CN103849602A - Bovine testicle cell line as well as establishment method and application thereof - Google Patents
Bovine testicle cell line as well as establishment method and application thereof Download PDFInfo
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Abstract
The invention provides a bovine testicle cell line and an establishment method thereof. The establishment method comprises the following main steps: (1) primary culture, namely collecting a bovine testicle of a newborn calf in a sterile manner, cleaning adhesive tissues, separating by virtue of a trypsinization method so as to obtain bovine testicle cells with high vitality, culturing the separated bovine testicle cells with high vitality in a DMEM (Dulbecco modified eagle medium), and culturing at 37 DEG C under the condition of 5% CO2 to obtain primary bovine testicle cells; (2) transfection and screening, namely extracting and purifying eukaryotic expression plasmids pCI-neo-hTERT containing hTERT genes, leading the extracted and purified eukaryotic expression plasmids pCI-neo-hTERT to the primarily cultured bovine testicle cells in the step (1) to perform transfection by adopting a lipidosome transfection method; (3) screening and enlarged culture. The bovine testicle cells provided by the invention are easy in culture and relatively fast in growth speed, can be stably inherited, are high in motility rate and purity, and can be well preserved; the method provided by the invention is simple and convenient in operation and convenient in popularization and application. The cell line can be used for producing hog cholera vaccines, sheep poxes, ecthyma vaccines and the like.
Description
Technical field
The invention belongs to System in Animal Cell Biotechnology technical field, relate to a kind of new clone, be specifically related to a kind of bull testis clone and establishment method and application.
Background technology
Vaccine is the main preventive measures of control swine fever.Produce vaccine with allos zooblast and can prevent that kind of the virulence from returning strong and avoiding using isogenic animal to cause other diseases to propagate, as primary cell propagation CSFV C strain vaccines such as bull testis, Testis Caprae seu Ovis, sheep kidneys.
Pestivirus suis (CSFV) can be bred in bull testis cell, but do not make cell produce pathology (CPE), and passage number and dimension that the propagation of virus can increase primary bull testis cell is treated the time.But for a long time, the hog cholera lapinised virus bull testis cell vaccine of China exists many problems aborning: in swine fever bovine testicle cell seedling production process, be raw materials for production because utilizing the superseded new-born calve testis in diary farm more, be subject to the restriction of donor amount and affect every batch of output, if only apply the bovine testicle of collection on the same day, often because the few and inconvenient scheduling of production of quantity.For meeting Production requirement, in production, conventionally adopt constant temperature to preserve, and the short period of time temperature too low (not constant temperature) usually causing due to the refrigeration mechanism of refrigerator affects, testicular cell is cultivated and the production of vaccine.
Along with the fast development of pig industry, swine fever bull testis cell vaccine demand is more and more large, relies on merely the primary cultivation of bovine testicle cell to be difficult to meet the needs of production, so it is extremely urgent to set up bull testis clone.Meanwhile, primary bull testis cell need to gather bovine testicle and organize to separate, and its source is very limited.Therefore, be badly in need of now its method to improve, bull testis cell can be stablized and go down to posterity down, reach high Cell viability, highly purified standard and can obtain good preservation and continue.
Summary of the invention
The object of this invention is to provide a kind of high reactivity, highly purified bull testis clone.
The present invention has set up a kind of bull testis clone, has reached foregoing invention object.The preserving number of described clone is: CCTCC C201399.
Above-mentioned bull testis clone can be used for producing swine Fever Vaccine, sheep pox vaccine and sheep aphtha vaccine, also can be in the application in Pestivirus suis, capripox virus, sheep of virus breed in testicular cell of research virus; Also can in isolation identification capripox virus and development capripox virus vaccine, apply; Also can in external source gene transfection, apoptosis and cytogamy research, apply.
