Exogenous-growth-factor-free co-culture method for normal colon organ and fibroblast of mouse and application of exogenous-growth-factor-free co-culture method

Abstract

The invention provides a non-exogenous growth factor co-culture method for normal colon organs and fibroblasts of a mouse, which comprises the following steps: amplifying the normal colon organs of the mouse: taking fresh normal colon epithelial tissues of the mouse, cutting into pieces, dissociating, and adding into an organ amplification culture medium for culturing; extracting and culturing normal colonic fibroblasts of the mice: taking fresh normal colonic tissues of the mice, cleaning, cutting into pieces, digesting, and adding into a fibroblast amplification culture medium for culturing; and co-culturing the normal colon organ and the fibroblast of the mouse: respectively preparing a single cell of the normal colon organ and a single cell of the normal colon fibroblast of the mouse, mixing the single cells, and co-culturing to obtain the normal colon organ of the mouse. According to the method disclosed by the invention, contact type co-culture of normal organoid and fibroblasts thereof can be realized, and the growth of the organoid completely depends on growth factors secreted by the fibroblasts, so that an in-vivo epithelium-interstitial interaction environment is better simulated; the method has great advantages in research work of intestinal disease mechanism research, target function verification, drug screening and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of organoid culture, and particularly relates to a method for co-culturing mouse normal colon organoids and fibroblasts without external growth factors and application thereof. BACKGROUND

[0002] Organoid technology uses three-dimensional cultures from stem cells to simulate the structure and function of natural organs, which is a breakthrough in the field of biomedical research in recent years, and provides a revolutionary perspective for human physiology and pathology. In 2009, Hans Clevers team in the Netherlands cultured crypt cells separated from mouse intestinal segments in three-dimensional Matrigel containing EGF, Noggin and R-Spondin1, and cultivated the first intestinal organoid, creating the word "Organoids" and opening the era of organoid research 1 . So far, researchers have successfully cultivated various tissue organoids including lung, brain, prostate and pancreas in vitro, which can realize the construction of normal tissues or tumor tissues. Organoid technology has a profound impact on various fields, especially oncology and regenerative medicine.

[0003] Organoids have unique advantages compared with existing biomedical models. They not only retain the individualized tissue characteristics of patients, enabling more reliable and efficient drug screening and functional verification, but also simulate the tissue microenvironment by adding different cells for co-culture, which is of great significance in studying epithelial-mesenchymal-immune interactions. The intestine is an organ that performs digestion and absorption functions in the human body. The intestinal epithelium is divided into two different regions, the villus region and the crypt region. The villus region is located on the top surface of the intestinal epithelium and contains a variety of differentiated cell types. The crypt region is located at the bottom of the intestinal epithelium and is in direct contact with the basement membrane. The crypt contains intestinal stem cells (ISCs), one of which is a mature intestinal proliferative stem cell population expressing LGR5 (a protein in the WNT signaling pathway) that can differentiate into all cell types of the mature intestine. LGR5+ stem cells maintain the ability to self-renew and regenerate into intestinal epithelium, mainly because they are in the stem cell microenvironment. The intestinal microenvironment contains a spatial gradient of high WNT and epithelial growth factor (EGF) and can inhibit bone morphogenetic protein (BMP) signaling. Intestinal fibroblasts are components of the lamina propria mesenchyme, composed of fibroblasts, myofibroblasts, pericytes, smooth muscle cells, and mesenchymal stem cells, and are key members of the intestinal microenvironment. Intestinal fibroblasts can secrete signal molecules such as EGF, Noggin, R-Spondin1, WNT, and are the main source of activated epithelial WNT pathway molecules. Recent studies have shown that intestinal fibroblasts have high heterogeneity and complexity, and different populations of fibroblasts play important roles in intestinal development, homeostasis, and disease.

[0004] Organoids co-cultured with fibroblasts in vitro is an important model for studying the interaction between epithelium and stroma. The establishment of tumor organoids-stromal biobank from patients can individually evaluate the influence of tumor microenvironment on drug treatment response. The co-culture system of normal organoids and fibroblasts can also be combined with CRISPR-Cas9 and other gene editing technologies to simulate genetic diseases or test treatment strategies, and can also be used for developmental biology, toxicology and other aspects of research, which has great application value. Tumor organoids can grow in reduced factor medium due to the activation of corresponding signaling pathways by gene mutations, such as APC gene mutation activating Wnt / β-catenin signaling pathway. The growth of normal organoids must depend on several key growth factors. The traditional normal organoid-fibroblast contact co-culture system uses organoid complete medium, which contains sufficient exogenous added growth factors EGF, Noggin, R-Spondin1, WNT3a and other supplements. Due to the great difference in the types and amounts of growth factors secreted by fibroblast subpopulations in different subpopulations and different disease states, the addition of exogenous growth factors in the medium will affect the signaling pathways of cells, which may lead to unclear experimental phenomena and inaccurate conclusions.

