CN105219702A

CN105219702A - The preparation method of a kind of newborn piglet intestinal villus epithelium primary cell strain

Info

Publication number: CN105219702A
Application number: CN201510766128.3A
Authority: CN
Inventors: 姚俊; 高林; 李华春
Original assignee: Yunnan Animal Science and Veterinary Institute
Current assignee: Yunnan Animal Science and Veterinary Institute
Priority date: 2015-11-11
Filing date: 2015-11-11
Publication date: 2016-01-06
Anticipated expiration: 2035-11-11
Also published as: CN105219702B

Abstract

The present invention relates to the preparation method of a kind of newborn piglet Epithelium of intestinal villus primary cell strain, belong to biological technical field.Preparation method of the present invention adopts the little intestinal segment of newborn piglet as material, provide the preparation method of a kind of quick, convenient, high efficiency newborn piglet Epithelium of intestinal villus primary cell strain, the chitterlings chorioepithelium primary cell strain of preparation can be passaged to more than 20-30 generation.Preparation method of the present invention can prepare the chitterlings chorioepithelium primary cell of a large amount of health, single, purifying at short notice, for the experimental studies such as cytobiology, Preventive Veterinary Medicine, virusology, immunology, animal nutrition or diarrhea of pigs virus type production of vaccine and antigen preparation.Meanwhile, the present invention prepares the chitterlings chorioepithelium primary cell strain of health, single, purifying first, has filled up the blank that life science research field lacks the chitterlings chorioepithelium primary cell strain of single, purifying, has had positive effect.

Description

The preparation method of a kind of newborn piglet intestinal villus epithelium primary cell strain

Technical field

The invention belongs to biological technical field, be specifically related to the preparation method of a kind of newborn piglet intestinal villus epithelium primary cell strain.

Background technology

Although existing cell culture technology goes through the development of decades, and achieve tremendous development, but the cell injuring model technical progress of some particular tissues organs is stagnated, still successfully do not prepare chitterlings chorioepithelium primary cell strain that is single, purifying by today, more need not carry and cultivate chitterlings chorioepithelium continuous cell line.After existing chitterlings villus epithelial cells preparation method normally aseptic taking-up tire chitterlings section, shred with scissors together with Epithelium of intestinal villus and basal layer tissue (comprising intestinal smooth muscle and reticular tissue), then by cultivating after the trysinization of 0.025% concentration, dispersion, therefore the chitterlings villus epithelial cells of obtained culture only containing minute quantity, more Ze Shi small intestine cell.This is that its gastrointestinal mucosal is not subject to food stimulus because tire pig is not in utero being sucked the breast, and causes intestinal villi not grow completely and caused by unfolding.Therefore when needing to carry out experimental study to Epithelium of intestinal villus cell that is single, purifying, can be few and be mixed with small intestine's cell because of Epithelium of intestinal villus cell concentration, and cause relatively large deviation, the error even mistake of experimental result.In addition, use existing Pollution control technology, namely add dual anti-, almost the pollution of this culture uncontrollable.

In the research field such as virusology or Preventive Veterinary Medicine, a lot of diarrhea virus is such as: Porcine epidemic diarrhea virus (PEDV), transmissible gastro-enteritis virus (TGEV), wheel turn viral etc. the cell receptor in a virus, coronavirus, bocavirus (BOV), storehouse cloth virus (KOV), enterovirus, norovirus, Sapporo, are all positioned on Epithelium of intestinal villus cell.Therefore in order to improve the Success rate of virus isolation of above diarrhea virus, and carry out the important research such as follow-up etiology, immunology, pathology, vaccine research and development, be necessary to work out a kind of method that can obtain single chitterlings chorioepithelium biography cell efficiently, easily completely.

Summary of the invention

The object of the invention is to solve the deficiencies in the prior art, the preparation method of a kind of newborn piglet intestinal villus epithelium primary cell strain is provided, the method can prepare chitterlings epithelium fine hair primary cell strain that is single, purifying simple and easy, convenient, efficiently, can be applicable to diarrhea of pigs virus purification, Animal nutrition regulation and control and the field such as Mechanism Study or cytobiology.

The technical solution used in the present invention is as follows:

A preparation method for newborn piglet Epithelium of intestinal villus primary cell strain, comprises the steps:

Step (1), gets the small intestine that age in days is the newborn sodium selenite in 1 age in days, and small intestine is cut into the little intestinal segment of long 5-10cm, then axially cut off by little intestinal segment, expose mucosal surface, for subsequent use;

Step (2), the little intestinal segment mucosal surface that will process through step (1) adopts washing fluid to rinse, to washing fluid limpid bright, then intestinal villi is cut, joined by intestinal villi in new washing fluid, every gram of intestinal villi needs 15-20mL washing fluid, more centrifugal, abandon supernatant liquor, obtain intestinal villi and organize agglomerate;

Step (3), organizes agglomerate to join the intestinal villi that step (2) obtains and is incubated in the trysinization liquid of 37 DEG C, and the add-on of every milliliter of trysinization liquid Small Intestine chorionic villi agglomerate is 0.075-0.1g; Then organize agglomerate to dispel intestinal villi and after making it suspend, add trysinization liquid, mixing, be placed in incubation digestion in 37 DEG C of water-baths and sustained oscillation, after 10-15min, take out, then add the first nutrient solution wherein, after mixing, organize agglomerate to dispel intestinal villi and adopt after making it suspend the aseptic copper mesh of 100 order to filter, getting filtrate, centrifugal, abandon supernatant liquor, obtain the first cell mass;

