Separation method of yak fallopian tube epithelial cells

Abstract

The invention discloses a yak fallopian tube epithelial cell separation method, and relates to the technical field of cell separation culture. According to the method, collagenase and Accutase are combined for use, so that the cell digestion process becomes milder and more efficient, and the problems that the cell surface antigen is damaged by pancreatin digestion, so that the separated cells cannot maintain the integrity of the cell antigen, the cell differentiation is influenced and the like are solved. When collagenase and Accutase are combined to hydrolyze collagen of an extracellular matrix, DNase I is added, so that DNA released in the cell separation process can be effectively reduced, the cell viscosity and agglutination are reduced, the cell separation efficiency is improved, the digestion efficiency is promoted to be improved, and the activity and adherence of cells are greatly improved.

Description

Technical Field

[0001] The invention relates to the technical field of cell separation and culture, and in particular to a method for separating yak fallopian tube epithelial cells. Background Art

[0002] Yaks are distributed in the high mountains and sub-alpine areas centered on the Qinghai-Tibet Plateau and its adjacent areas. They are one of the most precious animal genetic resources in my country and an extremely valuable gene bank. If animal husbandry is the pillar industry of the Qinghai-Tibet Plateau, then yaks are the dominant livestock species on the Qinghai-Tibet Plateau. The breeding level of yaks determines the development of animal husbandry on the Qinghai-Tibet Plateau. Among them, female yaks, as an important part of the breeding system, play an important role in genetic breeding.

[0003] The oviduct of yaks is an important organ of the reproductive system, connecting the ovaries and uterus. As one of the sites for early embryonic development, the oviduct fluid produced by the oviduct epithelial secretory cells, the swing of ciliated cells, and the movement of the smooth muscle of the tube wall provide necessary support for fertilization, gamete and early embryo transportation, and early embryonic development, and play an important role in reproductive physiology. Therefore, obtaining oviduct epithelial cells with high purity and good activity can provide experimental materials and research basis for in-depth analysis of the reproductive response mechanism of the yak oviduct; overcome the developmental block of early embryos in vitro; and improve the quality of embryonic development, thereby improving the pregnancy rate of yaks.

[0004] In 2013, Sun Yonggang et al. separated yak fallopian tube epithelial cells by mechanical scraping and 0.25% trypsin digestion for in vitro culture; in 2015, Yang Rong separated yak fallopian tube epithelial cells by digestion of cut fallopian tube tissue blocks with trypsin I (containing EDTA) plus collagenase I. To date, the isolation and culture methods of yak fallopian tube epithelial cells have not been updated. Among the current in vitro isolation methods of yak fallopian tube epithelial cells, the mechanical scraping method can only obtain tissue clumps of varying sizes, with a small number of dispersed single cells, and larger cell clumps are not easy to adhere to the wall and are suspended on the surface of the culture medium, and are easily mixed with other cell clumps, and epithelial cells with higher purity cannot be obtained; although the combined trypsin digestion method can obtain more single cells and smaller cell clumps, trypsin causes greater damage to epithelial cells, which slows down the proliferation rate of epithelial cells after adherence, increases the number of cell fragments in the cell suspension, and makes the prepared culture medium viscous. The method of first digesting the tissue block with trypsin I (containing EDTA) and then digesting it with collagenase I causes great damage to the epithelial cells. In addition, if the tissue block is first digested with trypsin, there will be many fibroblasts. After separation and culture, differential digestion and differential adhesion methods are required to remove the fibroblasts that are easily digested and adherent to the wall in order to obtain high-purity fallopian tube epithelial cells.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a method for isolating yak fallopian tube epithelial cells to solve the above technical problems.

