Mouse intestinal organ and culture method and application thereof

Abstract

The invention discloses a mouse intestinal tract organ and a culture method and application thereof. The method comprises the following steps: extracting fetal mouse colon tissues from pregnant mice, treating the fetal mouse colon tissues with digestive enzymes to prepare a cell mass suspension, and further mixing the cell mass suspension with a matrigel seed plate for culture. According to the method, the forming efficiency and success rate of the colon-like organs are remarkably improved, and the method has wide application prospects in the fields of disease modeling, drug screening and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell culture, in particular to a mouse intestinal organoid and a culture method and application thereof. BACKGROUND

[0002] An organoid is an in vitro cell model with organ function obtained by three-dimensional culture of pluripotent stem cells or adult stem cells, which can spontaneously proliferate, differentiate and have a certain spatial arrangement structure, and has core functions of organs such as nutrient absorption, hormone secretion and drug metabolism. It is an important cell model for studying disease development, drug screening and development research. The intestinal organoid is an in vitro three-dimensional model with a typical "crypt-villus" structure formed by culturing isolated intestinal crypt stem cells.

[0003] With the continuous research of intestinal organoids, a complete mouse adult intestinal organoid culture system has been initially formed. The basic culture medium is Dulbecco's modified Eagle's medium / Ham's F-12 (advanced DMEM / F12) supplemented with glutamine (2 mM), HEPES (10 mM), penicillin-streptomycin (0.11 mg / mL), N-2, B-27, n-acetylcysteine, epidermal growth factor (EGF), Noggin, R-spondin1 and Wnt3a.

[0004] Currently, the adult mouse intestine is often used to extract and culture mouse intestinal organoids. Due to the small number of adult mouse intestinal stem cells, the organoid derivation rate is low, and there are problems of slow growth and low success rate. Moreover, it has certain limitations for the study of intestinal development.

[0005] Therefore, it is of great significance to develop a method with high formation efficiency and fast molding speed. SUMMARY

[0006] Therefore, the main purpose of the present application is to provide a new extraction and culture method of mouse fetal intestinal organoids. The extraction method has the advantages of high success rate, rapid growth and less limitation for intestinal development research due to the large number of stem cells in the developing stage of fetal mouse intestine.

[0007] To achieve the above-mentioned purpose, the technical scheme of the present application is as follows: A culture method of mouse intestinal organoids, comprising: (1) isolating a fetal mouse; (2) taking out the complete fetal intestinal tissue of the fetal mouse, soaking in sterile PBS buffer for cleaning, slowly removing the omental fat and vascular tissue, and obtaining the fetal intestinal tissue without other tissues; (3) The fetal intestinal tissue is washed 2-3 times by soaking in sterile PBS buffer, cut into pieces in a culture dish with tissue digestion enzyme solution to obtain a fetal intestinal tissue mixture; (4) The fetal intestinal tissue mixture in step (3) is added to a multi-well plate for cell culture, 0.8-1.2 mL per well, and incubated at 37°C on a constant temperature shaking table; (5) After incubation, enzyme digestion termination solution is added, and the fetal rat intestinal tract cells are obtained by passing through a cell sieve; (6) The fetal rat intestinal tract cell suspension obtained in step (5) is centrifuged, the supernatant is discarded, and the matrix gel is resuspended to obtain a fetal rat intestinal tract cell matrix gel suspension; (7) The fetal rat intestinal tract cell matrix gel suspension obtained in step (6) is added to a multi-well plate, 20-40 μL per well, and incubated in a 37°C, 5% CO2 incubator until the matrix gel solidifies; (8) Then, mouse intestinal tract organoid culture medium is added, 400-600 μL per well, and fresh culture medium is replaced every 2-3 days; (9) After 2-3 days of culture, the mouse intestinal tract organoid culture medium in the multi-well plate in step (8) is replaced with differentiation culture medium, and incubated in a 37°C, 5% CO2 incubator for 5-7 days to obtain organoids containing crypt-villus structures.

