CN115717122A - Method for extracting mouse aortic fibroblast - Google Patents
Method for extracting mouse aortic fibroblast Download PDFInfo
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- CN115717122A CN115717122A CN202211560906.XA CN202211560906A CN115717122A CN 115717122 A CN115717122 A CN 115717122A CN 202211560906 A CN202211560906 A CN 202211560906A CN 115717122 A CN115717122 A CN 115717122A
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Abstract
The invention relates to a method for extracting mouse aortic fibroblast, which is characterized in that the whole mouse aortic blood vessel is extracted after a porcine trypsin model is made, a specially-prepared digestive solution and a stop solution are adopted for digestive culture, the mouse aortic fibroblast can be quickly and effectively extracted, the extraction technology is low in difficulty, the mouse aortic adventitia does not need to be stripped during extraction, the consumption time is short, the mouse aortic fibroblast can be obtained after Cheng Jinxu-4 hours, the digestion of vascular tissues is thorough, the cell activity is influenced little, and the purity of the extracted mouse aortic fibroblast is high.
Description
The technical field is as follows:
the invention belongs to the technical field of quantitative cell analysis, and particularly relates to a method for extracting mouse aortic fibroblast.
Background art:
flow cytometry is a single cell-based technique widely used in various aspects of basic research and clinical practice, such as immunophenotyping of mouse vascular cell membranes, and the preparation of single cell suspensions of mouse vascular tissues is a prerequisite for flow cytometry analysis. The existing preparation of the blood vessel tissue single cell suspension is difficult, single cells with high yield, high activity and strong functions are difficult to obtain, further experimental study is hindered, and the failure of experiments can be caused by insufficient cell quantity, excessive cell fragments, cell adhesion and agglomeration and the like. Therefore, how to prepare a single cell suspension with better quality is a problem to be solved at present. Patent application No. CN201910618214.8 discloses a method for preparing mouse aortic cell single cell suspension, which comprises the following steps: firstly, preparing 1ml of enzyme mixed liquor, a living healthy wild mouse, an operating table with a smooth ice surface on the whole table top and a plurality of instruments; then, 200-500 ul chloral hydrate is used for carrying out intraperitoneal injection anesthesia and death on the prepared wild mouse, 20ml of phosphate buffer saline solution is used for immediately carrying out perfusion washing on the blood vessel of the wild mouse, the dead wild mouse is integrally disinfected by alcohol with the volume fraction of 75%, thoracoabdominal operation is carried out after disinfection is finished, the heart and the aorta are exposed, and then the mouse blood vessel tissue single cell suspension is prepared by using a shearing and mixed enzyme combined digestion method. However, the cell extraction method has the disadvantages of complicated operation steps, long time consumption, large number of required mice, high trypsin consumption and high cost, can easily cause incomplete or excessive cell digestion, has a small number of extracted fibroblasts and low purity of single cells, and influences the feasibility and accuracy of single cell sequencing and flow detection experiments.
The invention content is as follows:
technical problem to be solved
Aiming at the background, the invention provides a method for extracting mouse aortic fibroblasts, and solves the problems that the operation steps for extracting the mouse aortic fibroblasts are complicated, the consumed time is long, the number of required mice is large, the trypsin consumption is high, the cell digestion is incomplete or excessive, the number of extracted fibroblasts is small, the purity is low, and the feasibility and the accuracy of single cell sequencing and flow detection experiments are influenced in the prior art.