[0007] The present invention is achieved in that:

[0008] In a first aspect, the present invention provides a method for isolating yak fallopian tube epithelial cells, which comprises the following steps:

[0009] S1: Take the oviducts of the yak from both sides and rinse them, then cut the oviducts longitudinally, scrape the upper tissue into a container with a knife, add the resuspension solution to resuspend, centrifuge, and discard the supernatant;

[0010] S2: Digestion: Add digestion solution to the container where the supernatant was discarded, and digest with shaking at 37℃±0.5℃ for 1-1.2h; the digestion solution includes collagenase I, Accutase digestion solution and DNase I, and the mixing ratio of collagenase I mass, Accutase digestion solution enzyme amount and DNase I enzyme amount in each ml of digestion solution is 0.1-0.2mg:100-200U:5-10U; the mixing volume ratio of digestion solution and tissue to be digested is 2-2.5:1-1.5;

[0011] S3: After terminating the digestion, the digestion mixture is filtered, the cell filtrate is collected and centrifuged, and the supernatant is discarded.

[0012] The present invention has the following beneficial effects:

[0013] The present invention uses collagenase and Accutase in combination, making the cell digestion process more gentle and efficient, avoiding the problem that pancreatic digestion causes damage to cell surface antigens, causing the separated cells to be unable to maintain the integrity of cell antigens, affecting cell differentiation, and other problems. When collagenase and Accutase jointly hydrolyze the collagen of the extracellular matrix, adding DNaseⅠ can effectively reduce the DNA released during cell separation, reduce cell viscosity and agglutination, improve cell separation efficiency, promote the improvement of digestion efficiency, and greatly improve cell activity and adhesion. The yak fallopian tube epithelial cells separated by the method provided by the present invention do not need to be subjected to differential digestion method, differential adhesion method and other methods to remove fibroblasts that are easily digested and easy to adhere to the wall. The preparation method is simpler, and high-purity fallopian tube epithelial cells can be obtained quickly and efficiently.

[0014] The method provided by the present invention is more applicable to mammalian cell lines. As a cell model for overcoming the developmental block of early embryos in vitro, promoting the development of early embryos and improving the quality of embryonic development, maintaining the integrity of cell antigens after separation is crucial to protecting the undifferentiated state and pluripotency of cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0016] Figure 1 This is a microscopic image of the cell morphology of the oviduct epithelial cells isolated by the method for isolating yak oviduct epithelial cells provided in Example 1 after being cultured for 4 days (magnification 4 times);

[0017] Figure 2 This is a microscopic image of the cell morphology of the oviduct epithelial cells isolated by the method for isolating yak oviduct epithelial cells provided in Example 1 after being cultured for 6 days (magnification 10 times);

[0018] Figure 3 The fallopian tube epithelial cells obtained by the separation method of Example 1 were cultured for the third generation, digested with 0.25% trypsin, and cultured on a cell slide for 24 hours, and then stained with hematoxylin and eosin for micrographs (magnification 20 times);

[0019] Figure 4 This is a picture of the immunofluorescence identification result of the fallopian tube epithelial cell suspension obtained by the separation method of Example 1 (magnification 10 times);

[0020] Figure 5 The cell morphology microscopic result of the oviduct epithelial cells isolated by the isolation method of yak oviduct epithelial cells provided in Comparative Example 1 after being cultured for 3 days (magnification 10 times);

[0021] Figure 6 This is a microscopic image of the cell morphology of the oviduct epithelial cells isolated by the isolation method of yak oviduct epithelial cells provided in Comparative Example 1 after being cultured for 6 days (magnification 10 times);

[0022] Figure 7 This is a microscopic image of the cell morphology of the oviduct epithelial cells isolated by the isolation method of yak oviduct epithelial cells provided in Comparative Example 2 after being cultured for 4 days (magnification 10 times);

[0023] Figure 8 This is a microscopic image of the cell morphology of the fallopian tube epithelial cells isolated by the isolation method of yak fallopian tube epithelial cells provided in Comparative Example 2 after being cultured for 7 days (magnification 10 times). DETAILED DESCRIPTION

[0024] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0025] In a first aspect, the present invention provides a method for isolating yak fallopian tube epithelial cells, which comprises the following steps:

[0026] S1: Take the oviducts of the yak from both sides and rinse them, then cut the oviducts longitudinally, scrape the upper tissue into a container with a knife, add the resuspension solution to resuspend, centrifuge, and discard the supernatant;

