CN110747159A - Mouse or rat kidney fibroblast cell separation and subculture method - Google Patents
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Abstract
The invention provides a method for separating and subculturing mouse or rat kidney fibroblast, which comprises the steps of cutting the complete kidney of a mouse or a rat, carrying out combined digestion by trypsin and IV type collagenase, culturing for 5-7 days in a complete culture medium, identifying the purity to be more than 95% by immunofluorescence, subculturing the separated fibroblast by using a special fibroblast culture medium, and realizing rapid cell proliferation and high purity. The method for separating and subculturing the mouse or rat kidney fibroblast provided by the invention has the advantages of convenient material acquisition, simple operation, rapid cell proliferation, high yield and high purity, is an ideal method for separating and subculturing the mouse or rat kidney fibroblast, and provides reliable cell resources for experiments.
Description
Technical Field
The invention relates to the field of primary cell culture, in particular to a method for separating and subculturing mouse or rat kidney fibroblasts.
Background
The renal interstitial fibroblasts are renal interstitial cells which are mainly positioned in renal bursa and renal tubular basement membrane and form a structural scaffold with capillary vessels around renal tubules, have vigorous functions and have various biological functions. The renal interstitium is the small amount of connective tissue between the small ducts of the kidney, where the major cellular component in loose connective tissue is fibroblasts. Renal interstitial fibroblasts are stimulated to activate and proliferate, and extra extracellular matrix is accumulated, which may cause renal injury.
Renal fibrosis is a common pathological process of various chronic renal diseases to end-stage renal failure, so that research on culturing renal fibroblasts as a cell model for in vitro research of various renal diseases is very necessary.
However, the related literature of domestic research is relatively few, and the mouse or rat kidney fibroblast isolation and culture method reported in the literature roughly comprises the following steps: 1. cutting the whole kidney of a mouse or a rat; 2. isolating renal medulla; 3. digesting the tissue fragment with trypsin; 4. cells were cultured with complete medium. Because the content of the kidney fibroblasts in the tissue is low, the difficulty in separating and extracting primary cells is high, the cell yield of a common separation culture method is extremely low, most epithelial cells or endothelial cells of glomeruli and renal tubules are obtained after 24 hours of culture, the number of the fibroblasts is extremely low, a small number of fibroblasts are obtained only by long-term culture, the culture success rate is low, the method has poor repeatability and the cost is high.
Disclosure of Invention
In view of the above, the invention provides a method for separating and culturing mouse or rat kidney fibroblast, which has the advantages of convenient material taking, simple operation, high yield, rapid proliferation and high purity of the separated and cultured mouse or rat kidney fibroblast.
The technical scheme of the invention is realized as follows:
in one aspect, the present invention provides a method for isolating mouse or rat renal fibroblasts, comprising the steps of:
s1, aseptically obtaining the complete kidney of a mouse or rat born for 1-30 days;
s2, repeatedly washing the kidney obtained in the step S1 with phosphate buffer solution containing 1% (v/v) penicillin/streptomycin double antibody for 2-3 times, and cutting into 1-3mm3A size tissue mass;
s3, digesting the tissue blocks by using digestive juice with the volume 3-5 times that of the tissue blocks;
s4, stopping digestion, sieving by a cell sieve, centrifuging, and removing supernatant to obtain cell sediment;
s5, resuspending the cell pellet in complete medium, inoculating the cell pellet into a culture dish, placing the culture dish at 37 ℃ and 5% CO2Carrying out static culture in a constant-temperature incubator, replacing a fresh culture medium after 24 hours to continue culture, then replacing the culture medium every 2-3 days, and culturing for 5-7 days to obtain cells with 80-90% confluency;
s6, inoculating the cells with the confluency of 80-90% in the step S5, digesting the cells with trypsin to obtain cell suspension, inoculating a slide, and performing Vimentin immunofluorescence identification.
Based on the above technical solution, preferably, the kidney in step S1 is a bilateral kidney of a mouse or a rat with a stripped kidney capsule.
Based on the above technical solution, preferably, the digestion solution in step S3 includes trypsin and collagenase type IV.
Still further, preferably, the trypsin concentration is 0.05% to 0.25% (w/v) and the collagenase type IV concentration is 0.05% to 0.2% (w/v).
