CN109294972A - A kind of lamb intestinal epithelial cell extracorporeal culturing method - Google Patents
A kind of lamb intestinal epithelial cell extracorporeal culturing method Download PDFInfo
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Abstract
The present invention discloses a kind of lamb intestinal epithelial cell extracorporeal culturing method, includes the following steps: the processes such as tissue sampling, degerming, digestion, cleaning, collection, resuspension, cell inoculation, purifying, passage.The present invention improves ruminant intestinal epithelial cell extracorporeal culturing method, offer operating procedure is simple, spends lower lamb intestinal epithelial cell extracorporeal culturing method, easily obtain that a large amount of purity are preferable, vigor is high, adherent fast intestinal epithelial cell, and the contaminated situation of cell substantially reduces, without the generation contaminated situation of cell in incubation.The common resulting cell of intestinal epithelial cell separation method after 4 days attached cell it is less, cell suspends rounded；And the resulting cell of intestinal epithelial cell isolated culture method of the invention is adherent more after 4 days, closely coupled between cell and cell, cellular morphology is grown in single layer " paver road " and polygonal.
Description
Technical field
The present invention relates to cells to be separately cultured field, especially a kind of lamb intestinal epithelial cell extracorporeal culturing method.
Background technique
The small intestine of ruminant is mainly carbohydrate, albumen, fat, the microprotein for digesting and absorbing rumen bypass
And amino acid etc..Many studies have shown that small intestine also plays important barrier and immunization, developmental condition is strong to animal is guaranteed
The very important effect of health.Therefore, Isolation and culture intestinal epithelial cell is for studying intestinal development, physiological function, nutrition
Material absorbing mechanism and immunization are of great significance.But divide in vitro about ruminant intestinal epithelial cell at present
Technology from culture is still not perfect.Mainly there is following two aspects factor, first is that the chyme in small intestine contains a large amount of microorganism,
It is not easy complete degerming in separation process, leads to vulnerable to pollution during the cultivation process；Second is that presently, there are some be separately cultured
Method and step is cumbersome, it is higher to spend, and it is undesirable to be separately cultured effect.
Goal of the invention
Summary of the invention: existing lamb intestinal epithelial cell extracorporeal culturing method there are aiming at the problem that, the present invention provide one
The novel lamb intestinal epithelial cell extracorporeal culturing method of kind, including tissue sampling, degerming, digestion, cleaning, collection, resuspension, cell
The processes such as inoculation, purifying, passage.Cultural method purpose of the invention is to improve ruminant intestinal epithelial cell in vitro culture
Method, provides that operating procedure is simple, spends lower lamb intestinal epithelial cell extracorporeal culturing method, easily obtain a large amount of purity compared with
Good, vigor height, adherent fast intestinal epithelial cell.
Technical solution: to achieve the goals above, a kind of lamb intestinal epithelial cell in vitro culture as described in the present invention
Method includes the following steps:
(1) small intestine is immediately disconnected and acquired after lamb is butchered, it will be clean with interior flushing outside intestinal tube；
(2) mesenterium and adipose tissue are rejected, intestinal tube is cast aside and is cut into segment, rinsed；
(3) intestinal tube is cut into fragment in a reservoir, continues cleaning until supernatant is limpid, centrifugation removes cleaning solution；
(4) fragment of tissue is poured into middle addition trypsin solution in sterile chamber, is digested, removes digestion after digestion
Liquid；
(5) tissue is poured into new sterile chamber by cleansing tissue fragment after digesting, and pours into trypsin solution digestion, so
After start collect cell；
(6) celliferous digestive juice is filled into the centrifuge tube for be previously added fetal calf serum with cell strainer and is collected carefully
Born of the same parents；
(7) supernatant is abandoned into the cell liquid centrifugation in centrifuge tube, culture medium is added in precipitating, piping and druming mixes；
(8) cell density plantation is adjusted to culture bottle, is cultivated in culture bottle incubator, is changed bottle culture to remove into fiber finer
Born of the same parents；Laying is completed, that is, cultivating resulting cell is lamb intestinal epithelial cell.
Wherein, step (1) is described with interior flushing to be completely by the D- containing penicillin and streptomysin outside intestinal tube
Hanks solution will be clean with interior flushing outside intestinal tube.Preferably, it is immediately disconnected after lamb being butchered and acquires the small of 30cm long
Intestines, will be clean with interior flushing outside intestinal tube with the D-Hanks solution containing 10 times of penicillin and 10 times of streptomysins.
