A method of co-culturing intestinal epithelial organoids with eosinophils

Abstract

This invention provides a method for co-culturing intestinal epithelial organoids with eosinophils. The method includes the following steps: culturing small intestinal crypts for day 3, then adding eosinophils and co-culturing for 1 day to detect organoid growth rate; or culturing small intestinal crypts for day 6, then adding eosinophils and co-culturing for 1 day to detect the differentiation rate of small intestinal goblet cells. The method provided by this invention allows for direct detection of the effect of eosinophils on small intestinal epithelial cells.

Description

Technical Field

[0001] This invention belongs to the field of intestinal epithelial organoid culture technology, specifically relating to a method for co-culturing intestinal epithelial organoids and eosinophils. Background Technology

[0002] Currently, the culture of mouse intestinal epithelial organoids cannot fully replicate the functions of in vivo intestinal epithelium. The culture conditions for intestinal epithelial organoids and the activity of purified eosinophils both affect the final co-culture results. The culture of intestinal epithelial organoids and the purification and collection of eosinophils both require significant financial and time investment.

[0003] Therefore, there is a need for a new method for culturing intestinal epithelial organoids that can fully replicate the full function of the in vivo intestinal epithelium and is cost-effective, thereby supporting the development of new drugs, providing personalized treatment options, and understanding individual differences in disease development. Summary of the Invention

[0004] Technical Purpose

[0005] One technical objective of this invention is to provide a method for co-culturing intestinal epithelial organoids and eosinophils, which enables in vitro stimulation of epithelial cells by eosinophils and analyzes the function of eosinophils by analyzing the proliferation and differentiation of organoids.

[0006] Technical solution

[0007] On one hand, the present invention provides a method for co-culturing intestinal epithelial organoids and eosinophils, the method comprising the following steps:

[0008] When the small intestinal crypts are cultured for 3 days, eosinophils are added and co-cultured for 1 day to detect the growth rate of organoids; or when the small intestinal crypts are cultured for 6 days, eosinophils are added and co-cultured for 1 day to detect the differentiation rate of small intestinal goblet cells.

[0009] In a specific implementation, the method includes the following steps:

[0010] 1) Gel the small intestinal crypts using organoid-specific matrix gel and add small intestinal organoid culture medium. Culture the crypts on day 3 or day 6, then discard the small intestinal organoid culture medium from the well plate.

[0011] 2) Dissolve the gel with PBS buffer, separate the organoids, and wash them;

[0012] 3) Resuspend the organoids obtained in step 2 using a mixture of organoid-specific matrix gel containing small intestinal eosinophils and small intestinal organoid culture medium;

[0013] 4) The resuspension system obtained in step 3) is gelled using an organoid matrix gel; and

[0014] 5) Add small intestinal organoid culture medium from the edge of the culture plate, place the plate in a cell culture incubator and co-culture for 1 day. Collect samples for analysis.

[0015] In the case of detecting organoid growth rate by area or volume, the culture time in step 1) is 3 days; in the case of detecting the differentiation rate of small intestinal goblet cells, the culture time in step 1) is 6 days.

[0016] In a specific implementation, in step 1), the separation of the small intestinal crypts is performed in accordance with conventional procedures in the art.

[0017] In a specific implementation, the isolation and pre-culture preparation of the small intestinal crypts in step 1) are carried out through the following steps:

[0018] Small intestine was removed from mice, longitudinally dissected, and the intestinal wall was cleaned. The small intestine was digested with intestinal villus digestion solution. The crypts of the intestinal segment were scraped off, sieved, centrifuged, and then resuspended in 1% BSA. The resuspended small intestine was sieved and centrifuged again. Matrigel-Medium MIX was then added for resuscitation, and the small intestine was cultured in a multiwell plate in small intestinal organoid culture medium.

[0019] In a specific implementation, the isolation and pre-culture preparation of the small intestinal crypts in step 1) are carried out through the following steps:

[0020] (1-1) Eight-week-old SPF-grade C57 mice were anesthetized and sacrificed, and their small intestines were removed.

[0021] (1-2) Use a syringe with a gavage needle to draw up PBS, flush out the contents of the small intestine, and cut about 10 cm of the small intestine from the beginning of the duodenum;

[0022] (1-3) Longitudinally cut open the small intestine, cut the intestinal segment into about 3cm pieces, and gently scrape the villi with a coverslip to remove the attached material from the intestinal wall.

