CN112458040B - Isolation culture method of rumen epithelial cells of adult yaks - Google Patents
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Abstract
The invention discloses a method for separating and culturing rumen epithelial cells of adult yaks, which comprises the steps of collecting rumen epithelial tissues of adult yaks, cleaning the rumen epithelial tissues by using 70% absolute ethyl alcohol, and digesting the tissues step by combining collagenase I and trypsin; filtering with nylon filter screen, collecting cell filtrate, adding MEM culture solution containing 10% fetal calf serum to stop digestion; washing the collected cell pellet; suspending cells by using low-sugar DMEM complete culture medium, and inoculating the cells into a culture bottle for culture; removing fibroblasts by a differential adherence method and a differential digestion method to obtain rumen epithelial cells of adult yaks. According to the method provided by the invention, the rumen epithelial cells of adult yaks can be successfully separated, and the cells which grow fast adherent to the wall and have higher activity and stable passage can be obtained. The invention can provide a test cell model for physiological regulation and control of rumen epithelium of yaks and a nutrient absorption mechanism.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for separating and culturing rumen epithelial cells of adult yaks.
Background
China is a world large yak breeding country, the stock space accounts for more than 92% of the total yak breeding amount in the world, yaks can adapt to the environment with high cold, low oxygen and lack of pasture, become one of the dominant animal breeds in the Qinghai-Tibet plateau area in China, can provide meat, milk, fur and fuel (excrement which is used as life fuel), and play an important role in the life of local herdsmen. Along with the improvement of living standard of people, the consumption of yak meat is continuously improved, and yak breeding becomes the economic pillar industry of pastoral area.
Rumen is an important organ for ruminant feed digestion and metabolism, nutrient absorption, nutrient deposition, immune barrier and other functions. Volatile fatty acid produced by rumen fermentation is mainly absorbed by rumen epithelium and taken as energy substance to various tissues and organs of the organism through blood circulation to provide energy for the organism. In addition, the rumen epithelium has four barrier protection functions, namely a biological barrier, a chemical barrier, a mechanical barrier and an immune barrier, so that toxic metabolites such as toxins, pathogenic bacteria, lipopolysaccharides and the like generated in the rumen cannot pass through the rumen barrier, thereby effectively preventing metabolic diseases. Healthy growth and proliferation of rumen epithelial cells are important for rumen epithelial tissues, and the in vitro cell model is an optimal in-vitro cell model for researching bovine rumen physiological functions from a molecular mechanism. The basis for the healthy and efficient yak breeding is to ensure the health of rumen epithelial tissues.
Primary culture is the only means for obtaining rumen epithelial cells, rumen epithelium is the main place for microorganism attachment, digestion and metabolism, and is easy to be polluted in the separation culture process, the methods for separating and culturing bovine rumen epithelial cells at home and abroad are few at present, the technology is immature, young bovine rumen tissues are mostly adopted, the survival time of cell culture is about 24 hours, stable passage rumen epithelial cells can be rarely obtained, the cost of young animals is high, and the success rate is low. A method for separating and culturing rumen epithelial cells of yaks does not exist. The method for culturing the rumen epithelial cells uses an enzyme digestion method at present, but the efficiency of cell collection is related to species, the size of tissue blocks, the type of enzyme, the enzyme digestion time, the enzyme digestion mode, the activity and the concentration of the enzyme, the yak living environment is special and is different from other yaks, and adult yak rumen epithelial cells are in an aging state, so the method for obtaining the rumen epithelial cells in the existing patent documents is not suitable for culturing the adult yak rumen epithelial cells, and the prior art does not report about culturing the adult yak rumen epithelial cells after retrieval.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for separating and culturing rumen epithelial cells of adult yaks, which has high reliability and feasibility.