[0005] Based on the above problems, the present application provides a method for co-culturing mouse normal colon organoids with their fibroblasts without exogenous growth factors, which can realize the contact co-culture of mouse normal organoids with their fibroblasts, and the growth of organoids completely depends on the growth factors secreted by fibroblasts, thereby better simulating the epithelial-stromal interaction environment in vivo.

[0006] REFERENCES

[0007] 1. Sato T, Vries RG, Snippert HJ, et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature. 2009; 459(7244): 262-265. doi: 10.1038 / nature07935. SUMMARY

[0008] The present application provides a method for co-culturing mouse normal colon organoids with their fibroblasts without exogenous growth factors and its application, which can realize the contact co-culture of normal organoids with their fibroblasts, and the growth of organoids completely depends on the growth factors secreted by fibroblasts, thereby better simulating the epithelial-stromal interaction environment in vivo.

[0009] The application discloses a method for co-culturing mouse normal colon organoids and fibroblasts of the mouse normal colon organoids without external growth factors.

[0010] (1) amplifying mouse normal colon organoids

[0011] Fresh mouse normal colon epithelial tissue is taken, cut into pieces, added with a cell dissociation agent, dissociated, and then added with a mouse normal colon organoid amplification culture medium for culture to obtain mouse normal colon organoids.

[0012] (2) extraction and culture of mouse normal colon fibroblasts

[0013] Fresh mouse normal colon tissue is taken, washed, cut into pieces, added with a digestion solution, digested, and then added with a fibroblast amplification culture medium for culture to obtain mouse normal colon fibroblasts.

[0014] (3) co-culture of mouse normal colon organoids and fibroblasts thereof

[0015] Mouse normal colon organoid single cells and mouse normal colon fibroblast single cells are prepared respectively, and the single cells are mixed, wherein the cell number ratio of the mouse normal colon organoid single cells to the mouse normal colon fibroblast single cells after mixing is 1:200-1:300, a co-culture medium is used for co-culture, and mouse normal colon organoids are obtained.

[0016] Further, the components of the mouse normal colon organoid amplification culture medium in the step (1) are as follows: 50% (V / V) of L-WRN cell line conditioned medium, Advanced DMEM / F12 basic culture medium, 2mM of GlutaMax, 1% (V / V) of double-antibiotic penicillin-streptomycin solution, 10mM of HEPES, 1% (V / V) of N2 additive, 1% (V / V) of B27 additive, 0.1mM of N-acetyl cysteine, 1mM of nicotinamide, 50ng / mL of mEGF and 10uM of Y-27632.

[0017] Further, the components of the fibroblast amplification culture medium in the step (2) are as follows: DMEM, 20% (V / V) of FBS, 1% (V / V) of double-antibiotic penicillin-streptomycin solution and 10uM of Y-27632.

[0018] Further, the components of the digestion solution in the step (2) are as follows: DMEM, 10% (V / V) of FBS, 1% (V / V) of double-antibiotic penicillin-streptomycin solution, 100mg / mL of collagenase II and 50U / mL of Dnase I.

[0019] Further, the components of the co-culture medium in step (3) are as follows: Advanced DMEM / F12 basal medium, 2 mM GlutaMax, 1% (V / V) penicillin-streptomycin solution, 10 mM HEPES, 1% (V / V) N2 additive, 1% (V / V) B27 additive, 0.1 mM N-acetylcysteine, 1 mM nicotinamide, 1% (V / V) insulin-transferrin-selenium ITS-G and 10 μM Y-27632.

[0020] Further, the step (3) for preparing single cells of normal mouse colon organoids is as follows: discard the culture medium from the organoids, add TrypLE digestive enzyme solution and digest in a 37°C incubator for 6 min, and neutralize the cells with DMEM / F12 to obtain a single cell suspension.

[0021] Further, the step (3) for preparing single cells of normal mouse colon fibroblasts is as follows: add Trypsin digestive enzyme solution to normal mouse fibroblasts and digest for 4 min, centrifuge at 4℃ and 400g for 5 min, discard the supernatant, and obtain single cell precipitate.

