CN112458040B - A method for isolating and culturing adult yak rumen epithelial cells - Google Patents

Abstract

The invention discloses a method for separating and culturing adult yak rumen epithelial cells. The adult yak rumen epithelial tissue is collected, cleaned with 70% absolute ethanol, and the tissue is digested step by step by combining type I collagenase and trypsin; The cell filtrate was collected by filtration and added to MEM medium containing 10% fetal bovine serum to terminate the digestion; the collected cell pellet was washed; the cells were resuspended in low-glucose DMEM complete medium and then inoculated in culture flasks; differential adhesion method and phase difference digestion method Fibroblasts were removed to obtain adult yak rumen epithelial cells. According to the method provided by the present invention, adult yak rumen epithelial cells can be successfully isolated, and cells with fast adherent growth, high vitality and stable passage can be obtained. The invention can provide a test cell model for the physiological regulation and nutrient absorption mechanism of yak rumen epithelium.

Description

A method for isolating and culturing adult yak rumen epithelial cells

technical field

The invention belongs to the field of biotechnology, in particular to a method for separating and culturing adult yak rumen epithelial cells.

Background technique

my country is the world's largest yak breeding country, and its stock accounts for more than 92% of the world's total yak breeding. The yak can adapt to the environment of high cold, low oxygen and lack of pasture, and has become one of the dominant livestock species in China's Qinghai-Tibet Plateau. It can provide meat and milk. , fur and fuel (dung, as living fuel), which play a vital role in the lives of local pastoralists. And with the improvement of people's living standards, the consumption of yak meat continues to increase, and the yak breeding industry has become a pillar industry of the pastoral economy.

The rumen is an important organ for ruminant feed digestion and metabolism, nutrient absorption, nutrient deposition, immune barrier and other functions. The volatile fatty acids produced by rumen fermentation are mainly absorbed through the rumen epithelium, and as energy substances are brought to various tissues and organs of the body through blood circulation to provide energy for the body. In addition, the rumen epithelium also has four barrier protection functions, namely biological barrier, chemical barrier, mechanical barrier and immune barrier, so that some toxic metabolites such as toxins, pathogenic bacteria, lipopolysaccharides, etc. produced in the rumen cannot pass through the rumen barrier. Effectively prevent the occurrence of some metabolic diseases. The healthy growth and proliferation of rumen epithelial cells is crucial for rumen epithelial tissue, and it is the best in vitro cell model for us to study the physiological function of bovine rumen from molecular mechanisms. Ensuring the health of rumen epithelial tissue is the basis for healthy and efficient breeding of yak.

Primary culture is the only way to obtain rumen epithelial cells. The rumen epithelium is the main place for microorganisms to attach, digest and metabolize. It is easy to be contaminated during the isolation and culture process. At present, there are few methods for isolation and culture of bovine rumen epithelial cells at home and abroad, and the technology is not enough. Mature, juvenile bovine rumen tissue is mostly used, and the cell culture survival time is mostly about 24 hours. Stable passage of rumen epithelial cells is rarely obtained. The cost of juvenile animals is high and the success rate is low. There is no method for the isolation and culture of yak rumen epithelial cells. The current method for culturing rumen epithelial cells uses enzymatic digestion, but the efficiency of collecting cells is related to the species, the size of the tissue block, the type of enzyme, the time of enzymatic digestion, the method of enzymatic digestion, the activity and concentration of the enzyme, and the living environment of yak is special. , different from other cattle, and the adult yak rumen epithelial cells are already in an aging state. Therefore, the methods for obtaining rumen epithelial cells in the existing patent documents are not suitable for culturing adult yak rumen epithelial cells. There are reports on the culture of adult yak rumen epithelial cells.

SUMMARY OF THE INVENTION

The purpose of the present invention is to overcome the shortcomings of the prior art, and to provide a method for separating and culturing adult yak rumen epithelial cells with high reliability and feasibility.

