CN115491344A - Method for separating and purifying primary mouse lung fibroblasts and constructing in-vitro activation model - Google Patents
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Abstract
The invention provides a method for separating and purifying primary mouse lung fibroblasts and constructing an in-vitro activation model, which mainly comprises the following steps of: separating mouse lung, separating lung fibroblast, purifying lung fibroblast, and constructing a lung fibroblast in-vitro activation model to obtain the lung fibroblast in-vitro activation model. The method can avoid the difficulty of cell screening caused by mixing a large amount of non-fibroblasts in the process of primary cell culture by innovatively and jointly using a differential wall sticking method, a differential wall sticking method and a cell nutrition screening method, is simple and convenient to operate and high in efficiency, can complete the separation and purification of the primary mouse lung fibroblasts and the construction of an in-vitro activation model without expensive instruments and equipment, and has important reference value for experimental research in the future.
Description
Technical Field
The invention relates to the field of cell culture, in particular to a method for separating and purifying primary mouse lung fibroblasts and constructing an in-vitro activation model.
Background
The lung is a key organ in the respiratory system of the human body, and provides an important place for gas exchange in the respiratory process. The lung fibroblasts are important cell compositions in lung interstitium, can be activated into myofibroblasts, so as to synthesize and secrete extracellular matrix proteins, and the proliferation, migration and transdifferentiation of the myofibroblasts are related to the maintenance of lung tissue structures and the repair and remodeling of damaged lungs, so that the lung function is influenced.
Pulmonary fibrosis is a chronic and progressive irreversible interstitial lung disease, is caused by various factors such as environmental, genetic and tissue abnormal repair, takes the activation and proliferation of fibroblasts, excessive deposition of extracellular matrix and chronic interstitial inflammation as main pathological features, and is also the final stage of development of a plurality of lung diseases. After a definitive diagnosis, the survival time of the patient is usually only 2-4 years. According to statistics, the global incidence rate of pulmonary fibrosis is 10-60 cases/10 ten thousand persons/year. There are studies showing that the probability of severe pulmonary fibrosis in late stage is significantly increased in 2019 patients with novel coronavirus pneumonia, especially in critically infected patients. Since pulmonary fibrosis is mainly clinically manifested by dry cough and progressive respiratory difficulty, once suffering from pulmonary fibrosis, the daily life of a patient is seriously affected for a long time, and the patient can die due to exhaustion of breath.
Abnormal continuous activation of lung fibroblasts is considered to be one of important processes in pulmonary fibrosis mechanism, and a new idea for treating or improving pulmonary fibrosis can be provided for people by simulating and researching the pulmonary fibrosis mechanism through mouse lung fibroblasts, but no effective culture method for mouse primary lung fibroblasts exists at present.
One of the existing culture methods: flow cytometry sorting or immunomagnetic bead sorting requires expensive instruments, magnetic beads and antibodies, has high requirements on experimental conditions and operation technologies, and is poor in economy and convenience.
If a traditional culture method is adopted, such as a nutrition screening method, purified primary fibroblasts can be obtained after cell passage generations need to be waited, so that the usable generation number of subsequent experiments of the fibroblasts is greatly reduced; besides, the proliferation speed of the fibroblasts is very slow, and the culture period is long.
