Construction method of co-culture model of gastric cancer organoid and functional human gastrointestinal tract organoid

Abstract

The invention relates to the technical field of cell engineering, in particular to a construction method of a co-culture model of gastric cancer organoid and functional human gastrointestinal tract organoid. According to the method disclosed by the invention, the gastric cancer organoid is injected into the cavity on one side of the mucous membrane layer of the functional human gastrointestinal tract organoid through microinjection equipment, the in-vitro gastric cancer tumor infiltration model is constructed, the gastric cancer invasion process is accurately simulated in vitro, and an observable platform and an intervention platform for gastric cancer invasion are provided. According to the construction method disclosed by the invention, the digestion time of the organoid dissociation agent is shortened, only the matrigel and the organoid are separated, the gastric cancer organoid and the functional human gastrointestinal tract organoid are not digested into single cells, the 3D structure of the organoid is reserved, and the advantages of the organoid simulating an organism are exerted. The co-culture system disclosed by the invention is more suitable for precision medical treatment, and is beneficial to predicting drug response and predicting recurrence and metastasis risks and prognosis of patients.

Description

Technical Field

[0001] The present invention relates to the field of cell engineering technology, and in particular to a method for constructing a co-culture model of gastric cancer organoids and functional human gastrointestinal organoids. Background Art

[0002] Gastric cancer has a high incidence rate and threatens national health, especially invasive ones. In order to better explore how gastric cancer invades in situ and further metastasizes, this field urgently needs to find a technical solution that can successfully simulate the first step of gastric cancer development in vitro - in situ invasion. Currently, there are two co-culture schemes with tumor organoids: co-culture of normal organoids with tumor organoids, and co-culture of normal cells with tumor organoids.

[0003] Co-culture scheme of normal organoids and tumor organoids: (1) The two organoids are digested into single cells, which destroys the structure of the organoids and cannot give full play to the advantage of organoids in simulating the in vivo environment. Taking this issue into consideration, this patent does not digest the functional human gastrointestinal organoids and gastric cancer organoids into single cells. (2) The two organoids are mixed in a 1:1 cell number ratio, which is not in accordance with the actual ratio in vivo and cannot simulate the in vivo situation well. Taking this issue into consideration, this patent injects gastric cancer organoids into the gastric mucosal cavity of the functional human gastrointestinal organoids, simulating the in vivo process of gastric cancer infiltrating the mucosa and muscular layer, providing an ideal experimental platform for understanding the progression mechanism of gastric cancer and discovering targeted therapies.

[0004] Co-culture scheme of normal cells and tumor organoids: (1) Simply co-culturing normal gastric cells with gastric cancer organoids cannot simulate the physiological structure and environment of the stomach in vitro, and thus cannot achieve the purpose of simulating gastric cancer infiltration in vivo.

[0005] Therefore, there is an urgent need to construct functional human gastrointestinal organoids that can simulate the structure and function of the stomach, co-culture them with gastric cancer organoids, and simulate the process of gastric cancer infiltration into the mucosa and muscularis in vivo. Summary of the invention

[0006] In response to the problems existing in the prior art, the present invention discloses a method for constructing a co-culture model of gastric cancer organoids and functional human gastrointestinal organoids, and constructs an in vitro organoid model that simulates the infiltration and growth of gastric cancer in the gastric mucosa and muscularis, providing an ideal experimental platform for understanding the progression mechanism of gastric cancer and discovering targeted treatments.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0008] The present invention discloses a method for co-culturing gastric cancer organoids and functional human gastrointestinal organoids, which is characterized by comprising the following steps: Step 1. Culture of gastric cancer organoids; Step 2. Cultivation of functional human gastrointestinal organoids; Step 3. Co-culture of gastric cancer organoids and functional human gastrointestinal organoids: (1) Collect the mature gastric cancer organoids obtained in step 1, digest them with a special dissociation agent for organoids, centrifuge, discard the gel, and centrifuge for 1×10 4 The density of cells / mL was resuspended in DMEM / F12 basal medium; (2) Inoculate the functional human gastrointestinal organoids obtained in step 2 into the gel droplets and place them in an incubator at 37°C for 10 min; (3) In a sterile workbench, use a glass microinjection needle with an inner diameter greater than 0.1 mm to aspirate the gastric cancer organoid suspension from step 2 and load it onto the microinjection device; (4) Under a stereomicroscope, the gastric cancer organoid suspension aspirated in (3) was injected into the cavity on the mucosal side of the functional human gastrointestinal organoid obtained in (2), and a co-culture culture medium was added and replaced every 3 days.