The object of the present invention is achieved through the following technical solutions: a method for separating and culturing adult yak rumen epithelial cells, which comprises the following steps:

S1. Collect adult yak rumen epithelial tissue, put it in a beaker and wash it with 70% ethanol; remove all the edges of the epithelial tissue, and put the remaining tissue in a centrifuge tube and cut it into a paste;

S2. Digest the shredded epithelial tissue with the EDTA mixture of type I collagenase and trypsin, then digest it with 0.5% trypsin four times, and collect the cell suspension for future use;

S3. Centrifuge the collected cell suspension to collect the cell pellet and resuspend the cells in MEM medium with 10% fetal bovine serum. The cell pellet collected by centrifugation is then resuspended in PBS buffer with antibiotics, and filtered with 45µm nylon. Filter the cell suspension through the mesh, disperse the cells, and centrifuge the collected single cell suspension to wash the cells;

Wherein, the PBS buffer of the antibiotic contains 200U/ml penicillin, 0.2mg/ml streptomycin, 100µg/ml gentamicin and 0.5ug/ml amphotericin B;

S4. The washed cell pellets were resuspended in low-sugar DMEM complete medium and then inoculated in culture flasks. The culture was cultured in a 37°C, 5% carbon dioxide incubator; new culture flasks were replaced every 40min. Fibroblasts were removed, and this was repeated four times. After culturing for two days, the medium was replaced, and the cells grew adherently;

Wherein, the medium contains 15% fetal bovine serum, 15ng/mL epidermal growth factor, 0.5% insulin-transferrin-selenium additive, 110 mg/L sodium pyruvate, 4mM L-glutamine, 200U/ml Penicillin, 0.2mg/ml streptomycin, 100µg/ml gentamicin, and 0.5ug/ml amphotericin B in low-sugar DMEM complete medium;

S5. When the cells adhere to the wall and grow 50% stably, digest the fibroblasts with 0.25% trypsin-0.02% EDTA for 3 minutes, and add low-glucose DMEM complete medium to continue the culture. When the cells grow to 80% of the culture flask, subculture or freeze Save the cells to obtain high-purity yak rumen epithelial cells.

Further, the mass percentage contents of type I collagenase, trypsin and EDTA in the mixed solution in step S2 are 0.1%, 0.25% and 0.02%, respectively.

Further, in step S2, the EDTA mixture of collagenase type I and trypsin was digested in a water bath shaker at 37°C for 30 min, and the volume ratio of the shredded epithelial tissue and the mixture was 1:2, and centrifuged to remove after digestion. The mixture was then washed by centrifugation with 4 ℃ pre-cooled PBS buffer containing antibiotics, the PBS buffer containing antibiotics was 200U/ml penicillin, 0.2mg/ml streptomycin, 100µg/ml gentamicin and 0.5 μg/ml gentamicin. ug/ml amphotericin B.

Further, the specific operation of the 0.5% trypsin digestion described in step S2 is as follows: transfer the epithelial tissue digested by the mixed solution to 2 times the volume of 0.5% trypsin, put it into a 37°C water bath shaker for 30 min, The trypsinized liquid was filtered through a 300-mesh nylon filter to collect the cell suspension. At the same time, the cell suspension was terminated with MEM medium containing 10% fetal bovine serum, and the tissue and filter were washed with D-Hanks solution. The remaining cells were collected, and new 0.5% trypsin was added for three cycles. The last three digestion times were 20 min, 20 min and 15 min in turn.

Further, the centrifugation method described in step S3 is that the rotation speed of the centrifuge at 4° C. is 3000 rpm, and the centrifugation time is 8 min.

Further, the cells were dispersed as described in step S3 using a 45 µm cell sieve to disperse the cells, the cell suspension was collected and the cells were dispersed twice.

Further, the passage in step S5 is that when the cells grow to 80% of the culture flask, they are digested with 0.25% trypsin-0.02% EDTA trypsin for 5-10 min to collect the cells for passage.

Further, the cryopreserved solution described in step S5 is DMEM culture solution, and fetal bovine serum and DMSO are configured in a volume ratio of 6:3:1.

The present invention has the following advantages:

1. The method of the present invention successfully separates and cultivates adult yak rumen epithelial cells in good condition, solves the problem that the prior art can only separate rumen epithelial cells from calves, significantly saves costs, and can be sampled in the slaughterhouse, It is convenient to use; the method of the invention provides a test cell model for the physiological regulation and nutrient absorption mechanism of yak rumen epithelium;

2. The present invention washes the collected adult yak rumen epithelial tissue with 70% absolute ethanol, which can effectively remove impurities and pollutants, saves cost and reduces pollution compared with traditional antibiotic washing;

3. The present invention firstly uses collagenase and trypsin combined digestion method to disperse the tissue cells preliminarily, and the effect is mild to protect the cells, and then the use of trypsin alone for digestion can further separate the cells in the tissue; using 0.5% Trypsin increases the efficiency of digestion and reduces the number of digestions, thus reducing cell damage. The collected epithelial cells have higher viability, earlier adherence and stable growth.