Wherein, step (3) cleaning is until supernatant is limpid mixed containing overturning after three anti-D-Hanks solution to be added
Even, the standing several seconds outwells supernatant, and so cleaning is until supernatant is limpid.Preferably, add the D-Hanks containing 1 times of three anti-concentration molten
It is mixed by inversion after liquid, the standing several seconds outwells supernatant, and so until supernatant is limpid, last time 1000r/min is centrifuged 7 points for cleaning
Clock, prevents that there are more D-Hanks solution.
Wherein, described three resist for penicillin, streptomysin and amphotericin B.
Wherein, step (4) the digestive juice trypsin solution, is digested, and removes digestive juice after digestion, repeats 5-6
It is secondary.
Preferably, the trypsin solution containing 0.25% is added when digestion, digestive juice is lost in 37 DEG C of digestion after ten minutes,
Wherein, cleansing tissue fragment is broken with the D-Hanks solution cleansing tissue resisted containing three after step (5) described digestion
Piece 3-4 times.
Wherein, step (6) it is described celliferous digestive juice is filled into cell strainer be previously added fetal calf serum from
It is to collect within every 10 minutes 1 time that cell is collected in heart pipe, is received 3-4 times altogether.
Wherein, step (7) centrifugation is that 1500-2000r/min is centrifuged 10-15 minutes, abandons supernatant.
Wherein, step (8) the adjusting cell density 106/ ml~107Culture bottle is arrived in plantation after/ml, and culture bottle is placed in 5%
CO2, 37 DEG C, cultivate in the incubator of 95% humidity.
Further, step (8) the resulting cell of culture, the secondary culture when cell converges in 80%, cell pass
It is commissioned to train and supports using trypsin digestion, the time is 4~7 minutes.
Mechanism: the present invention improves and optimizes to the isolated culture method of lamb intestinal epithelial cell, by intestines before digestion
The D-Hanks solution of 10 times of dual anti-concentration of external application is cleaned in managing, and reduces the interference of alien bacteria to the greatest extent.Disappearing
Intestinal tube is shredded before changing, and is cleaned repeatedly with the D-Hanks solution containing 1 times of three anti-concentration, has not only been washed inside intestinal tube
Mucus, further eliminate the microorganism of intestinal tube and mucous layer, prevent from polluting in cell cultivation process.By intestines
Pipe is easier to intestinal epithelial cell digestion after shredding falls off, to reduce digestion time to a certain extent, alleviate pancreas egg
Damage of the white enzyme to epithelial cell is conducive to shorten the growth adjustment period and adherent time after cell inoculation.Simultaneously using at fibre
The dimension cell adherent time, short characteristic carried out changing bottle culture, to remove fibroblast, carried out to required intestinal epithelial cell
Purifying.The intestinal epithelial cell of acquisition can be passed on, and reduced experimentation cost, facilitated Proliferation of Rat Intestinal Crypt Cells, apoptosis
And the development of metabolic test.
Penicillin in the present invention, streptomysin and amphotericin B and 0.25% tryptic digestive juice buy in
GIBCO company.
Wherein, the D-Hanks solution of 10 times of penicillin and 10 times of streptomysins is that penicillin concn is in D-Hanks solution
1000 units/ml, the concentration of streptomysin are 1mg/ml.
The D-Hanks solution of 1 times three anti-(penicillin, streptomysin and amphotericin B) is three anti-concentration in D-Hanks solution
For the amphotericin B of the streptomysin and 0.25 μ g/ml of 100 units/ml penicillin and 100 μ g/ml.
The utility model has the advantages that compared with prior art, the present invention has the advantages that
(1) the contaminated situation of cell substantially reduces, without the generation contaminated situation of cell in incubation.
(2) method and step of the invention is simple to operation, shortens the test period；And reduce cost cost, easily obtain
It is a large amount of that purity is preferable, vigor is high, adherent fast intestinal epithelial cell.The resulting cell 4 of common intestinal epithelial cell separation method
Attached cell is less after it, and cell suspends rounded；And the resulting cell of intestinal epithelial cell isolated culture method of the invention
Adherent more after 4 days, closely coupled between cell and cell, cellular morphology is grown in single layer " paver road " and polygonal.
