Primary culture method of mesenteric interstitial cells
Through the combined technology of mesothelial cell dissociation solution, EDTA solution, collagenase digestion solution and Ficoll/Percoll density gradient centrifugation, the problem of interfering mesothelial cell intravenous mesenchymal cell culture and cell adherence growth is solved, and efficient cell expansion and purity improvement is achieved.
Patent Information
- Application Number
- CN202411652275.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-19
- Publication Date
- 2025-03-28
AI Technical Summary
In the prior art, the primary culture method of mesenteric mesenchymal cells has the mixture of mesenchymal cells and endothelial cells, resulting in low cell expansion efficiency and long culture cycle, and the mesenteric membrane is rich in connective tissue, resulting in disturbed cell adherence growth.
Mesothelial cells were removed by using mesothelial cell dissociation solution, combined with EDTA solution and digestion solution containing type II collagenase and neutral protease for cell isolation and purification, and Ficoll/Percoll density gradient centrifugation was used to remove micro-tissue fragments, and finally itaconic acid derivatives were added to the amplification medium to promote cell expansion.
It effectively avoids the inclusion of mesothelial cells and endothelial cells, promotes the early adherence growth of mesenteric mesenchymal cells, improves cell amplification rate and purity, shortens the culture cycle, and delays the cellular aging process.
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Figure CN119709592A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of cell culture, and more particularly to a primary culture method of mesenteric stromal cells. Background Art
[0002] The mesentery is a double-layer peritoneal structure that fixes the intestine to the posterior abdominal wall. During development, multiple regions degenerate and then manifest as independent mesenteric regions. Traditional theory holds that the main function of the mesentery is to provide physical support and blood supply to the intestine. Recently, with the re-understanding of the anatomical structure and functional execution of the mesentery, more and more evidence has shown that the mesentery is an organ that is not only an incubator for all digestive organs in the abdomen during the embryonic period, but also provides functional support for tissues and organs after development. For example, mesenteric intrinsic cells can produce a large number of growth factors such as adiponectin, resistin, and lipophilin. Studies have shown that tissues and organs transplanted into the mesentery can maintain their vitality and function. The mesentery is also a common site for metastasis of abdominal tumors such as colorectal cancer, pancreatic cancer, and ovarian cancer. There is evidence that mesenteric intrinsic cells can provide a suitable niche environment for tumor cell implantation. Studies from colorectal cancer have also shown that surgical removal of the entire mesentery can significantly reduce the recurrence rate of tumors. Therefore, exploring the biological characteristics of mesenteric intrinsic cells can not only more clearly elucidate the role of the mesentery in the regeneration of abdominal digestive organs, but also provide more clues for the study of the mechanisms of mesenteric diseases and abdominal tumor invasion and metastasis.
[0003] Stromal cells are the main intrinsic cells in the mesenteric tissue, located in the submesothelial connective tissue and adipose connective diaphragm, and are a type of non-hematopoietic, non-epithelial, and non-endothelial mesenchymal cells. Cultivating mesenteric stromal cells is an important cytological basis for exploring the biological functions of the mesentery. However, in the prior art, there are few reports on primary culture of mesenteric stromal cells. If traditional culture methods, such as tissue block adhesion method, are used, it takes a lot of time to wait for cells to slowly crawl out of the tissue block, and there are mesothelial cells and mesothelial cells transformed from stromal cells. At the same time, although the conventional enzyme digestion method greatly shortens the culture time, there are many types of cells obtained by digestion. In order to obtain specific types of cells, cell purification is required; in addition, since the mesentery is rich in connective tissue, the connective debris produced by enzyme digestion will also interfere with cell adhesion and affect the expansion of primary cells.
[0004] Therefore, the present invention is directed to a primary culture method of mesenteric stromal cells to solve the above problems. Summary of the invention
[0005] The object of the present invention is a primary culture method of mesenteric stromal cells. The cell culture method of the present invention effectively avoids the mixing of other cells such as mesothelial cells and endothelial cells, and can promote the early adhesion growth of mesenteric stromal cells, improve the cell proliferation rate and shorten the cell culture cycle.