Description of drawings

Figure 1 is a schematic diagram of the growth of yak rumen epithelial cells isolated and cultured after high-concentration trypsin digestion in Comparative Example 1.

Fig. 2 is a schematic diagram showing the growth of yak rumen epithelial cells treated by the method of the present invention after two days of culture.

Figure 3 is a schematic diagram of the growth of yak rumen epithelial cells treated by the method of the present invention for four days in culture.

Figure 4 is a schematic diagram of the state of yak rumen epithelial cells after passage by the method of the present invention.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and embodiments, and the protection scope of the present invention is not limited to the following:

Embodiment 1: a method for separating and culturing adult yak rumen epithelial cells, which comprises the following steps:

S1. Using sterilized scissors and tweezers, after the yak was sacrificed, the rumen retrocaecum tissue was taken, and the rumen epithelial tissue of size 10cm×10cm was cut, rinsed several times with tap water, removed the rumen contents, and put it into a beaker with 70% The ethanol was rinsed up and down for 20 times, and then repeated six times with new 70% ethanol until the cleaning was clean, which can effectively remove the residual bacteria and fungi; hand with sterile gloves to manually separate the epithelial layer and the muscle layer, try to remove all the Remove all connective tissue, use sterilized scissors and tweezers to cut off all the edges of the epithelial layer, there will be residual microorganisms on the edges, and the remaining tissue is placed in a 50ml centrifuge tube and chopped into a paste;

S2. Digest with 2 times the volume of 0.1% type I collagenase and 0.25% trypsin-0.02% EDTA for 30 minutes, discard the supernatant, fill up with 4 ℃ pre-cooled PBS buffer containing antibiotics, and centrifuge at 1000 rpm for 3 minutes to wash twice. The PBS buffer of the antibiotics contains 200U/ml penicillin, 0.2mg/ml streptomycin, 100µg/ml gentamicin and 0.5ug/ml amphotericin B; after removing the supernatant, add 2 times the volume of 0.5% Trypsin digestion was performed step by step. The first digestion time was 30 min. The digestion solution was filtered through a 300-mesh nylon filter to collect the cell suspension. The digestion was terminated in MEM medium containing 10% fetal bovine serum. The tissue was washed with D-Hanks solution and filtered. The remaining cells on the Internet were still collected, and new trypsin was added for three cycles. The last three digestion times were 20min, 20min, and 15min. Finally, it was observed that the tissue was white and the viscosity was particularly large and completely stuck together, indicating that the digestion was complete;

S3. After centrifuging the collected cell suspension to collect the cell pellet, resuspend the cells with MEM medium containing 10% fetal bovine serum, and resuspend the cell pellet collected by centrifugation with PBS buffer of antibiotics for washing once. Antibiotics in PBS buffer containing 200U/ml penicillin, 0.2mg/ml streptomycin, 100µg/ml gentamicin and 0.5ug/ml amphotericin B; disperse cells with 45µm cell sieve, collect cell suspension and disperse The cells were washed twice, and the collected single-cell suspension was centrifuged to wash the cells; wherein the centrifugation was 4°C, the speed of the centrifuge was 3000 rpm, and the centrifugation time was 8 minutes;

S4. The washed cell pellets were resuspended in low-sugar DMEM complete medium and then inoculated in culture flasks. The culture was cultured in a 37°C, 5% carbon dioxide incubator; new culture flasks were replaced every 40min. Fibroblasts were removed, and this was repeated four times. After culturing for two days, the medium was replaced, and the cells grew adherently;

Wherein, the medium contains 15% fetal bovine serum, 15ng/mL epidermal growth factor, 0.5% insulin-transferrin-selenium additive, 110 mg/L sodium pyruvate, 4mM L-glutamine, 200U/ml Penicillin, 0.2mg/ml streptomycin, 100µg/ml gentamicin, and 0.5ug/ml amphotericin B in low-sugar DMEM complete medium;

S5. When the cells adhere to the wall and grow 50% stably, use 0.25% trypsin-0.02% EDTA to digest for 3 minutes to remove fibroblasts, and add low-glucose DMEM complete medium to continue the culture. When the cells grow to 80% of the culture flask, use 0.25% trypsin Protease-0.02% EDTA trypsin digestion for 5-10min to collect cells for passage;

When the cells grow to 80% of the culture flask, freeze the cells. The cryopreservation medium is DMEM medium, and the volume ratio of fetal bovine serum and DMSO is 6:3:1, as shown in Figure 4.