[0009] Furthermore, in step 3 (1), the dissociation agent digestion time is 4 minutes, followed by centrifugation to obtain complete gastric cancer organoids without matrix gel.

[0010] Furthermore, in step 3 (2), the inoculation ratio of functional human gastrointestinal organoids is 1:6.

[0011] Furthermore, the co-culture special culture medium in step 3 (4) comprises the following components: basal culture medium, glutamine, 4-hydroxyethylpiperazineethanesulfonic acid buffer, primary cell antibiotics, serum additive B27, serum additive N2, epidermal growth factor, Wnt-3a recombinant protein, R-Spondin1 recombinant protein, noggin, ROCK inhibitor, fibroblast growth factor 10, gastrin I, TGFβ inhibitor and glycogen synthase kinase 3 inhibitor; The total volume of the co-culture special medium includes: 1X basal medium, 2 mM glutamine, 13mM 4-hydroxyethylpiperazineethanesulfonic acid buffer, 50µg / mL primary cell antibiotics, 50X serum supplement B27, 100X serum supplement N2, 100ng / mL epidermal growth factor, 100ng / mL Wnt-3a recombinant protein, 1µg / mL R-Spondin1 recombinant protein, 100ng / mL noggin, 10µM ROCK inhibitor, 200ng / mL fibroblast growth factor 10, 10nM gastrin I, 2µM TGFβ inhibitor and 2µM glycogen synthase kinase 3 inhibitor.

[0012] The present invention also discloses an in vitro organoid model that simulates the infiltration and growth of gastric cancer in the gastric mucosa and muscularis, characterized in that it is constructed by the co-culture method of gastric cancer organoids and functional human gastrointestinal organoids as described in any of the above items.

[0013] The present invention also discloses a co-culture model of gastric cancer organoids and functional human gastrointestinal organoids, characterized in that it is constructed by the co-culture method of gastric cancer organoids and functional human gastrointestinal organoids as described in any of the above items.

[0014] The present invention also discloses an application of the above-mentioned in vitro organoid model simulating the infiltration and growth of gastric cancer in the gastric mucosa and muscularis or the above-mentioned co-culture model of gastric cancer organoid and functional human gastrointestinal organoid in studying the process of gastric cancer invasion of gastric mucosa and muscularis and related mechanisms.

[0015] The present invention also discloses a co-culture culture medium for gastric cancer organoids and functional human gastrointestinal organoids, characterized in that the co-culture special culture medium components consist of: basal culture medium, glutamine, 4-hydroxyethylpiperazineethanesulfonic acid buffer, primary cell antibiotics, serum additive B27, serum additive N2, epidermal growth factor, Wnt-3a recombinant protein, R-Spondin1 recombinant protein, noggin, ROCK inhibitor, fibroblast growth factor 10, gastrin I, TGFβ inhibitor and glycogen synthase kinase 3 inhibitor; The total volume of the co-culture special medium includes: 1X basal medium, 2mM glutamine, 13mM 4-hydroxyethylpiperazineethanesulfonic acid buffer, 50µg / mL primary cell antibiotics, 50X serum supplement B27, 100X serum supplement N2, 100ng / mL epidermal growth factor, 100ng / mL Wnt-3a recombinant protein, 1µg / mL R-Spondin1 recombinant protein, 100ng / mL noggin, 10µM ROCK inhibitor, 200ng / mL fibroblast growth factor 10, 10nM gastrin I, 2µM TGFβ inhibitor and 2µM glycogen synthase kinase 3 inhibitor.

[0016] Furthermore, the gastric cancer organoid and functional human gastrointestinal organoid co-culture medium described in any of the above items is used to construct a gastric cancer organoid and functional human gastrointestinal organoid co-culture model.

