Separation culture method and application of mouse intestinal fibroblasts

Abstract

The invention discloses an isolated culture method for mouse intestinal fibroblasts. According to the method, the intestinal tissue of an euthanasia mouse is used as a raw material, tissue pretreatment, compound enzyme digestion, filtration and centrifugation, adherent culture, purification and amplification and other in-vitro operations are performed in sequence, and efficient separation and culture of intestinal fibroblasts are realized by optimizing culture medium components and adding a TGF-beta inhibitor and a fibroblast growth factor. The prepared intestinal fibroblasts are high in purity (the proportion is larger than or equal to 99%), differentiation of the intestinal fibroblasts to CD146 + myofibroblasts (the proportion is smaller than 10%) can be effectively inhibited, CD34 + CD81 + fibroblast functional subgroups (the proportion is larger than or equal to 60%) are remarkably enriched, reliable cell resources are provided for research of intestinal disease mechanisms and development of related treatment technologies, and the application prospects are wide.

Description

Technical Field This invention relates to the field of cell culture technology, specifically to a method for isolating and culturing mouse intestinal fibroblasts and its application. Background Technology

[0001] The gut is a vital digestive and mucosal immune organ in the human body. Disruptions to its homeostasis are directly related to the development of diseases such as inflammatory bowel disease (IBD) and colon cancer. The interactions between intestinal fibroblasts, epithelial cells, immune cells, and gut microbiota not only regulate local intestinal immune responses but also play a crucial role in the formation, response, and maintenance of immune homeostasis. For a long time, due to the lack of subset-specific research tools, intestinal stromal cells have been considered a relatively simple type of functional cell lacking heterogeneity.

[0002] Recent single-cell sequencing studies have revealed highly complex heterogeneity in intestinal stromal cells. Researchers have identified several functionally specific and differentiated subpopulations, such as FOXL1. + Tro cells, small intestinal CD81 + Trophoblasts, colon CD34 + CD81 + Discovery of cells (MRISC). Among them, colonic CD34... + CD81 + Intestinal stromal cells (MRISCs) are a new subpopulation of special mesenchymal cells located near intestinal stem cells. They can sense damage-induced reactive oxygen species signals, increase Wnt signaling in intestinal stem cells, and enhance the damage repair function of the intestine. They are expected to become a new target cell for the treatment of inflammatory bowel disease.

[0003] Primary culture systems for intestinal fibroblasts are crucial for studying these fibroblast subsets. However, current in vitro isolation and culture systems have not kept pace with advancements in fibroblast subpopulation research, and still commonly employ a simple adherent culture method using DMEM medium containing 15% fetal bovine serum after enzymatic digestion. This method has the following limitations: for CD34... + CD81 + The enrichment efficiency of fibroblast subsets is low, and CD45 is easily contaminated in the culture system. + Immune cells, CD31 + Endothelial cells and CD326 + Non-target cells such as epithelial cells. Furthermore, fetal bovine serum in the culture medium easily leads to over-activation of fibroblasts, causing them to differentiate into CD146. + Myofibroblasts affect the reliability and accuracy of subsequent functional experiments. Summary of the Invention

[0004] To address the issues of low fibroblast purity, easy overactivation, and CD34 contamination in existing in vitro intestinal fibroblast isolation and culture systems, + CD81 + To address the technical problem of insufficient subpopulation enrichment, this invention provides a simple, efficient, and effective method for isolating and culturing mouse intestinal fibroblasts that also achieves high cell viability and good target subpopulation enrichment. This invention provides a method for isolating and culturing mouse intestinal fibroblasts, using in vitro intestinal tissue from euthanized mice as raw material, and achieving fibroblast isolation and culture through in vitro processes, including the following steps: Step 1: Tissue pretreatment: The mouse intestinal tissue was placed in a tissue pretreatment solution for pretreatment. Step 2, Enzymatic Digestion: The pretreated intestinal tissue is transferred to a tissue digester containing collagenase and deoxyribonuclease to obtain a single-cell suspension; Step 3, Cell Adherence Culture: After filtration and centrifugation, the single-cell suspension was inoculated into the optimized fibroblast culture medium for adherence culture for 24 hours; Step 4: Cell purification and expansion: Use cell washing solution to remove non-adherent cells, and after washing, add optimized fibroblast culture medium containing TGF-β inhibitor and fibroblast growth factor to purify and expand the cells to obtain the target intestinal fibroblasts.