(II) technical scheme
In order to solve the technical problems, the invention adopts the following technical scheme: a method for extracting mouse aortic fibroblast, comprising the following steps:
s1, after a mouse is anesthetized, cutting the abdominal skin and subcutaneous muscle tissues of the mouse, carrying out blunt separation on the fat and envelope tissues on the surfaces of abdominal aorta and vein by using micro forceps, opening an arteriovenous sheath, dissociating the front and side surfaces of the abdominal aorta, covering the surface of the abdominal aorta with cosmetic cotton soaked with porcine trypsin, recovering the surrounding envelope and fat tissues, timing for 40 minutes, taking out the cosmetic cotton after 40 minutes, flushing the abdominal cavity for 2 times by using normal saline, and suturing the abdominal part;
s2, on the 14 th day after operation, the mice are killed by anesthesia, the skin and the sternum are cut off, all organs of the pleuroperitoneal cavity except the cardiovascular system and the kidney are separated, the whole aortic blood vessels of the mice are separated under a microscope, the whole aortic blood vessels are placed into a culture dish filled with precooled PBS (phosphate buffered saline), the residual blood in the blood vessels is squeezed out by using a pair of micro-tweezers, and the two culture dishes filled with clean PBS (phosphate buffered saline) are sequentially cleaned;
s3, placing the cleaned aorta vessel on the wall of an EP (EP) tube, shearing the aorta vessel tissue by using a micro-shear, adding digestive juice into the sheared tissue block, and oscillating for 20 minutes at the temperature of 37 ℃ in a water bath kettle at 200 rpm; taking out the EP tube, standing for 2min, sucking out the supernatant by using a pipette, collecting the supernatant into the EP tube filled with the stop solution, and placing the collected EP tube on ice;
s4, adding the digestive juice into the remaining tissue-digestive juice mixture, shaking the mixture for 20 minutes at the speed of 200rpm of a water bath kettle at the temperature of 37 ℃, and repeatedly sucking and collecting supernatant;
s5, after the last digestion, filtering all liquid containing the cells in the collected EP tube into a new EP tube through a 70-micron filter screen, centrifuging the liquid at 4 ℃ for 5min at 1600rpm, and discarding the supernatant;
s6, resuspending the cell precipitate obtained in the step 5 by using a DMEM medium containing 15% serum, planting the cell precipitate in a 12-hole plate, placing the cell precipitate in a CO2 incubator for standing for 2 hours, removing a culture supernatant, softly washing the cell precipitate for 2-3 times by PBS, adding a complete culture medium, placing the cell precipitate in the incubator, and continuing culturing to obtain the fibroblast.
Further, the formula of the digestive juice is as follows: each milliliter of the digestive juice comprises 4-12.5U of collagenase XI, 2-6U of hyaluronic acid, 2-6U of deoxyribonuclease and 5363 ml of collagenase I, 15-45U, PBS; the formula of the stop solution is as follows: each ml of the stop solution comprises 900. Mu.l of DMEM medium and 100. Mu.l of bovine serum.
Further, the conditions of the culture described in step 6 are: culturing at 37 deg.C for 4-48 hr.
Further, the process of cutting the aorta vessel by using the micro scissors in the step 3 is controlled within 10 minutes.
Further, the method for separating the mouse aorta in the step 3 is to take down the whole mouse aorta from the aortic arch to the iliac bifurcation.
Further, the serum is bovine serum.
(III) advantageous effects
The invention has the following beneficial effects:
1. by adopting the technical scheme provided by the invention, the mouse aortic fibroblast can be quickly and effectively extracted, the extraction technical difficulty is low, and the mouse aortic adventitia does not need to be peeled off during extraction;
2. the technical scheme provided by the invention is applied to extract the mouse aortic fibroblast, the consumption time is short, and the mouse fibroblast can be obtained after Cheng Jinxu is used for 3-4 hours;
3. the digestive juice and the stop solution adopted by the technical scheme provided by the invention can completely digest vascular tissues and have small influence on cell activity;
4. the mouse aortic fibroblast extracted by the technical scheme provided by the invention has high purity, and the purity can be over 90% through cellular immunofluorescence staining identification.
Description of the drawings:
in order to more clearly illustrate the technical solution in the embodiments of the present invention, the drawings required to be used in the embodiments will be briefly described below.