[0017] Furthermore, the gastric cancer organoids and functional human gastrointestinal organoids co-culture medium described in any of the above items are used to construct an in vitro organoid model that simulates the infiltration and growth of gastric cancer in the gastric mucosa and muscularis.

[0018] The present invention proposes for the first time to prepare gastric cancer organoids and functional human gastrointestinal organoids separately. Then, the gastric cancer organoids are transfected with GFP and stable strains are selected. After that, mature and stably transfected gastric cancer organoids are collected, digested with a special organoid dissociation agent for 4 minutes, and then centrifuged. 4 Resuspend in DMEM / F12 basal medium at a density of 1 / mL. Functional human gastrointestinal organoids were inoculated in a new 50µL gel droplet at a ratio of 1:6 and placed in a 37°C incubator for 10 minutes. In a sterile workbench, use a glass microinjection needle with an inner diameter greater than 0.1mm to aspirate approximately 5µL of gastric cancer organoid suspension and load it onto the microinjection device. Under a stereomicroscope, inject the gastric cancer organoid suspension into the cavity of each mature functional human gastrointestinal organoid, add co-culture special culture medium, and replace it every 3 days. 24h after injection, fluorescence microscopy was used to verify the survival of gastric cancer organoids in functional human gastrointestinal organoids, and then confocal microscopy was used to observe the growth of gastric cancer organoids and their invasive behavior on functional human gastrointestinal organoids. After the co-cultured organoids were paraffin-embedded and sectioned, the invasion area was determined using a confocal microscope, the ROI function in the Fiji software was used to measure the area of ​​the invasion area, and the cell counting function was used to count GFP. + Cells were counted to determine the expression of GFP in the co-culture model. + Cell number and density.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0020] The present invention discloses an in vitro organoid model that simulates the infiltration and growth of gastric cancer in the gastric mucosa and muscularis, providing an ideal experimental platform for understanding the progression mechanism of gastric cancer and discovering targeted treatments.

[0021] The construction method disclosed in the present invention shortens the digestion time of the organoid dissociation agent, only separates the matrix gel and the organoid, does not digest the gastric cancer organoids and the functional human gastrointestinal organoids into single cells, retains the 3D structure of the organoid, and gives full play to the advantages of the organoid in simulating the body.

[0022] The existing technology simply co-cultures normal gastric cells with gastric cancer organoids, which cannot simulate the physiological structure and environment of the stomach in vitro, and thus cannot achieve the purpose of simulating gastric cancer infiltration in vivo. The present invention takes into account the spatial position relationship and injects gastric cancer organoids into the cavity on one side of the mucosal layer of functional human gastrointestinal organoids through a microinjection device, simulating the process of gastric cancer infiltration into the muscular layer. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Technical roadmap for primary culture of gastric cancer organoids.

[0024] Figure 2Technology roadmap for the culture of functional human gastrointestinal organoids.

[0025] Figure 3 Technical roadmap for co-culture of gastric cancer organoids and functional human gastrointestinal organoids.

[0026] Figure 4 Light microscopy of gastric cancer organoids co-cultured with functional human gastrointestinal organoids.

[0027] Figure 5 Growth size results of functional human gastrointestinal organoids at different inoculation ratios. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below with specific embodiments, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following embodiments, and all technologies realized based on the content of the present invention belong to the scope of the present invention.

[0029] Unless otherwise specified, all reagents and materials used in the present invention are commercially available.

[0030] Example 1. Co-culture of gastric cancer organoids and functional human gastrointestinal organoids.

[0031] This technology constructs an in vitro gastric cancer tumor infiltration model, accurately simulates the gastric cancer invasion process in vitro, and provides an observable platform and intervention platform for gastric cancer invasion. The co-culture system is more suitable for precision medicine and helps predict drug response, and predict the risk of recurrence and metastasis and prognosis of patients.

[0032] Table 1 Chinese and English list .

[0033] 1. Culture Protocol for Gastric Cancer Organoids Figure 1 ).

[0034] 1. Obtain gastric tumor tissue by surgery, with a volume of approximately 5mm×5mm×5mm. Fresh tissue should be placed in tissue preservation solution (DMEM high-glucose medium containing 10% FBS, 1.25 mM NAC, and 10µM Y27632) (4°C) within 30 minutes of ex vivo and transported to the laboratory at a constant temperature of 4°C.