In conclusion, the culture of primary lung fibroblasts of mice needs to solve the problems of long culture period, difficult cell purification, high price and the like.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a method for separating and purifying primary mouse lung fibroblasts and constructing an in-vitro activation model.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for separating and purifying primary mouse lung fibroblasts and constructing an in-vitro activation model comprises the following steps:
s1, separation of mouse lung: selecting 8-10 weeks of adult male mice, fixing the mice on a dissection plate after the mice die by cervical dislocation, sequentially cutting off epidermis, peritoneum and diaphragm, opening thoracic cavity from sternum to two sides, rapidly removing heart, cutting trachea up, cutting esophagus down, and taking out the intact lung to a 15mL centrifuge tube for later use;
s2, separating lung fibroblasts: in a biological safety cabinet, cleaning lung tissues by PBS, stripping trachea, esophagus, peripheral connective tissues and residual blood clots, shearing the lung tissues into paste in a 6cm cell culture dish by ophthalmic scissors, controlling the time to be about 5min, clamping the lung tissue blocks by 1 x 2-tooth tissue forceps, uniformly paving the lung tissue blocks in a six-hole plate, wherein the interval between the tissue blocks is about 3mm, and dripping a small amount of complete lung fibroblast culture medium timely to keep the tissues temporarily moist;
s3, culturing lung fibroblasts: after all tissues are paved, standing for a certain time, adding 2mL of complete culture medium into a six-hole plate, placing the six-hole plate into an incubator for culture, and changing the liquid every two days after two kinds of oval and long fusiform cells climb out around a tissue block seen under a mirror on about day 3;
s4, purifying lung fibroblasts: at the seventh day, the overall cell density reached 90%Then digesting with 0.25% pancreatin for 1.5-2 min, sieving with 200 mesh cell sieve, centrifuging at 1000rpm for 5min, discarding supernatant, using up complete culture medium to resuspend lung fibroblasts, inoculating in six-well plate, culturing for a certain time, discarding culture medium, adding new complete culture medium, and reacting at 37 deg.C and 5% CO 2 Culturing in an incubator to obtain purified lung fibroblasts;
s5, constructing a lung fibroblast in-vitro activation model: after the purified lung fibroblast cells are cultured overnight, the original culture medium is removed, 2mL of fresh lung fibroblast cell low serum culture medium is added into each hole, 3ng/mL of TGF-beta 1 is added into each hole, and the in-vitro activation model of the lung fibroblast cells is obtained after 48 hours.
Further, the complete medium has the following components: 33.6mL of H-DMEM basal medium, 6mL of Australian-fetal bovine serum and 0.4mL of the dual antibody solution.
Further, the low serum medium has the following components: 39.4mL of H-DMEM basal medium, 0.2mL of Australian-fetal bovine serum, and 0.4mL of the dual antibody solution.
Further, in S2, the six-well plate was incubated with 0.1% gelatin 30min in advance.
Further, in S3, the standing time is 10min.
Further, in S3, the culture conditions in the incubator are 5% CO 2 The humidity was 95% and the temperature was 37 ℃.
Further, in S4, the culture time of the incubator is 1h.
Further, the double antibody solution contained 10,000U/mL benzyl penicillin sodium and 10,000. Mu.g/mL streptomycin.
Compared with the prior art, the invention has the beneficial effects that:
1. by innovatively and jointly using a differential adherence method, a differential adherence method and a cell nutrition screening method, the method can avoid the cell screening difficulty caused by mixing a large number of non-fibroblasts in the primary cell culture process.
2. Convenient and fast, and short in period: the method can achieve excellent effects over the traditional method only by using easily-obtained conventional reagents, and improves the number of the cells creeping out and shortens the time required by the cell creeping out by adopting 0.1 percent of gelatin to incubate the six-hole plate for 30min in advance; after the lung tissue blocks are paved for 10min, the complete culture medium is added, so that the time for waiting for the tissue blocks to dry in the traditional tissue block method is greatly shortened; culturing fibroblasts in complete lung fibroblast culture medium containing 15% australian fetal bovine serum (AUS-FBS) allows more lung fibroblasts to climb out earlier and enter the logarithmic growth phase faster; 1h of liquid change after passage of the lung fibroblasts can remove the temporarily non-adherent impurity cells on the premise that as many lung fibroblasts are attached to the wall as possible; the lung fibroblast low serum culture medium only containing 0.5 percent of Australian fetal bovine serum (AUS-FBS) is used for culturing the fibroblast, so that the influence of serum on the action of a growth factor can be avoided, and the in-vitro activation model of the lung fibroblast is successfully constructed; TGF-beta 1 is proved to be a central factor in a pulmonary fibrosis generation mechanism and also a key factor in a fibroblast activation process, lung fibroblasts are stimulated by 3ng/mL TGF-beta 1 for 48h, WB detection shows that 3ng/mL and 6ng/mL TGF-beta 1 treatment can enable alpha-SMA expression to be obviously up-regulated, and the activation effect is obvious, so 3ng/mL TGF-beta 1 treatment for 48h is selected as a construction condition of a mouse lung fibroblast activation model.
