CN107164312A - A kind of Functional Maturation of Calf Intestinal Epithelial extracorporeal culturing method - Google Patents
A kind of Functional Maturation of Calf Intestinal Epithelial extracorporeal culturing method Download PDFInfo
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Abstract
The invention discloses a kind of Functional Maturation of Calf Intestinal Epithelial extracorporeal culturing method.Take calf small intestine, intestinal tube dilator is imported into intestinal tube and intestinal tube is strutted, enteron aisle meconium is rinsed with PBS liquid until the liquid of outflow is limpid, then intestinal tube one end is closed with haemostatic clamp, fill the re-closed intestinal tube other end after protease digestion liquid, it is placed in 37 DEG C of shaking table to digest 1 1.5 hours, collects liquid centrifuge washing twice, be eventually adding nutrient solution inoculated and cultured；Cell attachment grows after 12 24 hours, there is about 90% cell attachment, as intestinal epithelial cell after 34 days.Because protease digestion liquid is directly injected into enteric cavity in the present invention, not with enteron aisle wall contacts, so the fibroblast of enteron aisle outer wall and muscle cell will not be digested, the purity of epithelial cell farthest ensure that.
Description
Technical field
The present invention relates to a kind of biological culture technigue, specifically a kind of Functional Maturation of Calf Intestinal Epithelial extracorporeal culturing method.
Background technology
In mammal, small intestine is the main place that food is digested and assimilated, and small intestinal mucosa epithelial cell is intestines
The functioning cell in road, participates in digestion, absorption, immunization barrier and stress reaction etc. of enteron aisle chyme, and with the inside and outside secretion of enteron aisle
Function has substantial connection.Therefore, Isolation and culture intestinal epithelial cell is for research small intestine physiological function, absorption of nutrient ingredients
Pathophysiological change under mechanism, medicine effect and the effect of various pathogenic factors is significant.Some developed countries are
Through establishing a variety of small intestinal epithelial cell strains in fields such as pharmaceutical developmentses, but its animal species is only limitted to mouse, rat and people；And
For farm animals cytotrophy metabolism in terms of research it is just at the early-stage, research especially on ox report is more few
See, this is mainly due to the intestinal epithelial cell extracorporeal culturing method for not setting up maturation also so far.
At present, the culture of calf intestinal epithelial cell is primarily present two problems.(1) sampling ox only selects.Found in research, profit
Intestinal epithelial cell in vitro culture success rate is carried out with Adult Bovine extremely low because have in Adult Bovine enteron aisle a large amount of chymes and
Microorganism species, i.e., rinsed using a large amount of phosphate buffers (PBS) and be also possible to remaining bacteria, cause original cuiture to pollute；
, can hardly adherent growth and Adult Bovine intestinal epithelial cell break up by height.(2) epithelial cell purity is low.Original cuiture
It is main that crypts of small intestine epithelial cell is separated using protease digestion method.Small intestine is longitudinally cut off during operation, PBS liquid is added after rinsing
The processing of protease digestion liquid, nutrient solution termination digestion is added after enteron aisle crypts comes off from intestinal wall, liquid centrifuge washing is collected, most
Afterwards inoculated and cultured (Loret S, Rusu D, EI Moualij B, Taminiau B, Heinen E, Dandrifosse G,
Mainil J.Preliminary characterization of jejunocyte and colonocyte cell lines
isolated by enzymatic digestion from adult and young cattle.Res Vet Sci.2009,
87(1):123-32).Although this method can separate a large amount of epithelial cells of acquisition, shortcoming is also it will be apparent that because egg
During white enzymic digestion intestinal mucosa tissue, unavoidably the smooth muscle cell of intestinal tube outer wall, fibroblast can also be separated,
Existing epithelial cell in the cell of culture is caused there are fibroblast-like cellses again, epithelial cell mixes raw in fibroblast-like cellses
It is long.(3) separation fibroblast and epithelial cell complex steps and influence cytoactive.In order to separate fibroblast and epithelium
Cell, conventional method, which will typically add observation after pancreatin digestion, has fibroblast-like cellses loose or dislocation but when epithelial cell does not loosen
Digestion is terminated, adds gently to blow and beat after nutrient solution and as far as possible removes the fibroblast-like cellses come off, then pass the cell of residual
It is commissioned to train foster, after the fibroblast-like cellses for removing remnants after cell attachment 80-90% with pancreatin digestion process again, so causes purifying
During the multiple Secondary Culture of epithelial cell, reduce cytoactive and secreting function.