FIG. 1 is a graph of aortic blood vessels of mice after molding with porcine trypsin, in which it can be seen that aortic blood vessels of mice form aneurysms after molding with porcine trypsin;
FIG. 2 is a graph of the process of aortic vessel digestion in mice showing gradual vessel digestion completion;
FIG. 3 is a micrograph of fibroblasts obtained by extraction; the graph can show that after all the aortic vascular cells obtained by digestion are kept stand for two hours and suspension cells in supernatant are discarded, adherent cells can be seen to form fusiform and polygon in 24 hours, pseudopodium extends out from the periphery of the cells, the cell bodies are large, cytoplasm is transparent, the nucleus is clear, and the cells grow rapidly;
FIG. 4 is a diagram of immunofluorescence identification; the figure shows that identification of primary cells by immunofluorescence is carried out on cell plates of P2 and P3 generations, and that Vimentin in the extracted primary cells is high in expression, and alpha-SMA is low in expression and accords with the molecular biological characteristics of fibroblasts, so that the purity of the extracted primary fibroblasts is high.
The specific implementation mode is as follows:
in order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.
1. Experimental materials: tissue forceps 1, tissue scissors 1, microshearing 2, 3cm petri dishes, 12 well petri dishes 3, absorbent cotton pads, cotton swabs, stereotactic microscopes, porcine trypsin (british sigma), 4-0 sutures, suture needles, pipette tips, 10ml centrifuge tubes, water bath pots (wil laboratories ltd. Su), DMEM medium (Hyclone, usa), PBS (british sigma), physiological saline, sodium pentobarbital, DAPI (beijing china seikarika bridge), mouse anti-vimentin-anti (proteintech), mouse anti-a-SMA-anti (abcam, usa), alexa Fluor 647-labeled goat anti-IgG (H + L) (shanghai biyuntan), alexa Fluor-labeled goat anti-IgG (H + L) (shanghai biyuntan 488), chinese mukuri-labeled goat anti-IgG (H + L) (bshai yuntan pisma), 1-X-100 (trix-X), chinese mukuri-fixed-X solution (solyoto-X), and other high-pressure sterilized saline (solba) reagents (solba) for chinese experiment)
2. The experimental method comprises the following steps:
step 1, 3 8 week old C57 mice were taken and anesthetized with 200. Mu.l of 1% pentobarbital. After anaesthesia, the mice were anesthetized, the limbs were fixed on their back, the abdomen was depilated, the skin and subcutaneous muscle tissue of the abdomen were cut open, and a cotton swab was placed to absorb the liquid exuded from the abdominal cavity and push the abdominal viscera to one side. Blunt separating abdominal aorta and vein surface fat and enveloped tissue with micro forceps, opening arteriovenous sheath, dissociating front and side of abdominal aorta, covering two pieces of 1 × 1mm cosmetic cotton soaked with porcine trypsin on the surface of abdominal aorta, recovering surrounding enveloped and fat tissue, and timing for 40 minutes. After 40 minutes, the cotton wool was taken out, the abdominal cavity was washed with physiological saline 2 times, and the abdomen was sutured.
Step 2, on day 14 post-surgery, mice were anesthetized with 200 μ l of 1% pentobarbital and sacrificed by exsanguination of the eyeball. Mice were fixed supine, and the skin and sternum were cut open along the midline with tissue scissors, and all pleuroperitoneal organs except the cardiovascular system and kidneys were isolated. The mouse aorta was isolated under a microscope in a clean bench and placed in a petri dish with pre-cooled PBS from the aortic arch to the iliac bifurcation. Residual blood in the vessel was squeezed out with micro-forceps and washed in sequence in two additional dishes with clean PBS. The blood vessel of the mice after the model making is shown in figure 1.
And 3, placing the blood vessel on the wall of a 10ml EP tube, inclining the EP tube, shearing the tissue into pieces by using a micro-shear, adding 900 mu l of digestive juice into the tissue pieces, and shaking the tissue pieces for 20 minutes at 37 ℃ by 200rpm of a water bath kettle. The EP tube was removed, allowed to stand for 2min, the supernatant was aspirated off with a pipette and collected in a 10ml EP tube containing 3ml of stop solution, enzyme digestion was stopped, cell viability was maintained, and the EP tube was collected and placed on ice.
And 4, adding 800 mu l of digestive juice into the residual tissue-digestive juice mixture, shaking the mixture for 20 minutes at the speed of 200rpm of a water bath kettle at the temperature of 37 ℃, and repeatedly sucking and collecting supernatant. To the remaining mixture was added the last 800. Mu.l of digest and shaken in a 37 ℃ water bath at 200rpm for 20 minutes. The stepwise digestion method can almost completely digest the vascular tissues after three times of digestion, and the digestion effect of each time is shown in figure 2.