The establishment method that above-mentioned clone is also provided of the present invention, the method is carried out former culture by gathering nascent bovine testicle; Then carry out transfection to the eukaryon expression plasmid pCI-neo-hTERT of primary cell importing coding hTERT and neo gene, rear screening is containing the positive cell of this goal gene; Then screen and enlarged culturing, with the bull testis clone of being immortalized, finally this cell is carried out to frozen and preservation.
The establishment method of above-mentioned bull testis clone, is characterized in that, comprises the steps:
(1) former culture: gather nascent bovine testicle, be cut into 0.5～2.0mm
3tissue block, separate to obtain the bull testis cell of high vigor, cultivate, obtain primary bull testis cell,
(2) transfection and screening: extract, the purifying eukaryon expression plasmid pCI-neo-hTERT that contains hTERT gene, by extracting, purifying eukaryon expression plasmid pCI-neo-hTERT import the primary bull testis cell of step (1) gained and carry out transfection;
(3) screening and enlarged culturing: in the time that cell converges to 80%, reach in another 24 well culture plate after cell transfecting eukaryon expression plasmid pCI-neo-hTERT, carry out G418 screening with G418 screening liquid after 48h;
After the whole death of cell of control wells, the concentration reduction of G418 being screened to liquid maintains G418 screening, G418 is screened to positive clone cell and be inoculated in 96 well culture plates, then had digestive transfer culture to 24 well culture plate enlarged culturing, proceed G418 screening, obtain the positive cell of anti-G418;
(4) positive cell of the anti-G418 of step (3) gained is carried out to continuous passage culture in vitro in incubator, cultivate and exceed 60 generations, the bull testis clone of being immortalized; Described continuous passage culture in vitro adopts digestion method.
The establishment method of above-mentioned bull testis clone, is characterized in that, also comprises cell cryopreservation step, and this step is as follows in order:
Substratum in exchonge step (4) culturing bottle, continues to cultivate;
Use trysinization culturing cell, then add substratum termination reaction;
Counting;
Collect: centrifugal, remove supernatant liquor, add frozen storing liquid, mix, make cell resuspended, cultured cells is distributed into cryopreservation tube sealing;
Pre-freeze: 4 DEG C of pre-freeze cryopreservation tube 30～60min ,-20 DEG C of pre-freeze 1～2h ,-70 DEG C of pre-freeze 6～12h;
Frozen: the cryopreservation tube of-70 DEG C of pre-freezes is dropped into rapidly in liquid nitrogen cabinet;
The shared volume percent of each composition of described frozen storing liquid is: 10%DMSO, 60% foetal calf serum and 30%DMEM.
The establishment method of above-mentioned bull testis clone, is characterized in that, the bull testis cell cultures that the described cultivation of step (1) refers to high vigor is in DMEM substratum completely;
The described substratum of DMEM is completely that in liquid DMEM substratum, to add volume ratio be the DMEM substratum of 10% foetal calf serum;
Described liquid DMEM substratum can use this liquid nutrient medium of direct purchase, can be also to use the DMEM culture medium dry powder of buying formulated.
The establishment method of above-mentioned bull testis clone, is characterized in that, described transfection comprises the steps:
(1) 24h before transfection, with being digested to individual cells, is inoculated into bull testis cell in 24 orifice plates, makes cell can reach 80%～90% fusion the same day in transfection;
(2) 4h before transfection, DMEM substratum is replaced with liquid DMEM substratum completely;
(3) with liquid DMEM substratum, the pCI-neo-hTERT plasmid of three part of 5 μ L is all diluted to 50 μ L, obtains the plasmid DNA of dilution;
(4) Lipofectamine2000 that dilutes respectively 6 μ L, 9 μ L, 12 μ L with liquid DMEM substratum, to 50 μ L, obtains the Lipofectamine2000 of dilution;
(5) plasmid DNA of dilution of difference mixing step (3) and the Lipofectamine2000 of the dilution of step (4), obtain mixture;
(6) substratum in 24 orifice plates of sucking-off step (1), with D-Hank ' s cleaning, adds the liquid DMEM substratum of 900 μ L;
(7) add the mixture of step (5) in different holes, mix; Set up blank simultaneously;
(8) be 5%CO in volume fraction
2in saturated humidity incubator, hatch, discard substratum, add DMEM substratum completely;
(9) observation of cell, and change in time liquid.