[0008] Further, in step (1), the fetal rat is 17.5-19.5 days old.

[0009] Further, in step (3), the cutting is cutting the fetal intestinal tissue into 1-2 mm 3 in volume.

[0010] Further, in step (4), the incubation time is 30-50 min; In step (5), the size of the cell sieve is 70 μm, and the enzyme digestion termination solution is Advanced DMEM-F12 medium.

[0011] Further, in step (6), the centrifugation is at a speed of 300 g for 3 min, and the concentration of the fetal rat intestinal tract cell matrix gel suspension is 10 4 per 25 μL.

[0012] Further, in step (7), the incubation time is 30-50 min.

[0013] Further, in step (9), the differentiation medium comprises advanced DMEM / F12 medium, 1 x N2 supplement, 1 x B27 supplement, 2 mM L-glutamine, 15 mM HEPES, 1 x penicillin-streptomycin, 1 mM N-acetyl cysteine, 5 ng / mL cytokine EGF, 0.1 g / mL Noggin, 500 g / mL R-Spondin1.

[0014] The application also aims to provide the mouse colon organoid prepared by any of the above methods.

[0015] The application also aims to provide the application of the mouse colon organoid described above in disease development research, drug screening, and development research.

[0016] The application has at least the following advantages: (1) The application significantly improves the formation efficiency and molding speed of the intestinal organoid by optimizing the extraction of fetal mouse intestinal cells for organoid culture, and significantly improves the batch stability.

[0017] (2) The fetal intestinal organoid cultured by the differentiation medium provided by the application can increase the differentiation ratio, budding rate and other indicators of the organoid, and has broad prospects in the research of intestinal cell differentiation. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 It is the fetal intestinal organoid culture flowchart of Example 1.

[0019] Figure 2 It is the microscope observation chart of the organoid proliferation culture for 3 days of Example 1.

[0020] Figure 3 It is the microscope observation chart of the organoid proliferation culture for 3 days of Comparative Example 1.

[0021] Figure 4 It is the microscope observation chart of the organoid proliferation culture for 3 days of Comparative Example 2.

[0022] Figure 5 It is the organoid proliferation culture result statistical chart of Example 1, Comparative Example 1 and Comparative Example 2.

[0023] Figure 6 It is the microscope observation chart of the organoid differentiation medium culture for 7 days of Example 2.

[0024] Figure 7 It is the HE staining of the organoid differentiation medium culture for 7 days of Example 2.

[0025] Figure 8 It is the microscope observation chart of the organoid proliferation medium culture for 7 days of Comparative Example 3.

[0026] Figure 9 For Example 2, the organoid of Comparative Example 3 was differentiated and cultured. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work are within the protection scope of the present application.

[0028] In addition, the technical solutions in the embodiments of the present application can be combined with each other, but it must be based on the fact that a person of ordinary skill in the art can realize the combination. When the combination of technical solutions appears to be contradictory or unachievable, it should be considered that the combination of technical solutions does not exist and is not within the protection scope of the present application.

[0029] The reagents used in the experiments of the present application are shown in Table 1 below.

[0030] Table 1 Reagents and consumables used in the embodiments

[0031] The solutions proposed by the present application will be specifically described below through specific embodiments: Example 1 Extraction and culture method of a fetal intestine organoid Take a late-pregnancy C57BL / 6J mouse (pregnant for 17.5-19.5 days), anesthetize, and operate on ice. Take the uterus tissue of the pregnant mouse, dissect the uterus tissue, and separate the fetal mouse. Cut along the lower edge of the rib of the fetal mouse, and dissect down the midline. Cut off the complete fetal intestine tissue at the rectum and duodenum of the fetal mouse. Soak the fetal intestine tissue in sterile PBS buffer for cleaning, and slowly remove the omental fat and vascular tissue to obtain fetal intestine tissue without other tissues. Further separate the fetal mouse colon tissue. Isolate the fetal intestine tissue (colon segment) of the fetal mouse.