[0023] (1-4) Transfer the intestinal segment to the small intestinal villi digestion fluid, and place the centrifuge tube horizontally on ice;

[0024] (1-5) After digestion, the intestinal segment was transferred to PBS containing antibiotics, washed, and the waste liquid was discarded. This process was repeated until no obvious villi were shed from the solution.

[0025] (1-6) Transfer the intestinal segments to a culture dish rinsed with 1% BSA;

[0026] (1-7) Scrape off the crypts with a coverslip and transfer them through a 100μm sieve to a centrifuge tube rinsed with 1% BSA;

[0027] (1-8) Centrifuge the centrifuge tubes at 100×g, 5min, and 4℃.

[0028] (1-9) Remove the supernatant, add 10 mL of 1% BSA to resuspend, and transfer through a 70 μm sieve to a new 50 mL centrifuge tube;

[0029] (1-10) Centrifuge the centrifuge tubes at 70×g, 5min, and 4℃.

[0030] (1-11) Repeat steps (8)-(10) twice and then count the crypts. Then add Matrigel-Medium MIX to resuspend the crypts to 200 crypts / 50 μL.

[0031] (1-12) Add 50 μL of resuspended Matrigel-Medium MIX to the bottom of a 24-well plate preheated to 37°C, and then place the plate at 37°C for 5 min to observe the solidification of the matrix gel.

[0032] (1-13) Gently add small intestinal organoid culture medium from the edge of the culture plate.

[0033] In a specific embodiment, the Matrigel-Medium MIX is obtained by thoroughly mixing Matrigel and AdvancedDMEM / F12 in a 3:2 ratio.

[0034] In a specific embodiment, the small intestinal organoid culture medium is prepared as follows: 10 mM HEPES, 2 mM GlutaMAX, N2, B27, N-acetyl-l-cysteine, 50 ng / mL of ENR factor containing mouse EGF, 100 ng / mL of R-spondin, and 100 ng / mL of Noggin are added to Advanced DMEM / F12 culture medium.

[0035] In a specific implementation, in step 1), the small intestinal crypts are derived from mice; in step 3), the small intestinal eosinophils are derived from mice.

[0036] In a specific implementation, in step 2), the sample is centrifuged at 200×g for 5 min and washed twice with PBS buffer.

[0037] In a specific implementation, in step 3), the Matrigel-Medium MIX system containing 100,000 small intestinal eosinophils is used to resuspend the crypt system to 200-400 crypts / 50 μL.

[0038] In a specific implementation, in step 4), the resuspended crypt system obtained in step 3) is added at a rate of 50 μL per well to the bottom of a 24-well or 8-well plate preheated to 37°C within 5 seconds, and then the plate is placed in a 37°C incubator for 5 minutes until the matrix gel solidifies.

[0039] In a specific implementation, in step 5), 500 μL of small intestinal organoid culture medium is added from the edge of the culture plate, and the well plate is placed in a 37°C cell culture incubator for co-culture for 1 day to collect the samples.

[0040] In a specific embodiment, in step 5), the small intestinal organoid culture medium also contains NMU-23, and the concentration of NMU-23 can be 100 ng / mL.

[0041] Beneficial effects

[0042] This invention provides a more direct small intestinal epithelial-eosinophil interaction system to achieve the goal of intuitively detecting the effects of eosinophils on small intestinal epithelial cells.

[0043] This invention can also provide a more realistic eosinophil response function. By adding targeted stimulants to the system to activate eosinophils, the influence of the complex microenvironment of the small intestine can be avoided, the efficacy of the stimulants can be directly and accurately evaluated, and the development of new drugs can be accelerated.

[0044] In addition, the co-culture system of the present invention can create a personalized organoid and eosinophil co-culture system using a small number of patient intestinal epithelial cells, which is very important for developing personalized treatment plans and understanding individual differences in disease development in clinical practice. Attached Figure Description

[0045] Figure 1 The illustration shows a flowchart of the intestinal epithelial organoid-eosinophil co-culture method of this application.

[0046] Figure 2 The illustration shows the detection of organoid growth size (A) under the co-culture method of this application with a culture period of 3 days + co-culture of 1 day; and the detection of goblet cell density (B) under the culture period of 6 days + co-culture of 1 day. Detailed Implementation

[0047] The following detailed description of the operation process of the present invention through specific embodiments is intended to enable those skilled in the art to better understand the present invention. However, these embodiments are not intended to limit the scope of the present invention.

