Guangxi Bama miniature pig fetus kidney fibroblast subculture method
Through the combination of mechanical isoenzyme digestion method and commercial formula culture medium, the isolation and culture process of fetal renal fibroblasts of Bama minipigs in Guangxi has been simplified, and the problems of cumbersome and high cost in the existing technology have been solved, and stability has been achieved. Cell passage and cryogenic resuscitation provide important donor cells for nuclear transplantation and transgenic research.
Patent Information
- Application Number
- CN202411477596.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-22
- Publication Date
- 2025-02-14
AI Technical Summary
The existing research methods for pig kidney fibroblasts are cumbersome and expensive, making it difficult to establish effective donor cell lines.
The cell isolation and culture of small pig fetal renal fibroblasts were isolated and cultured by mechanical isoenzyme digestion method, and the finished commercial formula culture medium was used to simplify the cell isolation and culture process.
The in vitro sub-culture system of fetal renal fibroblasts of Bama minipigs in Guangxi was successfully established. The cells can be passaged for a long time and frozen resuscitation, providing a large number of donor cells for nuclear transplantation and transgenic research.
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Figure CN119432710A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of cell subculture, and in particular to a subculture method for Guangxi Bama miniature pig fetal kidney fibroblasts. Background Art
[0002] In modern medical research, especially in comparative medical research, miniature pigs are of great significance. Miniature pigs have great similarities with humans in organ morphology, structure, size, metabolism and physiological characteristics. They have more physiological advantages than traditional experimental rodents such as mice and rats, and are more economical than primates. The construction of human disease animal models in miniature pigs through gene editing technology is of great significance for the study of human genetic diseases, and gene-modified miniature pigs are now an important direction for xenotransplantation donor research. At present, the most economical and effective way to produce gene-edited miniature pigs is somatic cell nuclear transplantation. So far, many human disease model miniature pigs and xenotransplantation transgenic and modified miniature pigs have been born at home and abroad. However, the efficiency of somatic cell nuclear transplantation is affected by many factors, but the establishment of the donor nuclear cell line is the most important. There are great differences in the transfection efficiency of different types of donor nuclear cells and the development efficiency of the embryos produced as donor nuclei. At present, it has been reported that granulosa / cumulus cells, skin fibroblasts, fetal fibroblasts, heart cells, ear fibroblasts, testicular fibroblasts, kidney fibroblasts and other cells have been used to successfully obtain cloned surviving offspring in pigs.
[0003] Wang Xianwei and others successfully isolated and cultured renal fibroblasts of newborn Guangxi Bama miniature pigs for the first time. Many domestic scholars have also isolated, cultured and identified renal interstitial fibroblasts of humans and other animals, but most of them remain at the level of cell research or provide materials for the study of renal interstitial fibroblast mechanisms. There are very few studies on pig renal fibroblasts. Only Lu Shengsheng and others have conducted some research on pig fetal renal fibroblasts. The research mainly uses ordinary pig fetuses as experimental materials, which requires surgery or killing sows. The procedure is cumbersome and increases costs. Summary of the invention
[0004] The purpose of the present invention is to provide a convenient method for subculture of Guangxi Bama miniature pig fetal kidney fibroblasts, so as to solve the problem that the existing research methods of pig kidney fibroblasts are complicated and costly.
[0005] Mechanical and enzymatic digestion was used to separate and culture Guangxi Bama miniature pig fetal renal fibroblasts. The culture medium used a ready-made commercial formula, which eliminated the need for tedious compounding of various growth factors and was more conducive to studying their morphology and growth characteristics under in vitro culture conditions. It also provided a large number of abundant donor cells for somatic cell nuclear transplantation and transgenic nuclear transplantation, which is of great significance for improving the efficiency of nuclear transplantation and the materials for research.
[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:
[0007] A method for subculture of Guangxi Bama miniature pig fetal kidney fibroblasts, the method comprising the following steps:
[0008] Step 1: Obtain cell material;
[0009] Step 2: Subculture the cells;
[0010] Step 3: Cryopreservation and thawing;
[0011] Step 4: Draw the cell growth curve;
[0012] Step 5: Cell type identification.