Wherein, the volume of the trysinization liquid added is 4 times that former trysinization liquid amasss; The volume that adds of the first nutrient solution is 1.25 times that the trysinization liquid added amasss;

Step (4), after adding the second nutrient solution, dispels cell mass and makes it suspend in the first cell mass that step (3) obtains, and the volume that adds of the second nutrient solution is long-pending 0.5 times of the trysinization liquid added of step (3); Then add the second nutrient solution, mixing, obtains mixed solution, and the volume added is the half of the second nutrient solution volume that first time uses; Then mixed solution is transferred in the Tissue Culture Flask of band air-permeable envelope, overnight incubation in 37 DEG C of carbonic acid gas constant incubators;

Step (5), rock after Tissue Culture Flask to dead cell, mucus and the tissue block of step (4) incubated overnight suspends, incline nutrient solution, rejoin the second nutrient solution, continue to cultivate in 37 DEG C of carbonic acid gas constant incubators, the volume rejoining the second nutrient solution is 0.75 times of the second nutrient solution volume that step (4) first time uses;

Cultivate and change nutrient solution in 3-5 days, during replacing, if when cell attachment area accounts for culturing bottle culture area less than 20%, the nutrient solution inclined when changing liquid in 1/2 culturing bottle, then add the second fresh nutrient solution to identical with nutrient solution volume before replacing; If when cell attachment area accounts for culturing bottle culture area more than 20%, then the nutrient solution inclined in all culturing bottles, adds the second fresh nutrient solution to identical with nutrient solution volume before replacing;

After changing nutrient solution, continue to cultivate, until Growth of Cells becomes monolayer cell in culturing bottle, this monolayer cell and chitterlings chorioepithelium primary cell strain F1 generation;

Step (6), has the culturing bottle of monolayer cell to incline after nutrient solution by the growth that step (5) obtains, add trysinization liquid and rinse and wash monolayer cell, and the volume of the trysinization liquid added is long-pending 0.5 times of the trysinization liquid that uses step (3) first time;

Rinse and wash hypsokinesis and remove trysinization liquid, rejoin trysinization liquid again, the volume of the trysinization liquid rejoined is 0.25 times that the trysinization liquid used step (3) first time amasss, digestion is rocked when starting to occur flaking to cell monolayer in 37 DEG C of carbonic acid gas constant incubators, add the second nutrient solution, the adding volume and rinse the trysinization liquid washing monolayer cell and amass identical of second nutrient solution, and monolayer cell is blown to suspension, add the second nutrient solution, the volume adding the second nutrient solution rinses long-pending 5 times of the trysinization liquid of washing monolayer cell, then average mark is filled to two culturing bottles identical with former culturing bottle, again the culturing bottle after packing is placed in 37 DEG C of carbonic acid gas constant incubators and cultivates 2-3 days, when Growth of Cells becomes individual layer, this monolayer cell is chitterlings chorioepithelium primary cell strain F2 generation, repeat to incline in this step nutrient solution to the institute in when Growth of Cells becomes individual layer in steps, continue Secondary Culture,

Wherein, CO in step (4) and the carbonic acid gas constant incubator described in step (6) ₂volumetric concentration be 5%;

The preparation method of described washing fluid is as follows:

First get mass concentration be 10% ENR solution 1-3ml be dissolved in 97-99ml pure water, mixing, after 0.1 micron membrane filter filtration sterilization, namely obtain 100 × ENR solution;

Then get 100 × ENR solution 1mL, 40000IU/mL penicillin solution 1mL respectively to join in the MEM substratum of 97mL together with 40000 μ g/mL Streptomycin Solution 1mL, namely obtain washing fluid;

The preparation method of the first described nutrient solution is as follows:

Then get 100 × ENR solution 1mL, 40000IU/mL penicillin solution 1mL respectively joins in the MEM substratum of 87mL together with 40000 μ g/mL Streptomycin Solution 1mL, mix after adding 10mL new-born calve serum again, namely obtain the first nutrient solution;

The preparation method of the second described nutrient solution is as follows:

Then get 100 × ENR solution 1mL, 40000IU/mL penicillin solution 1mL respectively to join in the DMEM substratum of 87mL together with 40000 μ g/mL Streptomycin Solution 1mL, then mix after adding 10mL foetal calf serum, namely obtain the second nutrient solution;

The preparation method of described trysinization liquid is as follows: take 0.6 gram, Gibic pancreatin dry powder (1:250), sodium bicarbonate 0.64 gram, ETDA disodium 0.224 gram, 8.9 grams, sodium-chlor, 0.44 gram, Repone K and glucose 1.1 grams, then they are joined together in 1000ml pure water, after dissolving after 0.1 micron membrane filter filters, get filtrate, be trysinization liquid.。

Further, preferably described in step (2) cut intestinal villi adopt be eye scissors.

Further, preferably organize agglomerate to dispel intestinal villi in step (3) and make it suspend, cell mass dispelled in step (4) and make it suspend and in step (6), monolayer cell is blown to the instrument adopted that suspends to be common transfer pipet.

