CN108103004A - A kind of method of sika deer cud epithelial cell culture - Google Patents
A kind of method of sika deer cud epithelial cell culture Download PDFInfo
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Abstract
The present invention provides a kind of method of sika deer cud epithelial cell culture, is related to and is separately cultured cell field.To solve to cultivate the problems such as sika deer cud epithelial cell purity is low, and vigor is low, and success rate is low, easy to pollute, method and step provided by the invention is as follows：Sika deer cud tissue is taken, is rinsed with the PBS buffer solution containing antibiotic etc.；Epithelial tissue is taken to shred, adds in the DMEM/F12 culture solution adhere-wall cultures containing hyclone FBS, sodium selenite, antibiotic etc.；Add in the DMEM/F12 culture solution secondary cultures containing hyclone FBS, sodium selenite, antibiotic etc..Sika deer cud epithelial cell can be successfully separated out for the first time according to method provided by the invention, and purity and activity are higher and can stablize passage, and overcome the pollution problem that multiple-microorganism in cud is brought.Present invention can apply to the growth and development of sika deer cud epithelium, physiological function and the researchs in Nutrition and Metabolism etc..
Description
Technical field
The present invention relates to cell field is separately cultured, sika deer cud epithelial cell is particularly cultivated.
Background technology
Stomach and enteron aisle are the main digestive organs of animal, and ruminant to the digestion of feed mainly using ruminal digestion as
It is main, so the nutriment transhipment of research cud epithelial cell and mechanism of absorption are with progressively as the warm of Ruminant Nutrient Metabolism
One of point.The present invention can be used for growth and development, physiological function and the grinding in Nutrition and Metabolism etc. of sika deer cud epithelium
Study carefully.
Original cuiture is the only resource for obtaining cud epithelial cell, at home and abroad cud epithelial cell in major cell bank
System does not sell.Containing microorganisms such as bacterium, fungi, mycoplasma and protozoons in cud, therefore, pole during cell is cultivated
Easily polluted by microorganism.At present, the method for still ruminant tumor gastric epithelial cell isolation and culture is less both at home and abroad, with reference to
There is the cud primitive cell culture in document to carry out sika deer cell culture, sika deer can not be completely suitable for, ultimately resulted in
The cud epithelial cell of original cuiture is there is state labile, and vigor is low, and success rate is low, it is easy to pollute the shortcomings of.
The method of culture cud epithelial cell is mostly used enzyme digestion at present, and the application of tissue block method is less, utilizes enzymic digestion
The same species of efficiency, the size of tissue block, the type of enzyme, the activity of enzyme and the concentration that method collects cell are related, so existing special
The method of sharp Access to publication cud cell is not necessarily applied to culture sika deer epithelial cell, and has correlation test to prove profit
It is low compared with the culture of tissue block method with the cytoactive of enzyme digestion.
Therefore one kind is provided reliably, the high sika deer cud epithelial cell cultural method of feasibility is extremely urgent.
The content of the invention
Low to solve above-mentioned culture sika deer cud epithelial cell purity, vigor is low, and success rate is low, it is easy to pollute the problems such as,
The present invention provides a kind of method of sika deer cud epithelial cell culture.
A kind of method for being separately cultured sika deer cud epithelial cell, step are as follows：
(1) sika deer cud tissue is taken, after first rumen content is cleaned up with physiological saline, then with containing mould
Element, streptomysin, gentamicin, the PBS buffer solution of amphotericin B are rinsed 6-8 times, last adds PBS bufferings after rinsing
Liquid and static 10min；
(2) muscle layer of cud tissue and epithelial layer are separated, takes epithelial tissue spare；
(3) epithelial tissue is shredded, the even tissue shredded is laid in blake bottle, inversion is placed in the incubator
2h；
(4) add in containing hyclone FBS, EGF, L-Glutamine, insulin, transferrins, sodium selenite, mould
Element, streptomysin, gentamicin, the DMEM/F12 culture solutions of amphotericin B carry out adhere-wall culture；
Being observed after adding in culture solution, after 3d has cell to climb out of, over time, cell quantity showed increased after 8d.