[0013] Furthermore, the specific process of step 1 is:
[0014] Obtain the 35-day gestational age pig fetus of Guangxi Bama miniature pig, remove the placenta, store on ice for 1 hour and transport back to the laboratory for isolation and culture, rinse with pre-cooled 75% alcohol 5 times, thoroughly wash the fetus with PBS containing 5% penicillin and streptomycin antibiotics, then soak in 75% alcohol for 1 minute, and then thoroughly wash the fetus with PBS containing 2% penicillin and streptomycin. Dissect the fetus in a biosafety cabinet to separate the bilateral kidneys, rinse the fetal kidneys with sterile PBS, transfer to a 100 mm culture dish, separate the renal matrix with ophthalmic scissors and cut it into pieces, add 2 ml Wash twice with PBS by centrifugation, add a little DMEM culture medium, add preheated trypsin, and use a 1ml pipette tip to blow the tissue fragments in the culture dish to obtain a tissue suspension, add it to the culture medium and centrifuge to discard the supernatant, resuspend it with complete culture medium and transfer it to a 30mm culture dish, and culture it in a 5% CO2 incubator overnight. The incubator temperature is 37℃ and the humidity is 100%. Observe that the cells adhere to the wall, and fibroblasts are seen growing in about 3 days. Then change the medium every 3 days. When the cells grow to 70%-80% fusion, they are subcultured or frozen.
[0015] Furthermore, the specific process of step 2 is:
[0016] When the primary cultured cells grow to 90%-100% confluence, aspirate the original culture medium and rinse with PBS 1-2 times, then add the compound digestion solution and digest at 37℃ for 3min-5min. After tapping the culture dish until 80% of the cells begin to fall off, add complete culture medium to terminate digestion, and use the tip of the pipette to blow repeatedly to completely fall off the culture dish. Transfer the digested cell mixture to a centrifuge tube, centrifuge at 1000r / min for 5min, remove the supernatant, resuspend in PBS and centrifuge again, remove the supernatant, resuspend in complete culture medium, count the cells, and press 10 5Each culture flask was inoculated with 100 cells, and the culture medium was changed after 48 hours of culture at 37°C, 5% CO2, and 100% humidity. The medium was changed every 3 days, and the cells were passaged and frozen when they grew to 90%-100% confluence.
[0017] Further, the composite digestion solution includes 0·05% Trypsin and 0·02% EDTA.
[0018] Furthermore, the specific process of step 3 is:
[0019] When the primary cultured cells grow to 90%-100% confluence, aspirate the original culture medium and rinse with PBS 1-2 times, then add the compound digestion solution and digest at 37℃ for 3min-5min. After tapping the culture dish until 80% of the cells begin to fall off, add complete culture medium to terminate digestion, and use the tip of the pipette to blow repeatedly to completely fall off the culture dish. Transfer the digested cell mixture to a centrifuge tube and centrifuge at 1000r / min for 5min. Remove the supernatant, resuspend in PBS and centrifuge again. Count the cells and calculate the number of cells per 10 6 After adding 1 ml of cell freezing solution to each cell and beating evenly, the cells are divided into cell freezing tubes, sealed with sealing film, and the freezing tubes are placed in a cell freezing box with isopropanol, placed in a -80℃ refrigerator overnight, and then stored in a liquid nitrogen tank.
[0020] When reviving cells, take out the cryovials from liquid nitrogen, place them in a 37°C ultrasonic water bath, and sonicate at 20% intensity for 20 seconds. After all the ice has melted, add preheated complete culture medium and mix. Transfer the cell suspension to a centrifuge tube and centrifuge. After washing with PBS and centrifuging, inoculate the cells into a T25 culture flask for culture.
[0021] Furthermore, the specific process of step 4 is:
[0022] The cells that were cultured for 20 generations were collected and then 5×10 3 Cells were inoculated into each well of a 96-well culture plate and cultured in a CO2 incubator under saturated humidity and 37°C. Cell viability was determined by CCK8 method every 24 hours. The day of inoculation was recorded as 1 day, and a total of 7 days were measured with the number of culture days as the horizontal axis and the absorbance as the vertical axis to form a graph, namely the growth curve.
[0023] Furthermore, the specific process of step 5 is:
[0024] The cells seeded in the confocal dish were fixed with paraformaldehyde for 30 min, washed three times with pre-cooled PBS, dialyzed with 0.1% Triton X-100-PBS at room temperature for 5 min, blocked with 3% BSA-PBS for 30 min, washed twice with 0.2% BSA-PBS, incubated with mouse anti-vimentin monoclonal antibody diluted in 0.1% BSA-PBS at 37°C for 1 h or 4°C overnight, washed three times with 0.1% BSA-PBS, 1XPBS, and once with 0.1% BSA-PBS, stained with DAPI at a ratio of 1:1000 with 0.1% BSA-PBS for 5 min, washed three times with PBS, and incubated with FITC-labeled goat anti-rabbit IgG diluted 1:200 at room temperature for 30 min, washed three times with PBS, and the samples were mounted in low-fading mounting medium and examined by fluorescence microscopy.