Detailed description of the invention
Fig. 1 is the intestinal epithelial cell by commonsense method Isolation and culture 96h；
Fig. 2 is the intestinal epithelial cell by the method Isolation and culture 96h；
Specific embodiment
Below in conjunction with drawings and examples, the invention will be further described.
Embodiment 1
Lamb intestinal epithelial cell In-vitro separation culture method:
Tissue sampling: lamb is immediately disconnected and acquires the small intestine of 30cm long after butchering, with contain 10 times of penicillin and 10 times
The D-Hanks solution of streptomysin will be clean with interior flushing outside intestinal tube.
Degerming: carefully rejecting mesenterium and adipose tissue, cast aside intestinal tube with eye scissors and be cut into the segment of about 10cm long, uses
D-Hanks solution containing 10 times of penicillin and 10 times of streptomysins rinses 10 times.
Cleaning: intestinal tube is put into sterile beaker and is cut into fragment (< 0.5cm with Sterile ophthalmic2), it retransfers and sets 50ml's
It in centrifuge tube, is mixed by inversion after adding the D-Hanks solution containing 1 times of three anti-concentration, the standing several seconds outwells supernatant, and so cleaning is straight
Limpid to supernatant, last time 1000r/min is centrifuged 7 minutes, prevents that there are more D-Hanks solution.
Digestion: fragment of tissue being poured into sterile 150ml conical flask, and the trypsin solution containing 0.25% in right amount is added
(0.25% is mass percent) makes digestive juice not have tissue, and digestive juice is lost in 37 DEG C of digestion after ten minutes, so repeatedly 5-6
It is secondary.
Cleaning: three times with the D-Hanks solution cleansing tissue fragment containing 1 times of three anti-concentration.Tissue is put into new cone
In shape bottle, the trypsin solution containing 0.25% is poured into, 37 DEG C digest 10 minutes, then start to collect cell.
It collects: by celliferous digestive juice with 200 aim cell strainer filterings to being previously added 200 microlitres of fetal calf serums
Cell is collected in 50ml centrifuge tube.It collects within every 10 minutes 1 time, receives 3 times altogether, or increase to 4 times.
Be resuspended: the digestive juice 1500-2000r/min collected in centrifuge tube is centrifuged 10-15 minutes, abandons supernatant.It is heavy in gained
1ml DMEM culture medium is added in shallow lake, is blown and beaten and is mixed with pipettor.10% (10% is volume fraction) is added in DMEM culture medium
Fetal calf serum and 1% (1% is volume fraction) it is three anti-.
Cell inoculation: cell density is adjusted to 106/ ml, general 106/ml-107Between/ml, culture bottle is arrived in plantation
(25ml culture bottle adds 10ml complete medium, and the culture bottle of 75ml adds 25ml complete medium).Culture bottle is placed in 5%CO2, 37
DEG C, it is cultivated in the incubator of 95% humidity.
Purifying: 1h changes bottle culture after cell inoculation, and exchanging bottle repeatedly twice, to remove easily adherent fibroblast, changes bottle
After continue cultivate 96h, complete laying, that is, cultivate resulting cell be lamb intestinal epithelial cell.
Passage: the secondary culture when cell converges in 80%, cell secondary culture are disappeared using 0.25% trypsin solution
Change, the time is 4~7 minutes.
Comparative example 1
The separation method of common intestinal epithelial cell is referring to Jia (Jia G, Jiang R C, Wang K N.Effects of
glucagon-like peptide-2on morphology,proliferation and enzyme activity of
intestinal enterocyte cells of weaned piglets in vitro[J].Asian Australas J
Anim Sci, 2009,22:1160-1166.) method.
Cell growth status is observed under an optical microscope, at above-mentioned commonsense method processing and 1 method of the embodiment of the present invention
The intestinal epithelial cell growing state of reason is shown in Fig. 1 and Fig. 2.
The common resulting cell of intestinal epithelial cell separation method after 4 days attached cell it is less, cell suspends rounded；And
The resulting cell of intestinal epithelial cell separation method of the embodiment of the present invention 1 is adherent more after 4 days, tight between cell and cell
Close connected, cellular morphology is grown in single layer " paver road " and polygonal.Illustrate that digestion process can be effectively reduced in method of the invention
Damage to cell grows adjustment period after shortening cell inoculation, guarantees that the cell later period can be proliferated rapidly.