On the basis of the above technical scheme, preferably, the digestion conditions in step S3 are 25-45min for digestion time, 37 ℃ for digestion temperature, and water bath shaking digestion.
In addition to the above technical solutions, it is preferable that the number of the cell meshes in step S4 is 200 meshes.
On the basis of the above technical solution, preferably, the centrifugation rotation speed in step S4 is 800-.
On the basis of the above technical solution, preferably, the complete culture medium in step S5 is a special fibroblast culture medium or a DMEM culture medium containing 10% (v/v) fetal bovine serum and 1% (v/v) penicillin/streptomycin double antibody.
On the other hand, the invention also provides a mouse or rat kidney fibroblast subculture method, which comprises the steps of removing the culture medium of the cells with the confluence degree of 80-90% obtained in the step S5, washing the cells by using a phosphate buffer solution, adding trypsin for digestion, observing the cells under a microscope, adding a special fibroblast culture medium to stop digestion when the cells shrink and become round, obtaining a cell suspension, and dividing one bottle to a plurality of bottles for subculturing to obtain P2 generation cells.
Compared with the prior art, the mouse or rat kidney fibroblast separation and subculture method has the following beneficial effects:
(1) the invention directly takes the complete kidney tissue of a mouse or a rat to separate the whole kidney tissue into the fibroblasts, the materials are easy to obtain, the operation is simple, and the consumed time is short;
(2) compared with the method using single trypsin for digestion, the method adopts the combined digestion of the trypsin and the collagenase IV, explores the proper digestion time, has milder digestion effect, and obtains small cell damage, high activity and large quantity;
(3) after the method is adopted to separate cells and culture the cells for 24 hours, a large amount of fibroblasts can be obtained, and after the cells are cultured for 5-7 days, the purity is up to more than 95 percent through immunofluorescence identification, and the cell purity is high.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a microscopic view (100X) of mouse fibroblasts cultured for 5 days according to the isolation culture method of the present invention;
FIG. 2 is a graph (200X) showing immunofluorescence assay of mouse fibroblast cells Vimentin obtained by the isolation and culture method of the present invention;
FIG. 3 is a microscopic picture (100X) of mouse P2 fibroblast cells obtained by the isolation and culture method of the present invention;
FIG. 4 is a microscope photograph (200X) of rat fibroblasts cultured for 24 hours by the isolation culture method of the present invention, in which black arrows indicate rat fibroblasts;
FIG. 5 is a microscopic view (100X) of rat fibroblast cells cultured for 5d according to the isolation culture method of the present invention;
FIG. 6 is a chart of immunofluorescence assay of mouse fibroblast cells Vimentin obtained by the isolation and culture method of the invention (200X).
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
The experimental methods in the following examples, which are not specified under specific conditions, were selected according to the conventional methods and conditions, or according to the commercial instructions. The reagents and starting materials used in the present invention are well known in the art and are readily available on the market. The reagents and laboratory instruments described in the examples below were sterilized prior to use.
Example 1
Taking a mouse born for 30 days, injecting 0.5mL of 10% chloral hydrate into the thoracic cavity of each mouse, anesthetizing, soaking in 75% alcohol for 5min for sterilization, and transferring into an ultra-clean workbench.
Cutting skin from the center of the abdomen of the mouse by using sterile scissors, opening the abdominal cavity, taking out the kidneys at two sides, putting the kidneys into a sterile culture dish, and adding a proper amount of phosphate buffer solution containing 1% penicillin/streptomycin double antibody; peeling off kidney capsule with microscope forceps, washing kidney with 1% penicillin/streptomycin double-antibody-containing phosphate buffer solution for 3 times, transferring kidney into 1.5mL EP tube, shearing kidney tissue with ophthalmic scissors, and shearing tissue to 1mm3To size, 500. mu.L of phosphate buffer containing 1% penicillin/streptomycin diabody was added and the tissue transferred to a 15mL centrifuge tube using a pipette gun.