[0025] The present invention has the following beneficial effects due to the adoption of the above technical solution:
[0026] The present invention utilizes a mechanical method combined with an enzyme digestion method to separate and culture primary Guangxi Bama miniature pig fetal kidney fibroblasts, and obtains a primary cell line that can be routinely subcultured, frozen and revived; the proliferation potential is detected by a CCK8 method, and the cells are identified by immunofluorescence, and as a result, newborn Guangxi Bama miniature pig kidney fibroblasts are successfully separated, and a large number of subcultures and frozen can be performed in vitro, and currently more than 20 generations have been passed, and the growth is good; after freezing and resuscitation, normal culture and subculture can still be performed; cell immunofluorescence detection shows that vimentin expression is positive and keratin expression is negative, and the results show that an in vitro culture system of Guangxi Bama miniature pig embryonic kidney fibroblasts is successfully established, and the cells can be stably cultured and subcultured in vitro. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 This is a 35-day-old fetus of Guangxi Bama miniature pig of the present invention;
[0028] Figure 2 This is a diagram of the kidney of a 35-day-old Guangxi Bama miniature pig fetus of the present invention;
[0029] Figure 3 is a diagram of primary cultured cells of the present invention;
[0030] Figure 4 This is a diagram of cells cultured for 72 hours according to the present invention;
[0031] Figure 5 This is a diagram of the culture of cells after recovery according to the present invention;
[0032] Figure 6 is a growth curve diagram of the present invention;
[0033] Figure 7 is an anti-vimentin staining image of renal fibroblasts of the present invention;
[0034] Figure 8 It is a nuclear staining diagram of renal fibroblasts of the present invention;
[0035] Fig. 9 is the Merge diagram of renal fibroblasts of the present invention;
[0036] Fig.10 This is a picture of vimentin staining of embryonic skin fibroblasts of the present invention;
[0037] Fig.11 It is a nuclear staining diagram of embryonic skin fibroblasts of the present invention;
[0038] Fig.12 This is the Merge diagram of embryonic fibroblasts of the present invention. DETAILED DESCRIPTION
[0039] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and preferred examples. However, it should be noted that many details listed in the specification are only for the purpose of enabling the reader to have a thorough understanding of one or more aspects of the present invention, and these aspects of the present invention can be realized even without these specific details.
[0040] A method for subculture of Guangxi Bama miniature pig fetal kidney fibroblasts, the method comprising the following steps:
[0041] 1.1 Main Materials
[0042] Culture plates and dishes (COSTAR), trypsin (Gibco), fetal bovine serum (Gibco), DMEM (high glucose, GlutaMAX TM Additives (pyruvate, Gibco), penicillin and streptomycin (Sigma), fetal bovine serum (FBS) (Gibco), DMSO (Sigma), mouse anti-human cytokeratin monoclonal antibody, mouse anti-human vimentin monoclonal antibody, and goat anti-rabbit fluorescein antibody were purchased from Chengdu Zhengneng Biotechnology Co., Ltd.
[0043] 1.2 Material Acquisition
[0044] The pig fetus of Guangxi Bama miniature pig with a gestational age of 35 days was obtained from the College of Animal Science and Technology of Guangxi University. The fetal membrane was removed and the body length was 3 cm. It was stored on ice and transported back to the laboratory for isolation and culture within 1 hour. First, it was rinsed 5 times with pre-cooled 75% alcohol, and then the fetus was thoroughly washed with PBS containing 5% penicillin and streptomycin antibiotics. Then, it was soaked in 75% alcohol for 1 minute and transferred to a biosafety cabinet. Finally, the fetus was thoroughly rinsed with PBS containing 2% penicillin and streptomycin. The fetus was dissected in the biosafety cabinet, and the bilateral kidneys were separated and obtained. The fetal kidneys were rinsed with pre-cooled sterile PBS and transferred to a 30mm culture dish. The renal interstitium was separated and chopped with ophthalmic scissors, and 3 ml of pre-heated trypsin was added. The tissue fragments were blown in the culture dish with a 1 ml pipette to obtain a tissue suspension, which was transferred to an incubator for 10 minutes, added to the culture medium and centrifuged to discard the supernatant, and the complete DMEM (Gibco TM , DMEM, high glucose, GlutaMAX TM Additive, pyruvate) medium, resuspend in 30mm culture dish, put into 5% CO2 incubator and culture overnight. The temperature of incubator is 37℃, and the humidity is 100%. Cells adhere to the wall and fibroblasts are seen growing in about 3 days. Then the medium is changed every 3 days. When the cells grow to 70%-80% confluence, they are subcultured or frozen.