Further, the preparation method of preferably described newborn piglet Epithelium of intestinal villus primary cell strain, also comprises the steps:

Step (7), the growth obtained in selecting step (6) has 1 culturing bottle of monolayer cell within 10 generations to incline nutrient solution, add trysinization liquid to rinse and wash monolayer cell, the volume of the trysinization liquid added is long-pending 0.5 times of the trysinization liquid that uses step (3) first time;

Rinse and wash hypsokinesis and remove trysinization liquid, rejoin trysinization liquid again, the volume of the trysinization liquid rejoined is 0.25 times that the trysinization liquid used step (3) first time amasss, digestion is rocked when starting to occur flaking to cell monolayer in 37 DEG C of carbonic acid gas constant incubators, add the first nutrient solution, the adding volume and rinse the trysinization liquid washing monolayer cell and amass identical of first nutrient solution, and after monolayer cell to suspension will be blown, add the first nutrient solution, the volume adding the first nutrient solution rinses long-pending 3 times of the trysinization liquid of washing monolayer cell, mixing, centrifugal, abandon supernatant, obtain the second cell mass,

Step (8), cells frozen storing liquid is chilled to 4 DEG C in advance, then the second cell mass that step (7) obtains is added in cells frozen storing liquid, monolayer cell is blown to suspension, be sub-packed in cell cryopreservation tube, in 4 DEG C of standing 2h, transfer in freezing storing box, spend the night in-80 DEG C, be finally transferred to liquid nitrogen and preserve for a long time;

Wherein, the volume of cells frozen storing liquid is 0.45-0.55 times that the trysinization liquid used step (3) first time amasss; CO in described carbonic acid gas constant incubator ₂volumetric concentration be 5%;

The preparation method of described cells frozen storing liquid is: first get mass concentration be 10% ENR solution 1-3ml be dissolved in 97-99ml pure water, mixing, after 0.1 micron membrane filter filtration sterilization, namely obtain 100 × ENR solution;

Then get in DMEM substratum that 100 × ENR solution 1mL, 40000IU/mL penicillin solution 1mL and 40000 μ g/mL Streptomycin Solution 1mL, dimethyl sulfoxide (DMSO) 10mL join together with foetal calf serum 10-20mL respectively, control cumulative volume is 100mL, after mixing, namely obtain cells frozen storing liquid.

Further, preferably described centrifugal speed is 1000rpm, and centrifugation time is 5min.

Further, the preparation method of preferably described newborn piglet Epithelium of intestinal villus primary cell strain, comprises the steps:

Step (2), the little intestinal segment mucosal surface that will process through step (1) adopts washing fluid to rinse, to washing fluid limpid bright, then cut intestinal villi, then 1.5-2g intestinal villi is joined in the new washing fluid of 30mL, more centrifugal, abandon supernatant liquor, obtain intestinal villi and organize agglomerate;

Step (3), being organized by the intestinal villi that step (2) obtains agglomerate to join 20mL is incubated in the trysinization liquid of 37 DEG C, then agglomerate is organized to dispel intestinal villi and after making it suspend, add 80mL trysinization liquid, mixing, be placed in incubation digestion in 37 DEG C of water-baths and sustained oscillation, after 10-15min, take out, then add 100mL first nutrient solution wherein, after mixing, organize agglomerate to dispel intestinal villi and adopt after making it suspend the aseptic copper mesh of 100 order to filter, getting filtrate, centrifugal, abandon supernatant liquor, obtain the first cell mass;

Step (4), after joining 40mL second nutrient solution, dispels cell mass and makes it suspend in the first cell mass that step (3) obtains; Then add 20mL second nutrient solution, mixing, obtains mixed solution; Then mixed solution is transferred to the 150cm of band air-permeable envelope ²in Tissue Culture Flask, overnight incubation in 37 DEG C of carbonic acid gas constant incubators;

Step (5), rock after Tissue Culture Flask to dead cell, mucus and the tissue block of step (4) incubated overnight suspends, incline nutrient solution, rejoins 30mL second nutrient solution, continues to cultivate in 37 DEG C of carbonic acid gas constant incubators;

Cultivate and change nutrient solution in 3-5 days, during replacing, if when cell attachment area accounts for culturing bottle culture area less than 20%, the nutrient solution inclined when changing liquid in 15mL culturing bottle, then add the second fresh nutrient solution of 15mL; If when cell attachment area accounts for culturing bottle culture area more than 20%, then the nutrient solution inclined in all culturing bottles, adds the second nutrient solution that 30mL is fresh;

Step (6), has the culturing bottle of monolayer cell to incline after nutrient solution by the growth that step (5) obtains, add 10mL trysinization liquid and rinse and wash monolayer cell;

Rinse and wash hypsokinesis and remove trysinization liquid, then rejoin 5mL trysinization liquid, in 37 DEG C of carbonic acid gas constant incubators, rock digestion to cell monolayer 3-5min, add 10mL second nutrient solution, and monolayer cell is blown to suspension, add 50mL second nutrient solution; Then average mark is filled to 2 150cm ²in Tissue Culture Flask, then the culturing bottle after packing is placed in 37 DEG C of carbonic acid gas constant incubators cultivation 2-3 days, when Growth of Cells becomes individual layer, this monolayer cell is chitterlings chorioepithelium primary cell strain F2 generation; Repeat to incline in this step nutrient solution to the institute in when Growth of Cells becomes individual layer in steps, continue Secondary Culture;

Step (7), the growth obtained in selecting step (6) has 1 culturing bottle of monolayer cell within 10 generations to incline nutrient solution, adds 10mL trysinization liquid and rinses and wash monolayer cell;

Rinse and wash hypsokinesis and remove trysinization liquid, rejoin 5mL trysinization liquid again, digestion is rocked when starting to occur flaking to cell monolayer in 37 DEG C of carbonic acid gas constant incubators, add 10mL first nutrient solution, and after monolayer cell to suspension will be blown, add 30mL first nutrient solution, mixing, centrifugal, abandon supernatant, obtain the second cell mass;

Step (8), cells frozen storing liquid is chilled to 4 DEG C in advance, then the second cell mass that step (7) obtains is added in 9-11mL cells frozen storing liquid, monolayer cell is blown to suspension, be sub-packed in cell cryopreservation tube by 1.8mL/ pipe, in 4 DEG C of standing 2h, transfer in freezing storing box, spend the night in-80 DEG C, be finally transferred to liquid nitrogen and preserve for a long time.