Bull testis clone of the present invention has following characteristics: high cell growth speed, and healthy growth, form is good, and apoptosis rate is low.Cell purity is more than 99.9%, and Cell viability is not less than 92.8%.
The biological characteristics of bull testis clone of the present invention detects indices and all reaches American type culture collection (American Type CultureCollection, ATCC) expression rate of clone standard of perfection, and foreign gene in bull testis clone is more than 40%.
In the establishment method of bull testis clone of the present invention, the nascent bull testis tissue effect the best described in step (1).
In the establishment method of bull testis clone of the present invention, the concrete steps of trysinization are: in culturing bottle, add 2.5g/L pancreatin 1.5～2.5mL, rear upset digestion 30～60S after inversion culturing bottle is preheated to 37 DEG C in incubator.
In the establishment method of bull testis clone of the present invention, frozen cell can be recovered according to actual needs, concrete steps are: cryopreservation tube is taken out from liquid nitrogen and insert in 38 DEG C of water-baths, rock after 1 point of kind, cell is moved in the culturing bottle that is added with DMEM substratum completely to piping and druming evenly, place containing 37 DEG C 5%CO
2cO
2after continuing to cultivate 48h in incubator, can continue the cultivation of going down to posterity.
Advantage of the present invention and benefit: the present invention is for the improvement adjustment of adherent culture method and medium component, and adopt the method for cell monoclonal, can make the bull testis cell of turning out without impurity cells such as epithelial cells, cell purity compared with prior art has significantly raising; Make the cell of recovery compared with before frozen for the improvement of conditions of cryopreservation, cellular form and the speed of growth do not change, freeze-stored cell steady quality, and the Cell viability after cell cryopreservation can reach between 92.8%～96.7%, and passage growth is stable, be significantly increased compared with existing cell culture technology cultured cells, be applicable to large scale culturing.For the adjustment of substratum and the improvement of cultural method, can also make cost significantly reduce.The inventive method has made up the present situation that existing bull testis clone lacks, the lifting of bull testis clone survival rate and purity also makes the biotechnological formulation developments such as the research of swine fever and relative disease and vaccine that means are provided, and provides science material and instrument for the isolation identification of the viruses such as sheep pox and the development of vaccine; Also can be used for the use of foreign gene transfection, apoptosis and cytogamy research simultaneously.
Bull testis clone of the present invention not only can be produced swine Fever Vaccine, can also study virus value-added fundamental characteristics and rule in testicular cell, for the raising of Pestivirus suis output in production of vaccine provides new thinking.Meanwhile, bull testis cell can also be produced other virus vacciness: as sheep pox vaccine, sheep aphtha vaccine etc.
The inventive method is easy to cultivate, and the speed of growth is very fast, and working method is easy, and acquisition cell purity is high, survival rate is high and the growth of going down to posterity is stable, easy to utilize.
Biomaterial preservation information
Classification And Nomenclature: bull testis clone WWX-BTC02
Deposit number: CCTCC NO:C201399
Preservation mechanism: Chinese Typical Representative culture collection center (being called for short CCTCC)
Preservation mechanism address: China, Wuhan, Wuhan University's postcode: 430072
The preservation time: on June 24th, 2013
brief description of the drawings
Fig. 1 is primary bull testis microcytoscope figure;
Fig. 2 is positive bull testis cell clone after transfection;
Fig. 3 is bull testis clone the 5th generation microcytoscope figure;
Fig. 4 is bull testis clone the 30th generation microcytoscope figure;
Fig. 5 is bull testis clone the 50th generation microcytoscope figure;
Fig. 6 is iBTCs karyogram;
Fig. 7 is bull testis clone soft agar assay result figure;
Fig. 8 is HELA cell soft agar positive control;
Fig. 9 is iBTCs growth curve chart.