The purpose of the invention is realized by the following technical scheme: a method for separating and culturing rumen epithelial cells of adult yaks comprises the following steps:
s1, collecting rumen epithelial tissues of adult yaks, putting the rumen epithelial tissues into a beaker, and cleaning the rumen epithelial tissues with 70% ethanol; removing all the edges of the epithelial tissue, and putting the residual tissue in a centrifuge tube to be cut into paste;
s2, digesting the cut epithelial tissue with EDTA mixed solution of type I collagenase and trypsin, then digesting the tissue with 0.5% trypsin for four times, and collecting cell suspension for later use;
s3, centrifugally collecting cell sediment from the collected cell suspension, then resuspending the cells by using 10% MEM culture solution of fetal calf serum, resuspending the cell sediment from the centrifugally collected cell sediment by using PBS buffer solution of antibiotics, filtering the cell suspension by using a 45 mu m nylon filter screen, dispersing the cells, and centrifugally cleaning the cells by using the collected single cell suspension;
wherein the PBS buffer solution of the antibiotics contains 200U/ml penicillin, 0.2mg/ml streptomycin, 100 mug/ml gentamicin and 0.5ug/ml amphotericin B;
s4, suspending the washed cell sediment by using a low-sugar DMEM complete culture medium, inoculating the suspended cells into a culture bottle, and culturing in a 5% carbon dioxide incubator at 37 ℃; replacing a new culture bottle every 40min to culture and remove fibroblasts, repeating the steps for four times, replacing the culture medium after culturing for two days, and enabling the cells to grow in an adherent manner;
wherein the culture medium is a low-sugar DMEM complete culture medium containing 15% fetal calf 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, 100 mu g/mL gentamicin and 0.5ug/mL amphotericin B;
and S5, when the cells stably grow on the adherent surface by 50%, digesting for 3min by using 0.25% trypsin-0.02% EDTA to remove fibroblasts, adding a low-sugar DMEM complete culture medium to continue culturing, and carrying out passage or cryopreservation on the cells when the cells grow to 80% of a culture bottle, thereby obtaining the high-purity yak rumen epithelial cells.
Further, in the step S2, the mass percentages of the collagenase type I, the trypsin, and the EDTA in the mixed solution are 0.1%, 0.25%, and 0.02%, respectively.
Further, in the step S2, EDTA mixed solution digestion of collagenase type I and trypsin is carried out for 30min in a water bath oscillator at 37 ℃, the volume ratio of the minced epithelial tissue to the mixed solution is 1:2, after digestion, the mixed solution is removed by centrifugation, and then centrifugal washing is carried out by 4 ℃ precooling PBS buffer solution containing antibiotics, wherein the PBS buffer solution containing the antibiotics comprises 200U/ml penicillin, 0.2mg/ml streptomycin, 100 mu g/ml gentamicin and 0.5ug/ml amphotericin B.
Further, the specific operation of the 0.5% trypsin digestion in step S2 is: transferring the epithelial tissue digested by the mixed solution to 0.5% trypsin with the volume 2 times that of the epithelial tissue, placing the epithelial tissue into a 37 ℃ water bath oscillator for treatment for 30min, filtering the digestion solution by a 300-mesh nylon filter screen to collect cell suspension, simultaneously stopping cell digestion of the cell suspension by using MEM culture solution containing 10% fetal calf serum, cleaning the tissue and the cells remained on the filter screen by using D-Hanks solution and collecting the cells, adding new 0.5% trypsin for circulating three times, and sequentially performing three times of digestion for 20min, 20min and 15 min.
Further, the centrifugation method in step S3 is to use a 4 ℃ centrifuge with a rotation speed of 3000rpm and a centrifugation time of 8 min.
Further, the scattered cells are sieved using 45 μm cells in the step S3, and the cell suspension is collected and scattered twice.
Further, the passage described in step S5 is a passage of collecting cells by digesting with trypsin 0.25% trypsin-0.02% EDTA trypsin for 5-10min when the cells grow to 80% in the flask.
Further, the frozen stock solution in step S5 is a DMEM culture solution, and the fetal bovine serum and DMSO are prepared at a volume ratio of 6:3: 1.
The invention has the following advantages:
1. the method successfully separates and cultures the rumen epithelial cells of the adult yaks in good state, solves the problem that the rumen epithelial cells can only be separated from calves in the prior art, obviously saves the cost, can sample in slaughterhouses, and is convenient to use; the method provides a test cell model for physiological regulation and control of rumen epithelium and a nutrient absorption mechanism of the yak;
2. according to the invention, the collected rumen epithelial tissue of the adult yaks is washed by 70% absolute ethyl alcohol, so that impurities and pollutants can be effectively removed, and compared with the traditional antibiotic washing, the method saves the cost and reduces the pollution;
3. according to the invention, firstly, collagenase and trypsin are combined with a digestion method to disperse tissue cells primarily, the action effect is mild, the cells are protected, and then the trypsin is used alone for digestion, so that the cells in the tissue can be separated; by adopting 0.5 percent of trypsin, the digestion efficiency is increased, and the digestion times are reduced, so the damage of cells is reduced, the activity of the collected epithelial cells is higher, the adherence is earlier, and the growth is stable.