3. Is economical and convenient: the method is simple and convenient to operate, high in efficiency, capable of completing the separation and purification of the primary mouse lung fibroblasts and the construction of an in-vitro activation model without expensive instruments and equipment, and has important reference values for experimental research in the future.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a drawing of a six-well plate culture of lung tissue plated after cutting of the present invention;
FIG. 2 is a microscopic photograph of mouse lung fibroblasts on the third day of the present invention, wherein A is a white light photograph at a magnification of 5 times under a microscope, and B is a white light photograph at a magnification of 10 times under a microscope;
FIG. 3 is a cell microscopic image of P1 generation of mouse lung fibroblasts purified according to the present invention, wherein the image C is a 5 times magnified white light image under a mirror, and the image D is a 10 times magnified white light image under a mirror;
FIG. 4 is a graph of WB detection of mouse lung fibroblasts of the invention in low serum medium after 48h of treatment with 3ng/mL and 6ng/mL TGF- β 1, where α -SMA is α smooth muscle actin and GADPH is glyceraldehyde-3-phosphate dehydrogenase.
Detailed Description
The experimental procedures in the following examples are conventional unless otherwise indicated, and the starting materials and reagents used in the present invention are all commercially available products and are commercially available.
The technical features and characteristics of the present invention are described in detail by the following embodiments, which are not intended to limit the scope of the present invention.
The technical solution of the present invention will be described in further detail with reference to the accompanying drawings (fig. 1-4) and the detailed description.
Instruments and reagents
(1) The apparatus is as follows: dissection plate, biological safety cabinet, surgical scissors, 1 × 2 tooth tissue forceps clip, ophthalmic scissors, six-hole plate, incubator, high-speed centrifuge, 15mL centrifuge tube, 50mL centrifuge tube, high-pressure steam sterilization pot, oven, super clean bench, 10cm cell culture dish, 6cm cell culture dish, incubator
(2) Reagent: 0.1% gelatin, 0.25% pancreatin, complete medium, low serum medium, 3ng/mL TGF-beta 1, H-DMEM basal medium, australian-fetal bovine serum (AUS-FBS), benzylpenicillin sodium, streptomycin, 75% alcohol.
(3) Preparation of complete medium: to a 50mL sterile centrifuge tube was added 33.6mL H-DMEM basal medium, 6mL Australian fetal bovine serum (AUS-FBS), and 0.4mL double antibody (containing 10,000U/mL benzylpenicillin sodium and 10,000. Mu.g/mL streptomycin) (1% v/v). Mixing, and storing at 4 deg.C.
(4) Preparation of low serum medium: 39.4mL H-DMEM basal medium, 0.2mL Australian fetal bovine serum (AUS-FBS) and 0.4mL double antibody (containing 10,000U/mL benzylpenicillin sodium and 10,000. Mu.g/mL streptomycin) were added to a 50mL sterile centrifuge tube (1%. Mixing, and storing at 4 deg.C.
(5) Preparation of calcium and magnesium ion-free PBS (phosphate buffer solution):
the volume is fixed to 1L, the mixture is sterilized by high pressure steam (routine procedure), and then the mixture is put into a refrigerator at 4 ℃ for storage for later use.
(6) Preparation of TGF-beta 1 mother liquor: adding 3.3mL of sterile water into 2 mu g of TGF-beta 1 sterile powder in a biological safety cabinet, blowing and uniformly mixing, standing at 4 ℃ for 30min to fully dissolve the TGF-beta 1 powder to obtain 0.6 mu g/mL of TGF-beta 1 mother solution. Subpackaging into 200 μ L sterile EP tube, and storing in-80 deg.C refrigerator for use.
Example 1: isolation of primary mouse lung fibroblasts
1. Isolation of mouse Lung
All surgical instruments to be used were placed in an autoclave one day ahead of time and sterilized at 121 ℃ for 15min, and then the instruments were placed in a 56 ℃ oven for overnight drying. The six-well plate was coated with 0.1% gelatin for 30min in advance for use. Disinfecting the dissecting plate with 75% alcohol, placing in a super clean bench, and ultraviolet sterilizing for later use.
