CN104818238A - Isolated culture method for chicken enterocyte - Google Patents
Isolated culture method for chicken enterocyte Download PDFInfo
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Abstract
The invention relates to an isolated culture method for chicken enterocyte. The method comprises the following steps: step 1, digesting the intestinal tissue block of a chicken embryo with protease so as to obtain a digestion product; step 2, filtering the digestion product with a cell strainer and collecting cell aggregate with a size of 100 to 500 meshes; and step 3, subjecting the cell aggregate to centrifugation at a speed of 800 to 1500 r/min for 5 to 15 min and collecting cell sediment. The invention further provides the chicken enterocyte obtained by using the method. The method provided by the invention can substantially shorten testing time, save reagents and improve the purity of the isolated enterocyte.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of chicken intestinal epithelial cells isolation cultivation method.
Background technology
Intestinal epithelial cells is the medium of enteron aisle internal and external environment, is also the important component part of immunity of organism barrier simultaneously, has the physiological function that digestion, absorption, secretion, immunity etc. are important.Intestinal epithelial cells can carry out in-vitro separation and cultivation under certain condition.Isolation and culture intestinal epithelial cells is for research enteron aisle biological function, absorption of nutrient ingredients mechanism and regulation and control thereof and Intestinal epitheliual cell proliferation, differentiation and apoptosis provide simply, means fast and accurately, and provides desirable external model for research nutrient substance and the effect of other extraneous factors to enteric epithelium.But the separation and Culture difficulty of intestinal epithelial cells is comparatively large, and the report successfully setting up mono-clonal Intestine Epithelial Cell Lines is less.For a long time, at the Control factors of the research biological function of intestinal epithelial cells, cell proliferation and differentiation, many employings such as material absorbing and metabolism enteron aisle cancerous cell line is experimental model.But there is certain difference in enteron aisle cancer epithelial cell and normal epithelium cell, explain that normal behavior exists significant limitation with the result of study of cancerous cell line in cell basic structure, metabolism and functional performance etc.
The patent documentation had not yet to see about chicken enteric epithelium primary cell in-vitro separation or cultural method is open, although adopt routine cell culture techniques in existing chicken intestinal epithelial cells cultural method, the object of cultivation can be reached to a certain extent, but there is complex operation, cell is not easily adherent, other heteroproteose cells such as poor growth, inoblast are difficult to the problems such as separation, can not meet the requirement of cell experiment.
By long-term experiment research, the present inventor finds that intestinal epithelial cells is mingled with fibroblastic growth often owing to intestinal epithelial cells cannot be separated with inoblast when isolated cell, document is had to adopt the mode of differential velocity adherent or low-speed centrifugal to be separated into fibrocyte composition, but this process is comparatively loaded down with trivial details and consuming time, usually need 5-7 hour and cell purity is lower.
Therefore be necessary to develop the chicken intestinal epithelial cells In-vitro separation culture method that a kind of operating process is easy, cell attachment is effective, growth is rapid, cell purity is high.
Summary of the invention
The object of the invention is to overcome the above-mentioned defect existed in prior art, develop that a kind of operating process is easy, cell attachment is effective, it is rapid, in good condition to grow, purity is high chicken intestinal epithelial cells In-vitro separation culture method.
The present inventor's further investigation compares the method for domestic and international culture of isolated intestinal epithelial cells, is groped, improves the extracorporeal culturing method of chicken intestinal epithelial cells, for the subsequent biological Effect study of intestinal epithelial cells provides experimental model by great many of experiments.
The invention provides a kind of chicken intestinal epithelial cells isolation cultivation method, said method comprising the steps of:
Step 1: digest chicken embryo intestinal tissue block with proteolytic enzyme, obtains digestion product;
Step 2: with cell sieve, described digestion product is filtered, collect and be of a size of 100-500 object cell mass;
Step 3: by described cell mass centrifugal 5-15min under 800-1500r/min, collecting cell precipitates.
Optionally, described proteolytic enzyme is NTx proteolytic enzyme and/or Unidasa.
Optionally, when described proteolytic enzyme be I-type collagen enzyme and Unidasa time, the amount ratio of I-type collagen enzyme and Unidasa is 1-2:1.
Optionally, in the digestive process of step 1, in digestion system, the concentration of I-type collagen enzyme is 0.8-1.6mg/mL; The concentration of Unidasa is 0.4-1.6mg/mL.
Optionally, described method comprise chicken embryo intestinal tissue block is soaked in DMEM/F12 substratum after digest again.