The digestive juice and the stop solution in the step 3 are prepared in advance, 2.5ml of digestive juice and 3ml of stop solution are needed for one blood vessel on average, and the formula is as follows:
per ml of digestive juice: collagenase XI4-12.5U, hyaluronic acid 2-6U, deoxyribonuclease 2-6U, collagenase I15-45U, PBS ml. According to the experiment, a sufficient amount of enzyme can completely digest 3 blood vessels at most, so that the mixed enzyme prepared in the dosage range given by the formula can be used for digesting 1-3 blood vessels according to the method in step 3.
Per ml stop solution: 900 μ l DMEM medium, 100 μ l bovine serum.
Step 5, after the last digestion, all the cell-containing liquid in the collection tube was filtered through a 70 μm sieve into a new 10ml EP tube, centrifuged at 1600rpm for 5min at 4 ℃ and the supernatant discarded.
Step 6, the cell pellet obtained in step 5 was resuspended in DMEM medium containing 15% serum, seeded in 12-well plates, and 1 well per 3 vessels was suggested for cell seeding. Standing in a CO2 incubator for 2 hours, and removing the culture supernatant. And (3) after the PBS is washed for 2-3 times, adding a complete culture medium, placing the mixture in an incubator for continuous culture, and performing differential adherence according to the adherence time of the fibroblasts, wherein most adherent cells are fibroblasts.
When partial fusion of fibroblasts was observed under the microscope, passage was carried out with 0.25% trypsin digestion (1: 2); the 1 st to 3 rd generation cells can be used for experiments or identification.
Cell identification: and (3) taking the cells growing to 90% of confluence, adding 0.25% of trypsin, observing the cells under a microscope, adding 2ml of complete culture medium to stop digestion after the cells shrink and become round, obtaining cell suspension, inoculating the cell suspension to a slide by 2 x 105/ml, and performing Vimentin and alpha-SMA immunofluorescence staining identification, wherein the result shows that the Vimentin positive rate is 90%, namely the cell purity is 90%.
The experimental results are as follows:
as can be seen in FIG. 1, the mouse vascularization of the aneurysm after porcine trypsin modeling
As can be seen from FIG. 2, the vascular digestion was gradually completed after the stepwise digestion
As can be seen from FIG. 3, after all the obtained vascular cells are left to stand for two hours and suspension cells in the supernatant are discarded, adherent cells can be seen to form fusiform and polygon in 24 hours, pseudopodium extends out from the periphery of the cells, the cell bodies are large, the cytoplasm is transparent, the nucleus is clear, and the cells grow rapidly
As can be seen from FIG. 4, the identification of primary cells by immunofluorescence on cell plates of P2 and P3 generations revealed that Vimentin in the extracted primary cells is highly expressed, while alpha-SMA is lowly expressed, which is in line with the molecular biological characteristics of fibroblasts, thus proving that the extracted primary fibroblasts are highly pure. Vascular fibroblast purity (%) = Vimentin positive staining cell number/total number of nuclei 100%.
The statistics of the experimental results are as follows:
| Label | Areaofvimitin | Areaofα-SMA | ALL | Fibroblast％ |
| 1 | 26220 | 3750 | 29970 | 87.49％ |
| 2 | 14797 | 2024 | 16821 | 87.97％ |
| 3 | 13895 | 1397 | 15292 | 90.86％ |
| 4 | 13036 | 1213 | 14249 | 91.49％ |
| 5 | 18179 | 1786 | 19965 | 91.05％ |
| mean | 89.77％ | |||
| SD | 1.64％ | |||
| mean±SD | 89.77％±1.64％ |
in conclusion, the method for extracting mouse aortic fibroblasts provided by the invention solves the problems that the operation steps for extracting mouse aortic fibroblasts in the prior art are complicated, the time consumption is long, the number of required mice is large, the trypsin consumption is high, the incomplete cell digestion or the over cell digestion is easily caused, the number of extracted fibroblasts is small, the purity is low, and the feasibility and the accuracy of single cell sequencing and flow detection experiments are influenced.