[0005] In a preferred embodiment of the mouse intestinal fibroblast isolation and culture method provided by the present invention, the tissue pretreatment solution in step one includes: HBSS solution or PBS solution with 1%~5% fetal bovine serum, 1~5mM ethylenediaminetetraacetic acid and 0.5~2mM dithiothreitol added.

[0006] In a preferred embodiment of the mouse intestinal fibroblast isolation and culture method provided by the present invention, the pretreatment operation in step one is as follows: the cleaned intestinal tissue is placed in the tissue pretreatment solution and pretreated at 37°C for 20 min. After the pretreatment is completed, the tissue is vigorously shaken and cleaned for 1 min. The pretreatment operation is repeated once.

[0007] In a preferred embodiment of the method for isolating and culturing mouse intestinal fibroblasts provided by the present invention, the tissue digestion solution in step two comprises: a DMEM solution or RPMI 1640 solution containing 5%–20% fetal bovine serum, 10–100 U / ml penicillin, 10–100 μg / ml streptomycin, 100–1000 μg / mL collagenase, and 50–500 μg / mL deoxyribonuclease; wherein the collagenase is one or both of collagenase IV and collagenase VIII, and the deoxyribonuclease is one or both of deoxyribonuclease I and deoxyribonuclease II.

[0008] In a preferred embodiment of the mouse intestinal fibroblast isolation and culture method provided by the present invention, the enzymatic digestion in step two specifically involves: cutting the pretreated intestinal tissue into approximately 0.5 cm segments, transferring them to a tissue digestion solution, digesting them at 37°C and 5% CO2 for 40 minutes, and then vigorously shaking them until no obvious tissue fragments remain.

[0009] In a preferred embodiment of the mouse intestinal fibroblast isolation and culture method provided by the present invention, the cell adherence culture in step three specifically involves: filtering the single-cell suspension from step two through a cell filter, collecting the filtrate, centrifuging, discarding the supernatant, resuspending the cells in DMEM basal medium, centrifuging again, discarding the supernatant, adding optimized fibroblast culture medium to resuspend the cells, seeding them into culture dishes, and adhering to the culture medium at 37°C and 5% CO2 for 24 hours; the pore size of the cell filter is 70 μm or 100 μm; the centrifugation conditions are 4°C and 1200~2000 rpm for 2~10 min.

[0010] In a preferred embodiment of the mouse intestinal fibroblast isolation and culture method provided by the present invention, the optimized fibroblast culture medium in steps three and four includes: DMEM solution or RPMI 1640 solution supplemented with 5%~20% fetal bovine serum, 10~100U / mL penicillin, 10~100μg / mL streptomycin, 2~10μg / mL gentamicin and 2~10mM HEPES.

[0011] In a preferred embodiment of the method for isolating and culturing mouse intestinal fibroblasts provided by the present invention, the TGF-β inhibitor in step four is one or more of A77-01, A83-01, and ALK5-IN-10, with a working concentration of 100~2000nM; the fibroblast growth factor is one or more of FGF-1, FGF-2, and FGF-4, with a working concentration of 10~200ng / mL.

[0012] This invention also provides intestinal fibroblasts prepared using any of the isolation and culture methods described above, which, as identified by flow cytometry, have a fibroblast purity ≥99% and CD146 content. + Myofibroblast percentage ≤10%, CD34 + CD81 + The proportion of fibroblast subsets is ≥60%.

[0013] The present invention also provides an application of the above-mentioned intestinal fibroblasts in the screening of therapeutic targets for inflammatory bowel disease and in the study of intestinal damage repair mechanisms.

[0014] Compared with existing technologies, the mouse intestinal fibroblast isolation and culture method provided by this invention has the following beneficial effects: I. High cell purity: Flow cytometry analysis showed that the proportion of fibroblasts was ≥99%, and CD45 content was high. + Immune cells, CD31 + Endothelial cells and CD326 + The proportion of non-target cells, such as epithelial cells, is less than 1%, which is significantly better than existing technologies. II. Subgroup enrichment: CD34 + CD81 + The proportion of functional subgroups has increased to over 60%, providing a sufficient cell source for functional studies of this subgroup.

[0015] 3. Excellent cell viability: Through gentle digestion by compound enzymes and combined with optimized culture medium, cell damage is reduced, excessive activation of fibroblasts is inhibited, and the cell growth status is stable, which can meet the needs of subsequent experiments.