Adding a digestive juice with 5 times of tissue volume into the centrifugal tube, dissolving the digestive juice containing 0.25% of trypsin and 0.2% of collagenase IV by using a phosphate buffer solution, placing the centrifugal tube into a water bath shaker, and carrying out shaking digestion at the temperature of 37 ℃ for 25 min. After digestion, the centrifugal tube is taken out and the tissue is repeatedly blown and beaten until no obvious block exists, the tissue fluid is filtered by a 200-mesh cell sieve, and the filtrate is centrifuged for 5min at 1200r/min to obtain cell sediment.
Resuspending the cell pellet with a fibroblast-specific culture medium, inoculating the cell pellet in a culture dish, and placing the culture dish at 37 deg.C and 5% CO2And (3) performing static culture in a constant-temperature incubator, replacing a fresh culture medium after 24 hours, replacing the culture solution once after 2 days, culturing the cells for 5 days, namely, growing to 90% of confluence degree, performing subculture, cleaning the cells for 1 time by using a phosphate buffer solution during passage, adding 0.5mL of 0.25% trypsin, observing the cells under a microscope, adding 3mL of a special fibroblast culture medium to stop digestion after the cells shrink and become round, obtaining cell suspension, and uniformly distributing the cell suspension to 3 bottles for passage by using 1 bottle to obtain the cells of the P2 generation.
Cell identification: taking the cells grown to 90% confluence, digesting the cells with 0.25% trypsin to obtain a cell suspension at 2X 105one/mL is inoculated in
And climbing a slide, and performing Vimentin immunofluorescence identification, wherein the result shows that the Vimentin positive rate is 97%, namely the cell purity is up to 97%.
Example 2
Taking mice born for 1 day, injecting 0.5mL of 10% chloral hydrate into the thoracic cavity of each mouse, anesthetizing, soaking in 75% alcohol for 5min for sterilization, and transferring into an ultra-clean bench.
Cutting skin from the center of the abdomen of the mouse by using sterile scissors, opening the abdominal cavity, taking out the kidneys at two sides, putting the kidneys into a sterile culture dish, and adding a proper amount of phosphate buffer solution containing 1% penicillin/streptomycin double antibody; peeling off kidney capsule with microscope forceps, washing kidney with 1% penicillin/streptomycin double-antibody-containing phosphate buffer solution for 2 times, transferring kidney into 1.5mL EP tube, shearing kidney tissue with ophthalmic scissors, and shearing tissue to 2mm3To size, 500. mu.L of phosphate buffer containing 1% penicillin/streptomycin diabase was added and the tissue was pipettedTransfer into a 15mL centrifuge tube.
Adding 3 times of digestive juice containing 0.05% trypsin and 0.05% collagenase IV into the centrifugal tube, dissolving with phosphate buffer solution, placing the centrifugal tube into a water bath shaker, and vibrating and digesting at 37 deg.C for 45 min. After digestion, the centrifugal tube is taken out and the tissue is repeatedly blown and beaten until no obvious block exists, the tissue fluid is filtered by a 200-mesh cell sieve, and the filtrate is centrifuged for 8min at 800r/min to obtain cell sediment.
Resuspending the cell pellet in DMEM medium containing 10% fetal calf serum and 1% penicillin/streptomycin double antibody, inoculating to a culture dish, placing the culture dish at 37 deg.C and 5% CO2And (3) carrying out static culture in a constant-temperature incubator, replacing a fresh culture medium after 24h, replacing the culture medium once after 3d, growing the cells to reach 80% of confluence degree after 7d, carrying out subculture, cleaning the cells for 1 time by using a phosphate buffer solution during the subculture, adding 0.5mL of 0.25% trypsin, observing the cells under a microscope, adding 3mL of a special fibroblast culture medium to stop digestion after the cells shrink and become round, obtaining a cell suspension, and uniformly distributing the cell suspension to 5 bottles for subculture by 1 bottle to obtain P2 generation cells.
Cell identification: taking the cells grown to 80% confluence, digesting the cells with 0.25% trypsin to obtain a cell suspension at 2X 105one/mL is inoculated in
And climbing a slide, and performing Vimentin immunofluorescence identification, wherein the result shows that the Vimentin positive rate is 95%, namely the cell purity is 95%.
Example 3
The method comprises the steps of taking rats born for 30 days, injecting 1mL of 10% chloral hydrate into the thoracic cavity of each rat, anesthetizing, soaking in 75% alcohol for 5min for disinfection, and transferring into an ultra-clean workbench.