[0035] 2. Physically pre-treat the obtained sample tissue to obtain sample tissue fragments.

[0036] 3. Cut the sample tissue fragments into small pieces and transfer them to 5 mL~10 mL of DMEM / F12 culture medium containing 160 U / mL type XI collagenase, 80 U / mL type II collagenase, 2 U / mL type II dispase, and 50 µg / mL antibiotics for enzymatic digestion for 1.5 h.

[0037] 4. Terminate the digestion with an equal volume of DMEM / F12 medium containing 10% FBS, 2 mM glutamine, 2 mM 4-hydroxyethylpiperazineethanesulfonic acid buffer, 10 µM ROCK inhibitor, and 1.25 mM N-acetylcysteine.

[0038] 5. After centrifugation at 300g for 5 min, discard the supernatant and collect the precipitate as gastric cancer single cells. Add melted matrix gel to the precipitate and gently resuspend the cells. Add 100% matrix gel per 10 5 Add 100 μL of the matrix gel-cell mixture to each well of the 24-well plate, avoiding bubbles, and add 50 μL / well of the matrix gel-cell mixture to the 24-well plate. Incubate at 37°C for 10 minutes to allow it to solidify.

[0039] 6. Add 500µL / well of culture medium (preheated at 37°C in advance) to the culture wells for culture to obtain primary organoids; change the medium every 3-4 days and subculture every 7-9 days. The composition of the culture medium is: 1×Advanced DMEM / F12 medium, 100X GlutaMax, 1.25mM NAC, 1000Xprimocin, 10mM HEPES, 200ng / mL FGF-10, 50ng / mL EGF, 1ug / mL R-Spondin 1, 100ng / mL Noggin, 100ng / mL Wnt-3a, 500nM A83-01, 10nMGastrin, 50X B27, 100XN2 and 10mM Nicotinamide.

[0040] II. Culture Protocol for Functional Human Gastrointestinal Organoids ( Figure 2 ).

[0041] (A) Preparation of hindgut foregut spheroids.

[0042] 1. Culture of pluripotent stem cells (iPSC): Add 1 mL of DMEM / F12 containing 10-40 µL of matrix gel to each six-well plate for 1 hour, then slowly drip the iPSC cell suspension and replace the pre-warmed culture medium (mTeSR1 containing 10 µM Y27632) daily. When the monolayer cell confluence is 75-80%, add an appropriate amount of warm cell dissociation agent to dissociate for 7-10 minutes, then terminate the digestion, centrifuge and discard the supernatant, add warm mTeSR1 (containing 10 µM Y27632) to resuspend the cells, and add 500 µL to each 24-well plate to make the cell density reach 250,000-300,000 per well.

[0043] 2. Day 0~2: Differentiate embryonic stem cells into definitive endoderm (DE).

[0044] (1) Add 500 μL of pre-warmed Day 0 DE differentiation medium (RPMI 1640 medium containing 100× NEAA, 100 ng / mL Activin A, and 450 ng / mL BMP) to each well and return to the incubator.

[0045] (2) After 24 h, add Day 1 DE differentiation medium (RPMI 1640 containing 100×NEAA, 0.2% dFBS, and 100 ng / mL Activin A).

[0046] (3) After 24 hours, replace the Day 2 DE differentiation medium (RPMI1640 containing 100×NEAA, 0.2% dFBS, and 100 ng / mL ActivinA). At this time, the cell confluence should reach 90-100%. If this confluence is not reached, it means that the induction is unsuccessful and subsequent induction should not be performed.

[0047] 3. Day3~6: Induce DE into posterior foregut spheroids.

[0048] (1) Change the culture medium (RPMI 1640 containing 100×NEAA, 0.2% dFBS, 2μM CHIR99021, 500ng / mL FGF4, and 200ng / mL Noggin) every day from Day 3 to Day 4.

[0049] (2) After 24 h, the medium was replaced with Day 5 medium (RPMI 1640 medium containing 100×NEAA, 0.2% dFBS, 2 μM CHIR99021, 500 ng / mL FGF4, 200 ng / mL Noggin, and 2 μM RA).