[0016] IV. Effective isolation and culture of target cells through in vitro processes: The pretreatment optimization step uses a pretreatment solution containing EDTA and dithiothreitol to effectively remove most epithelial cells and reduce subsequent non-target cell contamination; the complex enzyme digestion step uses a digestion system combining collagenase and deoxyribonuclease to gently dissociate tissue blocks, reduce cell damage, and improve the quality of single-cell suspensions; the purification strategy removes non-adherent cells after 24 hours of adherent culture to further improve cell purity; the culture medium optimization adds gentamicin and HEPES to the basal medium to enhance antibacterial effects and maintain a stable culture environment; subsequently, TGF-β inhibitors are added to inhibit differentiation into myofibroblasts, and fibroblast growth factors are added to promote target cell proliferation. Attached Figure Description

[0017] Figure 1 This is a comparison diagram of the morphology of fibroblasts obtained by the original method and the optimized method of the present invention.

[0018] Figure 2 This is a flow cytometry comparison of the purity of fibroblasts obtained by the original method and the optimized method of this invention.

[0019] Figure 3 The original method and the optimized method of this invention are compared for CD146. + Comparative graphs of flow cytometry findings showing the influence of myofibroblast differentiation ratio on the overall cell differentiation rate.

[0020] Figure 4 The original method and the optimized method of this invention are compared for CD34. + Comparative graphs of flow cytometry findings showing the effect of fibroblast ratio on the detection of fibroblast proportion.

[0021] Figure 5 The original method and the optimized method of this invention are compared to CD81. +Comparative graphs of flow cytometry findings showing the effect of fibroblast ratio on the detection of fibroblast proportion. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0023] Example 1: Method for isolating and culturing mouse intestinal fibroblasts This embodiment uses the isolated colon tissue of euthanized mice as raw material, and achieves the isolation and culture of target cells through an in vitro process involving steps such as "pretreatment-enzyme digestion-cell adhesion culture-cell purification and amplification".

[0024] First, obtain isolated colon tissue from mice. 1) Tissue acquisition: The mice were euthanized, the abdominal cavity was opened, and the entire colon (isolated colon tissue) was removed. The mesentery and adipose tissue were separated, and the tissue was placed in a culture dish containing pre-cooled PBS solution. 2) Tissue washing: The colon lumen was thoroughly rinsed with PBS solution to remove feces. After being cut along the longitudinal axis, the tissue was transferred to a 50 mL centrifuge tube containing PBS solution and washed vigorously for 1 min.

[0025] Next, mouse intestinal fibroblasts were isolated and cultured, specifically including the following steps: S1. Tissue pretreatment: Mouse intestinal tissue was placed in a tissue pretreatment solution for pretreatment.

[0026] Specifically, the cleansed colon was transferred to a 50 mL centrifuge tube containing tissue pretreatment solution (HBSS solution supplemented with 2% fetal bovine serum, 3 mM EDTA, and 1 mM dithiothreitol), and pretreated at 37°C for 20 min. After pretreatment, the colon was vigorously shaken and washed for 1 min, and this step was repeated once. For the second washing, the colon was transferred to a 15 mL centrifuge tube containing PBS solution and gently inverted and washed for 1 min. The EDTA in the tissue pretreatment solution disrupts the junctions between epithelial cells, and the dithiothreitol dissociates the mucus layer, both reducing subsequent non-target cell contamination and laying the foundation for improved cell purity.

[0027] S2. Enzymatic digestion: The pretreated intestinal tissue is transferred to a tissue digestion solution containing collagenase and deoxyribonuclease to obtain a single-cell suspension.

[0028] Specifically, intestinal tissue was transferred to a 6cm culture dish and cut into segments of approximately 0.5cm. These segments were then transferred to 6-well plates, and 4mL of tissue digestion solution (DMEM solution containing 10% fetal bovine serum, 50U / ml penicillin, 50μg / ml streptomycin, 500μg / mL collagenase VIII, and 200μg / mL deoxyribonuclease I) was added. The plates were then incubated at 37°C with 5% CO2 for 40 minutes, followed by vigorous shaking until no obvious tissue fragments remained. The combination of collagenase VIII and deoxyribonuclease I gently dissociates intestinal tissue fibers and degrades nucleic acids released from cells, preventing cell aggregation and enhancing the activity of single-cell suspensions.

[0029] S3. Cell adherence culture: After filtration and centrifugation, the single-cell suspension is inoculated into the optimized fibroblast culture medium for adherence culture.