(5) adhere-wall culture discards culture solution after 4 days, adds in containing hyclone FBS, EGF, L-Glutamine, pancreas islet
Element, transferrins, sodium selenite, penicillin, the DMEM/F12 culture solutions of streptomysin, continue to cultivate, culture were changed every 4-5 days
Liquid, it is described to change culture solution operation to retain the 1/3-1/2 of original fluid, it adds in and identical with discarding nutrient solution volume contains tire ox
The DMEM/F12 cultures of serum FBS, EGF, L-Glutamine, insulin, transferrins, sodium selenite, penicillin, streptomysin
Liquid；Cell during cell confluent cultures bottom of bottle portion more than 80% is as the 0th generation cell；
(6) the 0th generation cells discard culture solution, and trypsase is added in after being rinsed with PBS buffer solution and is digested, when aobvious
During micro- Microscopic observation cell rounding, it is whole to add in the DMEM/F12 culture solution containing FBS5-10% (v/v) isometric with trypsase
Only digest, by adherent cell piping and druming into suspension, 1000r/min, which is centrifuged, abandons supernatant for 5 minutes, add in it is suitable containing FBS, EGF,
The DMEM/F12 culture solutions piping and druming of L-Glutamine, insulin, transferrins, sodium selenite, penicillin, streptomysin is resuspended, point
It adds same culture solution respectively into trisection to be cultivated respectively, in 37 DEG C of incubators after static 30min, culture solution is inhaled
Go out into another culture dish, utilize the epithelial cell method purifying cells different from the fibroblast adherent time, this operation weight
Identical culture solution is added after 3 times multiple to be cultivated, the sika deer cud epithelial cell that can be purified；Cell confluent cultures bottle
Cell during bottom more than 80% can be passed on next time.
Preferably, step (1) described physiological saline is the sodium chloride solution of quality volume fraction 0.9% (g/mL).
Preferably, concentration of step (1) the described penicillin in PBS is 700IU/mL, and concentration of the streptomysin in PBS is
700ug/mL, concentration of the gentamicin in PBS are 100ug/mL, and concentration of the amphotericin B in PBS is 2.5ug/mL.
Preferably, step (1) the addition PBS buffer solution, addition are that sika deer cud tissue is made to be fully immersed in PBS
In buffer solution.
Preferably, step (2) described separation method is to be separated by hand with tweezers.
Preferably, step (3) the placement condition is 37 DEG C, 5% (v/v) carbon dioxide.
Preferably, 5% (v/v) hyclone FBS, 10ng/mL is contained in step (4) the DMEM/F12 culture solutions
EGF, 2mmol/L L-Glutamine, 10ug/mL insulin, 5ug/mL transferrins, 5ug/mL sodium selenites, 700IU/mL are blue or green
Mycin, 700ug/mL streptomysins, 100ug/mL gentamicins, 5.0ug/mL amphotericin Bs；The condition of culture be 37 DEG C, 5%
(v/v) carbon dioxide.
Preferably, in step (5) or step (6) the DMEM/F12 culture solutions containing 5% (v/v) hyclone FBS,
10ng/mL EGF, 2mmol/L L-Glutamines, 10ug/mL insulin, 5ug/mL transferrins, 5ug/mL sodium selenites,
100IU/mL penicillin, 100ug/mL streptomysins；Step (5) or step (6) described condition of culture are 37 DEG C, 5% (v/v) dioxy
Change carbon.
Preferably, step (6) described trypsinase concentration is 0.25% (g/mL)
Detection：
By the 3rd generation of culture it is cells trypsinised after be resuspended using DMEM/F12 culture solutions, be inoculated with liquid-transfering gun
To being placed in the culture plate of coverslip, when cell covers with the 70%-80% of entire coverslip, cell is cleaned with PBS buffer solution,
4% paraformaldehyde fixes 30min, PBS cleaning 3 times, each 5min.The penetrating 30min of 1%Triton X-100, PBS cleaning 3 times,
Each 5min, 5%BSA close 30min, remove extra BSA, cover mouse CK18 antibody, negative control by equivalent PBS generations
It replaces, 4 DEG C are overnight, 37 DEG C of rewarming 30min, PBS flushings 3 times, each 10min of next day, add in the mountain sheep anti-mouse igg of fluorescent marker,
Room temperature is protected from light 60min, and DAPI mountings after finishing, photo are shot with fluorescence microscope.
Advantageous effect
Can be successfully separated out sika deer cud epithelial cell for the first time according to method provided by the invention, purity and activity compared with
Gao Bingke stablizes passage, and overcomes the pollution that multiple-microorganism in cud is brought.It, can't due to the particularity of sika deer
As other animals take infant animal tissue samples, the cud sample of this patent acquisition, sika deer has started the activity of ruminating, and cud is micro-
Biological flora gradually tends towards stability, and the pollution that multiple-microorganism in cud is brought is overcome when cultivating cell.The present invention can answer
For the growth and development of sika deer cud epithelium, physiological function and the research in Nutrition and Metabolism etc..