Optionally, the condition of described digestion is at 37 DEG C of concussion digestion 25-35min.
Optionally, described method also comprises carries out original cuiture to described cell precipitation after step 3, the condition of described original cuiture comprises: the DMEM/F12 added in described cell precipitation containing 20% foetal calf serum cultivates basic weight and revolves piping and druming and make cell suspension, by described cell suspension inoculation in Collagen type-I bag by good culture plate, be placed in 37 DEG C, 5%CO
2cultivate 24-48 hour in incubator, after adherent, change the DMEM/F12 substratum continuation cultivation that foetal calf serum concentration is 10%.
Optionally, described chicken embryo intestinal tissue block is of a size of 0.5-1mm
3.
Optionally, described chicken embryo intestinal tissue block takes from instar chicken embryo on the 15th.
Present invention also offers the chicken intestinal epithelial cells that the method described in utilization obtains.
The present invention utilizes intestinal epithelial cells agglomerate and the difference of inoblast in physics size, the cell of certain pore size sieve is adopted to filter digestion product, most of inoblast composition is taken out to be separated, the present invention shortens test period greatly, save a large amount of reagent, improve the purity being separated intestinal epithelial cells.
Accompanying drawing explanation
Fig. 1 is the tissue morphology figure of intestinal epithelial cells growth, and microscope amplifies 200 times and observes cultivation substantive dyeing after 24 hours, negative staining qualification result (control group is unstained).
Fig. 2 is the tissue morphology figure of intestinal epithelial cells growth, observes cultivation substantive dyeing after 24 hours, stained positive qualification result for microscope amplifies 200 times.
Fig. 3 is that microscope amplifies 200 times and observes cultivations and become individual cells poststaining negative identification result (control group is unstained) through trysinization after 24 hours.
Fig. 4 be microscope amplify 200 times observe cultivations after 24 hours and through trysinization become individual cells poststaining identify positive findings.
Embodiment
Below will the present invention is described in detail by embodiment.It will be appreciated that providing of following examples is only object in order to play explanation, being not used to limit scope of the present invention.Those skilled in the art, when not deviating from aim of the present invention and spirit, can carry out various amendment and replacement to the present invention.
The invention provides a kind of chicken intestinal epithelial cells isolation cultivation method, said method comprising the steps of:
Step 1: digest chicken embryo intestinal tissue block with proteolytic enzyme, obtains digestion product;
Step 2: with cell sieve, described digestion product is filtered, collect and be of a size of 100-500 object cell mass;
Step 3: by described cell mass centrifugal 5-15min under 800-1500r/min, collecting cell precipitates.
Wherein, the source of described chicken intestinal tissue block can be the chicken embryo intestinal tissue of 15 ages in days, cleans up after being rejected by the mesentery of described intestinal tissue.The method of cleaning can for rinsing repeatedly with containing antibiotic PBS solution, and it is 100U/L penicillin and 100 μ g/L Streptomycin sulphates that described microbiotic can comprise concentration.
Method provided by the present invention comprise chicken embryo intestinal tissue block is soaked in DMEM/F12 substratum after digest again, wherein, in digestive process, in digestion system, the concentration of I-type collagen enzyme is 0.8-1.6mg/mL; The concentration of Unidasa is 0.4-1.6mg/mL.
In one embodiment of the invention, the intestinal tissue after removal mesentery and cleaning can be soaked in DMEM/F12 substratum and carry out shearing acquisition chicken embryo intestinal tissue block, described chicken embryo intestinal tissue block is of a size of 0.5-1mm
3.
In method provided by the present invention, described proteolytic enzyme is NTx proteolytic enzyme and/or Unidasa.In preferred situation, described proteolytic enzyme is NTx proteolytic enzyme and Unidasa.
When described proteolytic enzyme be I-type collagen enzyme and Unidasa time, the amount ratio of I-type collagen enzyme and Unidasa is 1-2:1.
In the present invention, first I-type collagen enzyme can be dissolved as concentration is contact with tissue block to be digested after the enzyme solution of 1-2mg/mL again; Unidasa being dissolved as concentration is contact with tissue block to be digested after the enzyme solution of 0.5-2mg/mL again; Or I-type collagen enzyme and Unidasa are mixed with after mixed enzyme solution and contact with tissue block to be digested, wherein, in mixed enzyme solution, the concentration of I-type collagen enzyme is 1-2mg/mL, and the concentration of Unidasa is 0.5-2mg/mL.
Optionally, the condition of described digestion is at 37 DEG C of concussion digestion 25-35min, preferably digests 30min.