The content of the invention
In order to overcome the shortcoming of above-mentioned intestinal epithelial cell extracorporeal culturing method, the invention provides on a kind of new small intestine
The extracorporeal culturing method of chrotoplast.Selection just birth during (1) intestinal tissue sampling of the invention, do not inhale and permitted breast milk or other are any
The healthy calf of food.Because the intestinal environment of calf is theoretically sterile, and at calf intestinal epithelial cell major part
In undifferentiated state, multiplication capacity is strong, it is easy to adherent growth and Secondary Culture.(2) intestinal tube is not cut off longitudinally, but utilizes special
The dilator (Fig. 1) of system struts intestinal tube, obtains what is largely dissociated after fully rinsing meconium in enteron aisle, injection protease digestion liquid
Adherent growth is intestinal epithelial cell after crypts, inoculation.
The technical scheme is that：A kind of extracorporeal culturing method of Functional Maturation of Calf Intestinal Epithelial, it is characterized in that, take calf
Small intestine, imports intestinal tube by intestinal tube dilator and struts intestinal tube, with PBS liquid flushing enteron aisle meconium until the liquid of outflow is limpid,
Then intestinal tube one end is closed with haemostatic clamp, fills protease digestion liquid and (add 50 ± 5mg collagens in per 100ml DMEM liquid
Used after enzyme, fully 40 ± 4mg neutral proteinases, dissolving after the filtering of 0.22 μm of filter) after the re-closed intestinal tube other end, be placed in
37 DEG C of shaking table digests 1-1.5 hours, collects liquid centrifuge washing twice, is eventually adding nutrient solution inoculated and cultured；After 12-24 hours
Cell attachment grows, and has about 90% cell attachment, as intestinal epithelial cell after 3-4 days.
Specifically include following steps：
(1) nutrient solution is prepared
The blue or green chain of 50-60ml hyclones (FBS), 5-10ml is added in per 450-500ml cell culture fluids (DMEM/F12)
Mycin mixed liquor, 0.1-0.2g glutathione, 0.2-0.5mg insulin, 0.01-0.05mg IGF
With 0.01-0.05g sodium butyrate；Then it is dispensed into after being filtered through 0.22 micron membrane filter in the centrifuge tube of sterilizing, often pipe 40-45 millis
Rise, place 4 DEG C of preservations, be finished in two weeks.Nutrient solution was placed in 37 DEG C of water-baths in 0.5-1 hours before cell culture and preheated.
(2) intestinal epithelial cell culture
Just birth is gathered out of slaughterhouse, the small intestine for the healthy calf for permitting breast milk and other any foods is not inhaled, is placed on
Laboratory is taken back in PBS liquid containing 1% mycillin mixed liquor.Mesenterium, adipose tissue are removed with scissors in super-clean bench, so
Intestinal tube one end is clamped with tweezers afterwards, hand-held dilator slowly imports intestinal tube until intestinal tube is strutted completely, uses 10-20ml syringes
Extract PBS liquid and rinse intestinal tube, close intestinal tube one end with haemostatic clamp after the liquid flowed out is limpid, fill protease with syringe and disappear
Change liquid (to add in per 100ml DMEM liquid after 50 ± 5mg Collagenases, fully 40 ± 4mg neutral proteinases, dissolving through 0.22
Used after the filtering of μm filter), then closed the intestinal tube other end with haemostatic clamp, it is placed in 37 DEG C of shaking table and digests 1-1.5 hours.Collection liquid
Body simultaneously adds isometric nutrient solution and terminates digestion, is placed in centrifugation in 50ml centrifuge tubes (150-200 × g, 5 minutes), goes afterwards
Fall medium centrifugal of the supernatant addition containing 2% D-sorbite to wash twice, be eventually adding nutrient solution resuspension centrifugation bottom of the tube and sink
Starch, is inoculated into blake bottle by 800-1000 crypts/ml, is placed in 37 DEG C, 5%CO2Cultivated in saturated humidity incubator.