Description of the drawings
Photo under microscope after Fig. 1 is the 1 adherent 10d of cud tissue of embodiment；
Fig. 2 is photo under 1 2nd generation cud epithelial cell microscope of embodiment；
Fig. 3 CK18 identify the 3rd generation cud epithelial cell fluorescent microscopy images of embodiment 1.
Specific embodiment
Test reagent：PBS, mycillin, gentamicin, DMEM/F12, FBS, EGF, insulin, turn iron at anphotericin
Albumen, glutamine, sodium selenite, hydrocortisone, Triton X-100,5%BSA, mouse CK18 monoclonal antibodies, FITC-
Mountain sheep anti-mouse igg, DAPI.
Embodiment 1
It is separately cultured sika deer cud epithelial cell：
(1) sika deer cud tissue is taken, after first rumen content is cleaned up with physiological saline, then with containing mould
Element, streptomysin, gentamicin, the PBS buffer solution of amphotericin B are rinsed 6 times, last adds PBS buffer solution after rinsing
And static 10min；
(2) muscle layer of cud tissue and epithelial layer are separated, takes epithelial tissue spare；
(3) epithelial tissue is shredded, the even tissue shredded is laid in blake bottle, inversion is placed in the incubator
2h；
(4) add in containing hyclone FBS, EGF, L-Glutamine, insulin, transferrins, sodium selenite, mould
Element, streptomysin, gentamicin, the DMEM/F12 culture solutions of amphotericin B carry out adhere-wall culture；
Being observed after adding in culture solution, after 3d has cell to climb out of, over time, cell quantity showed increased after 8d.
(5) adhere-wall culture discards culture solution after 4 days, adds in containing hyclone FBS, EGF, L-Glutamine, pancreas islet
Element, transferrins, sodium selenite, penicillin, the DMEM/F12 culture solutions of streptomysin, continue to cultivate, and culture solution was changed every 4 days,
It is described to change culture solution operation to retain the 1/3 of original fluid, it adds in and identical with discarding nutrient solution volume contains hyclone
FBS, EGF, L-Glutamine, insulin, transferrins, sodium selenite, penicillin, the DMEM/F12 culture solutions of streptomysin；Carefully
Cell during born of the same parents confluent cultures bottom of bottle portion 80% is as the 0th generation cell；
(6) the 0th generation cells discard culture solution, and trypsase is added in after being rinsed with PBS buffer solution and is digested, when aobvious
During micro- Microscopic observation cell rounding, add in the DMEM/F12 culture solution containing FBS5% (v/v) isometric with trypsase and terminate
Digestion, by adherent cell piping and druming into suspension, 1000r/min, which is centrifuged, abandons supernatant for 5 minutes, and addition is suitable to contain FBS, EGF, L-
The DMEM/F12 culture solutions piping and druming of glutamine, insulin, transferrins, sodium selenite, penicillin, streptomysin is resuspended, and is divided into
Trisection is added same culture solution and is cultivated respectively respectively, and in 37 DEG C of incubators after static 30min, culture solution is suctioned out
Into another culture dish, using the epithelial cell method purifying cells different from the fibroblast adherent time, this operation repeats 3
It adds identical culture solution after secondary to be cultivated, the sika deer cud epithelial cell that can be purified；Cell confluent cultures bottom of bottle
Cell during portion more than 80% can be passed on next time.
Step (1) described physiological saline is the sodium chloride solution of quality volume fraction 0.9% (g/mL).
Concentration of step (1) the described penicillin in PBS is 700IU/mL, and concentration of the streptomysin in PBS is 700ug/
ML, concentration of the gentamicin in PBS are 100ug/mL, and concentration of the amphotericin B in PBS is 2.5ug/mL.
Step (1) the addition PBS buffer solution, addition are that sika deer cud tissue is made to be fully immersed in PBS buffer solution
In.
Step (2) described separation method is to be separated by hand with tweezers.
Step (3) the placement condition is 37 DEG C, 5% (v/v) carbon dioxide.