[0006] The above technical objectives of the present invention are achieved through the following technical solutions: A primary culture method of mesenteric stromal cells, comprising the following steps:
[0007] S1. Select mice with a growth cycle of more than 4 weeks, kill them by cervical dislocation, and disinfect them in 75% alcohol for 2 minutes. Then, cut the epidermis and peritoneum in turn in a biosafety cabinet, open the mouse abdominal cavity, remove the intestinal tissue completely and put it into a 50mL centrifuge tube filled with pre-cooled phosphate buffer for later use, wash to remove attached blood and tissue fragments, then transfer the intestinal tissue to a 10cm cell culture dish, add pre-cooled phosphate buffer to maintain a moist environment, use ophthalmic scissors to separate the mesenteric tissue, and continue to use phosphate buffer to wash the mesenteric tissue several times until the washing solution is clear;
[0008] S2, the separated mesenteric tissue was transferred to a cell culture dish, treated with mesothelial cell dissociation solution to remove mesothelial cells, then the tissue was minced and treated with EDTA solution, followed by digestion with a digestion solution containing type II collagenase and neutral protease, and finally the cells were purified by Ficoll / Percoll density gradient centrifugation;
[0009] S3. The purified cells are expanded and cultured in a culture medium containing itaconic acid derivatives, and subcultured when the cell confluence reaches 80%.
[0010] The present invention is further configured as follows: the specific process in step S2 includes the following steps:
[0011] S21. Transfer the separated mesenteric tissue to a 10 cm cell culture dish, lay it flat, and gently wipe the surface of the mesenteric tissue 2 to 5 times with a sterile cotton swab dipped in a mesothelial cell dissociation solution; turn the mesentery over, lay it flat, and gently wipe the surface of the mesenteric tissue 2 to 5 times with a sterile cotton swab dipped in a mesothelial cell dissociation solution; use phosphate buffer containing 10% double antibody to wash the mesenteric tissue several times to remove the dissociated mesothelial cells;
[0012] S22, transfer the mesenteric tissues without mesothelial cells to a 35 mm cell culture dish or vial, and mince the mesenteric tissues with ophthalmic scissors until they become a paste, and the time is controlled within 3 minutes;
[0013] S23. Add 10 mL of 0.005-0.05% EDTA solution to the chopped mesenteric tissue, mix well, and place on ice for 30-60 minutes.
[0014] S24, after centrifugation at 1000 rpm, add interstitial cell digestion solution and mix well, transfer to a 50 mL centrifuge tube, shake and digest on a shaker at 37°C and 140 rpm for 15 to 30 minutes until flocs appear on the liquid surface in the centrifuge tube; add the same volume of complete medium to terminate the digestion reaction, and centrifuge at 1000 rpm for 10 minutes;
[0015] S25, discard the supernatant, add complete culture medium containing 10% fetal bovine serum to resuspend the cells, and mix by pipetting; then filter the cell suspension through a 40 μm mesh into a 50 mL centrifuge tube, and centrifuge at a low speed of 800 rpm for 5 minutes; after discarding the supernatant, remove the red blood cells on the surface of the sediment layer; add culture medium to resuspend the cells, and filter through a 40 μm mesh again, and repeat this step 1 to 3 times;
[0016] S26, centrifuging the cell suspension obtained after digestion using Ficoll / Percoll separation solution to remove tiny tissue fragments, dead cells and red blood cells; collecting the mononuclear cell layer cells, washing them 2 to 3 times using phosphate buffer or serum-free medium after centrifugation;
[0017] S27. The washed cells were resuspended in DMEM medium containing 10% FBS, inoculated in a cell culture plate, and non-adherent cells were removed by differential adhesion method. Complete medium was added to continue culturing. The adherent cells were the purified mesenteric stromal cells.
[0018] The present invention is further configured as follows: the mesothelial cell dissociation solution in step S21 is a Hank's balanced salt solution to which EDTA and deoxyribonuclease I are added, the concentration of EDTA is 0.001% to 0.005%, and the concentration of deoxyribonuclease I is 0.05 to 0.5 mg / mL.