[0027] S2: Digestion: Add digestion solution to the container where the supernatant was discarded, and digest with shaking at 37℃±0.5℃ for 1-1.2h; the digestion solution includes collagenase I, Accutase digestion solution and DNase I, and the mixing ratio of collagenase I mass, Accutase digestion solution enzyme amount and DNase I enzyme amount in each ml of digestion solution is 0.1-0.2mg:100-200U:5-10U; the mixing volume ratio of digestion solution and tissue to be digested is 2-2.5:1-1.5;

[0028] S3: After terminating the digestion, the digestion mixture is filtered, the cell filtrate is collected and centrifuged, and the supernatant is discarded.

[0029] The present invention utilizes Accutase digestion solution instead of trypsin / EDTA digestion solution, and provides a new digestion method that is milder and more efficient. Compared with the traditional trypsin digestion that may damage cell surface antigens, this method is more applicable to mammalian cell lines. The cells obtained by digestion and separation have higher antigen integrity. The obtained cells can be used as a cell model for overcoming the developmental block of early embryos in vitro, promoting the development of early embryos and improving the quality of embryonic development. Maintaining the antigen integrity of the separated cells is of great significance for protecting the undifferentiated state and pluripotency of the cells.

[0030] When collagenase and Accutase are used together to hydrolyze collagen in the extracellular matrix, adding DNaseⅠ can effectively reduce the DNA released during cell separation, reduce cell viscosity and aggregation, and improve cell separation efficiency.

[0031] The combined use of collagenase with Accutase and DNaseⅠ improved the digestion efficiency, greatly increased cell yield and survival rate, and enhanced cell adhesion efficiency.

[0032] Accutase digestion solution (1×) was purchased from Life Science Company, USA.

[0033] The oviducts of the yak were flushed to remove residual blood, pollutants, and adhesion tissue of the oviduct. In order to remove bacterial and fungal contamination, alcohol can also be used for disinfection, and then the alcohol is removed to prevent residual alcohol from damaging the oviduct tissue.

[0034] In a preferred embodiment of the present invention, each mg of the digested material is mixed with 4-7 ml of digestive fluid.

[0035] In a preferred embodiment of the present invention, the resuspension solution in step S1 is a PBS solution containing 100-200 U / mL penicillin and 0.1-0.2 mg / ml streptomycin. The resuspension is performed in a solution containing antibiotics to remove bacterial or fungal contamination.

[0036] In a preferred embodiment of the present invention, the centrifugal parameters in step S1 are: 1500-1800r / min, centrifugation for 3-5min. For example, the centrifugal parameters are any one of 1500rpm, 1550rpm, 1600rpm, 1650rpm, 1700rpm, 1750rpm and 1800rpm. The centrifugal time is 3min, 4min or 5min. Under the above centrifugal conditions, a good separation effect is achieved.

[0037] In a preferred embodiment of the present invention, the flushing in step S1 refers to: firstly flushing the isolated yak oviducts on both sides with physiological saline, then disinfecting with alcohol, and then removing the alcohol with a buffer solution containing antibiotics.

[0038] Flushing with saline can save costs compared to antibiotics and remove residual blood and contaminants.

[0039] The alcohol concentration is 75% and the immersion and disinfection time is 45s.

[0040] In a preferred embodiment of the present invention, after flushing the isolated oviducts of the yak on both sides with physiological saline, the oviducts are immersed in a buffer solution containing antibiotics, and then disinfected with alcohol and the alcohol is removed.

[0041] In a preferred embodiment of the present invention, the digestion is terminated by using DMEM / F12 cell culture medium containing 10% fetal bovine serum.

[0042] In a preferred embodiment of the present invention, the filtration in step S3 is performed using a cell sieve with a mesh size of 40 μm.

[0043] In a preferred embodiment of the present invention, the centrifugation in step S3 is at 1500-1800 r / min for 3-5 min. For example, the centrifugation parameter is any one of 1500 rpm, 1550 rpm, 1600 rpm, 1650 rpm, 1700 rpm, 1750 rpm and 1800 rpm. The centrifugation time is 3 min, 4 min or 5 min. Under the above centrifugation conditions, a good separation effect is achieved.