[0030] Specifically, the digestion solution was transferred to a 15 mL centrifuge tube containing 4 mL of DMEM basal medium, shaken vigorously for 30 seconds, filtered through a cell filter into a 50 mL centrifuge tube, centrifuged at 4°C and 1500 rpm for 5 min, and the supernatant was discarded; 5 mL of DMEM basal medium was added to resuspend the solution, and centrifuged again under the same conditions. Adherent culture: Discard the supernatant, add 5 mL of optimized culture medium (DMEM solution with 15% fetal bovine serum, 50 U / mL penicillin, 50 μg / mL streptomycin, 5 μg / mL gentamicin and 5 mM HEPES) to resuspend the cells, transfer to a 6 cm culture dish, and incubate at 37°C in a 5% CO2 incubator for 24 h.

[0031] S4. Cell purification and expansion: Non-adherent cells are removed using cell washing solution. After washing, an optimized fibroblast culture medium containing TGF-β inhibitor and fibroblast growth factor is added to purify and expand the cells, obtaining the target intestinal fibroblasts.

[0032] Specifically, the culture supernatant was discarded, and PBS solution was added to gently remove non-adherent cells. After washing twice, an optimized culture medium containing 800 nM A83-01 (TGF-β inhibitor) and 80 ng / mL FGF-2 (fibroblast growth factor) was added, and the cells were cultured for another 5 days until the cell density reached about 90%, thus obtaining the target fibroblasts.

[0033] This embodiment uses two different culture systems, A and B, to culture fibroblasts and study the effects of different culture systems on the morphology and purity of fibroblasts: A. Existing fibroblast culture system: DMEM basal medium supplemented with 15% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, 10 μg / mL gentamicin and 10 mM HEPES.

[0034] B. Optimized fibroblast culture system: Based on DMEM basal medium (containing 15% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, 10 μg / mL gentamicin and 10 mM HEPES), 500 nM TGF-β inhibitor and 100 ng / mL fibroblast growth factor were added.

[0035] 1) Effects of optimized culture methods on fibroblast morphology and purity After 6 days of cultivation, it can be observed under an inverted microscope ( Figure 1 ): Existing cultivation system ( Figure 1 A) and the optimized culture system ( Figure 1 B) Both methods can effectively obtain spindle-shaped fibroblasts, and no significant difference was observed in their overall morphology.

[0036] Further flow cytometry analysis showed that ( Figure 2 Cells obtained from the original culture system contain a certain proportion of CD45. + Immune cells, CD31 + Endothelial cells or CD326 + Epithelial cells and other cell types ( Figure 2 A); and the proportion of the aforementioned non-target cells in the cells obtained after the optimized culture system was extremely low ( Figure 2 B). The results showed that the optimized culture system could significantly improve the purity of mouse intestinal fibroblasts.

[0037] 2) Optimized culture method for CD146 + Effect of mouse intestinal myofibroblast ratio Using the two fibroblast culture systems A and B described above, their effects on CD146 were studied. + Effects on myofibroblast formation. Flow cytometry results showed that in cells obtained from the original culture system, CD146... + The proportion of myofibroblasts was 17.5% ( Figure 3 A); however, in the optimized culture system, it decreased to 9.78% ( Figure 3 B). This indicates that the optimized culture system can significantly reduce the activation level of mouse intestinal fibroblasts and inhibit their differentiation into myofibroblasts.

[0038] 3) Optimized culture method for CD34 + CD81 + Effect of mouse intestinal fibroblast subset ratio Using the two fibroblast culture systems A and B described above, their effects on CD34 were evaluated. + CD81 +The effect of different cell subsets on the proportion of mouse intestinal fibroblasts. Flow cytometry analysis showed that under both culture systems, CD34... + The cell ratio was close to 100% ( Figure 4 , Figure 4 A represents the original system. Figure 4 B represents the optimized system. And in CD81... + Regarding the proportion of fibroblasts, the original culture system had 39.5% ( Figure 5 A), the optimized culture system increased the yield to 62.1% ( Figure 5 B) indicates that the optimized culture method can significantly enrich CD34. + CD81 + Mouse intestinal fibroblasts.

[0039] In summary, this invention employs a compound enzyme digestion method to gently dissociate mouse intestinal tissue into a single-cell suspension, resulting in minimal cell damage. Combined with an optimized culture medium and direct adherent culture, fibroblasts with higher purity can be obtained. Flow cytometry analysis of cell surface markers shows that the optimized culture system reduces the differentiation rate of fibroblasts into myofibroblasts and significantly enriches CD34. + CD81 + Mouse intestinal fibroblasts. Therefore, this method is particularly suitable for mouse CD34. + CD81 + Primary culture of intestinal fibroblasts; cultured intestinal fibroblasts have wide application value in screening therapeutic targets for inflammatory bowel disease and in studying intestinal damage repair mechanisms.