[0019] The present invention is further configured as follows: the mesenchymal cell digestion solution in step S24 is a DMEM culture medium added with type II collagenase and neutral protease, the concentration of type II collagenase is 2-5 mg / mL, and the concentration of neutral protease is 0.5-2 mg / mL.
[0020] The present invention is further configured as follows: the expansion culture medium in step S3 uses a single or multiple itaconic acid derivatives with high cell membrane permeability, including dimethyl itaconic acid, 4-octyl itaconic acid and 4-ethyl itaconic acid.
[0021] In summary, the present invention has the following beneficial effects:
[0022] 1. The present invention uses a mesothelial cell dissociation solution to remove mesothelial cells, thereby preventing the mixing of mesothelial cells and mesenchymal cells transformed from mesothelial cells during the culture process of primary mesenteric stromal cells;
[0023] 2. In the present invention, promoting the adherent growth of mesenteric stromal cells is an important step for efficiently expanding primary cells. To achieve this goal, neutral protease is added to the digestion solution of the stromal cells to effectively prevent cell aggregation and promote the adherence of separated cells. In addition, since the mesenteric tissue is rich in connective tissue, although the small-pore sieve can filter out most of the incompletely digested tissue, there are still a large number of tiny tissue fragments mixed with the separated cells, interfering with the cell adherent growth. The present invention can effectively purify the stromal cells by using Ficoll or Percoll density gradient centrifugation, avoid tissue fragments adhering to the bottom of the culture dish, thereby interfering with the adherent growth of the mesenteric stromal cells, and separate the mesenteric stromal cells with a high yield;
[0024] 3. In order to improve the expansion efficiency of mesenteric stromal cells and maintain their original phenotype, the present invention adds itaconic acid derivatives to the expansion culture medium to remove reactive oxygen species generated during cell metabolism, reduce the cell aging process, and improve the expansion efficiency of mesenteric stromal cells, thereby solving the problem that mesenteric stromal cells are very likely to stagnate due to aging during the expansion and passaging process, resulting in low cell expansion efficiency. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 is a schematic diagram of the separation of mesenteric stromal cells in an embodiment of the present invention;
[0026] Figure 2 is a schematic diagram of tissue floccules after mesenteric tissue is digested with collagenase in an embodiment of the present invention;
[0027] Figure 3 is a diagram of cell sedimentation layers after low-speed centrifugation in an embodiment of the present invention;
[0028] Figure 4 It is the liquid layer obtained by Ficoll density gradient centrifugation in the embodiment of the present invention;
[0029] Figure 5 is a microscopic image of cells obtained by Ficoll density gradient centrifugation in an embodiment of the present invention;
[0030] Figure 6 is a microscopic image of cells obtained without Ficoll density gradient centrifugation in an embodiment of the present invention;
[0031] Figure 7 This is a cell micrograph after 3 days of culture of interstitial cells in an embodiment of the present invention;
[0032] Figure 8 is a micrograph of mesenteric stromal cells after expansion in an embodiment of the present invention;
[0033] Fig. 9 It is an immunofluorescence image of the identification of mesenteric stromal cells in the example of the present invention. DETAILED DESCRIPTION
[0034] The following is combined with Figure 1-9 The present invention is described in further detail.
[0035] The experimental animals in this embodiment are healthy experimental mice over 4 weeks old, and the instruments and reagents used are as follows:
[0036] (1) Experimental instruments: biological safety cabinet (Thermo Fisher), surgical scissors, tissue forceps, ophthalmic scissors, 50 mL centrifuge tubes (Corning), six-well plates (Corning), 10 cm cell culture dishes (Corning), constant temperature CO2 cell culture incubator (ThermoFisher), centrifuge (Thermo Fisher), high pressure steam sterilizer (Panasonic), oven (Panasonic), constant temperature culture shaker (Shanghai Zhicheng), clean bench (Thermo Fisher), inverted fluorescence microscope (Olympus), cover slip (Shitai), slide (Shitai), 40 μm cell screen (NEST).