Finally, it should be noted that the above examples are only used for illustrating the present invention and do not limit the protection scope of the present invention. Further, various alterations, modifications and variations may be made by those skilled in the art after reading the technical content of the present invention, and all such equivalents may fall within the scope of the protection defined by the claims of the present application.
Claims (6)
1. A method for extracting mouse aortic fibroblasts is characterized by comprising the following steps:
s1, after a mouse is anesthetized, cutting the abdominal skin and subcutaneous muscle tissues of the mouse, carrying out blunt separation on the fat and envelope tissues on the surfaces of abdominal aorta and vein by using micro forceps, opening an arteriovenous sheath, dissociating the front and side surfaces of the abdominal aorta, covering the surface of the abdominal aorta with cosmetic cotton soaked with porcine trypsin, recovering the surrounding envelope and fat tissues, timing for 40 minutes, taking out the cosmetic cotton after 40 minutes, flushing the abdominal cavity for 2 times by using normal saline, and suturing the abdominal part;
s2, on the 14 th day after operation, the mice are killed by anesthesia, the skin and the sternum are cut off, all organs of the pleuroperitoneal cavity except the cardiovascular system and the kidney are separated, the whole aortic blood vessels of the mice are separated under a microscope, the whole aortic blood vessels are placed into a culture dish filled with precooled PBS (phosphate buffered saline), the residual blood in the blood vessels is squeezed out by using a pair of micro-tweezers, and the two culture dishes filled with clean PBS (phosphate buffered saline) are sequentially cleaned;
s3, placing the cleaned aorta vessel on the wall of an EP (EP) tube, shearing the aorta vessel tissue by using a micro-shear, adding digestive juice into the sheared tissue block, and oscillating for 20 minutes at 37 ℃ in a water bath kettle at 200 rpm; taking out the EP tube, standing for 2min, sucking out the supernatant by using a pipette, collecting the supernatant into the EP tube filled with the stop solution, and placing the collected EP tube on ice;
s4, adding the digestive juice into the remaining tissue-digestive juice mixture, shaking the mixture for 20 minutes at the speed of 200rpm of a water bath kettle at the temperature of 37 ℃, and repeatedly sucking and collecting supernatant;
s5, after the last digestion, filtering all liquid containing the cells in the collected EP tube into a new EP tube through a 70-micron filter screen, centrifuging the liquid at 4 ℃ for 5min at 1600rpm, and discarding the supernatant;
s6, resuspending the cell precipitate obtained in the step 5 by using a DMEM medium containing 15% serum, planting the cell precipitate in a 12-hole plate, placing the cell precipitate in a CO2 incubator for standing for 2 hours, removing a culture supernatant, softly washing the cell precipitate for 2-3 times by PBS, adding a complete culture medium, placing the cell precipitate in the incubator, and continuing culturing to obtain the fibroblast.
2. The method for extracting mouse aortic fibroblasts as claimed in claim 1, wherein the formulation of the digestive fluid is: each milliliter of the digestive juice comprises 4-12.5U of collagenase XI, 2-6U of hyaluronic acid, 2-6U of deoxyribonuclease and 5363 ml of collagenase I, 15-45U, PBS; the formula of the stop solution is as follows: each milliliter of the stop solution comprises 900. Mu.l of DMEM medium and 100. Mu.l of bovine serum.
3. The method for extracting mouse aortic fibroblasts as claimed in claim 1, wherein the culturing conditions in step 6 are: culturing at 37 deg.C for 4-48 hr.
4. The method for extracting mouse aortic fibroblasts as claimed in claim 1, wherein the process of cutting aortic blood vessels with the micro scissors in step 3 is controlled within 10 minutes.
5. The method for extracting mouse aortic fibroblasts of claim 1, wherein the method for isolating mouse aorta in step 3 is to take down the whole mouse aorta from aortic arch to iliac bifurcation.
6. The method for extracting mouse aortic fibroblasts as claimed in claim 1, wherein the serum is bovine serum.
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