[0044] In a preferred embodiment of the present invention, after centrifugation and discarding the supernatant in step S3, the cells are resuspended in DMEM / F12 cell culture medium containing 10% FBS and 100-200 U / ml penicillin, 0.1-0.2 mg / mL streptomycin, and 0.4-0.5 μg / mL amphotericin B. The DMEM / F12 cell culture medium containing the three antibodies is used to remove bacterial and fungal contamination.

[0045] For example, resuspend the cells in DMEM / F12 cell culture medium containing 10% FBS and 100U / ml, 110U / ml, 120U / ml, 130U / ml, 140U / ml, 150U / ml, 160U / ml, 170U / ml, 180U / ml, 190U / ml or 200U / ml penicillin, 0.1mg / mL, 0.15mg / mL or 0.2mg / mL streptomycin and 0.4μg / mL, 0.45μg / mL or 0.5μg / mL amphotericin B.

[0046] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0047] Example 1

[0048] (1) After the yaks were killed at the slaughterhouse, to avoid contamination, sterilized ophthalmic scissors and forceps were used to collect the oviducts on both sides of the female yaks, and the oviducts were rinsed with normal saline, which can save costs and remove residual blood and contaminants compared to the use of antibiotics. The oviducts were then immersed in a 4°C precooled PBS solution containing 150 U / mL penicillin and 0.2 mg / ml streptomycin and quickly brought back to the laboratory;

[0049] (2) Remove the fallopian tube and soak it in 75% alcohol for 45 seconds to remove bacterial and fungal contamination. Then rinse it three times with PBS solution containing 150 U / mL penicillin and 0.2 mg / ml streptomycin to remove the alcohol. Avoid residual alcohol from damaging the fallopian tube tissue. Move it to the clean bench and use sterilized ophthalmic scissors and forceps to remove the adhesion tissue around the fallopian tube. The adhesion tissue may be mixed with contaminants. Continue to rinse it three times with PBS solution containing 150 U / mL penicillin and 0.2 mg / ml streptomycin.

[0050] (3) Use a disposable scalpel to longitudinally cut the fallopian tube, tilt the blade at 45 degrees, and gently scrape the epithelial tissue from top to bottom. Repeat the scraping until the fallopian tube turns white.

[0051] (4) The scraped epithelial tissue was transferred to a 15 mL centrifuge tube, and 4 mL of PBS solution containing 150 U / mL penicillin and 0.2 mg / mL streptomycin was added and resuspended to remove bacterial or fungal contamination. The tissue suspension was centrifuged at 1500 r / min for 5 min, and the supernatant was discarded;

[0052] (5) Add digestion solution (Collagenase I purchased from Sigma, Accutase digestion solution (1× purchased from Life, USA), DNase I purchased from Sigma), the digestion solution contains collagenase I with a final concentration of 0.2 mg / ml, 200 U Accutase digestion solution and 5 U DNase I. Digest in a 37°C water bath for 1 h, inverting every 10 minutes to mix; mix 1 ml of the material to be digested with 2 ml of digestion solution;

[0053] (6) After visual inspection, if there are no obvious tissue fragments in the digestion solution, add DMEM / F12 cell culture medium containing 10% fetal bovine serum in an amount equal to the liquid in the centrifuge tube to terminate the digestion;

[0054] (7) Filtering with a cell sieve with a mesh size of 40 μm can effectively remove the remaining impurities. The filtered cell suspension is centrifuged at 1500 r / min for 5 min, the supernatant is discarded, and 4 mL of DMEM / F12 cell culture medium containing 10% FBS, 150 U / ml penicillin, 0.15 mg / mL streptomycin and 0.45 μg / mL amphotericin B is added to resuspend the cell suspension. After obtaining the cell suspension, the cells are inoculated and cultured in a 37°C incubator.