[0040] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent process transformations made using the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for isolating and culturing mouse intestinal fibroblasts, characterized in that, Using euthanized mouse intestinal tissue as raw material, fibroblasts were isolated and cultured via in vitro processes, including the following steps: Step 1: Tissue pretreatment: The mouse intestinal tissue was placed in a tissue pretreatment solution for pretreatment. Step 2, Enzymatic Digestion: The pretreated intestinal tissue is transferred to a tissue digestion solution containing collagenase and deoxyribonuclease to obtain a single-cell suspension; Step 3, Cell Adherence Culture: After filtration and centrifugation, the single-cell suspension was inoculated into the optimized fibroblast culture medium for adherence culture for 24 hours; Step 4: Cell purification and expansion: Use cell washing solution to remove non-adherent cells, and after washing, add optimized fibroblast culture medium containing TGF-β inhibitor and fibroblast growth factor to purify and expand the cells to obtain the target intestinal fibroblasts.

2. The isolation and culture method according to claim 1, characterized in that, The tissue pretreatment solution in step one includes: an HBSS solution or PBS solution containing 1%~5% fetal bovine serum, 1~5mM ethylenediaminetetraacetic acid and 0.5~2mM dithiothreitol.

3. The isolation and culture method according to claim 2, characterized in that, The pretreatment operation in step one is as follows: the cleaned intestinal tissue is placed in the tissue pretreatment solution and pretreated at 37°C for 20 minutes. After the pretreatment is completed, the tissue is vigorously shaken and cleaned for 1 minute. This pretreatment operation is repeated once.

4. The isolation and culture method according to claim 1, characterized in that, The tissue digestion solution in step two includes: a DMEM solution or RPMI 1640 solution containing 5%–20% fetal bovine serum, 10–100 U / ml penicillin, 10–100 μg / ml streptomycin, 100–1000 μg / mL collagenase, and 50–500 μg / mL deoxyribonuclease; the collagenase is one or both of collagenase IV and collagenase VIII, and the deoxyribonuclease is one or both of deoxyribonuclease I and deoxyribonuclease II.

5. The isolation and culture method according to claim 4, characterized in that, The enzymatic digestion in step two is as follows: the pretreated intestinal tissue is cut into pieces of about 0.5 cm / segment, transferred to a tissue digestion solution, and digested at 37°C and 5% CO2 for 40 minutes. After digestion, the tissue is shaken vigorously until there are no obvious tissue pieces.

6. The isolation and culture method according to claim 1, characterized in that, The cell adherence culture in step three is specifically as follows: the single-cell suspension from step two is filtered through a cell filter, the filtrate is collected, centrifuged, the supernatant is discarded, the cells are resuspended in DMEM basal medium, centrifuged again, and the supernatant is discarded. The cells are then resuspended in optimized fibroblast culture medium, seeded into culture dishes, and cultured at 37℃ and 5% CO2 for 24 hours. The pore size of the cell filter is 70μm or 100μm. The centrifugation conditions are 4℃ and 1200~2000rpm for 2~10min.

7. The isolation and culture method according to claim 1, characterized in that, The optimized fibroblast culture medium in steps three and four includes: DMEM solution or RPMI 1640 solution supplemented with 5%~20% fetal bovine serum, 10~100 U / mL penicillin, 10~100 μg / mL streptomycin, 2~10 μg / mL gentamicin and 2~10 mM HEPES.

8. The isolation and culture method according to claim 1, characterized in that, In step four, the TGF-β inhibitor is one or more of A77-01, A83-01, and ALK5-IN-10, with a working concentration of 100~2000nM; the fibroblast growth factor is one or more of FGF-1, FGF-2, and FGF-4, with a working concentration of 10~200ng / mL.

9. Intestinal fibroblasts prepared by the isolation and culture method according to any one of claims 1-8, characterized in that, Flow cytometry analysis showed that the fibroblasts had a purity of ≥99% and contained CD146. + Myofibroblast percentage ≤10%, CD34 + CD81 + The proportion of fibroblast subsets is ≥60%.

10. The application of intestinal fibroblasts as described in claim 9 in the screening of therapeutic targets for inflammatory bowel disease and the study of intestinal damage repair mechanisms.

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