Further, preferably described age in days is the newborn sodium selenite in 1 age in days is the newborn piglet of not sucking the breast.

The inverted light microscope figure of the newborn piglet Epithelium of intestinal villus primary cell strain that the inventive method is turned out is as shown in Fig. 1 ~ Fig. 3, and as can be seen from Figure 1, adherent chitterlings chorionic villi block periphery grows Epithelium of intestinal villus cell colony.As can be seen from Fig. 2 and Fig. 3, the newborn piglet Epithelium of intestinal villus primary cell strain that the inventive method obtains is healthy, single, and purifying, can be applicable to diarrhea of pigs virus purification, Animal nutrition regulation and control and the field such as Mechanism Study or cytobiology.

compared with prior art, its beneficial effect is in the present invention:

Tradition gathers the method that tire chitterlings shred, chitterlings chorioepithelium primary cell is prepared in trysinization cannot obtain purifying, single Epithelium of intestinal villus cell, the experimental result often made the mistake or experimental data deviation, error are increased.Gather Small Intestine of Piglets section material and prepare the epithelial words of intestinal villus, not only cannot obtain purifying, single Epithelium of intestinal villus cell, also effectively cannot control the pollution problem of culture, usually severe contamination causes the failure of an experiment, gets half the result with twice the effort often.

The invention provides the preparation method of a kind of quick, convenient, high efficiency newborn piglet Epithelium of intestinal villus primary cell strain, the chitterlings chorioepithelium primary cell strain of preparation can be passaged to more than 20-30 generation;

Preparation method of the present invention can prepare the chitterlings chorioepithelium primary cell of a large amount of health, single, purifying at short notice, for the experimental studies such as cytobiology, Preventive Veterinary Medicine, virusology, immunology, animal nutrition or diarrhea of pigs virus type production of vaccine and antigen preparation.

The present invention to prepare the chitterlings chorioepithelium primary cell strain of health, single, purifying first, has filled up the blank that domestic life science research field lacks the chitterlings chorioepithelium primary cell strain of single, purifying, has had positive effect.

Accompanying drawing explanation

Fig. 1 is the inverted light microscope figure of newborn piglet Epithelium of intestinal villus primary cell strain prepared by preparation method of the present invention; This figure is chitterlings chorioepithelium primary cell strain F1 generation, amplifies 100X;

Fig. 2 is the inverted light microscope figure of newborn piglet Epithelium of intestinal villus primary cell strain prepared by preparation method of the present invention; This figure is chitterlings chorioepithelium primary cell strain F6 generation, amplifies 40X;

Fig. 3 is the inverted light microscope figure of newborn piglet Epithelium of intestinal villus primary cell strain prepared by preparation method of the present invention; This figure is chitterlings chorioepithelium primary cell strain F6 generation, amplifies 100X.

Embodiment

Below in conjunction with embodiment, the present invention is described in further detail.

It will be understood to those of skill in the art that the following example only for illustration of the present invention, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition person in embodiment, according to the technology described by the document in this area or condition or carry out according to product description.Agents useful for same or the unreceipted production firm person of instrument, being can by buying the conventional products obtained.

Wherein, MEM substratum (1 × MEM, MinimumEssentialMedium) and DMEM substratum (1 × DMEM, Dulbecco ' sModifiedEagleMedium) are the liquid cell nutrient solution of U.S. Life Sci-Tech company limited Gibic brand.

Mass concentration is that the ENR solution of 10% is purchased from German Bayer company.

Containing 500ml Virahol in freezing storing box of the present invention.

Embodiment 1

Step (1), gets the small intestine that age in days is the newborn sodium selenite in 1 age in days, and small intestine is cut into the little intestinal segment of long 5cm, then axially cut off by little intestinal segment, expose mucosal surface, for subsequent use;

Step (2), the little intestinal segment mucosal surface that will process through step (1) adopts washing fluid to rinse, to washing fluid limpid bright, then intestinal villi is cut with eye scissors, then joined by intestinal villi in new washing fluid, every gram of intestinal villi needs washing fluid 15mL, more centrifugal, abandon supernatant liquor, obtain intestinal villi and organize agglomerate;

Step (3), organizes agglomerate to join the intestinal villi that step (2) obtains and is incubated in the trysinization liquid of 37 DEG C, and the add-on of every milliliter of trysinization liquid Small Intestine chorionic villi agglomerate is 0.075g; Then organize agglomerate to dispel intestinal villi and after making it suspend, add trysinization liquid, mixing, be placed in incubation digestion in 37 DEG C of water-baths and sustained oscillation, after 10min, take out, then add the first nutrient solution wherein, after mixing, organize agglomerate to dispel intestinal villi and adopt after making it suspend the aseptic copper mesh of 100 order to filter, getting filtrate, centrifugal, abandon supernatant liquor, obtain the first cell mass;

Cultivate and change nutrient solution in 3 days, during replacing, if when cell attachment area accounts for culturing bottle culture area less than 20%, the nutrient solution inclined when changing liquid in 1/2 culturing bottle, then add the second fresh nutrient solution to identical with nutrient solution volume before replacing; If when cell attachment area accounts for culturing bottle culture area more than 20%, then the nutrient solution inclined in all culturing bottles, adds the second fresh nutrient solution to identical with nutrient solution volume before replacing;

The preparation method of described washing fluid is as follows:

First get mass concentration be 10% ENR solution 1ml be dissolved in 99ml pure water, mixing, after 0.1 micron membrane filter filtration sterilization, namely obtain 100 × ENR solution;

The preparation method of the first described nutrient solution is as follows:

The preparation method of the second described nutrient solution is as follows:

Wherein, organize agglomerate to dispel intestinal villi in step (3) and make it suspend, cell mass dispelled in step (4) and make it suspend and in step (6), monolayer cell is blown to the instrument adopted that suspends to be common transfer pipet.