[0055] For cell identification:

[0056] A. Cell morphology identification

[0057] On the 4th day of culture, Figure 1 As shown in the figure, there are no other miscellaneous cells in the field of view, and the fallopian tube epithelial cells grow and aggregate into sheets. After culturing for 6 days, as shown in the figure, Figure 2 As shown, the fallopian tube epithelial cells have grown to confluence and grow in a cobblestone-like pattern with clear cell boundaries and close arrangement.

[0058] B. HE staining

[0059] Digest with 0.25% trypsin and resuspend in culture medium. Place the cell slide in a 24-well plate and adjust the cell density to 1×10 5Drop the solution on the cell slide and culture for 24 hours. Take out the cell slide, rinse 3 times with PBS, fix with 4% paraformaldehyde for 15 minutes, rinse twice with PBS, stain with hematoxylin for 15 minutes, rinse, stain with eosin for 5 minutes, rinse, dry the cell slide naturally, seal with neutral gum, and observe under a microscope.

[0060] like Figure 3 As shown, the fallopian tube epithelial cells are round or oval in shape, growing in a typical cobblestone pattern, with full cytoplasm and large round nuclei.

[0061] C. Immunofluorescence identification

[0062] Take the cell suspension to be identified and adjust the cell density to 3×10 4 Inoculate into 24-well plates, culture for 24 hours, wash 3 times with PBS, fix with 4% paraformaldehyde for 30 minutes, rinse with PBS, permeabilize with 0.5% Triton X-100 for 15 minutes, rinse with PBS, add 2% BSA, block at room temperature for 30 minutes, remove the blocking solution, add primary antibody (keratin antibody diluted 1:200), incubate at 4°C overnight, recover primary antibody, rinse with PBS, add 100 μL secondary antibody (protected from light), incubate at 37°C for 2 hours, rinse with PBS, stain nuclei with DAPI for 5 minutes, rinse with PBS, and observe under a microscope. Figure 4 As shown, the fallopian tube epithelial cells are positive for keratin expression.

[0063] Comparative Example 1

[0064] In this comparative example, trypsin treatment was used to separate yak oviduct epithelial cells.

[0065] (1) After the yaks were killed at the slaughterhouse, the oviducts on both sides of the female yaks were collected using sterilized ophthalmic scissors and forceps, and rinsed with physiological saline to remove residual blood and contaminants. The oviducts were then immersed in a 4°C precooled PBS solution containing 150 U / mL penicillin and 0.2 mg / mL streptomycin and quickly brought back to the laboratory;

[0066] (2) Remove the fallopian tube and soak it in 75% ethanol for 45 seconds to remove bacterial and fungal contamination. Then rinse it three times with PBS solution containing 150 U / mL penicillin and 0.2 mg / ml streptomycin to remove the ethanol. Avoid residual ethanol from damaging the fallopian tube tissue. Move it to the clean bench and use sterilized ophthalmic scissors and forceps to remove the adhesion tissue around the fallopian tube. The adhesion tissue may be mixed with contaminants. Continue to rinse it three times with PBS solution containing 150 U / mL penicillin and 0.2 mg / ml streptomycin.

[0067] (3) Use a disposable scalpel to longitudinally cut the fallopian tube, tilt the blade at 45 degrees, and gently scrape the epithelial tissue from top to bottom. Repeat the scraping until the fallopian tube turns white.

[0068] (4) The scraped epithelial tissue was transferred to a 15 mL centrifuge tube, and 4 mL of PBS solution containing 150 U / mL penicillin and 0.2 mg / mL streptomycin was added and resuspended to remove bacterial or fungal contamination. The tissue suspension was centrifuged at 1500 r / min for 5 min, and the supernatant was discarded;

[0069] (5) Add 2 mL of 0.25% pancreatin and place in a 37°C water bath for digestion for 15 min.