Contain 5% (v/v) hyclone FBS, 10ng/mL EGF, 2mmol/ in step (4) the DMEM/F12 culture solutions
L L-Glutamines, 10ug/mL insulin, 5ug/mL transferrins, 5ug/mL sodium selenites, 700IU/mL penicillin,
700ug/mL streptomysins, 100ug/mL gentamicins, 5.0ug/mL amphotericin Bs；The condition of culture is 37 DEG C, 5% (v/v)
Carbon dioxide.
Contain 5% (v/v) hyclone FBS, 10ng/mL in step (5) and step (6) the DMEM/F12 culture solutions
EGF, 2mmol/L L-Glutamine, 10ug/mL insulin, 5ug/mL transferrins, 5ug/mL sodium selenites, 100IU/mL are blue or green
Mycin, 100ug/mL streptomysins；Step (5) and step (6) described condition of culture are 37 DEG C, 5% (v/v) carbon dioxide.
Detection：
By the 3rd generation of culture it is cells trypsinised after be resuspended using DMEM/F12 culture solutions, be inoculated with liquid-transfering gun
To being placed in the culture plate of coverslip, when cell covers with the 70%-80% of entire coverslip, cell is cleaned with PBS buffer solution,
4% paraformaldehyde fixes 30min, PBS cleaning 3 times, each 5min.The penetrating 30min of 1%Triton X-100, PBS cleaning 3 times,
Each 5min, 5%BSA close 30min, remove extra BSA, cover mouse CK18 antibody, negative control by equivalent PBS generations
It replaces, 4 DEG C are overnight, 37 DEG C of rewarming 30min, PBS flushings 3 times, each 10min of next day, add in the mountain sheep anti-mouse igg of fluorescent marker,
Room temperature is protected from light 60min, and DAPI mountings after finishing, photo are shot with fluorescence microscope.Fig. 1 is adherent for 1 cud tissue of embodiment
Photo under microscope after 10d；Fig. 2 is photo under 1 2nd generation cud epithelial cell microscope of embodiment；Fig. 3 CK18 identify embodiment
1 the 3rd generation cud epithelial cell fluorescent microscopy images.As photo as can be seen that obtained by cell passage stablize, it is and adherent
Still no bacteria pollution after 10d, is proved by MTT experiment, and cytoactive is apparently higher than the cell of enzyme digestion culture.
Embodiment 2
It is separately cultured sika deer cud epithelial cell：
(1) sika deer cud tissue is taken, after first rumen content is cleaned up with physiological saline, then with containing mould
Element, streptomysin, gentamicin, the PBS buffer solution of amphotericin B are rinsed 8 times, last adds PBS buffer solution after rinsing
And static 10min；
(2) muscle layer of cud tissue and epithelial layer are separated, takes epithelial tissue spare；
(3) epithelial tissue is shredded, the even tissue shredded is laid in blake bottle, inversion is placed in the incubator
2h；
(4) add in containing hyclone FBS, EGF, L-Glutamine, insulin, transferrins, sodium selenite, mould
Element, streptomysin, gentamicin, the DMEM/F12 culture solutions of amphotericin B carry out adhere-wall culture；
Being observed after adding in culture solution, after 3d has cell to climb out of, over time, cell quantity showed increased after 8d.
(5) adhere-wall culture discards culture solution after 4 days, adds in containing hyclone FBS, EGF, L-Glutamine, pancreas islet
Element, transferrins, sodium selenite, penicillin, the DMEM/F12 culture solutions of streptomysin, continue to cultivate, and culture solution was changed every 5 days,
It is described to change culture solution operation to retain the 1/3 of original fluid, it adds in and identical with discarding nutrient solution volume contains hyclone
FBS, EGF, L-Glutamine, insulin, transferrins, sodium selenite, penicillin, the DMEM/F12 culture solutions of streptomysin；Carefully
Cell during born of the same parents' confluent cultures bottom of bottle portion more than 80% is as the 0th generation cell；
(6) the 0th generation cells discard culture solution, and trypsase is added in after being rinsed with PBS buffer solution and is digested, when aobvious
During micro- Microscopic observation cell rounding, add in the DMEM/F12 culture solution containing FBS10% (v/v) isometric with trypsase and terminate
Digestion, by adherent cell piping and druming into suspension, 1000r/min, which is centrifuged, abandons supernatant for 5 minutes, and addition is suitable to contain FBS, EGF, L-
The DMEM/F12 culture solutions piping and druming of glutamine, insulin, transferrins, sodium selenite, penicillin, streptomysin is resuspended, and is divided into
Trisection is added same culture solution and is cultivated respectively respectively, and in 37 DEG C of incubators after static 30min, culture solution is suctioned out
Into another culture dish, using the epithelial cell method purifying cells different from the fibroblast adherent time, this operation repeats 3
It adds identical culture solution after secondary to be cultivated, the sika deer cud epithelial cell that can be purified；Cell confluent cultures bottom of bottle
Cell during portion more than 80% can be passed on next time.