[0045] 1.3 Cell subculture method
[0046] When the primary cultured cells grow to 90%-100% confluence, discard the original culture medium and rinse 1-2 times with PBS, then add compound digestion solution (0.05% Trypsin + 0.02% EDTA) at 37℃ for 3-5 minutes. After 80% of the cells begin to fall off by gently tapping the culture dish, add complete culture medium to terminate digestion, and use the tip of the pipette to blow repeatedly to completely fall off the culture dish. Transfer the digested cell mixture to a centrifuge tube and centrifuge at 1000r / min for 5 minutes. Remove the supernatant, resuspend in PBS and centrifuge again, remove the supernatant, resuspend in complete culture medium, count the cells, and press 10 5 Each culture flask (T25) was inoculated with 100 cells and cultured at 37°C, 5% CO2, and 100% humidity for 48 hours before the medium was changed. The medium was changed every 3 days and the cells were passaged and frozen when they grew to 90%-100% confluence.
[0047] 1.4 Cryopreservation and thawing
[0048] When the primary cultured cells grow to 90%-100% confluence, discard the original culture medium and rinse 1-2 times with PBS, then add compound digestion solution (0.05% Trypsin + 0.02% EDTA) at 37℃ for 3-5 minutes, tap the culture dish gently, and when 80% of the cells begin to fall off, add complete culture medium to terminate digestion, use the tip of the pipette to blow repeatedly to completely fall off the culture dish, transfer the digested cell mixture to a centrifuge tube, and centrifuge at 1000r / min for 5 minutes. Remove the supernatant, resuspend in PBS and centrifuge again, count the cells, and calculate the number of cells per 10 6 Add 1 ml of cell freezing solution (10% DMSO, 20% FBS, 70% MAX DMEM) to each cell, pipette evenly, and dispense into cell freezing tubes, seal with sealing film. Place the freezing tubes in a cell freezing box with isopropanol, place in a -80℃ refrigerator overnight, and then store in a liquid nitrogen tank.
[0049] When reviving cells, take out the cryovials from liquid nitrogen, place them in a 37°C ultrasonic water bath, and sonicate at 20% intensity for 20 seconds. After all the ice has melted, add preheated complete culture medium and mix. Transfer the cell suspension to a centrifuge tube and centrifuge (1000r / min, 5min). After washing with PBS and centrifuging, inoculate the cells into T25 culture flasks for culture.
[0050] 1.5 Cell growth curve drawing
[0051] The cells that were cultured for 20 generations were collected and then 5×10 3 Cells were inoculated into each well of a 96-well culture plate and cultured in a CO2 incubator under saturated humidity and 37°C. Cell viability was determined by CCK8 method every 24 hours. The day of inoculation was recorded as 1 day, and a total of 7 days were measured with the number of culture days as the horizontal axis and the absorbance as the vertical axis to form a graph, namely the growth curve.
[0052] 1.6 Cell type identification
[0053] Fix the cells planted in the confocal dish with paraformaldehyde for 30 minutes, wash three times with pre-cooled PBS, dialyze the cells with 0.1% Triton X-100-PBS at room temperature for 5 minutes, and block with 3% BSA-PBS for 30 minutes (30 ml can be prepared); wash the cells twice with 0.2% BSA-PBS (prepare 50 ml), dilute the mouse anti-vimentin monoclonal antibody with 0.1% BSA-PBS (50 ml) (the dilution concentration is 1:200), and incubate at 37°C for 1 hour or 4°C overnight (dilution 1:500). The cells were gently washed three times with 0.1% BSA-PBS, 1XPBS, washed once with 0.1% BSA-PBS, stained with DAPI at a 1:1000 ratio with 0.1% BSA-PBS for 5 min, washed three times with PBS, and incubated with 1:200 diluted FITC-labeled goat anti-rabbit IgG at room temperature for 30 min; washed three times with PBS, and the samples were mounted in low-fading mounting medium and examined by fluorescence microscopy.