[0070] (6) After visual inspection, if there are no obvious tissue fragments in the digestion solution, add DMEM / F12 cell culture medium containing 10% fetal bovine serum in an amount equal to the liquid in the centrifuge tube to terminate the digestion;

[0071] (7) Filtering with a cell sieve with a mesh size of 40 μm can effectively remove the remaining impurities. The filtered cell suspension is centrifuged at 1500 r / min for 5 min, the supernatant is discarded, and 4 mL of DMEM / F12 cell culture medium containing 10% FBS, 150 U / ml penicillin, 0.15 mg / mL streptomycin and 0.45 μg / mL amphotericin B is added to resuspend the cell suspension. After obtaining the cell suspension, the cells are inoculated and cultured in a 37°C incubator.

[0072] Culture to 3 days, such as Figure 5 As shown in Figure 2, due to the greater damage of trypsin to epithelial cells, the cells obtained by digestion contain a large number of fibroblasts; after culturing for 6 days, as shown in Figure 2 Figure 6 As shown, fibroblasts that adhere quickly compete with epithelial cells that adhere slowly. In order to obtain high-purity and active fallopian tube epithelial cells, it is necessary to purify the epithelial cells through the differential adhesion method, which makes the separation and culture of fallopian tube epithelial cells complicated and difficult.

[0073] Comparative Example 2

[0074] In this comparative example, trypsin and collagenase digestion were used to separate yak oviduct epithelial cells.

[0075] (1) After the yaks were killed at the slaughterhouse, the oviducts on both sides of the female yaks were collected using sterilized ophthalmic scissors and forceps, and rinsed with physiological saline to remove residual blood and contaminants. The oviducts were then immersed in a 4°C precooled PBS solution containing 150 U / mL penicillin and 0.2 mg / mL streptomycin and quickly brought back to the laboratory;

[0076] (2) Remove the fallopian tube and soak it in 75% ethanol for 45 seconds to remove bacterial and fungal contamination. Then rinse it three times with PBS solution containing 150 U / mL penicillin and 0.2 mg / ml streptomycin to remove the ethanol. Avoid residual ethanol from damaging the fallopian tube tissue. Move it to the clean bench and use sterilized ophthalmic scissors and forceps to remove the adhesion tissue around the fallopian tube. The adhesion tissue may be mixed with contaminants. Continue to rinse it three times with PBS solution containing 150 U / mL penicillin and 0.2 mg / ml streptomycin.

[0077] (3) Use ophthalmic scissors to cut the fallopian tube into 1 mm pieces. 3 The tissue blocks were resuspended after adding 4 mL of PBS solution containing 150 U / mL penicillin and 0.2 mg / ml streptomycin to remove bacterial or fungal contamination. The tissue suspension was centrifuged at 1500 r / min for 5 min, and the supernatant was discarded. This was repeated three times.

[0078] (4) Add 3 mL of 0.25% trypsin to the tissue block and digest in a 37°C incubator for 10 min. Add complete culture medium to terminate digestion, centrifuge the tissue suspension at 1500 r / min for 5 min, and discard the supernatant. Add 3 mL of collagenase I and digest in a 37°C incubator for 2 h.

[0079] (5) After digestion, the digestion was terminated with an equal amount of complete medium. Filtering with a 0.22 μL cell sieve can effectively remove the remaining impurities. The filtered cell suspension was centrifuged at 1500 r / min for 5 min, the supernatant was discarded, and 4 mL of DMEM / F12 cell culture medium containing 10% FBS, 150 U / ml penicillin, 0.15 mg / mL streptomycin and 0.45 μg / mL amphotericin B was added to resuspend the cell suspension. After obtaining the cell suspension, the cells were inoculated and continued to be cultured in a 37°C incubator.

[0080] Culture to 4 days, such as Figure 7 As shown in Figure 2, cells began to grow attached to the wall, and the number of epithelial cells was relatively sparse. After 7 days of culture, as shown in Figure 2, Figure 8 Epithelial cells were grown to confluence as shown.

[0081] Combining the experimental results of Example 1, Comparative Example 1 and Comparative Example 2, it can be seen that the yak oviduct epithelial cells separated and purified by the present invention have typical epithelial cell morphology, and the keratin test is positive. The separated cells have good antigen integrity, good proliferation state, and high cell purity.