[0022] Further, the specific operation of co-culture in step (3) is as follows: add mouse normal colon organoid single cells and mouse normal colon fibroblast single cells to the matrix gel respectively, then mix them in equal volumes and apply them to the cell culture plate at a volume of 50uL per well. Place the cell culture plate in a 37℃ constant temperature cell culture incubator for 20min. After the matrix gel solidifies, add the co-culture medium and continue co-culturing in a 37℃ constant temperature cell culture incubator.

[0023] This invention also discloses the application of the above method in the study of intestinal disease mechanisms, target function verification, and drug screening.

[0024] Compared with the prior art, the beneficial effects that at least one technical solution adopted in the embodiments of this specification can achieve include at least:

[0025] First, the co-culture method of the present invention supports the growth of organoids with growth factors secreted by fibroblasts, eliminating the interference of exogenous addition of EGF, Noggin, R-Spondin1, and WNT3a growth factors in traditional co-culture, effectively simulating the dependent microenvironment of intestinal epithelial and stromal cells in vivo, and is an in vitro model that is closer to the in vivo model.

[0026] Secondly, this invention provides a simple, easy-to-prepare, stable, and efficient culture medium for expanding mouse normal colon organoids, which is beneficial for large-scale experiments. At the same time, the method for culturing mouse primary normal colon fibroblasts provided by this invention enables the extraction and culture of sufficient quantities of high-efficiency and high-viability fibroblasts.

[0027] Third, compared with cell lines, PDX models (patient-derived xenograft models), and individual organoid models, the co-culture system provided by this invention is a novel in vitro model derived from primary cells. It is simple to culture, grows rapidly, and retains the individual's traits. Mouse organoids and fibroblasts can be derived from the same individual or different individuals, thus allowing for different experimental designs to complete diverse studies. This method has significant advantages in research on the mechanisms of intestinal diseases, target function verification, and drug screening. Attached Figure Description

[0028] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on the drawings without creative effort.

[0029] Figure 1 A photograph taken under a conventional optical microscope before passage of normal colonic organoids from P3 generation mice in expansion culture medium.

[0030] Figure 2 Photograph of normal colon fibroblasts from P4 generation mice grown in expansion medium under a regular optical microscope before passage.

[0031] Figure 3 A photograph taken under a conventional optical microscope during the co-culture and growth of normal mouse colon organoids and fibroblasts.

[0032] Figure 4 This is a photograph of a normal mouse colon organoid from Example 2 under a conventional optical microscope without the addition of fibroblasts.

[0033] Figure 5 Immunofluorescence staining image for identification of normal mouse colon organoids and fibroblasts co-cultured. Detailed Implementation

[0034] The embodiments of this application will now be described in detail with reference to the accompanying drawings.

[0035] The following specific examples illustrate the implementation of this application. Those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. This application can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of this application. It should be noted that, in the absence of conflict, the following embodiments and features in the embodiments can be combined with each other. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0036] Example 1

[0037] A method for co-culturing normal mouse colon organoids with their fibroblasts without exogenous growth factors includes the following steps:

[0038] (1) Amplification of normal mouse colon organoids

[0039] Fresh normal mouse colonic epithelial tissue was collected, washed five times with PBS containing antibiotics, and cut into 0.5 mm pieces. The tissue was washed five times again. 2 g of the fragments were added to 10 mL of a mild cell dissociation agent (Stemcell), and the mixture was placed on a shaker at 37°C and 120 rpm for 30 min to digest. DMEM + 1% BSA was added to neutralize the cell dissociation agent, and the mixture was shaken vigorously and placed on ice. The supernatant was collected after large tissue fragments settled and labeled as fraction 1. DMEM + 1% BSA was added to fraction 1, and the mixture was shaken vigorously and placed on ice. The supernatant was collected after large tissue fragments settled and labeled as fraction 2. DMEM + 1% BSA was added to fraction 2, and the mixture was shaken vigorously and placed on ice. The supernatant was collected after large tissue fragments settled and labeled as fraction 3. The components of fractions 1, 2, and 3 were observed under a microscope. Fractions 2 or 3, containing more elongated crypts and fewer villi and debris, were suitable for further use.