Claims (10)
1. a kind of lamb intestinal epithelial cell extracorporeal culturing method, which comprises the steps of:
(1) small intestine is immediately disconnected and acquired after lamb is butchered, it will be clean with interior flushing outside intestinal tube；
(2) mesenterium and adipose tissue are rejected, intestinal tube is cast aside and is cut into segment, rinsed；
(3) intestinal tube is cut into fragment in a reservoir, continues cleaning until supernatant is limpid, centrifugation removes cleaning solution；
(4) fragment of tissue is poured into middle addition trypsin solution in sterile chamber, is digested, removes digestive juice after digestion；
(5) tissue is poured into new sterile chamber by cleansing tissue fragment after digesting, and is poured into trypsin solution digestion, is then opened
Begin to collect cell；
(6) celliferous digestive juice is filled into the centrifuge tube for be previously added fetal calf serum with cell strainer and collects cell；
(7) supernatant is abandoned into the cell liquid centrifugation in centrifuge tube, culture medium is added in precipitating, piping and druming mixes；
(8) cell density plantation is adjusted to culture bottle, is cultivated in culture bottle incubator, is changed bottle culture to remove fibroblast；
Continue culture completion laying after changing bottle, that is, cultivating resulting cell is lamb intestinal epithelial cell.
2. lamb intestinal epithelial cell extracorporeal culturing method according to claim 1, which is characterized in that step (1) is described
To completely be with interior flushing outside intestinal tube by the D-Hanks solution containing penicillin and streptomysin will outside intestinal tube with it is internal
It rinses well.
3. lamb intestinal epithelial cell extracorporeal culturing method according to claim 1, which is characterized in that step (3) is described
It cleans until supernatant is limpid to add such as containing being mixed by inversion after three anti-D-Hanks solution, the standing several seconds outwells supernatant, so clear
It washes until supernatant is limpid.
4. lamb intestinal epithelial cell extracorporeal culturing method according to claim 3, which is characterized in that described three is anti-preferred
For penicillin, streptomysin and amphotericin B.
5. lamb intestinal epithelial cell extracorporeal culturing method according to claim 1, which is characterized in that step (4) is described
Trypsin solution is added, is digested, removes digestive juice after digestion, repeats 5-6 times.
6. lamb intestinal epithelial cell extracorporeal culturing method according to claim 1, which is characterized in that step (5) is described
Cleansing tissue fragment is with containing three D-Hanks solution cleansing tissue fragment 3-4 times resisted after digestion.
7. lamb intestinal epithelial cell extracorporeal culturing method according to claim 1, which is characterized in that step (6) is described
Celliferous digestive juice being filled into the centrifuge tube for be previously added fetal calf serum with cell strainer and collecting cell is every 10 minutes
It collects 1 time, receives 3-4 times altogether.
8. lamb intestinal epithelial cell extracorporeal culturing method according to claim 1, which is characterized in that step (7) is described
Centrifugation is that 1500-2000r/min is centrifuged 10-15 minutes, abandons supernatant.
9. lamb intestinal epithelial cell extracorporeal culturing method according to claim 1, which is characterized in that step (8) is described
Adjust cell density 106/ ml~107Culture bottle is arrived in plantation after/ml.
10. lamb intestinal epithelial cell extracorporeal culturing method according to claim 1, which is characterized in that step (8) is described
Resulting cell is cultivated, the secondary culture when cell converges in 80%, cell secondary culture uses trypsin digestion, and the time is
4~7 minutes.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109097320A (en) * | 2018-07-23 | 2018-12-28 | 南京农业大学 | A kind of sheep lamb cud epithelial cell cultural method |
| CN112375730A (en) * | 2020-11-23 | 2021-02-19 | 创芯国际生物科技(广州)有限公司 | Small intestine tissue digestion composition, digestive juice system and application thereof |
| 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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| Publication number | Priority date | Publication date | Assignee | Title |
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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 |
| CN112375730A (en) * | 2020-11-23 | 2021-02-19 | 创芯国际生物科技(广州)有限公司 | Small intestine tissue digestion composition, digestive juice system and application thereof |
| 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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