[0082] The digestion results of Comparative Example 1 (pancreatin group) showed that fibroblasts and epithelial cells grew mixedly, and the amount of epithelial cells was small, and the cell survival rate was low (day 6), making the subsequent separation and culture more complicated. The digestion results of Comparative Example 2 (pancreatin collagenase group) showed that this method had the problem of long digestion time, and the obtained epithelial cells adhered slowly, and it took 7 days to grow and aggregate into sheets.

[0083] The method provided by the present invention has the advantages that Accutase is a digestive solution containing the dual activities of proteolytic enzyme and collagenase, does not contain any ingredients of animal or bacterial origin, and is based on the popular mixed digestion of collagenase and pancreatic enzyme, utilizing the characteristic of Accutase as a perfect substitute for pancreatic enzyme / EDTA, and using collagenase and Accutase together to make the cell digestion process more gentle and efficient. In addition, the addition of DNaseⅠ can better separate aggregated cells, promote digestion efficiency, and greatly improve cell activity and adhesion.

[0084]

[0085] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for isolating yak fallopian tube epithelial cells, It is characterized in that It includes the following steps: S1: Take the oviducts of the yak from both sides and rinse them, then cut the oviducts longitudinally, scrape the upper tissue into a container with a knife, add the resuspension solution to resuspend, centrifuge, and discard the supernatant; S2: Digestion: Add digestion solution to the container from which the supernatant has been discarded, and digest at 37°C±0.5°C with shaking for 1-1.2h; the digestion solution includes collagenase I, Accutase digestion solution and DNase I, and the mixing ratio of collagenase I mass, Accutase digestion solution enzyme amount and DNase I enzyme amount in each ml of the digestion solution is 0.1-0.2mg:100-200U:5-10U; the mixing volume ratio of the digestion solution to the tissue to be digested is 2-2.5:1-1.5; S3: After terminating the digestion, the digestion mixture is filtered, the cell filtrate is collected and centrifuged, and the supernatant is discarded.

2. The method for isolating yak fallopian tube epithelial cells according to claim 1, It is characterized in that Each mg of the digested material was mixed with 4-7 ml of the digestion solution.

3. The method for isolating yak fallopian tube epithelial cells according to claim 1, It is characterized in that The resuspension in step S1 is a PBS solution containing 100-200 U / mL penicillin and 0.1-0.2 mg / ml streptomycin.

4. The method for isolating yak fallopian tube epithelial cells according to claim 3, It is characterized in that The centrifugation parameters in step S1 are: 1500-1800 r / min, centrifugation for 3-5 min.

5. The method for isolating yak fallopian tube epithelial cells according to claim 1, It is characterized in that The flushing in step S1 refers to: first flushing the isolated yak oviducts on both sides with physiological saline, then disinfecting with alcohol, and then removing the alcohol with a buffer solution containing antibiotics.

6. The method for isolating yak fallopian tube epithelial cells according to claim 5, It is characterized in that After flushing the isolated yak oviducts on both sides with saline, the oviducts were immersed in a buffer solution containing antibiotics, and then disinfected with alcohol and the alcohol was removed.

7. The method for isolating yak fallopian tube epithelial cells according to claim 6, It is characterized in that The digestion was terminated by using DMEM / F12 cell culture medium containing 10% fetal bovine serum.

8. The method for isolating yak fallopian tube epithelial cells according to claim 1, It is characterized in that The filtration in step S3 is performed using a cell sieve with a mesh size of 40 μm.

9. The method for isolating yak fallopian tube epithelial cells according to claim 8, It is characterized in that The centrifugation in step S3 is at 1500-1800 r / min for 3-5 min.

10. The method for isolating yak fallopian tube epithelial cells according to claim 9, It is characterized in that After centrifugation and discarding the supernatant in step S3, the cells are resuspended in DMEM / F12 cell culture medium containing 10% FBS, 100-200 U / ml penicillin, 0.1-0.2 mg / mL streptomycin and 0.4-0.5 μg / mL amphotericin B.

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