Step (1) described physiological saline is the sodium chloride solution of quality volume fraction 0.9% (g/mL).
Concentration of step (1) the described penicillin in PBS is 700IU/mL, and concentration of the streptomysin in PBS is 700ug/
ML, concentration of the gentamicin in PBS are 100ug/mL, and concentration of the amphotericin B in PBS is 2.5ug/mL.
Step (1) the addition PBS buffer solution, addition are that sika deer cud tissue is made to be fully immersed in PBS buffer solution
In.
Step (2) described separation method is to be separated by hand with tweezers.
Step (3) the placement condition is 37 DEG C, 5% (v/v) carbon dioxide.
Contain 5% (v/v) hyclone FBS, 10ng/mL EGF, 2mmol/ in step (4) the DMEM/F12 culture solutions
L L-Glutamines, 10ug/mL insulin, 5ug/mL transferrins, 5ug/mL sodium selenites, 700IU/mL penicillin,
700ug/mL streptomysins, 100ug/mL gentamicins, 5.0ug/mL amphotericin Bs；The condition of culture is 37 DEG C, 5% (v/v)
Carbon dioxide.
Contain 5% (v/v) hyclone FBS, 10ng/mL in step (5) or step (6) the DMEM/F12 culture solutions
EGF, 2mmol/L L-Glutamine, 10ug/mL insulin, 5ug/mL transferrins, 5ug/mL sodium selenites, 100IU/mL are blue or green
Mycin, 100ug/mL streptomysins；Step (5) or step (6) described condition of culture are 37 DEG C, 5% (v/v) carbon dioxide.
Comparative example 1
With embodiment 1 difference lies in：Penicillin concn in step (1) and step (4) becomes 200IU/mL, streptomysin
Concentration becomes 200ug/mL.As a result only adhere-wall culture 3d just has found that germ contamination occurs in culture solution.
Claims (9)
- A kind of 1. method of sika deer cud epithelial cell culture, it is characterised in that：Step is as follows：(1) take sika deer cud tissue, after first rumen content is cleaned up with physiological saline, then with containing penicillin, chain Mycin, gentamicin, the PBS buffer solution of amphotericin B are rinsed 6-8 times, last adds PBS buffer solution simultaneously after rinsing Static 10min；(2) muscle layer of cud tissue and epithelial layer are separated, takes epithelial tissue spare；(3) epithelial tissue is shredded, the even tissue shredded is laid in blake bottle, 2h is placed in inversion in the incubator；(4) add in containing hyclone FBS, EGF, L-Glutamine, insulin, transferrins, sodium selenite, penicillin, chain Mycin, gentamicin, amphotericin B DMEM/F12 culture solutions in carry out adhere-wall culture；(5) adhere-wall culture is after 4 days, discards culture solution, adds in containing hyclone FBS, EGF, L-Glutamine, insulin, turns Ferritin, sodium selenite, penicillin, the DMEM/F12 culture solutions of streptomysin, continue to cultivate, and culture solution was changed every 4-5 days, described Culture solution operation is changed to retain the 1/3-1/2 of original fluid, adds in and identical with discarding nutrient solution volume contains hyclone FBS, EGF, L-Glutamine, insulin, transferrins, sodium selenite, penicillin, the DMEM/F12 culture solutions of streptomysin；Carefully Cell during born of the same parents' confluent cultures bottom of bottle portion more than 80% is as the 0th generation cell；(6) the 0th generation cells discard culture solution, and trypsase is added in after being rinsed with PBS buffer solution and is digested, when in microscope During lower observation cell rounding, add in DMEM/F12 culture solution containing FBS5-10% (v/v) termination isometric with trypsase and disappear Change, by adherent cell piping and druming into suspension, 1000r/min, which is centrifuged, abandons supernatant for 5 minutes, and addition is suitable to contain FBS, EGF, L- paddy The DMEM/F12 culture solutions piping and druming of glutamine, insulin, transferrins, sodium selenite, penicillin, streptomysin is resuspended, and is divided into three Decile is added same culture solution and is cultivated respectively respectively, in 37 DEG C of incubators after static 30min, by culture solution suction out to In another culture dish, using the epithelial cell method purifying cells different from the fibroblast adherent time, this operation is repeated 3 times It adds identical culture solution afterwards to be cultivated, the sika deer cud epithelial cell that can be purified；Cell confluent cultures bottom of bottle portion Cell when more than 80% can be passed on next time.