Embodiment
Below in conjunction with accompanying drawing and preferred forms, the present invention will be further described, so that the public has entirety and understands fully summary of the invention, and limiting the scope of the present invention not.Aforementioned part oneself through fully disclosing the protection domain that the present invention can implement, therefore all carry out according to the disclosure of invention any well known in the art are equal to replacement, all belong to infringement of the present invention.
G418 is a kind of aminoglycoside antibiotics, in molecular genetic test, is the most frequently used resistance screening reagent of stable transfection.
The value of concentration, temperature and its dependent variable of reagent of the present invention just illustrates application of the present invention, and is not construed as limiting the invention.
The source of experiment material of the present invention:
Biomaterial:
It is Gibco company that foetal calf serum is available commercially from foetal calf serum, article No.: 10099-141.
The eukaryon expression plasmid pCI-neo-hTERT that contains hTERT gene is presented by TanJin Agricultural College doctor Li Jixia.
Applicant's statement, above biomaterial all has preservation in applicant laboratory, from the applying date, in 20 years, can provide for proof test to the public.
Source and the specification of reagent are as follows:
DMSO is available commercially from Sigma, Dimethyl sulfoxide, cat.D8418, specification 100mL;
G418 is available commercially from Invitrogen, Cat no.11811023, specification: 1g;
DMEM culture medium dry powder is purchased from Dulbecco ' s Modified Eagle Medium, Gibco company, specification: 10 × 1L; Substratum is Powdered powder, low glucose low sugar; Article No.: Cat.no.31600-034, Lot No.843267;
Liquid DMEM substratum can use the liquid nutrient medium of direct purchase, and the present invention uses the DMEM culture medium dry powder of buying formulated, and compound method is shown in embodiment 1;
DMEM substratum is that in the liquid DMEM substratum of above-mentioned preparation voluntarily, to add volume ratio be 10% Gibco foetal calf serum completely;
G418 screening liquid is to add G418 to obtain in the liquid DMEM substratum of above-mentioned preparation voluntarily;
D-Hank ' s, i.e. D-Hank'S liquid: NaCl8g, KCl0.4g, Na
2hPO
4h
2o0.06g, KH
2pO
40.06g, NaHCO
30.35g is dissolved in 1000m1 distilled water, regulates pH value to 7.2-7.4, and autoclaving saves backup at 4 DEG C.Reagent is analytical pure;
Pancreatin, is purchased from pancreatin Invitrogen, article No.: 27250-018,10g, 2.5g/L;
Lipofectamine2000Reagent, is available commercially from invitrogen company, and Chinese is liposome 2000, Cat.no.11668-027;
Other the chemical reagent of not listing is all conventional chemical reagent, analytical pure level, and the approach that is purchased obtains, and generally chemical article company can buy.
Source and the specification of plant and instrument are as follows:
Endo-Free Plasmid Maxi Kit, without intracellular toxin plasmid extraction kit, is purchased from Omega company, article No.: D6926-01; When equivalent is large, plasmid extraction can adopt plasmid to extract in a large number test kit, company: QIAGEN, article No.: 12163;
DNA/ plasmid purification reclaims test kit, is purchased from Omega company, article No.: D2500-01, Gel Extraction Kit;
The present invention's substratum used has two kinds: liquid DMEM substratum and completely DMEM substratum, wherein liquid DMEM substratum is to use the DMEM culture medium dry powder being purchased (to be purchased from Dulbecco ' s Modified Eagle Medium, Gibco company) preparation voluntarily, and completely DMEM substratum is that in liquid DMEM substratum, to add volume ratio be 10% Gibco foetal calf serum;
Screening liquid used in the present invention is to have added G418 in liquid DMEM substratum, and 400 described μ g/mL G418 screening liquid refer to the DMEM substratum that contains 400 μ g G418 in every ml G418 screening liquid; 200 described μ g/mL G418 screening liquid refer to the DMEM substratum that contains 200 μ g G418 in every ml G418 screening liquid; G418 is a kind of microbiotic, adds G418 as G418 screening liquid herein in liquid DMEM substratum;
Trysinization of the present invention pancreatin used is the pancreatin that mass volume ratio is 2.5g/L;
DMEM is a kind of substratum containing each seed amino acid and glucose, is to develop on the basis of MEM substratum.Relatively increase various composition consumptions with MEM, be divided into again high glycoform (lower than 4500mg/L) and low-sugar type (lower than 1000mg/L) simultaneously.High glycoform is conducive to cell and berths in a position growth, is suitable for growing comparatively fast, adheres to more difficult tumour cell etc.