Cell attachment grows after 12-24 hours, there is about 90% cell attachment after 3-4 days.
The intestinal tube dilator of the present invention, it is sequentially connected including preposition ring, spiral steel ring, rearmounted ring and handle；Wherein hand
The connection of handle and rearmounted ring is to be detachably connected.In use, preposition ring to be put into one end of small intestine, rotating handle makes spiral steel ring
Rotation slowly enters small intestine, and rearmounted ring is stayed in the other end of small intestine.Handle is removed after injection protease digestion liquid, haemostatic clamp is used
Close intestinal tube.
The beneficial effects of the invention are as follows：(1) present invention is strutted intestinal tube using special enteron aisle dilator, injects PBS liquid
After quickly meconium in enteron aisle can be rinsed well；And after protease digestion liquid is added, digestive juice fully connects with intestinal mucosa
Touch, rapidly and uniformly can digest crypts, obtain largely free crypts, adherent growth is small intestine epithelium after inoculation
Cell.Because protease digestion liquid is directly injected into enteric cavity in the present invention, not with enteron aisle wall contacts, so will not be by parenterally
The fibroblast of wall and muscle cell digest, and farthest ensure that the purity of epithelial cell, also omit separation
The step of fibroblast and epithelial cell, it enormously simplify method.(2) present invention is added in nutrient solution increases to epithelial cell
Grow the energy matter glutathione for having the effect of being obviously promoted, promote insulin, IGF and the fourth of cell differentiation
Sour sodium, makes culture environment be more conducive to epithelial cell proliferation, differentiation, therefore can obtain 90% or so cell within 3-4 days after inoculation
It is adherent.(3) present invention is had the benefit that using intestinal tube dilator：Enteron aisle is all strutted, meconium in enteron aisle is rinsed and egg
The two steps of white enzymic digestion carry out ground ratio more thoroughly, it is not easy to stay dead angle, and can obtain largely free crypts；Rinse simultaneously
Speed with digestion is fast, does not influence cytoactive.
Brief description of the drawings
Fig. 1 is the structural representation of the special intestinal tube dilator of the present invention；
Fig. 2 is Functional Maturation of Calf Intestinal Epithelial using 100 × photo after the method culture 3 days of embodiment 1；
In figure：1st, preposition ring, 2, spiral steel ring, 3, rearmounted ring, 4, handle.
Embodiment
As shown in figure 1, the intestinal tube dilator of the present invention includes preposition ring 1, spiral steel ring 2, rearmounted ring 3 and handle 4 successively
Connection, wherein preposition ring 1, spiral steel ring (provided with 4 section spiral steel rings in spring-like, Fig. 1) 2 are to be fixedly connected with rearmounted ring；
Handle 4 is detachably connected by dismountable mode (be such as threadedly coupled, snap connection) and rearmounted ring 3.
In use, preposition ring to be put into one end of small intestine, the rotation of spiral steel ring slowly enters small intestine, rearmounted ring is stayed in small
The other end of intestines.It is easy to using form of annular rings by intestinal tube support to maximum at the two ends of above-mentioned intestinal tube dilator.Due to small intestine have it is flat,
Spiral steel ring form is used in the middle of the characteristics of bending, intestinal tube dilator, is easy to relatively easily enter small intestine using rotated versions,
Spiral steel ring form can improve the scalability of intestinal tube dilator simultaneously, be allowed to the small intestine suitable for different length, also allow for
Small intestine two ends tying.Handle is easy to remove handle after injection protease digestion liquid using detachable form, is closed with haemostatic clamp
Intestinal tube.