Described centrifugal speed is 1000rpm, and centrifugation time is 5min.

Embodiment 2

Step (1), gets the small intestine that age in days is the newborn sodium selenite in 1 age in days, and small intestine is cut into the little intestinal segment of long 10cm, then axially cut off by little intestinal segment, expose mucosal surface, for subsequent use;

Step (2), the little intestinal segment mucosal surface that will process through step (1) adopts washing fluid to rinse, to washing fluid limpid bright, then intestinal villi is cut with eye scissors, then joined by intestinal villi in new washing fluid, every gram of intestinal villi needs 20mL washing fluid, more centrifugal, abandon supernatant liquor, obtain intestinal villi and organize agglomerate;

Step (3), organizes agglomerate to join the intestinal villi that step (2) obtains and is incubated in the trysinization liquid of 37 DEG C, and the add-on of every milliliter of trysinization liquid Small Intestine chorionic villi agglomerate is 0.1g; Then organize agglomerate to dispel intestinal villi and after making it suspend, add trysinization liquid, mixing, be placed in incubation digestion in 37 DEG C of water-baths and sustained oscillation, after 15min, take out, then add the first nutrient solution wherein, after mixing, organize agglomerate to dispel intestinal villi and adopt after making it suspend the aseptic copper mesh of 100 order to filter, getting filtrate, centrifugal, abandon supernatant liquor, obtain the first cell mass;

Cultivate and change nutrient solution in 5 days, during replacing, if when cell attachment area accounts for culturing bottle culture area less than 20%, the nutrient solution inclined when changing liquid in 1/2 culturing bottle, then add the second fresh nutrient solution to identical with nutrient solution volume before replacing; If when cell attachment area accounts for culturing bottle culture area more than 20%, then the nutrient solution inclined in all culturing bottles, adds the second fresh nutrient solution to identical with nutrient solution volume before replacing;

The preparation method of described washing fluid is as follows:

First get mass concentration be 10% ENR solution 3ml be dissolved in 97ml pure water, mixing, after 0.1 micron membrane filter filtration sterilization, namely obtain 100 × ENR solution;

The preparation method of the first described nutrient solution is as follows:

The preparation method of the second described nutrient solution is as follows:

Described centrifugal speed is 1000rpm, and centrifugation time is 5min.

Embodiment 3

Step (1), gets the small intestine that age in days is the newborn sodium selenite in 1 age in days, and small intestine is cut into the little intestinal segment of long 8cm, then axially cut off by little intestinal segment, expose mucosal surface, for subsequent use;

Step (2), the little intestinal segment mucosal surface that will process through step (1) adopts washing fluid to rinse, to washing fluid limpid bright, then intestinal villi is cut with eye scissors, then joined by intestinal villi in new washing fluid, every gram of intestinal villi needs 17mL washing fluid, more centrifugal, abandon supernatant liquor, obtain intestinal villi and organize agglomerate;

Step (3), organizes agglomerate to join the intestinal villi that step (2) obtains and is incubated in the trysinization liquid of 37 DEG C, and the add-on of every milliliter of trysinization liquid Small Intestine chorionic villi agglomerate is 0.09g; Then organize agglomerate to dispel intestinal villi and after making it suspend, add trysinization liquid, mixing, be placed in incubation digestion in 37 DEG C of water-baths and sustained oscillation, after 13min, take out, then add the first nutrient solution wherein, after mixing, organize agglomerate to dispel intestinal villi and adopt after making it suspend the aseptic copper mesh of 100 order to filter, getting filtrate, centrifugal, abandon supernatant liquor, obtain the first cell mass;

Cultivate and change nutrient solution in 4 days, during replacing, if when cell attachment area accounts for culturing bottle culture area less than 20%, the nutrient solution inclined when changing liquid in 1/2 culturing bottle, then add the second fresh nutrient solution to identical with nutrient solution volume before replacing; If when cell attachment area accounts for culturing bottle culture area more than 20%, then the nutrient solution inclined in all culturing bottles, adds the second fresh nutrient solution to identical with nutrient solution volume before replacing;

The preparation method of described washing fluid is as follows:

First get mass concentration be 10% ENR solution 2ml be dissolved in 98ml pure water, mixing, after 0.1 micron membrane filter filtration sterilization, namely obtain 100 × ENR solution;

The preparation method of the first described nutrient solution is as follows:

The preparation method of the second described nutrient solution is as follows:

Described centrifugal speed is 1000rpm, and centrifugation time is 5min.