[0032] Use PBS to rinse twice, and transfer to a 60-mm culture dish. Add 1 ml of tissue enzyme solution to the culture dish, and use an ophthalmic scissors to cut the tissue into 1-2 mm 3 .

[0033] Transfer the above mixture to a low-adsorption 24-well plate, and use the tissue enzyme solution to rinse. Fix the 24-well plate in a 37℃ constant-temperature shaking incubator, shake horizontally, and incubate for 40 min.

[0034] Take Advanced DMEM-F12 medium as the enzyme digestion termination solution, and use a 70-μm cell sieve on a 50-mL centrifuge tube.

[0035] The enzymolyzed tissue and enzymolyzed solution are transferred to a cell filter screen with a pipette gun, and a disposable grinding rod is gently ground on the filter screen, and the filter screen is washed with an enzyme termination solution to form a cell cluster suspension.

[0036] After centrifugation of the cell cluster suspension at 300g for 3min, the supernatant is discarded.

[0037] After resuspension of the cell count with the mouse intestinal organoid culture medium, centrifugation at 300g for 3min, the supernatant is discarded, and resuspension is performed with Matrigel, and the concentration is adjusted to 10 4 cells / 25μL.

[0038] The resuspended Matrigel complex is dropped at the bottom of a 24-well plate, 30μL per well, and the culture plate is placed in a 37℃, 5%CO2 incubator for 30min to allow the Matrigel to solidify.

[0039] 500μL of mouse intestinal organoid culture medium is added to each well, and the culture plate is returned to the incubator for 37℃, 5%CO2 culture.

[0040] The mouse intestinal organoid culture medium is replaced every 2-3 days.

[0041] After 3 days, the organoids are observed under a microscope and photographed. The results are shown in Figure 2 , three experiments were performed under the same experimental conditions, the average organoid formation time was 2-3 days, the average organoid diameter was 202.167um, and the average 7-day survival rate of organoids was 92.30%, and the statistical results are shown in Figure 5 .

[0042] Example 2: Differentiation culture scheme of fetal intestinal organoids The differentiation culture medium is prepared according to the differentiation culture medium formula, including advanced DMEM / F12 as the base medium, adding 1xN2 supplement, 1xB27 supplement, L-glutamine (2mM), HEPES (10mM), penicillin-streptomycin (1x), N-acetyl cysteine (1mM), and cytokines EGF (5ng / mL), Noggin (0.1μg / mL), R-Spondin1 (500μg / mL).

[0043] The organoid samples cultured for 2-3 days in Example 1 are removed from the culture medium and replaced with differentiation culture medium, and cultured in a 37℃, 5%CO2 incubator for 5-7 days. The organoid morphology is observed after 7 days of culture, and the organoid tissue is collected for H&E staining. The results are shown in Figure 6 and 7 , three experiments were performed under the same experimental conditions, the average differentiation time of organoids was 2-3 days, the average differentiation rate was 82.34%, and the 7-day mortality rate was 8.63%, and the statistical results are shown in Figure 9shown.

[0044] Comparative Example 1: A conventional enteroid culture method Take adult C57BL / 6J mice (about 12 weeks old) that have been fasted for 12 hours, anesthetize, operate on ice, cut off the complete colon tissue at the rectal part and the lower part of the cecum; open and wash the contents.

[0045] Place the intestinal tissue in a new culture dish containing DPBS (Gibco) and wash, repeat washing 2 times. Cut the washed colon tissue into 2 mm wide pieces and transfer to a new culture dish, wash with DPBS 2 times.

[0046] Transfer the washed intestinal segments to pre-cooled DPBS containing 5 mM EDTA (Gibco) for digestion, incubate at 4°C for 20-30 min.

[0047] Gently blow the intestinal segments with a pipette, observe the supernatant under a microscope, and terminate the digestion when complete crypts appear in the supernatant.

[0048] After digestion is complete, transfer the tissue fragments to a new culture dish containing DPBS and wash, repeat 2 times to remove EDTA.