Optionally, described method also comprises carries out original cuiture to described cell precipitation after step 3, the condition of described original cuiture comprises: the DMEM/F12 added in described cell precipitation containing 20% foetal calf serum cultivates basic weight and revolves piping and druming and make cell suspension, by described cell suspension inoculation in Collagen type-I bag by good culture plate, be placed in 37 DEG C, 5%CO
2cultivate 24-48 hour in incubator, after adherent, change the DMEM/F12 substratum continuation cultivation that foetal calf serum concentration is 10%.
Wherein, carrying out in the substratum of original cuiture and continuation cultivation can also be the penicillin of 100U/L and the Streptomycin sulphate of 100 μ g/L containing concentration.
In one embodiment of the invention, method provided by the present invention comprises the following steps:
First digestion product is sieved by 100 object cells, collect respectively by the mixture 1 of 100 order cells sieves with not by the component 1 of 100 order cells sieves.
Wherein, the mixture 1 by 100 order cell sieves is collected supernatant liquor 1 and throw out 1 after 1000r/min is centrifugal 10 minutes.Wherein, the main component in supernatant liquor 1 is individual cells, NTx enzyme and/or Unidasa liquid.This supernatant liquor 1 may be used for follow-up digestion step.
Wherein, the main component of throw out 1 is enteric epithelium crypts unit and part individual cells, and it is resuspended to prevent cell dehydration dead to add DMEM/F12 substratum in the throw out 1 collected, and obtains re-suspension liquid 1.
Wherein, component 1 not by 100 order cells sieves to be reclaimed and to add DMEM/F12 substratum resuspended, mixture after this is resuspended is again crossed 100 order cells and is sieved after carrying out secondary digestion after mixing with the supernatant liquor 1 collected before, collect the mixture 2 by 100 order cell sieves, by described mixture 2 collecting precipitation thing 2 after 1000r/min is centrifugal 10 minutes by 100 order cell sieves, obtain re-suspension liquid 2. with after the resuspended described throw out 2 of DMEM/F12 substratum
Described re-suspension liquid 1 and re-suspension liquid 2 are mixed rear acquisition mixing suspension, described mixing suspension is crossed 500 order cell sieves and remove individual cells, collect not by the component of 500 order cell sieves, this component is abandoned supernatant collecting precipitation thing 3 after 1000r/min low-speed centrifugal, original cuiture is carried out to described throw out 3.
Present invention also offers the chicken intestinal epithelial cells utilizing method of the present invention to obtain.
Below by embodiment, the present invention will be described in detail:
In the examples below: subjects: instar chicken embryo on the 15th.
PBS (Hyclone company), DMEM/F12 (Hyclone company), foetal calf serum (Gibico company), NTx enzyme (Sigma company), Unidasa (Sigma company); BCIP/NBT alkaline phosphatase colouring reagents (green skies company), Collagen type-I (Sigma company), cell sieve (Suo Laibao company) CO
2incubator (Thermo Scientific company), inverted microscope (Olympus company).
Embodiment 1
1, original cuiture: get 2 pieces of instar chicken embryo intestinal tissues on the 15th under aseptic condition, carefully reject mesentery, rinses 3-4 time repeatedly with containing antibiotic PBS.Collection picks deimpurity intestinal tissue in 20mL small beaker, adds the DMEM/F12 of 5mL, is cut into is less than 1mm with eye scissors
3tissue block, be transferred to 50mL centrifuge tube, and add the Unidasa simultaneous digestion that NTx enzyme that 20mL concentration is 2mg/mL and 20mL concentration are 1mg/mL, in 37 water baths, 80r/min stirs 30min, then 100 order cell sieves are adopted to filter digestion product mixtures, the mixture 1000r/min sieved by cell is after centrifugal 10 minutes, supernatant liquor (main component is NTx enzyme and Unidasa liquid and individual cells) is transferred to new centrifuge tube stand-by, to throw out, (main component is enteric epithelium crypts unit, and part individual cells) to add the DMEM/F12 substratum of 5mL resuspended to prevent cell dehydration dead, stand-by.Not by the tissue block of 100 order cell sieves, continue to repeat a upper digestion step with the mixed enzyme Digestive system that previous step reclaims after recovery, and collect crypts unit with reference to aforesaid way, the crypts unit of collection mixes with crypts unit before, and add that DMEM/F12 heavily revolves, trim is to 50mL.Next, adopt 500 order cell sieved filter crypts mixed solution, to remove individual cells, reclaim not by the intestinal crypts unit of 500 order cell sieves with DMEM/F12, supernatant is abandoned after 1000r/min low-speed centrifugal, throw out 20mL is resuspended containing the DMEM/F12 substratum of 20%FBS, 100U/L penicillin and 100 μ g/L Streptomycin sulphates, and plants in 6 porocyte plates of Collagen type-I bag quilt, every hole 3mL.Finally be placed in 37 DEG C, 5%CO
2cultivate 24 hours in incubator, the DMEM/F12 substratum used instead after adherent containing 10%FBS, 100U/L penicillin and 100 μ g/L Streptomycin sulphates continues to cultivate, and changes liquid every 48 hours later.And every day observation of cell adherent growth situation under inverted microscope.