[0037] (2) Reagents: DMEM complete medium (Gibico), fetal bovine serum (Gibico), bovine serum albumin (Fushen Biotechnology), 0.25% trypsin (Gibico), penicillin-streptomycin solution (Gibico), 75% alcohol, PBS buffer (Shanghai Biotech), collagenase type II (Thermo Fisher), Ficoll solution (GE HealthCare), TritonX-100 (Sigma), paraformaldehyde (Sigma), anti-fluorescence quenching mounting medium containing DAPI (Shanghai Biotech), EDTA (MCE), Hank's balanced salt solution, neutral protease (Wobo Biotechnology), 4-OI (Glpbio), Vimentin antibody (Santa Cruz), CD31 (Abcam), CD45 (Abcam), E-cadherin (Proteintech), and anti-mouse fluorescent secondary antibody (Jackson Immune).
[0038] Embodiment: A primary culture method of mesenteric stromal cells comprises the following steps:
[0039] S1. Isolation of mouse mesentery: Select mice older than 4 weeks old, kill them by cervical dislocation, and disinfect them in 75% alcohol for 2 minutes. In a biosafety cabinet, cut the epidermis and peritoneum in turn, open the mouse abdominal cavity, remove the intestinal tissue completely and put it into a 50mL centrifuge tube filled with pre-cooled PBS for later use, and gently wash it to remove attached blood and tissue fragments. Subsequently, transfer the intestinal tissue to a 10cm cell culture dish, add pre-cooled PBS to maintain a moist environment, and use ophthalmic scissors to separate the mesentery tissue as completely as possible (see Figure 1 ); and wash the mesenteric tissue several times with PBS until the washing solution is clear.
[0040] S2. Isolation of mesenteric stromal cells, including the following steps:
[0041] (1) The separated mesenteric tissue was transferred to a 10 cm cell culture dish, spread flat, and the surface of the mesenteric tissue was gently wiped three times with a sterile cotton swab dipped in mesothelial cell dissociation solution; the mesenteric tissue was turned over, spread flat, and the surface of the mesenteric tissue was gently wiped three times with a sterile cotton swab dipped in mesothelial cell dissociation solution; the mesenteric tissue was washed several times with PBS containing 10% double antibody to completely remove the mesothelial cell dissociation solution.
[0042] In this example, the mesothelial cell dissociation solution is a Hank's balanced salt solution to which EDTA and DNase I are added; the concentration of EDTA is 0.002%, and the concentration of DNase I is 0.1 mg / mL.
[0043] (2) Transfer the mesenteric tissue without mesothelial cells to a 35 mm cell culture dish or vial and mince the mesenteric tissue with ophthalmic scissors until it becomes a paste. The time is controlled within about 3 minutes.
[0044] (3) Add 10 mL of 0.02% EDTA solution to the chopped mesenteric tissue, mix well, and place on ice for 30 to 60 minutes.
[0045] (4) After centrifugation at 1000 rpm, add interstitial cell digestion solution and mix well. Transfer to a 50 mL centrifuge tube and shake on a shaker at 37°C and 140 rpm for 15 to 30 minutes until flocs appear on the liquid surface of the centrifuge tube (see Figure 2 ); add the same volume of complete medium to terminate the digestion reaction, and centrifuge at 1000 rpm for 10 minutes.
[0046] In this embodiment, the mesenchymal cell digestion solution is a DMEM culture medium added with type II collagenase and neutral protease; the concentration of type II collagenase is 2 mg / mL, and the concentration of neutral protease is 1 mg / mL.
[0047] (5) Discard the supernatant, add complete culture medium containing 10% fetal bovine serum (FBS) to resuspend the cells, and mix by pipetting; then filter the cell suspension through a 40 μm mesh into a 50 mL centrifuge tube and centrifuge at a low speed of 800 rpm for 5 minutes; after discarding the supernatant, remove the red blood cells on the surface of the sediment layer (see Figure 3 ); add culture medium to resuspend the cells, filter again with a 40 μm mesh, and repeat this step 3 times.
[0048] In this embodiment, the complete culture medium was prepared by adding 50 mL of fetal bovine serum and 5 mL of dual antibody (containing 10000 U / mL penicillin sodium and 10000 μg / mL streptomycin) to 500 mL of DMEM complete culture medium, mixing well, and storing at 4°C for later use.