DMEM is the abbreviation of dulbecco's modified eagle medium, so that its feature mainly comprises is following:
(1) aminoacids content is 2 times of Yi Geer substratum, and contains non-essential amino acid, as glycine etc.;
(2) vitamin contents is 4 times of Yi Geer substratum;
(3) contain the important substance-pyruvic acid in glycolytic pathway;
(4) contain micro-iron ion.
BTC full name bovine testis cells, i.e. bull testis cell, the clone that this patent is set up, called after WWX-BTC01, the clone of building up after for cell transfecting telomerase gene by the bull testis histogen to separating exactly.Source: by bull testis separate tissue, the healthy newborn calf of Niu Shi Accessories during Binzhou man of peasant household.
Plasmid DNA is the pCI-neo-hTERT plasmid DNA of purifying;
The preparation of the liquid DMEM substratum of embodiment 1.
Liquid DMEM substratum is preparation voluntarily, and compound method is as follows:
(1) prepare fresh tri-distilled water or millipore ultrapure water, the preparation process of described millipore ultrapure water is as follows: tap water filters through first step strainer, filter through second stage CTO activated charcoal filter again, enter after the filtration of third stage security personnel filter, by ultra-clean high-pressure hydraulic pump processing, filtered by RO reverse osmosis membrane again, obtain ultrapure water finally by crossing after multistage pure water post is processed;
(2) a bag DMEM culture medium dry powder (being purchased from Dulbeccos Modified Eagle Medium, Gibco company) is added in the tri-distilled water of approximately whole half; Need wash packing bag inner face with water 2 times, import in substratum, ensure that all dry powder is all dissolved into substratum.Magnetic agitation or hand mixing make it to dissolve completely;
(3) according to the sodium bicarbonate that requires to add 3.7g in packing bag, the constant volume that adds water is to final volume 1L;
(4) with aseptic 0.22 μ m membrane filtration degerming, be sub-packed in aseptic serum bottle 4 DEG C of Refrigerator stores.
DMEM substratum is that in the DMEM substratum of serum-free of above-mentioned preparation voluntarily, to add volume ratio be 10% Gibco foetal calf serum completely;
The preparation of embodiment 2. bull testis clones is cultivated
(1) former culture: the aseptic collection bovine testicle of coming into being, to remove adhesion organization, is cut into 0.5～2.0mm with PBS rinsing 3～5 times
3tissue block, adopt trypsin digestion digestion to separate the primary bull testis cell that obtains high vigor; In the primary bull testis cell of the high vigor that separation is obtained, add DMEM substratum completely, after counting, be inoculated in (the liquid DMEM substratum that contains 10% foetal calf serum) 24 orifice plates according to the concentration of 5000 cell/mL, be placed in 37 DEG C, 5%CO
2under condition, cultivate, obtain primary bull testis cell;
The described substratum of DMEM is completely that in liquid DMEM substratum, to add volume ratio be the DMEM substratum of 10% foetal calf serum.