[0040] Fraction 3 was filtered using a 70μm cell filter. The filtrate was counted, and the suspension volume corresponding to 1800 crypts was collected. After centrifugation at 450g for 5 minutes at 4℃, the supernatant was discarded, and the precipitate was retained. 150µL of mouse normal colon organoid amplification medium was added to the precipitate, and after mixing, 150µL of Corning matrix gel was added and mixed thoroughly. 50µL of the gel was then dispensed into each well of a cell culture plate. The cell culture plates were placed in a 37℃ incubator for 20 minutes. After the matrix gel solidified, amplification medium was added. The composition of the mouse normal colon organoid amplification medium is shown in Table 1. The medium was changed every 2 days during culture. After 5-6 days, when the organoids reached a larger volume and higher density, they were passaged. The passaged organoids could be permanently amplified and co-cultured with fibroblasts.

[0041] The mouse normal colon organoid expansion medium consists of L-WRN cell line conditioned medium (brand name ATCC) providing Noggin, R-Spondin1, and WNT3a growth factors; Advanced DMEM / F12 basal medium; GlutaMax reagent to supplement L-glutamine; penicillin-streptomycin solution to inhibit bacterial growth; HEPES buffer to stabilize pH; serum substitutes N2 and B27 additives; antioxidants N-acetylcysteine ​​and nicotinamide; mouse-derived EGF growth factor; and Rock signaling pathway inhibitor Y-27632. The culture medium used in the same batch of experiments should be derived from the same batch of L-WRN cell line conditioned medium to minimize batch-to-batch variations in growth factor concentrations. Specifically, it consists of the following components at the following concentrations or volume percentages:

[0042] Table 1. Composition of culture medium for expanding normal mouse colon organoids

[0043]

[0044] (2) Extraction and culture of primary mouse colonic fibroblasts

[0045] Fresh, normal colonic tissue from 4-5 mice was washed three times with PBS containing penicillin-streptomycin, cut into 0.5 mm pieces, and digested for 40 min at 37°C and 120 rpm by adding 10 mL of mild cell dissociation agent to obtain colonic crypts. The remaining tissue was removed from the crypts and neutralized with DMEM + 10% FBS. After neutralization, the tissue was washed with PBS containing penicillin-streptomycin, and digestion solution (components of which are shown in Table 2) was added. The tissue was then digested for 40 min at 37°C and 120 rpm by shaking. After digestion, the digestion solution was neutralized, filtered through a 70 μm cell filter, and the filtrate was centrifuged at 4°C and 400 g for 5 min. The supernatant was discarded, and the cell pellet was resuspended and plated in fibroblast expansion medium (components of which are shown in Table 3) until the fibroblasts adhered. After 18-24 hours, fibroblasts were observed to adhere well under a microscope. The original culture medium was discarded, and the cells were washed and replaced with fresh culture medium. The medium was then changed every 2-3 days. Mouse intestinal fibroblasts extracted in this invention should be passaged after reaching a certain density. Cells from passages P10-P12 typically show better viability, while those from passages P4-P6 are more suitable for co-culture experiments. The digestion solution contains collagenase II and DNase I, with moderate strength, effectively dissociating tissue while preserving cell viability. Specifically, it consists of the following components at the following concentrations:

[0046] Table 2. Composition of Digestive Juices

[0047]

[0048] Table 3. Composition of fibroblast expansion culture medium

[0049]

[0050] (3) Co-culture of normal mouse colon organoids with their fibroblasts

[0051] When organoids are co-cultured at a 1:2 passage ratio, the optimal growth status is achieved when the ratio of single-cell colonic organoids to single-cell colonic fibroblasts is 1:200-1:300. This example uses a 1:250 ratio for co-culture. Normal mouse colonic organoids were cultured in expansion medium to passage P3. Microscopic images before passage are shown below. Figure 1As shown. Take 50 μL of organoid from one well of the dome, discard the culture medium, add 750 μL of TrypLE digestive enzyme solution (Gibco) preheated to 37°C, and digest in a 37°C incubator for 6 min. Observe under a microscope that the organoid has been digested into single cells. Neutralize the cells with DMEM / F12 to obtain a single-cell suspension. Centrifuge the single-cell suspension at 4°C and 400g for 5 min, discard the supernatant, retain the cell pellet, resuspend, and count the number of cells. Take the suspension volume corresponding to 2000 cell counts, centrifuge at 4°C and 400g for 5 min, discard the supernatant, and retain the cell pellet. Add 25 μL of matrix gel melted on ice to the cell pellet, mix well by pipetting, and temporarily store on ice.