[0049] Blow and resuspend the tissue in a pre-cooled 0.1% BSA DPBS culture dish or 50 mL centrifuge tube with a 5 mL pipette, take a portion of the suspension for microscopic examination, and stop blowing when a large number of crypt-like structures can be seen. Filter the blown tissue suspension through a 70 μm filter.

[0050] Collect the tissue suspension that passes through the filter, centrifuge at 300g for 3 min at 4°C.

[0051] Discard the supernatant, resuspend the tissue pellet with 1 mL of 0.1% BSA DPBS, take 20 μl of the suspension for microscopic examination and crypt counting, and after counting, take the suspension containing the desired amount of crypts, centrifuge at 300g for 3 min at 4°C.

[0052] Resuspend the cells after counting with mouse enteroid culture medium, centrifuge at 300g for 3 min, discard the supernatant, and resuspend with Matrigel, adjust the concentration to 10 4 cells / 25 μL.

[0053] Drop the resuspended Matrigel complex at the bottom of a 24-well plate, 30 μL per well, and place the culture plate in a 37°C, 5% CO2 incubator for 30 min to allow the Matrigel to solidify.

[0054] Observe and take pictures under a microscope at 3 days.

[0055] The results are shown in Table 1. Figure 3As shown, the average organoid formation time was 3-4 days under the same experimental conditions, the average organoid inner diameter was 183.83 um on the 3rd day, and the average 7-day survival rate of the organoids was 77.46%. The statistical results are as follows Figure 5 As shown.

[0056] Comparative Example 2: A conventional enteroid culture method Adult C57BL / 6J mice (about 12 weeks old) were fasted for 12 hours, anesthetized, and operated on ice. The complete colon tissue was cut off at the rectal part and the lower part of the cecum. The contents were cut open and washed.

[0057] The PBS was used to rinse twice and transferred to a 60 mm culture dish. 1 ml of tissue enzyme solution was added to the culture dish, and the tissue was cut into 1-2 mm 3 .

[0058] The above mixture was transferred to a low-adsorption 24-well plate, and the 24-well plate was fixed in a 37°C constant temperature shaker and incubated for 40 min.

[0059] The Advanced DMEM-F12 medium was used as the enzyme digestion termination solution, and a 70 μm cell screen was placed in a 50 mL centrifuge tube.

[0060] The enzyme-digested tissue and enzyme solution were transferred to the cell filter screen with a pipette, and a disposable grinding rod was used to gently grind on the filter screen. The enzyme digestion termination solution was added to rinse the filter screen to form a cell cluster suspension.

[0061] The cell cluster suspension was centrifuged at 300g for 3 min, and the supernatant was discarded.

[0062] The cell cluster suspension was centrifuged at 300g for 3 min, and the supernatant was discarded. 4 The concentration was adjusted to 10

[0063] The resuspended Matrigel complex was dropped at the bottom of the 24-well plate, 30 μL per well, and the culture plate was placed in a 37°C, 5% CO2 incubator for 30 min to solidify the Matrigel.

[0064] 500 μL of mouse enteroid culture medium was added to each well, and the culture plate was returned to the incubator for 37°C, 5% CO2 culture.

[0065] The new mouse enteroid culture medium was replaced every 2-3 days.

[0066] The results are as follows Figure 4As shown, the average organoid formation time was 3-4 days under the same experimental conditions, the average organoid inner diameter was 182.39 um on the 3rd day, and the average 7-day survival rate of the organoids was 89.15%. The statistical results are shown in Table 1. Figure 5 As shown in Table 2.

[0067] Comparative Example 3: Differentiation culture scheme of existing fetal intestine organoids The organoid samples cultured for 2-3 days in Example 1 were removed from the culture medium and replaced with fresh mouse intestinal organoid culture medium. The organoids were cultured in a 37°C, 5% CO2 incubator for 7 days, and the morphology of the organoids was observed.

[0068] The results are shown in Table 3. Figure 8 As shown, the average differentiation time of the organoids was 2-3 days under the same experimental conditions, the average differentiation rate was 66.47%, and the 7-day mortality rate was 13.69%. The statistical results are shown in Table 3. Figure 9 As shown in Table 4.