(2) transfection and screening: the DH5 а bacterium that contains pCI-neo-hTERT plasmid that is kept at-80 DEG C is taken out, be seeded in the LB substratum that contains ammonia benzyl, shaking culture 10h, get bacterium liquid and extract plasmid, with plasmid extraction kit extract (plasmid extraction adopt plasmid extract in a large number test kit, company: QIAGEN, article No.: 12163); Adopt DNA/ plasmid purification to reclaim test kit plasmid is carried out to purifying, must contain the eukaryon expression plasmid pCI-neo-hTERT of hTERT gene, adopt liposome transfection method, the bull testis cell that the eukaryon expression plasmid pCI-neo-hTERT of coding hTERT and neo gene is imported to the former culture of step (1) carries out transfection, specifically the comprising the steps: of transfection
24h before A, transfection, becomes individual cells by bull testis cell with 2.5g/L trysinization, with 1 × 10
5individual cells/well is inoculated in 24 orifice plates, makes cell can reach 80%～90% fusion the same day in transfection;
4h before B, transfection, DMEM substratum is replaced with liquid DMEM substratum completely;
C, to dilute 5 μ L pCI-neo-hTERT plasmid to volumes with liquid DMEM substratum be 50 μ L, mixes gently, and plasmid concentration reaches 300ng/ μ L～400ng/ μ L room temperature effect 5min, obtains the plasmid DNA of dilution;
D, dilute respectively Lipofectamine2000 to 6 μ L, 9 μ L, 12 μ L to 50 μ L with liquid DMEM substratum, mix gently, room temperature effect 5min, obtains the Lipofectamine2000 of dilution;
The plasmid DNA of dilution of E, mixed step C and the dilution of step D Lipofectamine2000, now cumulative volume is 100 μ L.Mix gently, room temperature effect 20min, obtains mixture;
F, by the old substratum sucking-off in 24 orifice plates, clean with D-Hank ' s, add the liquid DMEM substratum of 900 μ L;
G, dropwise add the mixture of step e in different holes, before and after the edged of limit, waggle culture plate, mixes gently; Set up 2 hole untransfecteds as blank simultaneously;
H, be 5%CO at 37 DEG C, Volume fraction
2in saturated humidity incubator, hatch 4～6h, discard substratum, add DMEM substratum completely;
I, 24h～48h observation of cell, and change in time liquid.；
(3) screening and enlarged culturing: until cell transfecting pCI-neo-hTERT eukaryon expression plasmid after 48 hours in the time that cell converges to 80%, use 2.5g/L trysinization, reach in another 24 well culture plate with 1:1, after 48h, add 400 μ g/mL G418 screening liquid (adding G418 in liquid DMEM substratum) to carry out G418 screening, within every three days, change once DMEM substratum completely, and add the G418 screening liquid of same concentration to carry out G418 screening, the 7th day time, available pancreatin is in foramen primum peptic cell, discard Digestive system and add G418 screening liquid, cultured continuously two weeks, commutation in every two days is with concentration screening liquid 1 time, every day, observation of cell was grown and death condition, after the whole death of cell of control wells, the concentration that G418 is screened to liquid is down to 200 μ g/mL and is maintained G418 screening, continue G418 screening cell about one month, within every two days, change 1 not good liquor, until positive colony cell is visible, after G418 is screened to positive clone cell and digests and count, cell is carried out to infinite dilution, be inoculated in 96 porocyte culture plates by every hole number≤2, the hole of each cell of mark, grow after cloning cluster until unicellular, digest with 2.5g/L pancreatin, and cell is reached to 24 well culture plates, in 37 DEG C, 5% CO
2under condition, carry out enlarged culturing, continue to utilize G418 screening, obtain the positive cell of anti-G418, herein, in 96 well culture plates, be equivalent to utilizing monoclonal method purifying cells.
Under (4) 37 DEG C of conditions, adopt digestion method by step (3) gained positive cell in 5% CO
2in incubator, carry out continuous passage culture in vitro, after sorting, cell presents long shuttle shape monolayer adherence growth, and iuntercellular exists contact inhibition, cultivates and exceedes 60 generations, the bull testis clone of being immortalized.