Drawings
FIG. 1 is a schematic diagram showing the growth of rumen epithelial cells of yaks separately cultured after digestion with trypsin at a high concentration in comparative example 1.
Fig. 2 is a schematic diagram of the growth situation of the rumen epithelial cells of yaks treated by the method of the invention cultured for two days.
Fig. 3 is a schematic diagram of the four-day growth of rumen epithelial cells of yaks treated by the method.
Fig. 4 is a schematic diagram of the state of rumen epithelial cells of yaks after passage by the method.
Detailed Description
The invention is further described with reference to the following figures and examples, without limiting the scope of the invention to the following:
example 1: a method for separating and culturing rumen epithelial cells of adult yaks comprises the following steps:
s1, using sterilized scissors and tweezers to make a yak dead, taking a rumen posterior abdominal cecum tissue, cutting a 10cm multiplied by 10cm rumen epithelial tissue, washing the rumen epithelial tissue for multiple times by using tap water to remove rumen contents, putting the rumen epithelial tissue into a beaker, washing the rumen epithelial tissue for 20 times up and down by using 70% ethanol, and repeating the steps for six times until the rumen epithelial tissue is cleaned by using new 70% ethanol, so that residual bacteria and fungi can be effectively removed; manually separating an epithelial layer and a muscle layer by using a hand-carried sterile glove, completely removing all connective tissues to the greatest extent, completely shearing the edges of the epithelial layer by using sterilized scissors and tweezers, wherein microorganisms are remained on the edges, and cutting the remained tissues into paste in a 50ml centrifuge tube;
s2, digesting for 30min by using 0.1% type I collagenase and 0.25% trypsin-0.02% EDTA in 2 times of volume, discarding the supernatant, and adding PBS buffer solution pre-cooled at 4 ℃ and containing antibiotics and centrifuging for 3min at 1000rpm for twice, wherein the PBS buffer solution containing the antibiotics contains 200U/ml penicillin, 0.2mg/ml streptomycin, 100 mu g/ml gentamicin and 0.5ug/ml amphotericin B; removing supernatant, adding 0.5% trypsin with 2 times volume for fractional digestion, wherein the first digestion time is 30min, filtering the digestion solution by a 300-mesh nylon filter screen to collect cell suspension, stopping digestion by using MEM (minimum medium) containing 10% fetal calf serum, cleaning tissues and collecting residual cells on the filter screen by using D-Hanks solution, adding new trypsin for circulating for three times, and sequentially carrying out 20min, 20min and 15min on the last three times of digestion time, and finally observing that the tissue is very large in whitening viscosity and completely stuck together, thereby indicating that the digestion is complete;
s3, after centrifugally collecting cell precipitates from the collected cell suspension, resuspending the cells by using 10% MEM culture solution of fetal calf serum, and washing the cell precipitates after centrifugally collecting the cell precipitates by resuspending the cell precipitates by using PBS buffer solution of antibiotics once, wherein the PBS buffer solution of the antibiotics contains 200U/ml penicillin, 0.2mg/ml streptomycin, 100 mug/ml gentamicin and 0.5ug/ml amphotericin B; screening scattered cells by using cells of 45 mu m, collecting cell suspension, dispersing the cells twice, and centrifugally cleaning the collected single cell suspension; wherein the centrifugation is performed at 4 ℃ with the rotation speed of a centrifuge of 3000rpm and the centrifugation time of 8 min;
s4, suspending the washed cell sediment by using a low-sugar DMEM complete culture medium, inoculating the suspended cells into a culture bottle, and culturing in a 5% carbon dioxide incubator at 37 ℃; replacing new culture bottles every 40min to culture and remove fibroblasts, repeating the steps for four times, replacing the culture medium after culturing for two days, and enabling cells to grow in an adherent manner;
wherein the culture medium is a low-sugar DMEM complete culture medium containing 15% fetal calf 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, 100 mu g/mL gentamicin and 0.5ug/mL amphotericin B;
s5, when the cells stably grow at 50% adherent, digesting for 3min by using 0.25% trypsin-0.02% EDTA to remove fibroblasts, adding low-sugar DMEM complete culture medium to continue culturing, and digesting for 5-10min by using 0.25% trypsin-0.02% EDTA trypsin when the cells grow to 80% of the culture bottle to collect the cells for passage;
when the cells grow to 80% of the culture flask, the cells are frozen, the frozen stock solution is a DMEM culture solution, and the fetal calf serum and the DMSO are prepared in a volume ratio of 6:3:1, as shown in FIG. 4.