Embodiment 4

Step (2), the little intestinal segment mucosal surface that will process through step (1) adopts washing fluid to rinse, to washing fluid limpid bright, then intestinal villi is cut with eye scissors, then joined by intestinal villi in new washing fluid, every gram of intestinal villi needs 18mL washing fluid, more centrifugal, abandon supernatant liquor, obtain intestinal villi and organize agglomerate;

Step (8), cells frozen storing liquid is chilled to 4 DEG C in advance, then the second cell mass that step (7) obtains is added in cells frozen storing liquid, monolayer cell is blown to suspension, be sub-packed in cell cryopreservation tube, in 4 DEG C of standing 2h, transfer in freezing storing box, spend the night in-80 DEG C, be finally transferred to liquid nitrogen and preserve for a long time; The volume of cells frozen storing liquid is 0.45 times that the trysinization liquid used step (3) first time amasss;

Wherein, CO in step (4), step (6) and the carbonic acid gas constant incubator described in step (7) ₂volumetric concentration be 5%;

The preparation method of described washing fluid is as follows:

The preparation method of the first described nutrient solution is as follows:

The preparation method of the second described nutrient solution is as follows:

The preparation method of described cells frozen storing liquid is: first get mass concentration be 10% ENR solution 1ml be dissolved in 99ml pure water, mixing, after 0.1 micron membrane filter filtration sterilization, namely obtain 100 × ENR solution;

Then get in 77mLDMEM substratum that 100 × ENR solution 1mL, 40000IU/mL penicillin solution 1mL and 40000 μ g/mL Streptomycin Solution 1mL, dimethyl sulfoxide (DMSO) 10mL join together with foetal calf serum 10mL respectively, after mixing, namely obtain cells frozen storing liquid.

Described centrifugal speed is 1000rpm, and centrifugation time is 5min.

Embodiment 5

Step (1), gets the small intestine that age in days is the sodium selenite that the new life in 1 age in days does not suck the breast, and small intestine is cut into the little intestinal segment of long 5cm, is then axially cut off by little intestinal segment, exposes mucosal surface, for subsequent use;

Step (2), the little intestinal segment mucosal surface that will process through step (1) adopts washing fluid to rinse, to washing fluid limpid bright, then intestinal villi is cut with eye scissors, then 1.5g intestinal villi is placed in 50ml sterile centrifugation tube internal wall of pipe orifice place, then with the washing fluid that 30mL is new, the flushing of the intestinal villi at tube wall place is fallen into bottom centrifuge tube, more centrifugal, abandon supernatant liquor, obtain intestinal villi and organize agglomerate;

Step (3), being organized by the intestinal villi that step (2) obtains agglomerate to join 20mL is incubated in the trysinization liquid of 37 DEG C, then agglomerate is organized to dispel intestinal villi and after making it suspend, be transferred in the aseptic triangular flask of 300ml, add 80mL trysinization liquid, mixing, be placed in incubation digestion in 37 DEG C of water-baths and sustained oscillation, after 10min, take out, add 100mL first nutrient solution more wherein, after mixing, intestinal villi is organized agglomerate to dispel and is adopted after making it suspend the aseptic copper mesh of 100 order to filter by 10ml large mouth transfer pipet gently, get filtrate, filtrate is transferred in 4 50ml centrifuge tubes respectively, centrifugal, abandon supernatant liquor, obtain the first cell mass,

Step (4), after adding 10mL second nutrient solution, to dispel cell mass with the common transfer pipet of 10ml and makes it suspend respectively to 4 in step (3) in the centrifuge tube filling intestinal villi cell mass; Collect cell suspension in all centrifuge tubes in the triangular flask of a new 200ml size, then add 20mL second nutrient solution, mixing, obtains mixed solution; Then mixed solution is transferred to the 150cm of band air-permeable envelope ²in Tissue Culture Flask, overnight incubation in 37 DEG C of carbonic acid gas constant incubators;

Step (5), rock after Tissue Culture Flask to dead cell, mucus and the tissue block of step (4) incubated overnight suspends, incline nutrient solution, to dispose the dead cell residued in culturing bottle, mucus and tissue block, rejoin 30mL second nutrient solution, continue to cultivate in 37 DEG C of carbonic acid gas constant incubators;

Cultivate and change nutrient solution in 3 days, during replacing, if when cell attachment area accounts for culturing bottle culture area less than 20%, the nutrient solution inclined when changing liquid in 15mL culturing bottle, then add the second fresh nutrient solution of 15mL; If when cell attachment area accounts for culturing bottle culture area more than 20%, then the nutrient solution inclined in all culturing bottles, adds the second nutrient solution that 30mL is fresh;

Rinse and wash hypsokinesis and remove trysinization liquid, rejoin 5mL trysinization liquid again, in 37 DEG C of carbonic acid gas constant incubators, rock digestion 3min, add 10mL second nutrient solution, and with 10mL common transfer pipet, monolayer cell is blown to suspension, add 50mL second nutrient solution; Then average mark is filled to 2 150cm ²in Tissue Culture Flask, then the culturing bottle after packing is placed in 37 DEG C of carbonic acid gas constant incubators cultivation 2-3 days, when Growth of Cells becomes individual layer, this monolayer cell is chitterlings chorioepithelium primary cell strain F2 generation; Repeat to incline in this step nutrient solution to the institute in when Growth of Cells becomes individual layer in steps, continue Secondary Culture; Chitterlings chorioepithelium primary cell strain prepared by the application can be passaged to more than 20-30 generation;

Rinse and wash hypsokinesis and remove trysinization liquid, rejoin 5mL trysinization liquid again, digestion is rocked when starting to occur flaking to cell monolayer in 37 DEG C of carbonic acid gas constant incubators, add 10mL first nutrient solution, after blowing monolayer cell to suspension with the common transfer pipet of 10ml, add 30mL first nutrient solution, mixing, centrifugal, abandon supernatant, obtain the second cell mass;

Step (8), cells frozen storing liquid is chilled to 4 DEG C in advance, then the second cell mass that step (7) obtains is added in 9mL cells frozen storing liquid, monolayer cell is blown to suspension, be sub-packed in cell cryopreservation tube by 1.8mL/ pipe, in 4 DEG C of standing 2h, transfer in freezing storing box, spend the night in-80 DEG C, be finally transferred to liquid nitrogen and preserve for a long time.

