Application of capsaicin in intestinal organ culture and culture solution containing capsaicin

Abstract

The invention discloses an application of capsaicin in intestinal organ culture and a culture solution containing capsaicin, and belongs to the technical field of tissue culture. The intestinal organ culture solution comprises capsaicin with the concentration of 1.25-5 [mu] mol / L and a stem cell culture solution. When the culture solution is used in pig jejunum organ culture, generation and budding of jejunum crypt organs can be remarkably promoted, and cell growth and intestinal organ amplification are effectively promoted. According to the method for promoting amplification of the intestinal organs provided by the invention, many limitations of a traditional model can be overcome, and the development of intestinal pathology is assisted.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of tissue culture, and particularly relates to application of capsaicin in intestinal organoid culture and culture solution containing capsaicin. BACKGROUND

[0002] The intestine is an important part of the human digestive system, and undertakes important tasks such as food digestion, nutrient absorption and metabolism. Moreover, the intestine is not only a digestive organ of the human body, but also an important immune organ of the human body. When the intestine is damaged, it can cause various intestinal diseases, including inflammatory bowel disease, celiac disease, colorectal cancer and the like. Traditional in vitro intestinal models are mainly divided into three categories: animal models, two-dimensional intestinal cells or immortalized cell lines. Due to the defects of the models, the in vitro research of the intestine and the mechanism exploration of intestinal diseases cannot be further deepened. The emergence and development of intestinal organoids overcome many limitations of traditional models and help the development of intestinal pathology. Intestinal organoids are three-dimensional tissue analogues formed by crypts or single stem cells from the intestine in an in vitro culture system, and have self-renewal and differentiation capabilities. In 2009, Hans Clevers' team successfully formed a three-dimensional cell cluster with crypt-villus epithelial regions by in vitro culture of Lgr5 + -rich small intestinal stem cells, producing the first intestinal organoid. This has opened a new era of organoid construction and promoted the research of new in vitro intestinal models into a new stage of rapid development. At present, research on promoting the expansion of organoids has made some progress, but there are still many technical bottlenecks.

[0003] The separation methods currently used on organoids include EDTA method and collagenase method. The separation efficiency is higher by using the EDTA method, but the damage to the intestinal crypt is greater, and there are more impurities in the separation, and the activity of the crypt is poorer. The culture system of pig intestinal organoids mostly follows the mouse or human scheme, and the core cytokines are mainly human or mouse origin, lacking pig-specific recombinant proteins. Due to the difference in affinity of signal molecules between species (such as the reduced binding efficiency of pig Wnt receptor and human Wnt3a), it can lead to incomplete differentiation of organoids. SUMMARY

[0004] In view of the above prior art, the application provides application of capsaicin in intestinal organoid culture and culture solution containing capsaicin, which solves the problems of lack of regulation of intestinal organoid culture expansion technology and different sources of core cytokines between different species in the prior art.

[0005] In order to achieve the above purpose, the technical scheme adopted by the application is to provide application of capsaicin in intestinal organoid culture.

[0006] On the basis of the above technical scheme, the application can also be improved as follows.

[0007] Furthermore, capsaicin is used to promote the formation of crypt organoids and budding.

[0008] Furthermore, an intestinal organoid culture medium contains capsaicin.

[0009] Furthermore, the intestinal organoid culture medium includes capsaicin at a concentration of 1.25–5 mol / L and stem cell culture medium.

[0010] Furthermore, the concentration of capsaicin was 2.5 μmol / L.

[0011] Furthermore, the stem cell culture medium comprises the following components: 45% by volume of Advanced DMEM / F12, 45% by volume of WRN cell supernatant, 10% by volume of FBS, 1% by volume of N2 and 1% by volume of B27, 1 mmol / L of N-acetylcysteine, 10 μmol / L of SB202190 and 0.5 μmol / L of LY2157299.

[0012] Furthermore, the method for preparing intestinal organoid culture medium includes the following steps: (1) Preparation of high-concentration capsaicin stock solution: Dissolve capsaicin in PBS buffer preheated at 37°C and filter using a 0.22μm filter to obtain the solution; (2) Preparation of stem cell culture medium: Mix 45% Advanced DMEM / F12, 45% WRN cell supernatant and 10% fetal bovine serum evenly by volume. Then dissolve N2 and B27 in the mixture at a volume ratio of 1:100. Then add N-acetylcysteine, SB202190 and LY2157299, vortex and mix evenly. (3) Preparation of intestinal organoid culture medium: According to the required capsaicin concentration, add the high-concentration capsaicin stock solution to the stem cell culture medium in proportion to obtain the culture medium.

