were mixed with the mouse small intestinal crypt-like structures and resuspended in matrix gel.

8. The method for culturing primary small intestinal tumor organoids according to claim 1, characterized in that: In step S4, the complete culture medium is 10% FBS-DMEM culture medium supplemented with Y27632 and chir99021, the working concentration of Y27632 is 10 μM, and the working concentration of chir99021 is 10 μM.

9. The method for culturing primary small intestinal tumor organoids according to claim 1, characterized in that: The matrix gel is a three-dimensional matrix gel Matrigel containing EGF, Noggin and R-spondin.

10. The method for culturing primary small intestinal tumor organoids according to claim 1, characterized in that: The 24-well cell culture plate is used in combination with a culture rack.

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