Taking an adult male mouse of 8-10 weeks, killing the mouse by a cervical dislocation method, soaking and disinfecting the mouse in 75% alcohol solution for 2min, fixing the mouse on a dissection plate, sequentially cutting off epidermis, peritoneum and diaphragm by using surgical scissors, opening the thoracic cavity from the sternum to two sides, rapidly removing the heart, then cutting off the trachea and the esophagus, and taking out the complete lung to a centrifuge tube of 15mL for later use.
2. Isolation of pulmonary fibroblasts
The isolated lung was transferred to a biosafety cabinet, lung tissue was washed in PBS in a 10cm cell culture dish, each leaf of lung tissue was isolated, connective tissue around trachea, esophagus, lung was stripped using sterile ophthalmic scissors and forceps, and residual blood clots were thoroughly washed. Shearing lung tissue into paste in 6cm cell culture dish with ophthalmic scissors for 5 min. After fully shearing, clamping the sheared lung tissue blocks by using 1 multiplied by 2 tooth tissue forceps, uniformly spreading the lung tissue blocks in a six-hole plate which is pre-coated by gelatin, enabling the interval between the tissue blocks to be about 3mm, and dropping a small amount of lung fibroblast complete culture medium at proper time to keep the tissues temporarily moist.
The minced lung tissue was plated in a six-well plate as shown in fig. 1, with the tissue blocks spaced approximately 3mm apart.
3. Culture of lung fibroblasts
After all tissue plating was complete, the six well plates were placed in a 37 ℃ incubator for 10min, after which 2mL of lung fibroblast complete medium per well was added, placed at 5% CO 2 And culturing in an incubator with 95% humidity and 37 ℃. On day 3, both oval and spindle shaped cells crawled out around the visible tissue mass under the mirror, after which the fluid was changed every two days.
On the third day, the mouse lung fibroblasts crawled out and then seen in the cell microscopic image of FIG. 2, and the cells mainly appeared oval and long spindle-shaped.
Example 2: purification of primary mouse lung fibroblasts
After the cultured lung fibroblasts in example 1 reached 90% of the total density of the crawled cells on the about 7 th day, the original culture medium was discarded, and the cells were washed twice with PBS without calcium and magnesium ions; adding 1mL of preheated 0.25% pancreatin per well, adding at 37 deg.C, 5% 2 Digesting for 1.5-2 min in an incubator; after the cells were observed to shrink and become round under the microscope, 2mL of digestion stop solution was added to stop the digestion, the cells were gently blown down, and the cell suspension was collected. Adding 3mL of PBS for washing, blowing off again and collecting cell suspension; passing the collected cell suspension through a 200-mesh cell screen to remove tissue blocks, collecting the cell suspension in a 15mL centrifuge tube, centrifuging at 1000rpm for 5min, discarding supernatant, carrying out resuspension by using a lung fibroblast complete culture medium, then carrying out passage on the cells according to the proportion of 1, inoculating in a six-hole plate, and shaking up; at 37 ℃ C, 5% CO 2 Standing in incubator for 1 hr, discarding culture medium, addingAnd (4) putting the new complete culture medium into an incubator for culture to obtain the purified lung fibroblasts.
A P1 generation cell microscopic image of purified mouse lung fibroblast cells is shown in FIG. 3, and the cells mainly present an oval shape and a long spindle shape.
Example 3: construction of mouse primary lung fibroblast in-vitro activation model
After the lung fibroblast purified in the embodiment 2 is used for overnight, the density reaches about 50%; taking out the six-hole plate in the incubator, removing the original culture medium by suction, and adding 2mL of fresh lung fibroblast low-serum culture medium into each hole; TGF-. Beta.1 stock solutions were added at concentrations of 3ng/mL and 6ng/mL, respectively, and six-well plates were placed at 37 ℃ and 5% CO 2 Continuously culturing for 48h in an incubator with the concentration; and after 48 hours, taking out the six-hole plate from the incubator, detecting by WB, and treating by 3ng/mL TGF-beta 1 for 48 hours to obtain the mouse lung fibroblast in-vitro activation model.