After observation, the yak rumen epithelial cells obtained by the above method began to adhere to the wall in 24-48 hours, and completed the layering in 3-4 days. As shown in Figure 2 and Figure 3, the obtained cells were uniform in morphology and paving stone-like, indicating the present invention. The method can effectively remove the contamination of microorganisms and reduce the damage to the cells. The combined use of collagenase and trypsin can effectively reduce the number of digestions, and the cell activity is high, ensuring that the cells can quickly enter the logarithmic growth phase.

Comparative Example 1

(1) Using sterilized scissors and tweezers, after the yak was killed, the rumen retro-caecum tissue was taken, and the rumen epithelial tissue of size 10cm×10cm was cut, rinsed several times with tap water to remove the rumen contents, and then used a rumen containing 700U/ml. Wash carefully with penicillin, 0.7mg/ml streptomycin, 350µg/ml gentamicin and 1.5ug/ml amphotericin B in 4°C pre-chilled PBS buffer;

(2) The rumen tissue was placed in a ziplock bag containing antibiotics pre-cooled PBS buffer at 4°C, placed on ice, and brought back to the laboratory; after careful cleaning with distilled water in the laboratory, pre-cooled at 4°C containing antibiotics Wash with PBS buffer, put it into a beaker containing antibiotics with pre-cooled PBS buffer at 4°C and bring it to the ultra-clean bench;

(3) Put the tissue into a 15cm petri dish containing antibiotics with pre-cooled PBS buffer at 4°C, and manually separate the muscle layer and rumen epithelium on the rumen tissue;

(4) Continue to wash the separated rumen epithelial tissue three times with PBS buffer containing 4 ℃ of pre-cooled antibiotics, and cut the tissue into a paste;

(5) Transfer the chopped epithelial tissue into a 50 mL centrifuge tube containing 5% trypsin, the volume ratio of tissue to collagenase is 1:2, and put it into a 37°C water bath shaker for 30 min. The cell filtrate from the first two digestions was discarded, and the third time the trypsinized liquid was filtered through a 300-mesh nylon filter to collect the cell suspension. The MEM medium was used to stop cell digestion. After filtering through a nylon filter, a small amount of D'Hanks solution containing 200U/ml penicillin, 0.2mg/ml streptomycin and 0.5ug/ml amphotericin B was added to wash the tissue and the remaining cells on the filter. collected, and thus digested six times;

(6) Centrifuge the cell suspension at 3000 rpm for 8 min, collect the cell pellet, resuspend the cells in MEM medium with 10% fetal bovine serum, and wash the cells by centrifugation;

(7) After washing the cell pellet, resuspend the cells in MEM complete medium containing 10% fetal bovine serum and 1x antibiotics, and inoculate them in culture flasks;

(8) Repeat this four times for a new culture flask every 40 minutes to remove fibroblasts; after 24 hours, replace the culture medium and observe the cell state, as shown in Figure 1.

It can be seen from the above experiments that the trypsin digestion in Comparative Example 1 is too strong, which seriously damages the cells and prevents the cells from adhering to the wall. Most of the collected cells are dead cells, and the use of a large number of antibiotics increases the cost. The cells obtained by the method for separating yak rumen epithelial cells in Example 1 of the present invention begin to adhere to the wall in 2-3 days, can be in the logarithmic phase of cell proliferation in 4 days, and complete the layering in 4-5 days, resulting in high cell viability and stable cell growth. Passaged rumen epithelial cells.

The above description is only a preferred embodiment of the present invention, but the protection scope of the present invention is not limited to this. Equivalent replacements or changes to the inventive concept thereof are all included within the protection scope of the present invention.

