Culture method of chicken intestinal tract organoid

Abstract

The invention provides a culture method of a chicken intestinal tract organoid, and belongs to the technical field of organoid culture. According to the method, chicken small intestine crypt stem cells are used for mouse lung organoid culture, after precooled Matrigel matrigel and 3D organoid cell culture fluid are mixed, the crypt stem cells are resuspended, the number of crypts reaches 8-30 / mu L, and the crypts are laid on a cell culture plate; culturing the cell culture plate at 37 DEG C for 10 minutes to polymerize the matrigel; and after the matrigel is completely solidified, adding a preheated 3D organoid cell culture solution, and culturing for 7 days under the conditions of 37 DEG C and 5% CO2 to obtain the chicken intestinal tract organoid. According to the invention, the problem of cell loss in the existing poultry intestinal crypt cell culture process is solved, and the prepared chicken intestinal organ has huge application potential in the aspect of intestinal disease model establishment.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of organoid culture, and particularly relates to a culture method of chicken intestinal tract organoids. BACKGROUND

[0002] Intestinal tract organoids are three-dimensional (3D) in vitro models based on self-organization of intestinal tract stem cells, which can simulate the "crypt-villus" structure of intestinal tract epithelium and various cell types (such as intestinal absorption cells, goblet cells, Paneth cells, etc.), and have significant advantages in nutritional absorption, pathogen infection, immune response and drug screening research. At present, the culture technology of human and mouse intestinal tract organoids has been relatively mature, and the culture medium formula (such as adding growth factors such as Wnt3A, R-spondin1 and Noggin) and culture process have been standardized.

[0003] Although the mammalian organoid technology develops rapidly, the culture of avian intestinal tract organoids, especially chicken, still faces multiple technical obstacles, mainly in the following aspects: 1. Low crypt separation efficiency and high contamination rate: Chicken intestinal tract tissue separation is prone to mix fibroblasts and endothelial cells, and the crypt cell clusters obtained by traditional enzyme digestion method (such as type I collagenase) have poor integrity and high proportion of single cells. The existing method relies on antibiotic washing (such as penicillin / streptomycin double antibiotic), but cannot completely solve the pollution problem, resulting in a primary culture success rate of less than 30%. 2. Organoid growth is slow and the shape is abnormal: Compared with mammalian organoids (3 days to form a cystic structure), chicken intestinal tract crypt cells proliferate and differentiate slowly: cystic structure needs to be formed for more than 5 days, which takes much longer than mice (2-3 days); the rate of appearance of bud-like crypt structure is low (<20%), and the diameter is mostly less than 200 um; cells are prone to premature apoptosis, and the subculture is difficult. Therefore, it is necessary to propose a culture method of chicken intestinal tract organoids. SUMMARY

[0004] Therefore, the present application provides a culture method of chicken intestinal tract organoids to solve the above problems.

[0005] In order to achieve the above application purposes, the present application provides the following technical solutions: The application provides a 3D organoid cell culture solution, which comprises the following components in volume percentage: Advanced DMEM / F12 91-92%, streptomycin-zeocin mixture (100x) 0.8-1.2%, GlutaMAX (100x) 0.8-1.2%, HEPES (1M) 0.8-1.2%, B27 (50x) 1.8-2.2%, N2 0.8-1.2%, N-acetyl cysteine 0.2-0.3%, EGF 0.04-0.06%, recombinant mouse Noggin 0.3-0.5%, recombinant R-Spondin1 0.4-0.6%, Y-27632 0.2-0.3%, chicken serum 0.2-0.3%, SB202190 0.08-0.12%, CHIR99021 0.2-0.4%, VPA 0.15-0.25%, and A8301 0.04-0.06%.

[0006] The application further provides application of the 3D organoid cell culture solution in chicken intestinal tract organoid culture.

[0007] The application further provides a culture method of chicken intestinal tract organoids, comprising the following steps: (1) isolating chicken small intestinal crypt stem cells; (2) mixing pre-cooled Matrigel matrix glue and the 3D organoid cell culture solution according to claim 1, resuspending the crypt stem cells, so that the number of crypts reaches 8-30 / μL, and then spreading on a cell culture plate; (3) placing the cell culture plate at 37℃ for 10 min to make the matrix glue polymerize; (4) after the matrix glue completely solidifies, adding pre-heated 3D organoid cell culture solution and culturing at 37℃ under 5% CO2; and replacing the culture solution every 2-3 days.