[0013] Furthermore, a method for culturing intestinal organoids involves culturing crypts using the aforementioned intestinal organoid culture medium.

[0014] Furthermore, the method for culturing intestinal organoids includes the following steps: mixing crypts with matrix gel on ice, dropping the mixture into a culture plate, adding preheated intestinal organoid culture medium after the matrix gel has solidified, and culturing at 37°C and 5% CO2, replacing the culture medium with fresh medium every 2 days.

[0015] Furthermore, the crypts originate from the jejunal tissue of piglets.

[0016] The beneficial effects of this invention are: the capsaicin-containing culture medium provided by this invention can significantly improve the viability of porcine intestinal epithelial cells (IPEC-J2) and the generation and budding efficiency of intestinal organoids in piglets. The scheme provided by this invention can promote the expansion and differentiation of intestinal organoids, solving the problem of different sources of core cytokines among different species during organoid culture, and has great application potential in actual production. The method for promoting the expansion of intestinal organoids provided by this invention helps overcome many limitations of traditional models and contributes to the development of intestinal pathology. Attached Figure Description

[0017] Figure 1 To determine the optimal concentration of capsaicin for promoting IPEC-J2 cell growth using the MTT assay; Figure 2 The effect of different concentrations of capsaicin on the growth of jejunal organoids in piglets was investigated. Among them, A represents the growth of organoids in each group (magnification 40×), B represents the forming efficiency, C represents the budding efficiency, and D represents the branching coefficient (n=3). Detailed Implementation

[0018] The specific embodiments of the present invention will be described in detail below with reference to examples.

[0019] Example 1 Determine the optimal dosage of capsaicin. Porcine intestinal epithelial cells (IPEC-J2) in the logarithmic growth phase were digested, centrifuged, and counted. 2000–3000 cells were seeded into each well of three 96-well plates and cultured at 37°C and 5% CO2 for 24 h. Each 96-well plate was then divided into four treatment groups: a control group, a 1.25 μmol / L capsaicin-added group, a 2.5 μmol / L capsaicin-added group, and a 5 μmol / L capsaicin-added group, with 10 wells in each group. Complete culture medium and complete culture medium containing 1.25 μmol / L, 2.5 μmol / L, and 5 μmol / L capsaicin were added, respectively (the culture medium formulations for each treatment group are shown in Table 1). Cells were cultured at 37°C and 5% CO2, and cell viability was assessed using the MTT assay at 0 h, 24 h, and 48 h.

[0020] Table 1. Composition of the culture medium in each group in Example 1

[0021] The results are as follows Figure 1As shown, the addition of capsaicin can promote the growth of IPEC-J2 cells, and the culture medium containing 2.50 μmol / L capsaicin has a significant promoting effect.

[0022] Example 2 Effects of capsaicin on intestinal organoids in piglets (1) Isolation of intestinal crypts in piglets Collect 10 cm of fresh piglet jejunum tissue and place it in a culture dish containing pre-chilled DPBS buffer. After removing the mesentery, longitudinally dissect the intestine to expose the mucosal layer. Then, repeatedly rinse the inside of the intestine with pre-prepared, chilled DPBS buffer. Cut the cleaned intestinal fragments into small pieces, add pre-chilled DPBS buffer, and shake for 5 min. Repeat the DPBS buffer replacement until the supernatant is clear. Transfer the tissue fragments to 50 mL centrifuge tubes and add pre-chilled crypt separation buffer (a buffer containing 2 mM EDTA prepared with DPBS buffer). Place the centrifuge tubes in an icebox on a shaker (120 rpm / min). After 30 min, remove the centrifuge tubes from the icebox, discard the separation buffer, and add approximately 15 mL of pre-chilled DPBS buffer to the centrifuge tubes containing the tissue fragments. After vigorously shaking the centrifuge tube for 3 minutes, let it stand for a while, then pour it into a culture dish and observe the crypt separation under a microscope. If a large number of clean and structurally intact crypts appear in the field of view, the crypt separation is successful. Otherwise, transfer the tissue fragments to a new centrifuge tube, add pre-chilled DPBS buffer, and shake again until the crypts are successfully separated. Transfer the crypt-rich supernatant to a new 50 mL pre-chilled centrifuge tube and incubate on ice for 10 minutes to allow the crypts to accumulate at the bottom of the tube.