WB detection of mouse lung fibroblasts in low serum medium after 48h of treatment with 3ng/mL and 6ng/mL TGF-beta 1 is shown in FIG. 4, and it can be seen that the alpha smooth muscle actin (alpha-SMA) content is obviously increased after 48h of treatment with 3ng/mL and 6ng/mL TGF-beta 1.
While the preferred embodiments of this patent have been described in detail, this patent is not limited to the embodiments described above, and variations and modifications in other forms may occur to those skilled in the art, within the knowledge of the person skilled in the art. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (8)
1. A method for separating and purifying primary mouse lung fibroblasts and constructing an in-vitro activation model comprises the following steps:
s1, separation of mouse lung: selecting 8-10 weeks old male mice, fixing the mice on a dissection plate after the mice die by cervical dislocation, sequentially cutting off epidermis, peritoneum and diaphragm, opening thoracic cavity from sternum to two sides, rapidly removing heart, cutting trachea up and esophagus down, and taking out complete lung to a 15mL centrifuge tube for later use;
s2, separating lung fibroblasts: cleaning lung tissue by PBS (phosphate buffer solution), stripping trachea, esophagus, peripheral connective tissue and residual blood clots, shearing the lung tissue into paste in a 6cm cell culture dish by ophthalmic scissors, controlling the time to be about 5min, clamping the lung tissue blocks by 1 x 2 teeth tissue forceps, uniformly paving the lung tissue blocks in a six-hole plate, wherein the interval between the tissue blocks is about 3mm, and dripping a small amount of complete lung fibroblast culture medium in the period to keep the tissue to be temporarily moist;
s3, culturing lung fibroblasts: after all tissues are paved, standing for a certain time, adding 2mL of complete culture medium into a six-hole plate, placing the six-hole plate into an incubator for culture, and changing the solution every two days after two kinds of oval and long fusiform cells climb out around a tissue block seen under a mirror on about day 3;
s4, purifying lung fibroblasts: on the seventh day, after the cell density reached 90%, digesting with 0.25% pancreatin for 1.5-2 min, sieving with 200 mesh cell sieve, centrifuging at 1000rpm for 5min, discarding the supernatant, using the complete medium to resuspend the lung fibroblasts, inoculating in a six-well plate, culturing for a certain time, discarding the medium, adding a new complete medium, and reacting at 37 ℃ and 5% CO 2 Culturing in an incubator to obtain purified lung fibroblasts;
s5, constructing a lung fibroblast in-vitro activation model: after the purified lung fibroblast cells are cultured overnight, the original culture medium is removed, 2mL of fresh lung fibroblast cell low serum culture medium is added into each hole, 3ng/mL of TGF-beta 1 is added into each hole, and the in-vitro activation model of the lung fibroblast cells is obtained after 48 hours.
2. The method of claim 1, wherein the complete medium has the following composition: 33.6mL of H-DMEM basal medium, 6mL of Australian-fetal bovine serum and 0.4mL of the dual antibody solution.
3. The method of claim 1, wherein the low serum medium has the following composition: 39.4mL of H-DMEM basal medium, 0.2mL of Australian-fetal bovine serum, and 0.4mL of the dual antibody solution.
4. The method of claim 1, wherein in S2, the six-well plate is incubated with 0.1% gelatin 30min in advance.
5. The method of claim 1, wherein in S3, the standing time is 10min.
6. The method according to claim 1, wherein in S3, the incubator culture conditions are 5% CO 2 The humidity was 95% and the temperature was 37 ℃.
7. The method of claim 1, wherein in S4, the cultivation time of the incubator is 1h.
8. The method of claim 2 or 3, wherein the double antibody solution comprises 10,000U/mL benzyl penicillin sodium and 10,000 μ g/mL streptomycin.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113736737A (en) * | 2021-09-18 | 2021-12-03 | 四川大学华西医院 | Primary glioma-related fibroblast culture method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113736737A (en) * | 2021-09-18 | 2021-12-03 | 四川大学华西医院 | Primary glioma-related fibroblast culture method |
| CN113736737B (en) * | 2021-09-18 | 2023-09-08 | 四川大学华西医院 | Primary glioma-related fibroblast culture method |
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