According to observation, 24-48 hours of the rumen epithelial cells of the yak obtained by the method begin to adhere to the wall, layering is completed in 3-4 days, and as shown in figures 2 and 3, the obtained cells are uniform in shape and are in a paving stone shape, so that the method can effectively remove the pollution of microorganisms, reduce the damage to the cells, effectively reduce the digestion times due to the combined use of collagenase and trypsin, have higher cell activity, and ensure that the cells can quickly enter a logarithmic phase.
Comparative example 1
(1) Using sterilized scissors and tweezers, taking abdominal cecum tissue behind a rumen after a yak is killed, cutting rumen epithelial tissue with the size of 10cm multiplied by 10cm, washing for multiple times by using tap water to remove rumen content, and carefully washing clean by using 4-DEG C precooled PBS buffer solution containing 700U/ml penicillin, 0.7mg/ml streptomycin, 350 mu g/ml gentamicin and 1.5ug/ml amphotericin B;
(2) putting rumen tissue into a self-sealing bag containing antibiotics and precooling PBS buffer solution at 4 ℃, putting the self-sealing bag on ice and bringing the self-sealing bag back to a laboratory; carefully cleaning the materials in a laboratory by using distilled water, then cleaning the materials by using a 4 ℃ precooled PBS buffer solution containing antibiotics, putting the materials into a beaker of the 4 ℃ precooled PBS buffer solution containing the antibiotics and bringing the beaker into a super clean bench;
(3) placing the tissue in a 15cm petri dish containing antibiotics in 4 ℃ pre-cooled PBS buffer, and manually separating the muscle layer from the rumen epithelium on the rumen tissue;
(4) continuously washing the separated rumen epithelial tissue with PBS buffer solution containing 4 deg.C precooled antibiotic for 3 times, and cutting the tissue into paste;
(5) minced epithelial tissue was transferred to a 50mL centrifuge tube containing 5% trypsin, at a tissue to collagenase volume ratio of 1:2, placing the mixture into a water bath oscillator with the temperature of 37 ℃ for treatment for 30 min. Discarding the cell filtrate obtained in the previous two times of digestion, filtering the digestion solution of the liquid obtained after the trypsin digestion through a 300-mesh nylon filter screen for the third time, collecting cell suspension, simultaneously stopping the cell digestion of the cell suspension by using MEM culture solution containing 10% fetal calf serum and 2 times of antibiotics, cleaning tissues and residual cells on the filter screen by adding a small amount of D' Hanks solution containing 200U/ml penicillin, 0.2mg/ml streptomycin and 0.5ug/ml amphotericin B after the nylon filter screen is filtered, and collecting the cleaned tissues and the residual cells on the filter screen, thus digesting for six times;
(6) centrifuging the cell suspension at 3000rpm for 8min, collecting cell precipitate, resuspending the cells in 10% fetal bovine serum MEM culture solution, and centrifuging and cleaning the cells;
(7) after the washed cell sediment is re-suspended in MEM complete medium containing 10% fetal calf serum and 1 time of antibiotics, the cell sediment is inoculated into a culture flask for culture;
(8) replacing a new culture flask every 40min, repeating the steps for four times, and removing fibroblasts; after 24h, the medium was changed and the cell status was observed, as shown in FIG. 1.
As can be seen from the above experiments, the trypsin digestion in comparative example 1 is too strong, cells are seriously damaged, cells cannot grow adherently, most of the collected cells are dead cells, and the use of a large amount of antibiotics increases the cost. The cells obtained by the separation method of the rumen epithelial cells of the yak in the embodiment 1 start adherent growth in 2-3 days, can be in the logarithmic phase of cell proliferation in 4 days, and can be layered in 4-5 days, so that the rumen epithelial cells with higher cell activity and stable passage can be obtained.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can substitute or change the technical solution of the present invention and the inventive concept within the technical scope of the present invention.