[0023] (2) Organoid culture and expansion The isolated crypts were mixed with matrix gel on ice and dropped into culture plates to form droplets. After the droplets solidified in an incubator for 20 min, preheated intestinal organoid culture medium was added. Intestinal organoids were then cultured in vitro at 37°C and 5% CO2, with the culture medium replaced every two days. The formulations of the intestinal organoid culture medium are shown in Table 2, containing concentrations of 0, 1.25, 2.5, and 5 μmol / L capsaicin, respectively. Intestinal organoids were photographed and observed every 24 h to detect formation rate, budding rate, and number of buds (formation rate: the percentage of organoids that became round on day 2 out of the number of organoids inoculated on day 1; budding rate: the percentage of organoids that sprouted on day 5 out of the number of organoids that became round on day 2; number of buds: the number of organoids that sprouted in each field of view; these three indicators reflect the growth advantage of the organoids).

[0024] The specific preparation methods for the intestinal organoid culture medium in each treatment group are as follows: 1) Preparation of high-concentration capsaicin stock solution: Dissolve capsaicin in PBS buffer preheated at 37°C, mix well, filter through a 0.22 μm filter, and transfer to centrifuge tubes to obtain the stock solution; 2) Preparation of stem cell culture medium: Mix 45% Advanced DMEM / F12, 45% WRN cell supernatant and 10% fetal bovine serum (FBS) by volume. Then dissolve N2 and B27 in the mixture at a volume ratio of 1:100. Add N-acetylcysteine ​​(1 mmol / L), SB202190 (10 μmol / L) and LY2157299 (0.5 μmol / L), and vortex to mix thoroughly. 3) Prepare stem cell culture medium containing capsaicin: Add the high-concentration capsaicin stock solution to the stem cell culture medium according to the required capsaicin concentration (1.25, 2.5 or 5 μmol / L).

[0025] Table 2. Composition of the culture medium in each group in Example 2

[0026] Depend on Figure 2 It can be seen that the crypt cells become rounded on day 1 and fully sprout on day 4. Compared with the control group, the addition of capsaicin can improve the jejunal crypt organoid generation efficiency and sprouting efficiency. Among them, the 2.5 μmol / L capsaicin group showed significantly improved jejunal crypt organoid generation efficiency and sprouting efficiency, indicating that capsaicin can effectively promote cell growth and intestinal organoid expansion.

[0027] Although specific embodiments of the present invention have been described in detail with reference to examples, they should not be construed as limiting the scope of protection of this patent. Various modifications and variations that can be made by those skilled in the art without inventive effort within the scope described in the claims are still within the scope of protection of this patent.

Claims

1. Use of capsaicin in the culture of intestinal organoids.

2. The use of capsaicin in the culture of intestinal organoids according to claim 1, characterized in that: The capsaicin is used to promote crypt organoid generation and outgrowth.

3. An enteroid culture solution, characterized by: The capsaicin is contained.

4. The intestinal organoid culture of claim 3, wherein, The capsaicin is contained at a concentration of 1.25-5 μmol / L and the stem cell culture solution.

5. The intestinal organoid culture of claim 4, wherein: The concentration of the capsaicin is 2.5 μmol / L.

6. The intestinal organoid culture of claim 5, wherein, The stem cell culture solution comprises the following components: 45% (v / v) Advanced DMEM / F12, 45% WRN cell supernatant, 10% FBS, 1% N2 and 1% B27, 1 mmol / L N-acetyl cysteine, 10 μmol / L SB202190 and 0.5 μmol / L LY2157299.

7. The intestinal organoid culture of claim 6, wherein, The preparation method of the intestinal organoid culture solution comprises the following steps: (1) Preparation of high-concentration capsaicin stock solution: dissolve capsaicin in 37°C preheated PBS buffer, filter with a 0.22 μm filter, and obtain the stock solution; (2) Preparation of stem cell culture solution: mix 45% Advanced DMEM / F12, 45% WRN cell supernatant and 10% fetal bovine serum in a volume ratio, then dissolve N2 and B27 in the mixture at a volume ratio of 1:100, and then add N-acetyl cysteine, SB202190 and LY2157299, vortex and mix well; (3) Preparation of intestinal organoid culture solution: add the high-concentration capsaicin stock solution to the stem cell culture solution in a proportion according to the required capsaicin concentration, and obtain the intestinal organoid culture solution.

8. A method of culturing an intestinal organoid, characterized by: The intestinal organoid culture solution of any one of claims 3-7 is used to culture crypts.

9. The culture method of intestinal organoids according to claim 8, characterized in that, The method comprises the following steps: Mix the crypts with Matrigel on ice, drop into the culture plate, and add preheated intestinal organoid culture solution after the Matrigel solidifies, and culture at 37°C, 5% CO2, and replace the fresh culture solution every 2 days.

10. The method of culturing an intestinal organoid according to claim 8, wherein: The crypts are derived from the jejunum tissue of piglets.

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