[0050] (3) After 24 hours, there should be a large number of posterior foregut spheroids floating freely in the culture medium in each well. Under a stereomicroscope, collect the posterior foregut spheroids.

[0051] (B) Preparation of smooth muscle layer.

[0052] 1. Dissociate partially fused embryonic stem cell colonies into single cells using a cell dissociation agent, add culture medium (mTesR1 containing 1 μM ROCK inhibitor), and subculture at a ratio of 1:20 into 24-well plates pre-coated with gelatin. Replace Day 0 culture medium (Advanced DMEM / F12 containing Activin A 30 ng / mL, BMP4 40 ng / mL, CHIR99021 6 μM, FGF2 20 ng / mL, PIK90 100 nM).

[0053] 2. Day 1, replace the culture medium (Advanced DMEM / F12 containing A83-01 1μM, BMP4 30ng / mL, C59 1μM).

[0054] 3. Day 2-4, replace the culture medium (Advanced DMEM / F12 containing A8301 1 μM, BMP4 30 ng / mL, C59 1 μM, FGF2 20 ng / m, RA 2 μM).

[0055] 4. Day 5-6, replace the culture medium (Advanced DMEM / F12 containing RA 2μM, PMA 2μM).

[0056] 5. On Day 7, replace the culture medium (Advanced DMEM / F12 containing RA 2μM, PMA 2μM, NOG 100ng / mL). Use a cell dissociation agent to dissociate for 6-10 minutes, dissociate the fused cells into single cells, resuspend them in Advanced DMEM / F12, and immediately combine with hAGO and hFGO.

[0057] (III) Assemble organoids.

[0058] 1. Count the smooth muscle cell suspension and collect it in a 2.5 mL tube at a ratio of 1 hindgut foregut spheroid and 1500 smooth muscle cells.

[0059] 2. Gently pipette the cell mixture, centrifuge at 300g for 3-5min, gently resuspend in 50μL of Matrigel, and then pipette into a 24-well culture dish in the form of gel droplets for three-dimensional in vitro culture.

[0060] 3. Day 6-20: Induce the posterior foregut spheroids into gastric organoids (hGOs).

[0061] (1) Add Day 6 medium (GM growth medium containing CHIR99021 2μM, Noggin 200ng / mL, RA 2μM, EGF100ng / mL) without changing the medium. GM growth medium is DMEM-F12 basal medium containing 1×N2, 1×B27 (without vitamin A), 2mM L-glutamine, 100u / mL penicillin-streptomycin, and 15mM 4-hydroxyethylpiperazineethanesulfonic acid buffer.

[0062] (2) Three days later, add Day 9 medium (GM growth medium containing CHIR99021 2 μM and EGF 100 ng / mL) and change the medium every three days until Day 20.

[0063] 4. Day 20~34: Induce hGOs into human gastric antrum (hAGOs) / gastric body tissue organoids (hFGOs) containing differentiated glands.

[0064] (1) On Day 20, hGOs were collected and added to Matrigel. They were seeded in 24-wells in the form of gel drops, with approximately 15 spheres per well. GM growth medium containing 2 μM CHIR99021 and 100 ng / mL EGF was added (50 ng / mL FGF10 was additionally added for hFGOs). The medium was changed every four days for 9 days.

[0065] (2) On Day 30-31, the culture medium was replaced with GM growth medium containing 2 μM CHIR99021 and 10 ng / mL EGF (hFGOs were supplemented with 50 ng / mL FGF10, 2 μM PD0325901, and 50 ng / mL BMP4).

[0066] (3) On Day 32-34, the medium was changed to GM growth medium containing 2 μM CHIR99021 and 10 ng / mL EGF (hFGOs were additionally supplemented with 50 ng / mL FGF10, 2 μM PD0325901, and 50 ng / mL BMP4).

[0067] 3. Achieving co-culture of gastric cancer organoids and functional human gastrointestinal organoids ( Figure 3 ).

[0068] 1. The specific steps of the co-culture model of gastric cancer organoids and functional human gastrointestinal organoids are as follows.

[0069] (1) Gastric cancer organoids were transfected with GFP and stable strains were selected.