Claims (5)

1. a method for separating and culturing adult yak rumen epithelial cells, is characterized in that, it comprises the following steps: S1. Collect adult yak rumen epithelial tissue, put it in a beaker and rinse it with 70% ethanol; remove all the edges of the epithelial tissue, and put the remaining tissue in a centrifuge tube and cut it into a paste; S2. The shredded epithelial tissue is digested with the EDTA mixture of type I collagenase and trypsin and then digested four times with 0.5% trypsin, and the cell suspension is collected for subsequent use; wherein, type I collagenase in the mixture The mass percentages of , trypsin and EDTA were 0.1%, 0.25% and 0.02%, respectively; the EDTA mixture of collagenase I and trypsin was digested in a water bath shaker at 37 °C for 30 min, and the shredded epithelial tissue and The volume ratio of the mixed solution is 1:2. After digestion, the mixed solution is removed by centrifugation and then centrifuged and washed with 4 ℃ of pre-cooled PBS buffer containing antibiotics. The PBS buffer containing antibiotics contains 200 U/ml penicillin, 0.2 mg/ml ml streptomycin, 100µg/ml gentamicin and 0.5ug/ml amphotericin B; the specific operation of the 0.5% trypsin digestion is: transfer the epithelial tissue digested by the mixed solution to 2 times the volume of 0.5% trypsin was placed in a 37°C water bath shaker for 30 min, and the trypsinized liquid was filtered through a 300-mesh nylon filter to collect the cell suspension. The culture medium stops cell digestion, and D-Hanks solution is used to wash the tissue and the remaining cells on the filter and collect them, and then add new 0.5% trypsin for three cycles, and the last three digestion times are 20min, 20min and 15min in turn; S3. Centrifuge the collected cell suspension to collect the cell pellet and resuspend the cells in MEM medium with 10% fetal bovine serum. The cell pellet collected by centrifugation is then resuspended in PBS buffer containing antibiotics, using 45µm nylon Filter the cell suspension with a filter, disperse the cells, and centrifuge the collected single cell suspension to wash the cells; Wherein, the antibiotic-containing PBS buffer contains 200U/ml penicillin, 0.2mg/ml streptomycin, 100µg/ml gentamicin and 0.5ug/ml amphotericin B; S4. The washed cell pellets were resuspended in low-sugar DMEM complete medium and then inoculated in culture flasks. The culture was cultured in a 37°C, 5% carbon dioxide incubator; new culture flasks were replaced every 40min. Fibroblasts were removed, and this was repeated four times. After culturing for two days, the medium was replaced, and the cells grew adherently; Wherein, the medium contains 15% fetal bovine serum, 15ng/mL epidermal growth factor, 0.5% insulin-transferrin-selenium additive, 110 mg/L sodium pyruvate, 4mM L-glutamine, 200U/ml Penicillin, 0.2mg/ml streptomycin, 100µg/ml gentamicin, and 0.5ug/ml amphotericin B in low-sugar DMEM complete medium; S5. When the cells adhere to the wall and grow 50% stably, digest the fibroblasts with 0.25% trypsin-0.02% EDTA for 3 minutes, and add low-glucose DMEM complete medium to continue the culture. When the cells grow to 80% of the culture flask, subculture or freeze Save the cells to obtain high-purity yak rumen epithelial cells. 2 . The method for separating and culturing adult yak rumen epithelial cells according to claim 1 , wherein, in step S3, the method for centrifugation is that a 4° C. centrifuge rotating speed is 3000 rpm, and the centrifugation time is 8 min. 3 . 3 . The method for separating and culturing adult yak rumen epithelial cells according to claim 1 , wherein in step S3 , the dispersed cells are dispersed by using a 45 μm cell sieve, and the cell suspension is collected and dispersed twice. 4 . 4. the method for separating and culturing a kind of adult yak rumen epithelial cell according to claim 1, is characterized in that, in step S5, described passage is to use 0.25% trypsin-0.02% when cell grows to 80% of culture flask Cells were collected and passaged by trypsinization with EDTA for 5-10 min. 5. the separation and culture method of a kind of adult yak rumen epithelial cell according to claim 1, is characterized in that, in step S5, the cryopreservation solution of described cryopreservation is DMEM culture fluid, and fetal bovine serum and DMSO are by volume ratio Configured for a 6:3:1 ratio.

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