[0049] (6) To remove tiny tissue fragments, dead cells, and red blood cells, the cell pellet was resuspended in serum-free medium to 5 mL and slowly dripped into a centrifuge tube containing 5 mL of Ficoll solution. Centrifuge at 2000 rpm for 30 min. After centrifugation, the tube was divided into three layers. The white cloudy layer in the middle was aspirated with a sterile pipette (see Figure 4 ) were placed in another centrifuge tube; PBS was added to resuspend the cells and washed twice.
[0050] (7) The washed cells were resuspended in DMEM medium containing 10% FBS, inoculated in a cell culture plate, and non-adherent cells were removed by differential adhesion method, and then complete medium was added to continue culturing. It can be observed that after Ficoll density gradient centrifugation, tiny tissue fragments can be effectively removed (see Figure 5 ), while the cell suspension obtained without Ficoll density gradient centrifugation contains a large amount of tissue fragments, which seriously hinders the cell adhesion growth (see Figure 6 ).
[0051] S3. Proliferation culture of mesenteric stromal cells: After the cells adhered for 24 hours, in order to promote the proliferation of mesenteric stromal cells and maintain their original phenotype, the adherence medium was changed to the proliferation medium and continued to be cultured. When the cell confluence reached 80%, the cell passage operation was performed. In this embodiment, the proliferation medium used a single or multiple itaconic acid derivatives with high cell membrane permeability, including dimethyl itaconate, 4-octyl itaconate (4-Octyl itaconate, 4-OI), 4-ethyl itaconate, etc.
[0052] (1) Observe the growth status of mesenteric stromal cells. The newly isolated primary cells are spherical (see Figure 7 A); After 3 days of culture, the mesenteric stromal cells were completely attached to the wall. Microscopically, the cell bodies were large and mostly spindle-shaped or irregularly triangular in shape (see Figure 7 B).
[0053] (2) During the expansion of mesenteric stromal cells, a DMEM medium containing an itaconic acid derivative was used. In this example, by adding an itaconic acid derivative 4-OI (100 μM) to the expansion medium, the results showed that 4-OI can effectively remove reactive oxygen species and promote the expansion of mesenteric stromal cells (see Figure 8 ).
[0054] In this example, in order to further confirm the purity of the obtained mesenteric stromal cells, the cultured primary cells were taken for immunofluorescence staining to identify their cell phenotypes.
[0055] The specific process is as follows: wash the coverslip in a biosafety cabinet and dry it, place it on the bottom of a 24-well plate, inoculate the expanded mesenteric stromal cells in the 24-well plate, remove the coverslip when the confluence reaches about 60%, wash with PBS three times, and fix with 4% paraformaldehyde solution at room temperature for 20 minutes; after washing with PBS three times, add 0.2% TritonX-100 to permeabilize the cells for 15 minutes; after washing with PBS three times, use 5% bovine serum albumin (BSA) to block for 1 hour; then add mesenchymal marker Vimentin, endothelial marker CD31, leukocyte marker CD45 and epithelial marker E-cadherin respectively, and incubate at 4°C overnight; after washing with PBS three times, add species-specific fluorescent secondary antibodies and incubate at room temperature for 1 hour; after washing with PBS three times, DAPI is used for background staining. Under a fluorescence microscope, almost all cells express Vimentin, with a positive cell ratio of >95%, while endothelial cell, leukocyte, and epithelial cell markers are all negative (see Fig. 9 ), the results showed that the mesenteric stromal cells separated by this method were of high purity.
[0056] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.