[0070] (2) Mature and stably transfected gastric cancer organoids were collected and digested with a special organoid dissociation agent for 4 minutes, centrifuged at 300g for 5 minutes, and the gel was discarded and 1×10 4 The cells were resuspended in DMEM / F12 basal medium at a density of 100 / mL.

[0071] (3) Inoculate the functional human gastrointestinal organoids in a 50 µL gel droplet at a ratio of 1:6 and place in a 37°C incubator for 10 min. The ratio is the ratio of the amount of gel added in the previous generation to the amount of gel added after subculture, which can also be said to be the ratio of the number of wells in the previous generation to the number of wells in the next generation.

[0072] (4) On a sterile workbench, use a glass microinjection needle with an inner diameter greater than 0.1 mm to aspirate 5 µL of the gastric cancer organoid suspension prepared in (2). Under a stereomicroscope, inject it into the cavity on one side of the mucosal layer of each mature functional human gastrointestinal organoid and add co-culture culture medium (Table 2). Change it every 3 days.

[0073] (5) 24 hours after injection, fluorescence microscopy was used to verify the survival of gastric cancer organoids in functional human gastrointestinal organoids.

[0074] Imaging techniques such as confocal microscopy were used to observe the growth of gastric cancer organoids and their invasion behavior on functional human gastrointestinal organoids. After paraffin embedding and sectioning of the co-cultured organoids, the invasion area was determined using a confocal microscope, the area of ​​the invasion area was measured using the ROI function in the Fiji software, and the GFP+ cells were counted using the cell counting function in the Fiji software. This provided the number of GFP+ cells and the density of GFP+ cells in the co-culture model.

[0075] 2. The culture medium used for the co-culture model of gastric cancer organoids and functional human gastrointestinal organoids is a DMEM / F12 culture medium containing the following components: B27 serum supplement, N2 serum supplement, HEPES, L-glutamine, primary cell antibiotics, Y-27632, R-Spondin 1, FGF-10, EGF, Gastrin I, A83-01, CHIR99021, Noggin and Wnt-3a. The concentrations of the components are shown in the following table.

[0076] Table 2 Culture media used in the co-culture model of gastric cancer organoids and functional human gastrointestinal organoids .

[0077] 4. Results. Figure 4 As shown, the morphology under light microscopy of gastric cancer organoids injected into functional human gastrointestinal organoids and co-cultured one week later showed that most of the gastric cancer organoids were located in the cavity of the functional human gastrointestinal organoids, and a small number of gastric cancer organoids invaded from the mucosal layer to the muscular layer.

[0078] Example 2: Effects of experimental conditions on the co-culture effect in the co-culture model of gastric cancer organoids and functional human gastrointestinal organoids.

[0079] 1. A co-culture system of gastric cancer organoids and functional human gastrointestinal organoids was constructed according to the construction method described in Example 1, except that in the co-culture scheme of gastric cancer organoids and functional human gastrointestinal organoids, different inoculation ratios of functional human gastrointestinal organoids affected the co-culture effect. The different inoculation ratios were 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 and 1:10.

[0080] The results are as follows Figure 5 As shown, different inoculation ratios were used, and the organoid growth was best at an inoculation ratio of 1:6.

[0081] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for co-culturing gastric cancer organoids and functional human gastrointestinal organoids, characterized in that: The specific steps include: Step 1. Culture of gastric cancer organoids; Step 2. Cultivation of functional human gastrointestinal organoids; Step 3. Co-culture of gastric cancer organoids and functional human gastrointestinal organoids: (1) Collect the mature gastric cancer organoids obtained in step 1, digest them with a special dissociation agent for organoids, centrifuge, discard the gel, and centrifuge for 1×10 4 The density of cells / mL was resuspended in DMEM / F12 basal medium; (2) Inoculate the functional human gastrointestinal organoids obtained in step 2 into the gel droplets and place them in an incubator at 37°C for 10 min; (3) In a sterile workbench, use a glass microinjection needle with an inner diameter greater than 0.1 mm to aspirate the gastric cancer organoid suspension from step 2 and load it onto the microinjection device; (4) Under a stereomicroscope, the gastric cancer organoid suspension aspirated in (3) was injected into the cavity on the mucosal side of the functional human gastrointestinal organoid obtained in (2), and a co-culture culture medium was added and replaced every 3 days.