[0054] 2 Results Analysis
[0055] 2.1 Primary culture
[0056] The fetus of Guangxi Bama miniature pig at 35 days of gestation is about 3 cm long, and the kidney is about 0.5 cm × 0.5 cm long ( Figure 1 ). In the early stage of kidney tissue fragment culture, a large number of tubular objects can be observed floating, with renal tubules or collecting ducts floating. After 4 hours, the epithelial-like cells can be seen growing around the tissue block. From the second day, fibroblasts will be freed from the tissue block to form a growth circle. In primary culture, the cells are spindle-shaped, elongated, polygonal or irregular ( Figure 1 ); After 3-5 cell passages, the cell bodies were observed under an inverted microscope to be long spindle-shaped, which were fibroblasts. They gradually became larger and the distance between them finally decreased, and the cells merged ( Figure 3 ). A large number of subcultures found that the cells can become confluent after subculturing at a ratio of 1:4 for about 4 days ( Figure 4 ).
[0057] 2.2 Cell cryopreservation, thawing, and recovery
[0058] Starting from the second generation, fetal fibroblasts were cryopreserved every three generations. After a period of time, the cells of the 10th generation were randomly thawed using the conventional cryopreservation method. After the cells recovered overnight, the cells were observed to be attached to the wall and growing in good condition. There was no significant difference in morphology between the passaged cells and the primary cells ( Figure 5 ).
[0059] 2.3 Growth curve
[0060] The porcine fetal kidney fibroblasts inoculated by the digestion method were in a slow growth period from the 2nd to the 3rd day, and the cells began to grow rapidly on the 3rd day. The exponential growth of the cells from the 3rd to the 5th day was a rapid growth period. After the 6th day, the cells returned to a plateau period. The growth curve is shown in Figure 6 .
[0061] 2.4 Cell type identification
[0062] After culturing cells for 3 generations, all single cells were spindle cells. Anti-vimentin immunofluorescence staining showed green cytoplasm and blue nuclei ( Figure 7 , 8 , 9) were positive. The fluorescent positive control group was embryonic skin fibroblasts, with the nucleus stained blue and the cytoplasm stained green ( Fig.10 , 11 , 12).
[0063] Primary Bama miniature pig fetal renal fibroblasts were isolated and cultured by mechanical method combined with enzyme digestion method, and primary cell lines that can be routinely subcultured, frozen and revived were obtained; the proliferation potential was detected by CCK8 method, and the cells were identified by immunofluorescence. Results Newborn Bama miniature pig fetal renal fibroblasts were successfully isolated, which can be subcultured and frozen in large quantities in vitro. They have been subcultured for more than 20 generations and have grown well; they can still be cultured and subcultured normally after freezing and thawing; cell immunofluorescence detection showed that vimentin expression was positive and keratin expression was negative. The results showed that this experiment successfully established an in vitro culture system for Bama miniature pig fetal renal fibroblasts in Guangxi, which can be stably cultured and subcultured in vitro.
[0064] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A method for subculturing Guangxi Bama miniature pig fetal kidney fibroblasts, characterized in that: The method comprises the following steps: Step 1: Obtain cell material; Step 2: Subculture the cells; Step 3: Cryopreservation and thawing; Step 4: Draw the cell growth curve; Step 5: Cell type identification.
2. The method for subculture of Guangxi Bama miniature pig fetal kidney fibroblasts according to claim 1, characterized in that: The specific process of step 1 is: Obtain the 35-day gestational age pig fetus of Guangxi Bama miniature pig, remove the placenta, store on ice for 1 hour and transport back to the laboratory for isolation and culture. First, rinse with pre-cooled 75% alcohol for 5 times, then thoroughly wash the fetus with PBS containing 5% penicillin and streptomycin antibiotics, then soak with 75% alcohol for 1 minute, transfer to a biosafety cabinet, and finally thoroughly rinse the fetus with PBS containing 2% penicillin and streptomycin. Dissect the fetus in the biosafety cabinet, separate and obtain bilateral kidneys, rinse the fetal kidneys with pre-cooled sterile PBS, transfer to a 30mm culture dish, separate the renal interstitium with ophthalmic scissors and cut it into pieces, add 3ml of pre-heated trypsin, and use a 1ml pipette tip to blow the tissue fragments in the culture dish to obtain a tissue suspension, transfer to an incubator and place for 10 minutes, add the culture medium and centrifuge and discard the supernatant, and use complete DMEM (Gibco) containing 10% serum. TM , DMEM, high glucose, GlutaMAX TM Additive, pyruvate) medium, resuspend in 30mm culture dish, put into 5% CO2 incubator and culture overnight. The temperature of incubator is 37℃, and the humidity is 100%. Cells adhere to the wall and fibroblasts are seen growing in about 3 days. Then the medium is changed every 3 days. When the cells grow to 70%-80% confluence, they are subcultured or frozen.