The preparation method of described cells frozen storing liquid is: first get mass concentration be 10% ENR solution 2ml be dissolved in 98ml pure water, mixing, after 0.1 micron membrane filter filtration sterilization, namely obtain 100 × ENR solution;

Then get in 67mLDMEM substratum that 100 × ENR solution 1mL, 40000IU/mL penicillin solution 1mL and 40000 μ g/mL Streptomycin Solution 1mL, dimethyl sulfoxide (DMSO) 10mL join together with foetal calf serum 20mL respectively, after mixing, namely obtain cells frozen storing liquid.

The preparation method of described washing fluid is as follows:

The preparation method of the first described nutrient solution is as follows:

The preparation method of the second described nutrient solution is as follows:

Described centrifugal speed is 1000rpm, and centrifugation time is 5min.

Embodiment 6

Step (1), gets the small intestine that age in days is the sodium selenite that the new life in 1 age in days does not suck the breast, and small intestine is cut into the little intestinal segment of long 10cm, is then axially cut off by little intestinal segment, exposes mucosal surface, for subsequent use;

Step (2), the little intestinal segment mucosal surface that will process through step (1) adopts washing fluid to rinse, to washing fluid limpid bright, then intestinal villi is cut with eye scissors, then 2g intestinal villi is placed in 50ml sterile centrifugation tube internal wall of pipe orifice place, then with the washing fluid that 30mL is new, the flushing of the intestinal villi at tube wall place is fallen into bottom centrifuge tube, more centrifugal, abandon supernatant liquor, obtain intestinal villi and organize agglomerate;

Step (3), being organized by the intestinal villi that step (2) obtains agglomerate to join 20mL is incubated in the trysinization liquid of 37 DEG C, then agglomerate is organized to dispel intestinal villi and after making it suspend, be transferred in the aseptic triangular flask of 300ml, add 80mL trysinization liquid, mixing, be placed in incubation digestion in 37 DEG C of water-baths and sustained oscillation, after 15min, take out, add 100mL first nutrient solution more wherein, after mixing, intestinal villi is organized agglomerate to dispel and is adopted after making it suspend the aseptic copper mesh of 100 order to filter by 10ml large mouth transfer pipet gently, get filtrate, filtrate is transferred in 4 50ml centrifuge tubes respectively, centrifugal, abandon supernatant liquor, obtain the first cell mass,

Cultivate and change nutrient solution in 5 days, during replacing, if when cell attachment area accounts for culturing bottle culture area less than 20%, the nutrient solution inclined when changing liquid in 15mL culturing bottle, then add the second fresh nutrient solution of 15mL; If when cell attachment area accounts for culturing bottle culture area more than 20%, then the nutrient solution inclined in all culturing bottles, adds the second nutrient solution that 30mL is fresh;

Rinse and wash hypsokinesis and remove trysinization liquid, rejoin 5mL trysinization liquid again, in 37 DEG C of carbonic acid gas constant incubators, rock digestion 5min, add 10mL second nutrient solution, and with 10mL common transfer pipet, monolayer cell is blown to suspension, add 50mL second nutrient solution; Then average mark is filled to 2 150cm ²in Tissue Culture Flask, then the culturing bottle after packing is placed in 37 DEG C of carbonic acid gas constant incubators cultivation 2-3 days, when Growth of Cells becomes individual layer, this monolayer cell is chitterlings chorioepithelium primary cell strain F2 generation; Repeat to incline in this step nutrient solution to the institute in when Growth of Cells becomes individual layer in steps, continue Secondary Culture; Chitterlings chorioepithelium primary cell strain prepared by the application can be passaged to more than 20-30 generation;

Step (8), cells frozen storing liquid is chilled to 4 DEG C in advance, then the second cell mass that step (7) obtains is added in 11mL cells frozen storing liquid, monolayer cell is blown to suspension, be sub-packed in cell cryopreservation tube by 1.8mL/ pipe, in 4 DEG C of standing 2h, transfer in freezing storing box, spend the night in-80 DEG C, be finally transferred to liquid nitrogen and preserve for a long time.

The preparation method of described cells frozen storing liquid is: first get mass concentration be 10% ENR solution 3ml be dissolved in 97ml pure water, mixing, after 0.1 micron membrane filter filtration sterilization, namely obtain 100 × ENR solution;

Then get in 72mLDMEM substratum that 100 × ENR solution 1mL, 40000IU/mL penicillin solution 1mL and 40000 μ g/mL Streptomycin Solution 1mL, dimethyl sulfoxide (DMSO) 10mL join together with foetal calf serum 15mL respectively, after mixing, namely obtain cells frozen storing liquid.