2, the qualification of intestinal epithelial cells: intestinal epithelial cell can secrete mucus, alkaline phosphatase is the significant enzyme on intestinal epithelial cells microvillus.After cell confluent culture plate, with trysinization to individual cells, make cell suspension and with BCIP/NBT alkaline phosphatase colouring reagents box, dyeing qualification carried out to cell, basis of microscopic observation staining conditions, be black to occur that result shows positive epithelial cell, negative control does not develop the color, and counts under the microscope and take pictures.
3, result: original cuiture result: chicken intestinal epithelial cells crypts is generally adherent at 24 hours, in " island " shape.Continue cultivation after changing liquid medium 24 hours, as seen the radiation growth towards periphery from island, 48-60 as a child converged in flakes gradually, in " paving stone " shape.
The qualification of intestinal epithelial cells: after cell confluent culture plate, application BCIP/NBT alkaline phosphatase colouring reagents box carries out dyeing qualification to cell, and positive cell is dyed black-and-blue, and positive rate, to more than 80%, confirms that most cell is intestinal epithelial cells.
Be the tissue morphology figure of intestinal epithelial cells growth see Fig. 1 to Fig. 4: Fig. 1, microscope amplifies 200 times and observes cultivation substantive dyeing after 24 hours, negative staining qualification result (control group is unstained).Fig. 2 is the tissue morphology figure of intestinal epithelial cells growth, observes cultivation substantive dyeing after 24 hours, stained positive qualification result for microscope amplifies 200 times.
Fig. 3 is that microscope amplifies 200 times and observes cultivations and become individual cells poststaining negative identification result (control group is unstained) through trysinization after 24 hours.Fig. 4 be microscope amplify 200 times observe cultivations after 24 hours and through trysinization become individual cells poststaining identify positive findings.
Comparative example 1
According to document (chicken enteric epithelium primary cell Isolation and culture and qualification, Shi Run, Yang Xia, Chen Lu, etc., Jiangsu's agriculture science, 2013,41 (1): 34-37.) in the method that provides carry out the separation and Culture of chicken enteric epithelium myocyte:
Aseptic taking-up instar chicken embryo intestinal tissue on the 15th, PBS repetitive scrubbing, rejects mesentery, is cut into and is less than 1mm
3tissue block, I type neutral protease and Ⅺ Collagenase Type simultaneous digestion intestinal tissue, at 37 DEG C of water bath low rate mixings, 80r/min 30min.With the centrifugal 10min of 1000r/min after having digested, a small amount of DMEM/F12 substratum suspension cell is added in cell precipitation, after centrifuge washing three times, the cell culture fluid (DMEM/F12 (pH7.0) 12g/L+20 μ g/L Urogastron+100mg/L heparin sodium+110mg/L Sodium.alpha.-ketopropionate+2.5mg/L Regular Insulin+200mmol/L L-glutamic acid ammonia+100U/L penicillin+100 μ g/L Streptomycin sulphate) of the cell collected containing 10%FBS suspends, and plants in glass cell bottle.After 2.5 hours, nutrient solution and not adherent cell are transferred in centrifuge tube respectively, with the centrifugal 10min of 1000r/min, be inoculated in the mistake disposable T25 cell bottle of 20%FBS bag quilt add the cell culture fluid (DMEM/F12 (pH7.0) 12g/L+20 μ g/L Urogastron+100mg/L heparin sodium+110mg/L Sodium.alpha.-ketopropionate+2.5mg/L Regular Insulin+200mmol/L (2mmol/L) L-glutamic acid ammonia+100U/L penicillin+100 μ g/L Streptomycin sulphate) containing 2.5%FBS in cell precipitation after, observe after 24h and change liquid.Sharp comparative example 1 is compared with the isolation cultivation method of embodiment 1, the results are shown in table 1.