[0052] Normal mouse fibroblasts were cultured to passage P4. Microscopic images were taken before passage. Figure 2 As shown. Take three 10cm dishes, discard the culture medium, wash once with PBS, add 2mL of Trypsin digestive enzyme solution (Gibco brand) and digest for 4min. Neutralize with culture medium and centrifuge at 4℃, 400g for 5min. Discard the supernatant and retain the cell pellet. Add 1mL of culture medium to the cell pellet and mix well. Use a cell counter to accurately count the cells. Take the volume of liquid corresponding to 250k cells, centrifuge at 4℃, 400g for 5min, discard the supernatant and retain the cell pellet. Add 25uL of matrix gel to the cell pellet, mix well and temporarily store on ice. Add the matrix gel containing organoid single cells to the matrix gel containing fibroblast single cells, quickly pipette to mix well, and dispense 50uL of the gel into each well of the cell culture plate. Place the cell culture plate in a 37℃ incubator for 20min. After the matrix gel solidifies, add the co-culture medium shown in Table 4 and continue culturing in a 37℃ incubator. Optionally or preferably, after placing the cell culture plate upright in the incubator for 5-6 minutes until the matrix gel slightly solidifies, invert it for another 15 minutes to reduce cell settling. During culture, change the culture medium every 2 days. Spherical growth of organoids can be observed after 8 days, and they reach their maximum size after 14-20 days.

[0053] The co-culture medium for mouse colon organoids includes Advanced DMEM / F12 basal medium, GlutaMax (a reagent to supplement L-glutamine), a penicillin-streptomycin solution to inhibit bacterial growth, HEPES (a pH-stabilizing buffer), B27 and N2 serum substitutes, nicotinamide and N-acetylcysteine ​​(antioxidants), insulin-transferrin-selenium ITS-G (a nutritional supplement), and Y-27632 (an anti-apoptotic Rock signaling pathway inhibitor).

[0054] Table 4. Composition of Co-culture Medium

[0055]

[0056] Figure 3 The image shows a conventional optical microscope image of the growth status of mouse organoids during co-culture with fibroblasts. As can be seen from the image, the spherical organoids are growing rapidly on day 15; on day 18, the organoids have grown to their maximum volume, and the organoids are dense spherical with a maximum diameter of about 250 μm.

[0057] Example 2: Co-culture of normal mouse colon organoids without fibroblasts

[0058] The difference between this embodiment and Example 1 is that fibroblast co-culture is not added. The specific operation is as follows: Normal mouse colon organoids are cultured in expansion medium to the P2 generation. Microscopic images before passage are shown below. Figure 1 As shown. Take 50 μL of organoids from one well of the dome, discard the culture medium, add 750 μL of TrypLE digestive enzyme solution preheated to 37℃, and digest in a 37℃ incubator for 6 min. Observe under a microscope that the organoids have been digested into single cells. Neutralize the cells with DMEM / F12 to obtain a single-cell suspension. Centrifuge the single-cell suspension at 4℃ and 400g for 5 min, discard the supernatant, add 50 μL of matrix gel to the cell pellet, and quickly pipette to mix. Dispense 50 μL of the mixture into each well of a cell culture plate. Place the cell culture plate in a 37℃ incubator for 20 min. After the matrix gel solidifies, add the co-culture medium shown in Table 4. Optionally or preferably, after the cell culture plate is placed upright in the incubator for 5-6 min and the matrix gel has slightly solidified, invert it for 15 min to reduce cell settling. Change the culture medium every 2 days during the culture process.

[0059] Figure 4 The image shows a bright-field micrograph of the organoid growth status without fibroblast co-culture. As can be seen, no significant organoid growth was observed on day 4, and all cells died. This indicates that when cultured using the same method without fibroblasts, the epithelial cells lack the essential growth factors for survival, cannot grow into organoids, and die.

[0060] Example 3: Identification by Immunofluorescence Staining of Co-cultured Normal Mouse Organoids and Fibroblasts

[0061] Collect co-cultured cells on day 6, remove the plate and discard the culture medium. Wash once with PBS, add 1 mL of 4% paraformaldehyde, and fix overnight at room temperature. The next day, remove the plate, discard the fixative, add 1 mL of 70% ethanol and soak for 1 day. Scrape the co-culture, along with the matrix gel, from the plate with a blade and place it on the surface of the solidified Histogel pre-embedding gel. Add another 150 μL of Histogel on top and solidify on ice to form a sandwich structure. Place the Histogel block into the embedding cassette for embedding. Subsequent steps, including embedding, sectioning, dewaxing, fluid replenishment, antigen retrieval, blocking, primary antibody incubation, and secondary antibody incubation, are the same as routine immunofluorescence staining procedures. In this embodiment, the primary antibodies used were rabbit anti-αSMA antibody (brand name: Abcam), diluted 1:200, and mouse anti-panCK antibody (brand name: Santa Cruz), diluted 1:200; the secondary antibodies used were goat anti-mouse 647 antibody (brand name: Invitrogen) and donkey anti-rabbit 488 antibody (brand name: Invitrogen), diluted 1:500.