Cutting skin from the middle of the abdomen of the rat by using sterile scissors, opening the abdominal cavity, taking out the kidneys at two sides, putting the kidneys into a sterile culture dish, and adding a proper amount of phosphate buffer solution containing 1% penicillin/streptomycin double antibody; the kidney envelope was stripped with a pair of tweezers and washed with 1% penicillin/streptomycin double antibody phosphate bufferViscera 3 times, kidney transferred into 1.5mL EP tube, and kidney tissue cut into pieces with ophthalmic scissors, and tissue cut into 3mm pieces3To size, 500. mu.L of phosphate buffer containing 1% penicillin/streptomycin diabody was added and the tissue transferred to a 15mL centrifuge tube using a pipette gun.
Adding a digestive juice with 5 times of tissue volume into the centrifugal tube, dissolving the digestive juice containing 0.25% of trypsin and 0.2% of collagenase IV by using a phosphate buffer solution, placing the centrifugal tube into a water bath shaker, and carrying out shaking digestion at the temperature of 37 ℃ for 25 min. After digestion, the centrifugal tube is taken out and the tissue is repeatedly blown and beaten until no obvious block exists, the tissue fluid is filtered by a 200-mesh cell sieve, and the filtrate is centrifuged for 3min at 1500r/min to obtain cell sediment.
Resuspending the cell pellet with a fibroblast-specific culture medium, inoculating the cell pellet in a culture dish, and placing the culture dish at 37 deg.C and 5% CO2And (3) performing static culture in a constant-temperature incubator, replacing a fresh culture medium after 24 hours, replacing the culture solution once after 2 days, culturing the cells for 5 days, namely, growing to 90% of confluence degree, performing subculture, cleaning the cells for 1 time by using a phosphate buffer solution during passage, adding 0.5mL of 0.25% trypsin, observing the cells under a microscope, adding 3mL of a special fibroblast culture medium to stop digestion after the cells shrink and become round, obtaining cell suspension, and uniformly distributing the cell suspension to 3 bottles for passage by using 1 bottle to obtain the cells of the P2 generation.
Cell identification: taking the cells grown to 90% confluence, digesting the cells with 0.25% trypsin to obtain a cell suspension at 2X 105one/mL is inoculated in
And climbing a slide, and performing Vimentin immunofluorescence identification, wherein the result shows that the Vimentin positive rate is 97%, namely the cell purity is up to 97%.
Example 4
Injecting 1mL 10% chloral hydrate into the thoracic cavity of each rat, anesthetizing, soaking in 75% alcohol for 5min, sterilizing, and transferring to an ultra-clean bench.
Cutting skin from the center of mouse abdomen with sterile scissors, opening abdominal cavity, taking out bilateral kidney, and placing into sterile culture dishAdding a proper amount of phosphate buffer solution containing 1% penicillin/streptomycin double antibody; peeling off kidney capsule with microscope forceps, washing kidney with 1% penicillin/streptomycin double-antibody-containing phosphate buffer solution for 2 times, transferring kidney into 1.5mL EP tube, shearing kidney tissue with ophthalmic scissors, and shearing tissue to 1mm3To size, 500. mu.L of phosphate buffer containing 1% penicillin/streptomycin diabody was added and the tissue transferred to a 15mL centrifuge tube using a pipette gun.
Adding 3 times of digestive juice containing 0.05% trypsin and 0.05% collagenase IV into the centrifugal tube, dissolving with phosphate buffer solution, placing the centrifugal tube into a water bath shaker, and vibrating and digesting at 37 deg.C for 45 min. After digestion, the centrifugal tube is taken out and the tissue is repeatedly blown and beaten until no obvious block exists, the tissue fluid is filtered by a 200-mesh cell sieve, and the filtrate is centrifuged for 6min at 1000r/min to obtain cell sediment.