Table 1
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. a chicken intestinal epithelial cells isolation cultivation method, is characterized in that, said method comprising the steps of:
Step 1: digest chicken embryo intestinal tissue block with proteolytic enzyme, obtains digestion product;
Step 2: with cell sieve, described digestion product is filtered, collect and be of a size of 100-500 object cell mass;
Step 3: by described cell mass centrifugal 5-15min under 800-1500r/min, collecting cell precipitates.
2. method according to claim 1, is characterized in that, described proteolytic enzyme is NTx proteolytic enzyme and/or Unidasa.
3. method according to claim 2, is characterized in that, when described proteolytic enzyme be I-type collagen enzyme and Unidasa time, the amount ratio of I-type collagen enzyme and Unidasa is 1-2:1.
4. according to the method in claim 2 or 3, it is characterized in that, in step 1, in digestion system, the concentration of I-type collagen enzyme is 0.8-1.6mg/mL; The concentration of Unidasa is 0.4-1.6mg/mL.
5. method according to claim 4, is characterized in that, described method comprise chicken embryo intestinal tissue block is soaked in DMEM/F12 substratum after digest again.
6. method according to claim 5, is characterized in that, the condition of described digestion is at 37 DEG C of concussion digestion 25-35min.
7. method according to claim 6, it is characterized in that, described method also comprises carries out original cuiture to described cell precipitation after step 3, the condition of described original cuiture comprises: the DMEM/F12 added in described cell precipitation containing 20% foetal calf serum cultivates basic weight and revolves piping and druming and make cell suspension, by described cell suspension inoculation in Collagen type-I bag by good culture plate, be placed in 37 DEG C, 5%CO
2cultivate 24-48 hour in incubator, after adherent, change the DMEM/F12 substratum continuation cultivation that foetal calf serum concentration is 10%.
8. the method according to claim 6 or 7, is characterized in that, described chicken embryo intestinal tissue block is of a size of 0.5-1mm
3.
9. method according to claim 8, is characterized in that, described chicken embryo intestinal tissue block takes from instar chicken embryo on the 15th.
10. utilize the chicken intestinal epithelial cells that the method in claim 1-9 described in any one obtains.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106884002A (en) * | 2015-12-15 | 2017-06-23 | 上海市针灸经络研究所 | A kind of colon epithelial cell cultural method of stabilization |
| CN107217040A (en) * | 2017-06-16 | 2017-09-29 | 中国农业大学 | One kind immortalizes rabbit intestinal epithelial cell line and its construction method |
| CN110396496A (en) * | 2018-09-30 | 2019-11-01 | 湖北省农业科学院畜牧兽医研究所 | A kind of cultural method of duck intestinal epithelial cell and application |
| CN114480255A (en) * | 2022-03-10 | 2022-05-13 | 吉林农业大学 | Method for isolating intestinal epithelial cells under contaminated conditions |
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| US20120190100A1 (en) * | 2009-07-21 | 2012-07-26 | Transgene AS | Enzymatic composition for the digestion of chicken embryos |
| CN104357393A (en) * | 2014-11-14 | 2015-02-18 | 中国农业科学院饲料研究所 | Isolated culture method of chicken intestinal tract epithelial GammaDeltaT cells |
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| US20120190100A1 (en) * | 2009-07-21 | 2012-07-26 | Transgene AS | Enzymatic composition for the digestion of chicken embryos |
| CN104357393A (en) * | 2014-11-14 | 2015-02-18 | 中国农业科学院饲料研究所 | Isolated culture method of chicken intestinal tract epithelial GammaDeltaT cells |
| CN105483078A (en) * | 2015-12-24 | 2016-04-13 | 山西农业大学 | Isolation and primary culture methods of chicken small intestinal epithelial cells |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106884002A (en) * | 2015-12-15 | 2017-06-23 | 上海市针灸经络研究所 | A kind of colon epithelial cell cultural method of stabilization |
| CN106884002B (en) * | 2015-12-15 | 2021-03-19 | 上海市针灸经络研究所 | Stable colon epithelial cell culture method |
| CN107217040A (en) * | 2017-06-16 | 2017-09-29 | 中国农业大学 | One kind immortalizes rabbit intestinal epithelial cell line and its construction method |
| CN110396496A (en) * | 2018-09-30 | 2019-11-01 | 湖北省农业科学院畜牧兽医研究所 | A kind of cultural method of duck intestinal epithelial cell and application |
| CN114480255A (en) * | 2022-03-10 | 2022-05-13 | 吉林农业大学 | Method for isolating intestinal epithelial cells under contaminated conditions |
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