[0008] Preferably, the Matrigel matrix glue and the cell culture solution are mixed according to a volume ratio of 1:0.8-1.2.

[0009] Preferably, the 3D organoid cell culture solution in step (4) is pre-heated to 37℃.

[0010] Preferably, the isolation method of the chicken small intestinal crypt stem cells is as follows: washing the small intestine, cutting it into pieces, removing villus cells and adipose tissue, adding EDTA, oscillating and digesting at 3-5℃ for 20-50 min, centrifuging after digestion, filtering the supernatant, and the filtrate is the crypt stem cell suspension.

[0011] Preferably, the concentration of the EDTA is 2-3 mM.

[0012] Preferably, the oscillation speed is 60-80 rpm.

[0013] Preferably, the centrifugation speed is 900-1100 rpm, and the centrifugation time is 4-6 min.

[0014] Preferably, the small intestine is derived from a 17-day-old embryonic broiler.

[0015] By adopting the technical scheme, the present application has the following beneficial effects: the present application solves the problem of cell loss in the culture process of the existing poultry intestinal crypt cells, and can better represent the animal intestinal tissue and cell structure. In addition, the separated small intestine crypts can be continuously proliferated in the culture of the matrix glue, small molecule compounds, cell additives and various growth factors, have self-renewal and differentiation ability, can differentiate to form various mature intestinal cells, and the morphological structure is embodied in cell budding and other organoid characteristics, and has great application potential in the establishment of intestinal disease models. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The isolated chicken crypt cells.

[0017] Figure 2 The chicken small intestine organoid cells cultured for different time.

[0018] Figure 3 The chicken small intestine organoid cell skeleton immunofluorescence staining diagram. DETAILED DESCRIPTION

[0019] The technical scheme provided by the present application will be described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application.

[0020] Example 1 1. Isolation and culture of chicken small intestine crypt stem cells (1) Isolate a 17-day-old chicken embryo, take the jejunum 5-7 cm, and remove the mesentery and peripheral blood vessels; (2) Use a syringe to suck clean ice PBS to repeatedly and gently wash the inside of the intestine, remove the intestinal contents, cut the intestinal tissue into small pieces about 1 cm, and place them in a 20 mL centrifuge tube; (3) Add appropriate amount of DPBS, and blow 3-4 times with a pipette gun, 20 times each time, until the liquid is clear, to remove villus cells and floating adipose tissue; (4) Discard the supernatant, add 2.5 mM EDTA, and digest for 20 min at 4°C on a 70 rpm shaker; (5) After digestion, centrifuge at 1000 rpm for 5 min; (6) Discard the supernatant, add 50 mL of Advanced DMEM / F12 medium, blow, and filter through a 100 μm filter screen to obtain a crypt stem cell suspension; 2. Chicken small intestine organoid culture (1) Centrifuge the crypt stem cell suspension at 1000 rpm for 5 min, and discard the supernatant.

[0021] (2) Add an appropriate amount of pre-cooled Advanced DMEM / F12 medium to resuspend the crypts.

[0022] (3) Take 20 μL of the suspension on a coverslip, and count the crypts under a microscope.

[0023] (4) Mix the pre-cooled Matrigel matrix glue and cell culture medium in equal volumes, mix well, and the crypt count is 30 per μL, then inoculate the cell plate for culture. The Matrigel matrix glue is provided by Corning Company.

[0024] (5) Place the culture plate in a 37°C incubator for 10 min.

[0025] (6) After the matrix glue is completely solidified, add 500 μL of pre-heated 3D organoid cell culture medium, and culture in a 5% CO2 incubator at 37°C for 7 days, and replace the culture medium every 2 days during the period. The composition of the 3D organoid cell culture medium is: Advanced DMEM / F12 91.65%, penicillin-streptomycin mixture (100x) 1%, GlutaMAX (100x) 1%, HEPES (1M) 1%, B27 (50x) 2%, N2 1%, N-acetylcysteine 0.25%, EGF 0.05%, recombinant mouse Noggin 0.4%, recombinant R-Spondin1 0.5%, Y-27632 0.25%, chicken serum 0.25%, SB202190 0.1%, CHIR99021 0.3%, VPA 0.2%, A8301 0.05%.