- 2. the method for sika deer cud epithelial cell culture according to claim 1, it is characterised in that：Step (1) is described Physiological saline is the sodium chloride solution of quality volume fraction 0.9% (g/mL).
- 3. the method for sika deer cud epithelial cell culture according to claim 1, it is characterised in that：Step (1) is described Concentration of the penicillin in PBS is 700IU/mL, and concentration of the streptomysin in PBS is 700ug/mL, and gentamicin is in PBS Concentration is 100ug/mL, and concentration of the amphotericin B in PBS is 2.5ug/mL.
- 4. the method for sika deer cud epithelial cell culture according to claim 1, it is characterised in that：Step (1) is described PBS buffer solution is added in, addition is that sika deer cud tissue is made to be fully immersed in PBS buffer solution.
- 5. the method for sika deer cud epithelial cell culture according to claim 1, it is characterised in that：Step (2) is described Separation method is to be separated by hand with tweezers.
- 6. the method for sika deer cud epithelial cell culture according to claim 1, it is characterised in that：Step (3) is described Placement condition is 37 DEG C, 5% (v/v) carbon dioxide.
- 7. the method for sika deer cud epithelial cell culture according to claim 1, it is characterised in that：Step (4) is described Contain 5% (v/v) hyclone FBS, 10ng/mL EGF, 2mmol/L L-Glutamines, 10ug/ in DMEM/F12 culture solutions ML insulin, 5ug/mL transferrins, 5ug/mL sodium selenites, 700IU/mL penicillin, 700ug/mL streptomysins, 100ug/mL Gentamicin, 5.0ug/mL amphotericin Bs；The condition of culture is 37 DEG C, 5% (v/v) carbon dioxide.
- 8. the method for sika deer cud epithelial cell culture according to claim 1, it is characterised in that：Step (5) or step Suddenly 5% (v/v) hyclone FBS, 10ng/mL EGF, 2mmol/L L- glutamy are contained in (6) described DMEM/F12 culture solutions Amine, 10ug/mL insulin, 5ug/mL transferrins, 5ug/mL sodium selenites, 100IU/mL penicillin, 100ug/mL streptomysins； Step (5) or step (6) described condition of culture are 37 DEG C, 5% (v/v) carbon dioxide.
- 9. the method for sika deer cud epithelial cell culture according to claim 1, it is characterised in that：Step (6) is described Trypsinase concentration is 0.25% (g/mL).
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| CN109097320A (en) * | 2018-07-23 | 2018-12-28 | 南京农业大学 | A kind of sheep lamb cud epithelial cell cultural method |
| CN109943521A (en) * | 2019-04-17 | 2019-06-28 | 安徽省农业科学院畜牧兽医研究所 | A kind of intestine of young pigs epithelial cell fast preparation method suitable for Porcine epidemic diarrhea virus culture |
| CN111838134A (en) * | 2020-07-31 | 2020-10-30 | 江苏莱森生物科技研究院有限公司 | Tumor tissue transfer liquid and application |
| CN112458040A (en) * | 2020-12-23 | 2021-03-09 | 四川农业大学 | Isolation culture method of rumen epithelial cells of adult yaks |
| CN113930383A (en) * | 2021-09-28 | 2022-01-14 | 黑龙江八一农垦大学 | Method for constructing in-vitro culture system of rumen epithelial tissue of dairy cow |
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| CN111838134A (en) * | 2020-07-31 | 2020-10-30 | 江苏莱森生物科技研究院有限公司 | Tumor tissue transfer liquid and application |
| CN112458040A (en) * | 2020-12-23 | 2021-03-09 | 四川农业大学 | Isolation culture method of rumen epithelial cells of adult yaks |
| CN112458040B (en) * | 2020-12-23 | 2022-07-05 | 四川农业大学 | Isolation culture method of rumen epithelial cells of adult yaks |
| CN113930383A (en) * | 2021-09-28 | 2022-01-14 | 黑龙江八一农垦大学 | Method for constructing in-vitro culture system of rumen epithelial tissue of dairy cow |
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