2. The culture method according to claim 1, characterized in that The dissociation agent digestion time in step 3 (1) is 4 minutes, followed by centrifugation to obtain complete gastric cancer organoids without matrix gel.

3. The culture method according to claim 1, characterized in that Step 3 (2) The inoculation ratio of functional human gastrointestinal organoids is 1:

6.

4. The culture method according to claim 1, characterized in that The co-culture special culture medium in step 3 (4) comprises the following components: basal culture medium, glutamine, 4-hydroxyethylpiperazineethanesulfonic acid buffer, primary cell antibiotics, serum additive B27, serum additive N2, epidermal growth factor, Wnt-3a recombinant protein, R-Spondin1 recombinant protein, noggin, ROCK inhibitor, fibroblast growth factor 10, gastrin I, TGFβ inhibitor and glycogen synthase kinase 3 inhibitor; The total volume of the co-culture special medium includes: 1X basal medium, 2 mM glutamine, 13mM 4-hydroxyethylpiperazineethanesulfonic acid buffer, 50µg / mL primary cell antibiotics, 50X serum supplement B27, 100X serum supplement N2, 100ng / mL epidermal growth factor, 100ng / mL Wnt-3a recombinant protein, 1µg / mL R-Spondin1 recombinant protein, 100ng / mL noggin, 10µM ROCK inhibitor, 200ng / mL fibroblast growth factor 10, 10nM gastrin I, 2µM TGFβ inhibitor and 2µM glycogen synthase kinase 3 inhibitor.

5. An in vitro organoid model simulating the infiltration and growth of gastric cancer in the gastric mucosa and muscularis, characterized in that: The gastric cancer organoid is constructed by the co-culture method of gastric cancer organoid and functional human gastrointestinal organoid as described in any one of claims 1 to 4.

6. A gastric cancer organoid and functional human gastrointestinal organoid co-culture model, characterized in that: The gastric cancer organoid is constructed by the co-culture method of gastric cancer organoid and functional human gastrointestinal organoid as described in any one of claims 1 to 4.

7. An application of the in vitro organoid model simulating gastric cancer invading and growing in gastric mucosa and muscularis according to claim 5 or the co-culture model of gastric cancer organoid and functional human gastrointestinal organoid according to claim 6, characterized in that: The applications include applications in studying the process of gastric cancer invading the gastric mucosa and muscularis and related mechanisms.

8. A co-culture medium for gastric cancer organoids and functional human gastrointestinal organoids, characterized in that: The co-culture special culture medium components include: basal culture medium, glutamine, 4-hydroxyethylpiperazineethanesulfonic acid buffer, primary cell antibiotics, serum additive B27, serum additive N2, epidermal growth factor, Wnt-3a recombinant protein, R-Spondin1 recombinant protein, noggin, ROCK inhibitor, fibroblast growth factor 10, gastrin I, TGFβ inhibitor and glycogen synthase kinase 3 inhibitor; The total volume of the co-culture special medium includes: 1X basal medium, 2mM glutamine, 13mM 4-hydroxyethylpiperazineethanesulfonic acid buffer, 50µg / mL primary cell antibiotics, 50X serum supplement B27, 100X serum supplement N2, 100ng / mL epidermal growth factor, 100ng / mL Wnt-3a recombinant protein, 1µg / mL R-Spondin1 recombinant protein, 100ng / mL noggin, 10µM ROCK inhibitor, 200ng / mL fibroblast growth factor 10, 10nM gastrin I, 2µM TGFβ inhibitor and 2µM glycogen synthase kinase 3 inhibitor.

9. Use of the co-culture medium of gastric cancer organoids and functional human gastrointestinal organoids according to any one of claims 8 to 9 in constructing a co-culture model of gastric cancer organoids and functional human gastrointestinal organoids.

10. Use of the co-culture medium of gastric cancer organoids and functional human gastrointestinal organoids according to any one of claims 8 to 9 in constructing an in vitro organoid model simulating the infiltration and growth of gastric cancer in the gastric mucosa and muscularis.

Images