Embodiment 1
(1) nutrient solution is prepared
55ml hyclones (FBS), 5ml mycillin mixed liquors are added in per 500ml cell culture fluids (DMEM/F12)
[Pen .- Strep mixed liquor (100X), Penicillin Content is 10000U/ml, and the content of streptomysin is 10mg/ml], 0.1g
Glutathione, 0.3mg insulin, 0.05mg IGF solution and 0.05g sodium butyrate；Then through 0.22
It is dispensed into after micron membrane filter filtering in the centrifuge tube of sterilizing, often 40 milliliters of pipe, places 4 DEG C of preservations, be finished in two weeks.Cell culture
Nutrient solution was placed in 37 DEG C of water-baths in first 1 hour and preheated.
(2) intestinal epithelial cell culture
Just birth is gathered out of slaughterhouse, the small intestine for the healthy calf for permitting breast milk or other any foods is not inhaled, is placed on
Laboratory is taken back in PBS containing 1% mycillin mixed liquor.Mesenterium, adipose tissue are removed with scissors in super-clean bench, then
Intestinal tube one end is clamped with tweezers, hand-held intestinal tube dilator slowly imports intestinal tube until intestinal tube is strutted completely, uses 20ml syringes
PBS liquid flushing intestinal tube is extracted (slowly to move intestinal tube dilator in flushing process, make the place contacted with intestinal tube dilator
Fully rinsed).Intestinal tube one end is closed with haemostatic clamp after the liquid flowed out is limpid, protease digestion liquid is filled with syringe
(filtered after 50mg Collagenases, fully 40mg neutral proteinases, dissolving are added in per 100ml DMEM liquid through 0.22 μm of filter
After use), then the intestinal tube other end is closed with haemostatic clamp, is placed in 37 DEG C of shaking table and digests 1.5 hours.Collect the body such as liquid and addition
Long-pending nutrient solution terminates digestion, is placed in centrifugation in 50ml centrifuge tubes (200 × g, 5 minutes), removes supernatant afterwards and adds and contains 2%
The medium centrifugal of D-sorbite is washed twice, be eventually adding nutrient solution be resuspended centrifuge tube bottom sediment, by 1000 crypts/
Ml is inoculated into blake bottle, is placed in 37 DEG C, 5%CO2Cultivated in saturated humidity incubator.Cell attachment grows after 18 hours, 3 days
After have about 90% cell attachment (Fig. 2).Observed under inverted microscope, cellular morphology is in irregular polygonal, just as cobblestone
Shape, and the growth of clear border, cluster, these are all the characteristic features of epithelial cell.Intestinal epithelial cell purity up to 98% with
On.
Claims (6)
1. a kind of extracorporeal culturing method of Functional Maturation of Calf Intestinal Epithelial, it is characterized in that, calf small intestine is taken, intestinal tube dilator is imported
Intestinal tube simultaneously struts intestinal tube, rinses enteron aisle meconium with PBS liquid until the liquid of outflow is limpid, then with haemostatic clamp closing intestinal tube one
End, fills the re-closed intestinal tube other end after protease digestion liquid, is placed in 37 DEG C of shaking table and digests 1-1.5 hours, collects liquid centrifugation
Washing, is eventually adding nutrient solution inoculated and cultured；The protease digestion liquid is：50 ± 5mg glue is added in per 100ml DMEM liquid
Former protease, is used after 40 ± 4mg neutral proteinases, fully dissolving, filtering.
2. a kind of extracorporeal culturing method of Functional Maturation of Calf Intestinal Epithelial as claimed in claim 1, it is characterized in that, the intestinal tube support
Device is driven, it is sequentially connected including preposition ring, spiral steel ring, rearmounted ring and handle；The connection of wherein handle and rearmounted ring is removable
Unload connection.