[0048] Experimental reagents:

[0049] PBS: Weigh 8g NaCl, 5.78g Na2HPO4·12H2O2, 0.24g KH2PO4 and 0.2g KCl powder using a weighing instrument and dissolve them in 800mL of deionized water. Adjust the pH of the solution to 7.4 using HCl or NaOH. Finally, bring the volume to 1L with deionized water at pH 7.4. After autoclaving, store at room temperature or 4°C.

[0050] 1% BSA: BSA was prepared to a concentration of 1% by weight in Advanced DMEM / F12 medium.

[0051] Small intestinal villus digestion fluid: Add penicillin / streptomycin and 200 μL EDTA (0.5 M) to 2 mL PBS.

[0052] Small intestinal organoid culture medium: Advanced DMEM / F12 medium supplemented with 10mM HEPES, 2mM GlutaMAX, N2, B27, N-acetyl-l-cysteine, ENR factors containing murine EGF (50ng / mL), R-spondin (100ng / mL), and Noggin (100ng / mL).

[0053] Preparation of Matrigel-Medium MIX: Simply mix Matrigel and AdvancedDMEM / F12 thoroughly at a ratio of 3:2 according to the required total liquid volume.

[0054]

[0055]

[0056] Example 1. Preparations for small intestinal crypt isolation and organoid culture

[0057] (1) Eight-week-old SPF-grade C57 mice were anesthetized and sacrificed, and their small intestines were removed.

[0058] (2) Use a 10mL syringe to attach an enema needle to draw up PBS (pre-cooled on ice for 1 hour beforehand), flush out the contents of the small intestine twice, and cut about 10cm of the small intestine from the beginning of the duodenum.

[0059] (3) Cut the small intestine longitudinally, cut the intestinal segment into about 3cm pieces, and use a coverslip to gently scrape the villi to remove the attached material from the intestinal wall.

[0060] (4) Transfer the intestinal segment to the small intestinal villus digestion solution and place the 50mL centrifuge tube horizontally on ice for 40-50 minutes (observe the degree of villus shedding according to the actual situation).

[0061] (5) After digestion, the intestinal segment was transferred to 20 mL of PBS containing antibiotics (penicillin-streptomycin mixture, diluted 100 times to 1 concentration), and washed 15 times. The waste liquid was discarded, and the process was repeated until no obvious villi were shed in the solution.

[0062] (6) Transfer the intestinal segment to a 10cm culture dish rinsed with 1% bovine serum albumin (BSA).

[0063] (7) Scrape off the crypts with a coverslip and transfer them through a 100μm sieve to a 50mL centrifuge tube rinsed with 1% BSA.

[0064] (8) Centrifuge the centrifuge tube at 100×g, 5min, 4℃.

[0065] (9) Remove the supernatant, add 10 mL of 1% BSA to resuspend, and transfer through a 70 μm sieve to a new 50 mL centrifuge tube.

[0066] (10) Centrifuge the centrifuge tube at 70×g, 5min, 4℃.

[0067] (11) Repeat steps (8)-(10) twice and then count the crypts. Then add Matrigel-Medium MIX to resuspend the crypts to 200 crypts / 50 μL.

[0068] (12) Add 50 μL of resuspended Matrigel-Medium MIX (containing 200 crypts / 50 μL) to the bottom of a 24-well plate preheated to 37°C. Then place the plate at 37°C for 5 min and observe the solidification of the matrix gel.

[0069] (13) Gently add 500 μL of small intestinal organoid culture medium per well from the edge of the culture plate.

[0070] Example 2: Culture of small intestinal organoids and co-culture with eosinophils (for detecting organoid growth size)

[0071] (2-1) Place the well plate from step (13) of Example 1 in a 37°C cell culture incubator and culture it until day 3. Then, aspirate the organoid culture medium from the well plate along the culture wells.

[0072] (2-2) Gently rinse the organoid matrix gel Matrigel with PBS pre-cooled to 4°C, transfer it to a 15mL centrifuge tube, and wash twice by centrifuging at 200×g for 5min.

[0073] (2-3) Resuspend the organoid system in Matrigel-Medium MIX (with or without 100,000 small intestinal eosinophils) to a 200-400 crypt / 50 μL system. Specifically, when resuspending the crypt system in Matrigel-Medium MIX containing 100,000 small intestinal eosinophils, add 50 μL of Matrigel-Medium MIX to eosinophils of 95% or higher purity isolated from the small intestine at a density of 100,000 cells per sample, and resuspend repeatedly by pipetting. Then, resuspend the crypts isolated from the small intestinal epithelium in Matrigel-Medium MIX containing small intestinal eosinophils.