3. The method for subculture of Guangxi Bama miniature pig fetal kidney fibroblasts according to claim 1, characterized in that: The specific process of step 2 is: When the primary cultured cells grow to 90%-100% confluence, the original culture medium is aspirated and the cells are rinsed with PBS 1-2 times. Then, the complex digestion solution is added and digested at 37℃ for 3min-5min. After the culture dish is tapped until 80% of the cells begin to fall off, complete culture medium is added to terminate the digestion. The cells are repeatedly blown with a pipette tip to completely fall off the culture dish. The digested cell mixture is transferred to a centrifuge tube and centrifuged at 1000r / min for 5min. The supernatant is removed, the cells are resuspended in PBS and centrifuged again. The supernatant is removed, the cells are resuspended in complete culture medium and counted. The cells are counted according to 10 5 Each culture flask was inoculated with 100 cells and cultured at 37°C, 5% CO2, and 100% humidity for 48 hours, followed by medium change every 3 days. When the cells grew to 90%-100% confluence, they were passaged and cryopreserved.
4. The method for subculture of Guangxi Bama miniature pig fetal kidney fibroblasts according to claim 3, characterized in that: The complex digestion solution included 0.05% Trypsin and 0.02% EDTA.
5. The method for subculture of Guangxi Bama miniature pig fetal kidney fibroblasts according to claim 1, characterized in that: The specific process of step 3 is: When the primary cultured cells grow to 90%-100% confluence, the original culture medium is aspirated and the cells are rinsed with PBS 1-2 times. Then, the compound digestion solution is added and digested at 37℃ for 3min-5min. After the culture dish is tapped until 80% of the cells begin to fall off, complete culture medium is added to terminate the digestion. The cells are repeatedly blown with a pipette tip to completely fall off the culture dish. The digested cell mixture is transferred to a centrifuge tube and centrifuged at 1000r / min for 5min. The supernatant is removed, the cells are resuspended in PBS and centrifuged again. The cells are counted and the number of cells is calculated according to the number of cells per 10 6 Add 1 ml of cell freezing solution to each cell, pipet evenly and dispense into cell freezing tubes, seal with sealing film, place the freezing tubes in a cell freezing box with isopropanol, place in a -80℃ refrigerator overnight, and then store in a liquid nitrogen tank. When reviving cells, take out the cryovials from liquid nitrogen, place them in a 37°C ultrasonic water bath, and sonicate at 20% intensity for 20 seconds. After all the ice has melted, add preheated complete culture medium and mix. Transfer the cell suspension to a centrifuge tube and centrifuge. After washing with PBS and centrifuging, inoculate the cells into a T25 culture flask for culture.
6. The method for subculture of Guangxi Bama miniature pig fetal kidney fibroblasts according to claim 1, characterized in that: The specific process of step 4 is as follows: take cells that have been cultured for 20 generations and add 5×10 3 Cells were inoculated into each well of a 96-well culture plate and cultured in a CO2 incubator at saturated humidity and 37°C. Cell viability was determined by CCK8 method every 24 hours. The day of inoculation was recorded as 1 day, and a total of 7 days were measured with the number of culture days as the horizontal axis and the absorbance as the vertical axis, which was the growth curve.
7. The method for subculture of Guangxi Bama miniature pig fetal kidney fibroblasts according to claim 1, characterized in that: The specific process of step 5 is: Cells seeded in confocal dishes were fixed with paraformaldehyde for 30 min, washed three times with pre-cooled PBS, dialyzed with 0.1% Triton X-100-PBS at room temperature for 5 min, blocked with 3% BSA-PBS for 30 min, washed twice with 0.2% BSA-PBS, incubated with mouse anti-vimentin monoclonal antibody diluted in 0.1% BSA-PBS at 37°C for 1 h or 4°C overnight, washed three times with 0.1% BSA-PBS, 1XPBS, washed once with 0.1% BSA-PBS, stained with DAPI at a ratio of 1:1000 in 0.1% BSA-PBS for 5 min, washed three times with PBS, and incubated with FITC-labeled goat anti-rabbit IgG diluted 1:200 at room temperature for 30 min, washed three times with PBS, and the samples were mounted in low-fading mounting medium and examined by fluorescence microscopy.