3. a kind of extracorporeal culturing method of Functional Maturation of Calf Intestinal Epithelial as claimed in claim 1 or 2, it is characterized in that, the training
Nutrient solution is：50-60ml hyclones, the mixing of 5-10ml mycillins are added in per 450-500ml DMEM/F12 cell culture fluids
Liquid, 0.1-0.2g glutathione, 0.2-0.5mg insulin, 0.01-0.05mg IGF and 0.01-
0.05g sodium butyrate.
4. a kind of extracorporeal culturing method of Functional Maturation of Calf Intestinal Epithelial as claimed in claim 3, it is characterized in that, out of slaughterhouse
Collection just birth, the small intestine for not inhaling the healthy calf for permitting breast milk and other any foods, are placed on containing 1% mycillin mixed liquor
PBS liquid in take back laboratory；Mesenterium, adipose tissue are removed with scissors in super-clean bench, intestinal tube one is then clamped with tweezers
End, hand-held dilator slowly imports intestinal tube until intestinal tube is strutted completely, extracts PBS liquid with 10-20ml syringes and rinses intestinal tube,
Intestinal tube one end is closed with haemostatic clamp after the liquid flowed out is limpid, protease digestion liquid is filled with syringe, then will with haemostatic clamp
The intestinal tube other end is closed, and is placed in 37 DEG C of shaking table and is digested 1-1.5 hours；Collect liquid and add isometric nutrient solution and terminate and disappear
Change, be placed in 50ml centrifuge tubes and centrifuge, remove medium centrifugal of the supernatant addition containing 2% D-sorbite afterwards and wash twice,
It is eventually adding nutrient solution and centrifuge tube bottom sediment is resuspended, is inoculated into by 800-1000 crypts/ml in blake bottle, is placed in 37
DEG C, 5%CO2Cultivated in saturated humidity incubator.
5. a kind of extracorporeal culturing method of Functional Maturation of Calf Intestinal Epithelial as claimed in claim 4, it is characterized in that, the protease
Digestive juice is：Passed through after 50 ± 5mg Collagenases, fully 40 ± 4mg neutral proteinases, dissolving are added in per 100ml DMEM liquid
Used after 0.22 μm of filter filtering.
6. a kind of extracorporeal culturing method of Functional Maturation of Calf Intestinal Epithelial as claimed in claim 4, it is characterized in that, the centrifugation
For：150-200 leaves the heart 5 minutes.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111849868A (en) * | 2020-08-03 | 2020-10-30 | 青岛农业大学 | Epithelial cell culture solution |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN2140239Y (en) * | 1992-11-10 | 1993-08-18 | 中国人民解放军第251医院 | Dilating stand for rectum |
| CN106676059A (en) * | 2016-12-23 | 2017-05-17 | 湖南师范大学 | Piglet small intestine epithelial cell classification and separation method |
-
2017
- 2017-07-24 CN CN201710607158.9A patent/CN107164312B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN2140239Y (en) * | 1992-11-10 | 1993-08-18 | 中国人民解放军第251医院 | Dilating stand for rectum |
| CN106676059A (en) * | 2016-12-23 | 2017-05-17 | 湖南师范大学 | Piglet small intestine epithelial cell classification and separation method |
Non-Patent Citations (3)
| Title |
|---|
| 刘芳宁等: "哺乳动物肠上皮细胞的原代培养", 《动物医学进展》 * |
| 欧阳万金 等: "几种氨基酸及其衍生物对猪肠上皮细胞", 《饲料工业》 * |
| 赵倩明等: "奶牛小肠上皮细胞的原代培养和鉴定", 《中国农业大学学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111849868A (en) * | 2020-08-03 | 2020-10-30 | 青岛农业大学 | Epithelial cell culture solution |
| CN111849868B (en) * | 2020-08-03 | 2022-02-08 | 青岛农业大学 | Epithelial cell culture solution |
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