Wherein, CO in described carbonic acid gas constant incubator ₂volumetric concentration be 5%;

The preparation method of described washing fluid is as follows:

The preparation method of the first described nutrient solution is as follows:

The preparation method of the second described nutrient solution is as follows:

Described centrifugal speed is 1000rpm, and centrifugation time is 5min.CO in described carbonic acid gas constant incubator ₂volumetric concentration be 5%

Embodiment 7

Step (1), gets the small intestine that age in days is the sodium selenite that the new life in 1 age in days does not suck the breast, and small intestine is cut into the little intestinal segment of long 8cm, is then axially cut off by little intestinal segment, exposes mucosal surface, for subsequent use;

Step (2), the little intestinal segment mucosal surface that will process through step (1) adopts washing fluid to rinse, to washing fluid limpid bright, then intestinal villi is cut with eye scissors, then 1.8g intestinal villi is placed in 50ml sterile centrifugation tube internal wall of pipe orifice place, then with the washing fluid that 30mL is new, the flushing of the intestinal villi at tube wall place is fallen into bottom centrifuge tube, more centrifugal, abandon supernatant liquor, obtain intestinal villi and organize agglomerate;

Step (3), being organized by the intestinal villi that step (2) obtains agglomerate to join 20mL is incubated in the trysinization liquid of 37 DEG C, then agglomerate is organized to dispel intestinal villi and after making it suspend, be transferred in the aseptic triangular flask of 300ml, add 80mL trysinization liquid, mixing, be placed in incubation digestion in 37 DEG C of water-baths and sustained oscillation, after 12min, take out, add 100mL first nutrient solution more wherein, after mixing, intestinal villi is organized agglomerate to dispel and is adopted after making it suspend the aseptic copper mesh of 100 order to filter by 10ml large mouth transfer pipet gently, get filtrate, filtrate is transferred in 4 50ml centrifuge tubes respectively, centrifugal, abandon supernatant liquor, obtain the first cell mass,

Cultivate and change nutrient solution in 4 days, during replacing, if when cell attachment area accounts for culturing bottle culture area less than 20%, the nutrient solution inclined when changing liquid in 15mL culturing bottle, then add the second fresh nutrient solution of 15mL; If when cell attachment area accounts for culturing bottle culture area more than 20%, then the nutrient solution inclined in all culturing bottles, adds the second nutrient solution that 30mL is fresh;

Rinse and wash hypsokinesis and remove trysinization liquid, rejoin 5mL trysinization liquid again, in 37 DEG C of carbonic acid gas constant incubators, rock digestion 4min, add 10mL second nutrient solution, and with 10mL common transfer pipet, monolayer cell is blown to suspension, add 50mL second nutrient solution; Then average mark is filled to 2 150cm ²in Tissue Culture Flask, then the culturing bottle after packing is placed in 37 DEG C of carbonic acid gas constant incubators cultivation 2-3 days, when Growth of Cells becomes individual layer, this monolayer cell is chitterlings chorioepithelium primary cell strain F2 generation; Repeat to incline in this step nutrient solution to the institute in when Growth of Cells becomes individual layer in steps, continue Secondary Culture; Chitterlings chorioepithelium primary cell strain prepared by the application can be passaged to more than 20-30 generation;

Step (8), cells frozen storing liquid is chilled to 4 DEG C in advance, then the second cell mass that step (7) obtains is added in 10mL cells frozen storing liquid, monolayer cell is blown to suspension, be sub-packed in cell cryopreservation tube by 1.8mL/ pipe, in 4 DEG C of standing 2h, transfer in freezing storing box, spend the night in-80 DEG C, be finally transferred to liquid nitrogen and preserve for a long time.

The preparation method of described washing fluid is as follows:

The preparation method of the first described nutrient solution is as follows:

The preparation method of the second described nutrient solution is as follows:

More than show and describe ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and specification sheets just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and these changes and improvements all fall in the claimed scope of the invention.Application claims protection domain is defined by appending claims and equivalent thereof.

Claims

1. a preparation method for newborn piglet Epithelium of intestinal villus primary cell strain, is characterized in that, comprise the steps:

The preparation method of described washing fluid is as follows:

The preparation method of the first described nutrient solution is as follows:

The preparation method of the second described nutrient solution is as follows:

The preparation method of described trysinization liquid is as follows: take 0.6 gram, Gibic pancreatin dry powder (1:250), sodium bicarbonate 0.64 gram, ETDA disodium 0.224 gram, 8.9 grams, sodium-chlor, 0.44 gram, Repone K and glucose 1.1 grams, then they are joined together in 1000ml pure water, after dissolving after 0.1 micron membrane filter filters, get filtrate, be trysinization liquid.

2. the preparation method of newborn piglet Epithelium of intestinal villus primary cell according to claim 1 strain, is characterized in that, described in step (2) cut intestinal villi adopt be eye scissors.

3. the preparation method of newborn piglet Epithelium of intestinal villus primary cell according to claim 1 strain, is characterized in that, organizes agglomerate to dispel intestinal villi and make it suspend to adopt large mouth transfer pipet in step (3); In step (4), cell mass is dispelled and make it suspend and in step (6), monolayer cell is blown to suspend adopt instrument be common transfer pipet.

4. the preparation method of newborn piglet Epithelium of intestinal villus primary cell according to claim 1 strain, is characterized in that, also comprise the steps:

5. the preparation method of the newborn piglet Epithelium of intestinal villus primary cell strain according to claim 1-4, it is characterized in that, described centrifugal speed is 1000rpm, and centrifugation time is 5min.

6. the preparation method of newborn piglet Epithelium of intestinal villus primary cell according to claim 1 strain, is characterized in that, comprise the steps:

7. the preparation method of newborn piglet Epithelium of intestinal villus primary cell according to claim 1 strain, is characterized in that, described age in days is the newborn sodium selenite in 1 age in days is the newborn piglet of not sucking the breast.