[0069] It should be noted that in this document, the terms "comprising", "including", or any other variant thereof are intended to cover non-exclusive inclusions, so that processes, methods, articles, or devices that include a series of elements not only include those elements, but also include other elements not explicitly listed, or inherent to such processes, methods, articles, or devices. Without more limitations, the element defined by the statement "comprising a" does not exclude the presence of other identical elements in the process, method, article, or device that includes the element.

[0070] The above-mentioned embodiment numbers of the present application are only for description, not representing the advantages and disadvantages of the embodiments.

[0071] The above embodiments are only used to illustrate the technical solutions of the present application and not limit the present application. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced equivalently without departing from the purpose and scope of the present application, and they should be covered in the scope of the claims of the present application.

Claims

1. A method of culturing mouse intestinal organoids, characterized by, The method comprises the following steps: (1) separating a fetal mouse; (2) taking out the complete fetal intestinal tissue of the fetal mouse, cleaning in sterile PBS buffer solution, slowly removing the omental fat and blood vessel tissue, and obtaining the fetal intestinal tissue without other tissues; (3) cleaning the fetal intestinal tissue in sterile PBS buffer solution for 2-3 times, cutting into pieces in a culture dish added with a tissue digestion enzyme solution, and obtaining a fetal intestinal tissue mixture; (4) adding the fetal intestinal tissue mixture in step (3) into a multi-well plate for cell culture, 0.8-1.2 mL per well, and incubating at 37 DEG C in a constant-temperature shaking bed; (5) after the incubation is completed, adding an enzyme digestion termination solution, passing through a cell sieve, and obtaining a fetal mouse intestinal tract cell suspension; (6) centrifuging the fetal mouse intestinal tract cell suspension obtained in step (5), discarding the supernatant, resuspending by adding a Matrigel, and obtaining a fetal mouse intestinal tract cell Matrigel suspension; (7) taking the fetal mouse intestinal tract cell Matrigel suspension obtained in step (6), adding into a multi-well plate, 20-40 muL per well, and incubating in a 37 DEG C, 5% CO2 incubator until the Matrigel solidifies; (8) then adding a mouse intestinal tract organoid culture medium, 400-600 muL per well, and continuing to culture, and replacing fresh culture medium every 2-3 days; (9) after 2-3 days of culture, replacing the mouse intestinal tract organoid culture medium in the multi-well plate in step (8) with a differentiation culture medium, incubating in a 37 DEG C, 5% CO2 incubator for 5-7 days, and obtaining an organoid comprising crypt-villus structure.

2. The method of claim 1, wherein, In step (1), the gestational age of the fetal mouse is 17.5-19.5 days.

3. The method of claim 1, wherein, In step (3), mincing is mincing the fetal intestinal tissue into 1-2 mm in volume of tissue. 3 In step (3), mincing is mincing the fetal intestinal tissue into 1-2 mm in volume of tissue.

4. The method according to claim 1, wherein, In step (4), the incubation time is 30-50 min; In step (5), the size of the cell sieve is 70 mu m, and the enzyme digestion termination solution is an Advanced DMEM-F12 culture medium.

5. The method of claim 1, wherein, In step (6), centrifugation was performed at 300g for 3 minutes, and the concentration of the fetal rat intestinal cell matrix suspension was 10. 4 25μL per cell.

6. The method of claim 1, wherein, In step (7), the incubation time is 30-50 min.

7. The method of claim 1, wherein, In step (9), the differentiation culture medium comprises an advanced DMEM / F12 culture medium, 1 x N2 additive, 1 x B27 additive, 2 mM L-glutamine, 15 mM HEPES, 1 x penicillin-streptomycin, 1 mM N-acetyl cysteine, 5 ng / mL cytokine EGF, 0.1 mu g / mL Noggin, and 500 mu g / mL R-Spondin1.

8. A mouse colon organoid prepared by the method according to any one of claims 1-7.

9. Use of the mouse colon organoid according to claim 8 in disease development research, drug screening, and development research.

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