[0026] 3. Immunofluorescence staining Immunofluorescence staining was performed on the prepared chicken small intestine organoids to observe whether the cells of the small intestine organoids presented a three-dimensional structure, and the specific steps are as follows: (1) Fixation: The cells were fixed with 4% paraformaldehyde for 15 min, and washed with PBS twice.

[0027] (2) Permeabilization: Permeabilize with 0.2% Triton X-100 on ice for 15 min, and wash 2 times.

[0028] (3) Blocking: Add 5% BSA-containing PBS on the coverslips, and block at 4°C for 30 min.

[0029] (4) Antibody incubation: Incubate the sections with Actin-Tracker Red-Rhodamine (Thermo Scientific) at 37℃ for 2 h, and wash with PBS for 3 times.

[0030] (5) Mounting: Add anti-quenching mounting medium containing DAPI, cover with a glass slide, and seal the edges with nail polish. (6) Observation: Obtain images using corresponding spectral channels (detection channels: DAPI 350nm, green 488nm, red 647nm) using a confocal microscope (LSM 800).

[0031] The chicken small intestine organoid cytoskeleton immunofluorescence staining diagram is as shown in Figure 3 The results show that the small intestine organoid cells present a three-dimensional structure after culture.

[0032] It can be seen from the above examples that the present application provides a culture method of chicken intestinal tract organoids. The culture method of chicken intestinal tract organoids can differentiate into various mature intestinal cells, and at the same time solves the problem of cell loss in the culture process of poultry intestinal crypt cells.

[0033] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A 3D organoid cell culture solution, characterized by, The 3D organoid cell culture solution comprises the following components in volume percentage: Advanced DMEM / F12 91~92%, streptomycin mixture (100x) 0.8~1.2%, GlutaMAX (100x) 0.8~1.2%, HEPES (1M) 0.8~1.2%, B27 (50x) 1.8~2.2%, N2 0.8~1.2%, N-acetyl cysteine 0.2~0.3%, EGF 0.04~0.06%, recombinant mouse Noggin 0.3~0.5%, recombinant R-Spondin1 0.4~0.6%, Y-27632 0.2~0.3%, chicken serum 0.2~0.3%, SB202190 0.08~0.12%, CHIR99021 0.2~0.4%, VPA 0.15~0.25%, A8301 0.04~0.06%.

2. The 3D organoid cell culture solution of claim 1 is used in chicken intestinal tract organoid culture.

3. A method of culturing chicken enteroids, characterized by, The method comprises the following steps: (1) isolating chicken small intestinal crypt stem cells; (2) mixing pre-cooled Matrigel matrix glue and the 3D organoid cell culture solution of claim 1, resuspending the crypt stem cells, so that the number of crypts is 8~30 / μL, and then spreading on a cell culture plate; (3) placing the cell culture plate at 37℃ for 10 min to make the matrix glue polymerize; (4) after the matrix glue is completely solidified, adding preheated 3D organoid cell culture solution, and culturing at 37℃, 5% CO2 for 7 days to obtain chicken intestinal tract organoids; during the period, the culture solution is replaced every 2~3 days.

4. The culture method according to claim 3, characterized by, The Matrigel matrix glue and the cell culture solution are mixed at a volume ratio of 1:0.8~1.

2.

5. The culturing method according to claim 3, wherein The 3D organoid cell culture solution of step (4) is preheated to 37℃.

6. The culturing method according to claim 3, wherein The isolation method of chicken small intestinal crypt stem cells is as follows: washing the small intestine, cutting it into pieces, removing villus cells and adipose tissue, adding EDTA, oscillating and digesting at 3~5℃ for 20~50 min, centrifuging after digestion, filtering the supernatant, and the filtrate is the crypt stem cell suspension.

7. The culture method according to claim 6, characterized by, The concentration of EDTA is 2~3mM.

8. The culture method according to claim 7, wherein The rotation speed of oscillation is 60~80 rpm.

9. The culturing method according to claim 8, wherein, The centrifugation speed is 900~1100 rpm, and the centrifugation time is 4~6 min.

10. The culturing method according to claim 3, wherein The small intestine is derived from a 17-day-old embryonic broiler.

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