[0074] (2-4) Quickly add 50 μL of the above-mentioned resuspended crypt system to the bottom of a 24-well or 8-well plate preheated to 37°C, and then place the plate at 37°C for 5 min to observe the solidification of the matrix gel.

[0075] (2-5) Gently add 500 μL of small intestinal organoid culture medium from the edge of the culture plate. Place the well plate in a 37°C cell culture incubator and co-culture for 1 day before collecting the samples.

[0076] Example 3: The operation is the same as in Example 2 above, except that the small intestinal organoid culture medium in step (2-5) contains 100 ng / mL of the target stimulant NMU-23.

[0077] Example 4: Culture of small intestinal organoids and co-culture with eosinophils (for detecting organoid differentiation)

[0078] (4-1) Place the well plate from step (13) of Example 1 in a 37°C cell culture incubator and culture until day 3. Aspirate the organoid culture medium from the well plate along the culture wells, place the contents in a centrifuge tube, add 10 mL of 1% BSA to resuspend, and replace the culture medium and matrix gel according to steps (10)-(13) of Example 1. Continue culturing until day 6, and aspirate the organoid culture medium from the well plate along the culture wells.

[0079] (4-2) The operation is as described in step (2-2) above.

[0080] (4-3) The operation is the same as the steps (2-3) described above.

[0081] (4-4) The operation is the same as the steps (2-4) described above.

[0082] (4-5) The operation is the same as the steps (2-5) described above.

[0083] Example 5: The operation is the same as in Example 4 above, except that the small intestinal organoid culture medium in step (4-5) contains 100 ng / mL of the target stimulant NMU-23.

[0084] Figure 1 The diagram illustrates a flowchart of the intestinal epithelial organoid-eosinophil co-culture method used in this application. First, small intestinal crypts are isolated from wild-type mice for organoid culture. After 3 or 6 days of culture, the organoids are cultured for another day with or without the addition of NmU-23 and small intestinal eosinophils, and samples are collected for analysis.

[0085] Figure 2 The growth of organoids and the differentiation of goblet cells are shown under four experimental conditions (including: the original culture condition control group (Medium), the culture colloid and culture medium with NMU (NMU-23), the culture colloid and culture medium with eosinophils (EOS), and the culture colloid and culture medium with both eosinophils and NMU (NMU-23+EOS)).

[0086] Figure 2 The left image (A) is a representative image obtained by laser confocal microscopy, and the right image is a statistical graph. Experimental results show that in experimental samples obtained after 1 day of co-culture following 3 days of organoid culture (such as the samples obtained in Examples 2 and 3 above), the area of ​​organoid growth was significantly increased in the eosinophil-only group compared to the control group and the NMU-only group. This indicates that eosinophils themselves release stimuli to accelerate organoid growth. Furthermore, the simultaneous addition of eosinophils and NMU further promotes organoid growth, the formation of organoid bud structures, and more complex organoid structures, suggesting that the NMU stimulant activates eosinophils, causing them to produce factors that more strongly regulate goblet cell growth.

[0087] Figure 2 The left image (B) is a representative image obtained by fluorescence microscopy, and the right image is a statistical graph. On day 6, the organoids in which eosinophils and NMU were added simultaneously to the colloid showed a higher goblet cell density after one day of culture compared to the group with only NMU added (as in the samples obtained in Examples 4 and 5 above). These results indicate that eosinophils stimulated by NMU not only stimulate organoid growth but also promote the differentiation of small intestinal goblet cells.

Claims

1. A method for co-culturing intestinal epithelial organoids and eosinophils, the method comprising the following steps: When the small intestinal crypts are cultured for 3 days, eosinophils are added and co-cultured for 1 day to detect the growth rate of organoids; or when the small intestinal crypts are cultured for 6 days, eosinophils are added and co-cultured for 1 day to detect the differentiation rate of small intestinal goblet cells.