[0062] Figure 5 This is an immunofluorescence staining image of a normal mouse colon organoid co-cultured with fibroblasts. The organoids are visible as rings in the slice, surrounded by fibroblasts.

[0063] The above embodiments are merely illustrative of the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made based on the essence of the content of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method of co-culturing mouse normal colon organoids with their fibroblasts without exogenous growth factors, characterized in that, The method comprises the following steps: (1) amplifying mouse normal colon organoids Fresh mouse normal colon epithelial tissue is taken, cut into pieces, and then added with a cell dissociation agent. After dissociation, the mouse normal colon organoids are obtained by culturing in a mouse normal colon organoid amplification medium. (2) extraction and culture of mouse normal colon fibroblasts Fresh mouse normal colon tissue is taken, washed, cut into pieces, and then added with a digestion solution. After digestion, the mouse normal colon fibroblasts are obtained by culturing in a fibroblast amplification medium. (3) co-culture of mouse normal colon organoids and fibroblasts Mouse normal colon organoid single cells and mouse normal colon fibroblast single cells are prepared, and the single cells are mixed, wherein the cell number ratio of the mouse normal colon organoid single cells to the mouse normal colon fibroblast single cells is 1:200-1:

300. The co-culture medium is used for co-culture to obtain mouse normal colon organoids.

2. The method of claim 1, wherein, In the step (1), the mouse normal colon organoid amplification medium comprises the following components: 50% (V / V) of L-WRN cell line conditioned medium, Advanced DMEM / F12 basal medium, 2mM of GlutaMax, 1% (V / V) of double-antibiotic penicillin-streptomycin solution, 10mM of HEPES, 1% (V / V) of N2 supplement, 1% (V / V) of B27 supplement, 0.1mM of N-acetyl cysteine, 1mM of nicotinamide, 50ng / mL of mEGF and 10μM of Y-27632.

3. The method of claim 1, wherein, In the step (2), the fibroblast amplification medium comprises the following components: DMEM, 20% (V / V) of FBS, 1% (V / V) of double-antibiotic penicillin-streptomycin solution and 10μM of Y-27632.

4. The method of claim 1, wherein, In the step (2), the digestion solution comprises the following components: DMEM, 10% (V / V) of FBS, 1% (V / V) of double-antibiotic penicillin-streptomycin solution, 100mg / mL of collagenase II and 50U / mL of Dnase I.

5. The method of claim 1, wherein, In the step (3), the co-culture medium comprises the following components: Advanced DMEM / F12 basal medium, 2mM of GlutaMax, 1% (V / V) of double-antibiotic penicillin-streptomycin solution, 10mM of HEPES, 1% (V / V) of N2 supplement, 1% (V / V) of B27 supplement, 0.1mM of N-acetyl cysteine, 1mM of nicotinamide, 1% (V / V) of insulin-transferrin-selenium ITS-G and 10μM of Y-27632.

6. The method of claim 1, wherein, In the step (3), the mouse normal colon organoid single cells are prepared by taking the organoids, adding TrypLE enzyme solution, and digesting in a 37℃ incubator for 6min. The cells are neutralized in DMEM / F12 to obtain a single cell suspension.

7. The method of claim 1, wherein, The step (3) is to add Trypsin digestive enzyme solution to the mouse normal fibroblasts and digest for 4 min, centrifuge at 4 ℃ and 400 g for 5 min, discard the supernatant, and obtain the single cell precipitate.

8. The method of claim 1, wherein, The step (3) is to add the mouse normal colon organoid single cells and the mouse normal colon fibroblast single cells into the Matrigel respectively, then mix them in equal volume, and spot them in the cell culture well plate with a volume of 50 uL per well, place the cell culture well plate in a 37 ℃ constant temperature cell incubator for 20 min, after the Matrigel solidifies, add the co-culture medium, and continue the co-culture in the 37 ℃ constant temperature cell incubator.

9. The use of the method according to any one of claims 1-8 in the research of intestinal disease mechanism, the verification of target function and the drug screening.

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