Claims (5)
1. A method for separating and culturing rumen epithelial cells of adult yaks is characterized by comprising the following steps:
s1, collecting rumen epithelial tissues of adult yaks, putting the tissues into a beaker, and washing the tissues clean with 70% ethanol; removing all the edges of the epithelial tissue, and putting the residual tissue in a centrifuge tube to be cut into paste;
s2, digesting the cut epithelial tissue with EDTA mixed solution of type I collagenase and trypsin, then digesting the cut epithelial tissue with 0.5% trypsin for four times, and collecting cell suspension for later use; wherein, the mass percentage contents of the collagenase type I, the trypsin and the EDTA in the mixed solution are respectively 0.1%, 0.25% and 0.02%; the digestion of EDTA mixed solution of collagenase type I and trypsin is carried out for 30min in a water bath oscillator at 37 ℃, the volume ratio of the minced epithelial tissue to the mixed solution is 1:2, the mixed solution is centrifugally removed after digestion, and then the mixed solution is centrifugally cleaned by PBS buffer solution which is precooled at 4 ℃ and contains antibiotics, wherein the PBS buffer solution contains 200U/ml penicillin, 0.2mg/ml streptomycin, 100 mu g/ml gentamicin and 0.5ug/ml amphotericin B; the specific operation of the 0.5% trypsin digestion is as follows: transferring the epithelial tissue digested by the mixed solution to 0.5% trypsin with the volume 2 times that of the epithelial tissue, placing the epithelial tissue into a 37 ℃ water bath oscillator for treatment for 30min, filtering the digestion solution of the liquid digested by the trypsin through a 300-mesh nylon filter screen to collect cell suspension, simultaneously stopping cell digestion of the cell suspension by using MEM culture solution containing 10% fetal calf serum, cleaning the tissue and the cells remained on the filter screen by using D-Hanks solution and collecting the cells, adding new 0.5% trypsin, circulating the steps for three times, and sequentially performing digestion for the three times of 20min, 20min and 15 min;
s3, after centrifugally collecting cell sediment from the collected cell suspension, re-suspending the cells by using 10% MEM culture solution of fetal calf serum, re-suspending the cell sediment from the centrifugally collected cell sediment by using PBS buffer solution containing antibiotics, filtering the cell suspension by using a 45 mu m nylon filter screen, dispersing the cells, and centrifugally cleaning the collected single cell suspension;
wherein the PBS buffer solution containing antibiotics contains 200U/ml penicillin, 0.2mg/ml streptomycin, 100 mug/ml gentamicin and 0.5ug/ml amphotericin B;
s4, suspending the washed cell sediment by using a low-sugar DMEM complete culture medium, inoculating the suspended cells into a culture bottle, and culturing in a 5% carbon dioxide incubator at 37 ℃; replacing new culture bottles every 40min to culture and remove fibroblasts, repeating the steps for four times, replacing the culture medium after culturing for two days, and enabling cells to grow in an adherent manner;
wherein the culture medium is a low-sugar DMEM complete culture medium containing 15% fetal calf 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, 100 mu g/mL gentamicin and 0.5ug/mL amphotericin B;
and S5, when the cells stably grow on the adherent surface by 50%, digesting for 3min by using 0.25% trypsin-0.02% EDTA to remove fibroblasts, adding a low-sugar DMEM complete culture medium to continue culturing, and carrying out passage or cryopreservation on the cells when the cells grow to 80% of a culture bottle, thereby obtaining the high-purity yak rumen epithelial cells.
2. The method for isolating and culturing rumen epithelial cells of adult yaks according to claim 1, wherein in step S3, the centrifugation method comprises a 4 ℃ centrifuge rotating speed of 3000rpm and a centrifugation time of 8 min.
3. The method for separately culturing the adult yak rumen epithelial cells according to claim 1, wherein in the step S3, the dispersed cells are sieved by using 45 μm cells, and a cell suspension is collected and dispersed twice.
4. The method for isolating and culturing rumen epithelial cells of adult yaks according to claim 1, wherein in step S5, the passage is performed by collecting cells for passage after the cells are digested with trypsin 0.25% trypsin-0.02% EDTA trypsin for 5-10min when the cells grow to 80% of the culture flask.
5. The method for isolating and culturing rumen epithelial cells of adult yaks according to claim 1, wherein in step S5, the frozen stock solution is DMEM culture solution, and fetal bovine serum and DMSO are prepared in a volume ratio of 6:3: 1.
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| CN113088482B (en) * | 2021-04-16 | 2022-12-23 | 四川农业大学 | Method for separating and culturing rumen epithelial cells of calves |
| CN113388570B (en) * | 2021-06-16 | 2023-02-14 | 浙江大学 | Bovine rumen epithelial tissue dissociation method for single cell sequencing |
| CN113717932B (en) * | 2021-09-16 | 2023-03-14 | 四川农业大学 | Primary isolation culture and induced differentiation method for intramuscular precursor adipocytes of adult yaks |
| CN114058592B (en) * | 2021-11-16 | 2023-07-04 | 四川农业大学 | Immortalized yak rumen epithelial cell line and construction method thereof |
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