Resuspending the cell pellet in DMEM medium containing 10% fetal calf serum and 1% penicillin/streptomycin double antibody, inoculating to a culture dish, placing the culture dish at 37 deg.C and 5% CO2And (3) carrying out static culture in a constant-temperature incubator, replacing a fresh culture medium after 24h, replacing the culture medium once after 3d, growing the cells to reach 80% of confluence degree after 7d, carrying out subculture, cleaning the cells for 1 time by using a phosphate buffer solution during the subculture, adding 0.5mL of 0.25% trypsin, observing the cells under a microscope, adding 3mL of a special fibroblast culture medium to stop digestion after the cells shrink and become round, obtaining a cell suspension, and uniformly distributing the cell suspension to 4 bottles for subculture by 1 bottle to obtain P2 generation cells.
Cell identification: taking the cells grown to 80% confluence, digesting the cells with 0.25% trypsin to obtain a cell suspension at 2X 105one/mL is inoculated in
And climbing a slide, and performing Vimentin immunofluorescence identification, wherein the result shows that the Vimentin positive rate is 96%, namely the cell purity reaches 96%.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (9)
1. A method for separating mouse or rat kidney fibroblasts, which is characterized by comprising the following steps:
s1, aseptically obtaining the complete kidney of a mouse or rat born for 1-30 days;
s2, repeatedly washing the kidney obtained in the step S1 with phosphate buffer solution containing 1% (v/v) penicillin/streptomycin double antibody for 2-3 times, and cutting into 1-3mm3A size tissue mass;
s3, digesting the tissue blocks by using digestive juice with the volume 3-5 times that of the tissue blocks;
s4, stopping digestion, sieving by a cell sieve, centrifuging, and removing supernatant to obtain cell sediment;
s5, resuspending the cell pellet in complete medium, inoculating the cell pellet into a culture dish, placing the culture dish at 37 ℃ and 5% CO2Carrying out static culture in a constant-temperature incubator, replacing a fresh culture medium after 24 hours to continue culture, then replacing the culture medium every 2-3 days, and culturing for 5-7 days to obtain cells with 80-90% confluency;
s6, inoculating the cells with the confluency of 80-90% in the step S5, digesting the cells with trypsin to obtain cell suspension, inoculating a slide, and performing Vimentin immunofluorescence identification.
2. A method of isolating mouse or rat kidney fibroblasts as claimed in claim 1 wherein: step S1 the kidney is a bilateral kidney of a mouse or rat with a stripped kidney capsule.
3. A method of isolating mouse or rat kidney fibroblasts as claimed in claim 1 wherein: the digestive fluid in step S3 includes trypsin and collagenase type IV.
4. A mouse or rat kidney fibroblast isolation method of claim 3 wherein: the concentration of the trypsin is 0.05-0.25% (w/v), and the concentration of the type IV collagenase is 0.05-0.2% (w/v).
5. A method of isolating mouse or rat kidney fibroblasts as claimed in claim 1 wherein: in the step S3, the digestion conditions are that the digestion time is 25-45min, the digestion temperature is 37 ℃, and the digestion is carried out in a water bath oscillation mode.
6. A method of isolating mouse or rat kidney fibroblasts as claimed in claim 1 wherein: the cell mesh number in step S4 is 200 mesh.
7. A method of isolating mouse or rat kidney fibroblasts as claimed in claim 1 wherein: in the step S4, the centrifugal rotation speed is 800-1500r/min, and the centrifugal time is 3-8 min.
8. A method of isolating mouse or rat kidney fibroblasts as claimed in claim 1 wherein: the complete culture medium in the step S5 is a special fibroblast culture medium or a DMEM culture medium containing 10% (v/v) fetal calf serum and 1% (v/v) penicillin/streptomycin double antibody.
9. A method of subculturing mouse or rat kidney fibroblasts isolated from the method of claim 1, comprising: and (3) discarding the culture medium of the cells with the confluence degree of 80-90%, washing by using a phosphate buffer solution, adding trypsin for digestion, observing the cells under a microscope, adding a special fibroblast culture medium to stop digestion when the cells shrink and become round, obtaining a cell suspension, and uniformly dividing one bottle to a plurality of bottles for passage to obtain the cells of the P2 generation.
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| CN114231479A (en) * | 2021-11-09 | 2022-03-25 | 中国医科大学附属第四医院 | Kit for extracting human primary liver cancer fibroblast and extraction method |
| CN114045255A (en) * | 2021-11-23 | 2022-02-15 | 广州华越肾科再生医学科技有限公司 | Method for separating and culturing renal tubular epithelial cells |
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