Claims
1. A primary culture method of mesenteric stromal cells, characterized by: The following steps are involved: S1. Select mice with a growth cycle of more than 4 weeks, kill them by cervical dislocation, and disinfect them in 75% alcohol for 2 minutes. Then, cut the epidermis and peritoneum in turn in a biosafety cabinet, open the mouse abdominal cavity, remove the intestinal tissue completely and put it into a 50mL centrifuge tube filled with pre-cooled phosphate buffer for later use, wash to remove attached blood and tissue fragments, then transfer the intestinal tissue to a 10cm cell culture dish, add pre-cooled phosphate buffer to maintain a moist environment, use ophthalmic scissors to separate the mesenteric tissue, and continue to use phosphate buffer to wash the mesenteric tissue several times until the washing solution is clear; S2, the separated mesenteric tissue was transferred to a cell culture dish, treated with mesothelial cell dissociation solution to remove mesothelial cells, then the tissue was minced and treated with EDTA solution, followed by digestion with a digestion solution containing type II collagenase and neutral protease, and finally the cells were purified by Ficoll / Percoll density gradient centrifugation; S3. The purified cells are expanded and cultured in a culture medium containing itaconic acid derivatives, and subcultured when the cell confluence reaches 80%.
2. The primary culture method of mesenteric stromal cells according to claim 1, characterized in that: The specific process in step S2 includes the following steps: S21. Transfer the separated mesenteric tissue to a 10 cm cell culture dish, lay it flat, and gently wipe the surface of the mesenteric tissue 2 to 5 times with a sterile cotton swab dipped in a mesothelial cell dissociation solution; turn the mesentery over, lay it flat, and gently wipe the surface of the mesenteric tissue 2 to 5 times with a sterile cotton swab dipped in a mesothelial cell dissociation solution; use phosphate buffer containing 10% double antibody to wash the mesenteric tissue several times to remove the dissociated mesothelial cells; S22, transfer the mesenteric tissues without mesothelial cells to a 35 mm cell culture dish or vial, and mince the mesenteric tissues with ophthalmic scissors until they become a paste, and the time is controlled within 3 minutes; S23. Add 10 mL of 0.005-0.05% EDTA solution to the chopped mesenteric tissue, mix well, and place on ice for 30-60 minutes. S24, after centrifugation at 1000 rpm, add interstitial cell digestion solution and mix well, transfer to a 50 mL centrifuge tube, shake and digest on a shaker at 37°C and 140 rpm for 15 to 30 minutes until flocs appear on the liquid surface in the centrifuge tube; add the same volume of complete medium to terminate the digestion reaction, and centrifuge at 1000 rpm for 10 minutes; S25, discard the supernatant, add complete culture medium containing 10% fetal bovine serum to resuspend the cells, and mix by pipetting; then filter the cell suspension through a 40 μm mesh into a 50 mL centrifuge tube, and centrifuge at a low speed of 800 rpm for 5 minutes; after discarding the supernatant, remove the red blood cells on the surface of the sediment layer; add culture medium to resuspend the cells, and filter through a 40 μm mesh again, and repeat this step 1 to 3 times; S26, centrifuging the cell suspension obtained after digestion using Ficoll / Percoll separation solution to remove tiny tissue fragments, dead cells and red blood cells; collecting the mononuclear cell layer cells, washing them 2 to 3 times using phosphate buffer or serum-free medium after centrifugation; S27. The washed cells were resuspended in DMEM medium containing 10% FBS, inoculated in a cell culture plate, and non-adherent cells were removed by differential adhesion method. Complete medium was added to continue culturing. The adherent cells were the purified mesenteric stromal cells.
3. The primary culture method of mesenteric stromal cells according to claim 2, characterized in that: The mesothelial cell dissociation solution in step S21 is a Hank's balanced salt solution to which EDTA and deoxyribonuclease I are added, wherein the concentration of EDTA is 0.001% to 0.005%, and the concentration of deoxyribonuclease I is 0.05 to 0.5 mg / mL.
4. The primary culture method of mesenteric stromal cells according to claim 2, characterized in that: The mesenchymal cell digestion solution in step S24 is a DMEM culture medium added with type II collagenase and neutral protease, the concentration of type II collagenase is 2-5 mg / mL, and the concentration of neutral protease is 0.5-2 mg / mL.
5. The primary culture method of mesenteric stromal cells according to claim 1, characterized in that: The expansion medium in step S3 uses a single or multiple itaconic acid derivatives with high cell membrane permeability, including dimethyl itaconic acid, 4-octyl itaconic acid and 4-ethyl itaconic acid.