2. The method according to claim 1, wherein, The method includes the following steps: 1) The isolated small intestinal crypts were gelled using organoid matrix gel and cultured in small intestinal organoid culture medium until day 3 or day 6. The small intestinal organoid culture medium in the well plate was then aspirated. 2) Dissolve the gel with PBS buffer, separate the organoids, and wash them; 3) Resuspend the organoids obtained in step 2) using a mixture of organoid-specific Matrigel containing small intestinal eosinophils and small intestinal organoid culture medium (Matrigel-Medium MIX); 4) The resuspension system obtained in step 3) is gelled using an organoid matrix gel; and 5) Add small intestinal organoid culture medium from the edge of the culture plate, place the plate in a cell culture incubator and co-culture for 1 day. Collect samples for analysis. In the case of detecting organoid growth rate by area or volume, the culture time in step 1) is 3 days; in the case of detecting the differentiation rate of small intestinal goblet cells, the culture time in step 1) is 6 days.

3. The method according to claim 2, wherein, In step 1), the isolation and pre-culture preparation of the small intestinal crypts are carried out through the following steps: Small intestine was removed from mice, longitudinally dissected, and the intestinal wall was cleaned. The small intestine was digested with intestinal villus digestion solution. The crypts of the intestinal segment were scraped off, sieved, centrifuged, and then resuspended in 1% BSA. The resuspended small intestine was sieved and centrifuged again. Matrigel-Medium MIX was then added for resuscitation, and the small intestine was cultured in a multiwell plate in small intestinal organoid culture medium.

4. The method according to claim 3, wherein, In step 1), the isolation and pre-culture preparation of the small intestinal crypts are carried out through the following steps: (1-1) Eight-week-old SPF-grade C57 mice were anesthetized and sacrificed, and their small intestines were removed. (1-2) Use a syringe with a gavage needle to draw up PBS, flush out the contents of the small intestine, and cut about 10 cm of the small intestine from the beginning of the duodenum; (1-3) Longitudinally cut open the small intestine, cut the intestinal segment into about 3cm pieces, and gently scrape the villi with a coverslip to remove the attached material from the intestinal wall. (1-4) Transfer the intestinal segment to the small intestinal villi digestion fluid, and place the centrifuge tube horizontally on ice; (1-5) After digestion, the intestinal segment was transferred to PBS containing antibiotics, washed, and the waste liquid was discarded. This process was repeated until no obvious villi were shed from the solution. (1-6) Transfer the intestinal segments to a culture dish rinsed with 1% BSA; (1-7) Scrape off the crypts with a coverslip and transfer them through a 100μm sieve to a centrifuge tube rinsed with 1% BSA; (1-8) Centrifuge the centrifuge tubes at 100×g, 5min, and 4℃. (1-9) Remove the supernatant, add 10 mL of 1% BSA to resuspend, and transfer through a 70 μm sieve to a new 50 mL centrifuge tube; (1-10) Centrifuge the centrifuge tubes at 70×g, 5min, and 4℃. (1-11) Repeat steps (8)-(10) twice and then count the crypts. Then add Matrigel-Medium MIX to resuspend the crypts to 200 crypts / 50 μL. (1-12) Add 50 μL of resuspended Matrigel-Medium MIX to the bottom of a 24-well plate preheated to 37°C, and then place the plate at 37°C for 5 min to observe the solidification of the matrix gel. (1-13) Gently add small intestinal organoid culture medium from the edge of the culture plate.

5. The method according to claim 2, wherein, The small intestinal organoid culture medium was prepared as follows: 10 mM HEPES, 2 mM GlutaMAX, N2, B27, N-acetyl-l-cysteine, 50 ng / mL of ENR factor containing mouse EGF, 100 ng / mL of R-spondin, and 100 ng / mL of Noggin were added to Advanced DMEM / F12 medium.

6. The method according to claim 2, wherein, In step 1), the small intestinal crypts are derived from mice; in step 3), the small intestinal eosinophils are derived from mice.

7. The method according to claim 2, wherein, In step 2), centrifuge at 200×g for 5 min and wash twice with PBS buffer.

8. The method according to claim 2, wherein, In step 3), the crypt system containing 100,000 small intestinal eosinophils is resuspended in 200-400 crypts / 50 μL.

9. The method according to claim 2, wherein, In step 4), the resuspended crypt system obtained in step 3) is added at a rate of 50 μL per well to the bottom of a 24-well or 8-well plate preheated to 37°C within 5 seconds. The plate is then placed in a 37°C incubator for 5 minutes until the matrix gel solidifies.

10. The method according to claim 2, wherein, In step 5), add 500 μL of small intestinal organoid culture medium from the edge of the culture plate, place the well plate in a 37°C cell culture incubator and co-culture for 1 day to collect the samples.

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