(5) cell cryopreservation:
A, reject are also changed liquid DMEM substratum 5～8mL in culturing bottle, continue to cultivate 24h;
B, use trysinization culturing cell, then add liquid DMEM substratum 5～8mL termination reaction;
C, use red blood cell count(RBC) plate calculate frozen front total cellular score;
Centrifugal 5～the 8min of D, collection: 1000rpm, removes supernatant liquor, adds the frozen storing liquid 1mL of 4 DEG C of precoolings, mixes to blow and beat gently and make cell resuspended with suction pipe afterwards; The shared volume percent of the each composition of described frozen storing liquid is: namely, in 100mL frozen storing liquid, DMSO accounts for 10% of cumulative volume to 10%DMSO 10 60% foetal calf serum+30%DMEM(, and foetal calf serum accounts for 60% of cumulative volume, and DMEM cultivates fiduciary point 30%);
E, cell culture is distributed in the cryopreservation tube of sterilizing and seals;
F, pre-freeze: 4 DEG C, by cryopreservation tube pre-freeze 30～60min, transfer to-20 DEG C of pre-freeze 1～2h, then proceed to-70 DEG C of pre-freeze 6～12h;
G, frozen: the cryopreservation tube of-70 DEG C of pre-freezes is dropped into rapidly in liquid nitrogen cabinet, complete cell cryopreservation.
Above-mentioned obtained bull testis clone is delivered to the Chinese Typical Representative culture collection center that is deposited in, and it is referred to as CCTCC, and preservation enters volume logins volume and be numbered CCTCC C201399, and depositary institution address is: Wuhan City, Hubei Province Wuhan University preservation center.
Postcode: 430072; Phone: 027-68754712
Preservation day: on June 24th, 2013, preserving number: CCTCC C201399, the Classification And Nomenclature of suggestion is bull testis clone.
The compliance test result of embodiment 3. embodiment 2 gained bull testis clones
The bull testis clone that embodiment 2 is cultivated is carried out biological characteristics inspection, and method and conclusion are as follows.
One, morphological observation
Method: cell in culturing process, carries out routine examination to cell in vitro, and whether observation of cell growth conditions, substratum colour-change situation and cell pollute.
Conclusion: as shown in Figures 1 to 4, in the time that cell growth state is good, its form presents fusiformis or sealene triangle.The overall picture of cell colony is observed, and that institute's cultured cells is all is radial, flamboyancy or whirlpool shape tendency, proves Growth of Cells health, and form is good.
Two, microorganism detection
1, bacterium, fungi detect
Method:
(1) get the freeze-stored cell of freeze-stored cell amount 0.5%, in 8mL nonreactive substratum, mix, cell suspension is with the centrifugal 20min of 1000rpm, and repeats twice, to eliminate antibiotic impact.Be resuspended in again in the substratum of 2mL antibiotic-free.
(2) cell is inoculated in Trypsin beans peptone and malt extract medium with 0.8mL, the incubator that is placed in respectively 37 DEG C and 26 DEG C is cultivated two weeks.
(3) establishing contrast is:
A. positive control: bacillus subtilis and Candida albicans bacterium are inoculated into respectively in the old and malt extract medium of Trypsin beans and cultivate, the incubator that is placed in 37 DEG C and 26 DEG C is cultivated two weeks.
B. negative control: the old substratum of Trypsin beans and malt extract medium, the incubator that is placed in respectively 37 DEG C and 26 DEG C is cultivated two weeks.
Conclusion: visual inspection inoculation has soybean Trypsin arteries and veins substratum and the malt extract medium of bull testis cell, all presents limpid transparence, and test tube is placed in to micro-Microscopic observation, and the rounded transparence bright spot of cell swims in substratum, without other foreign matter.Prove that in the whole process of and cell recovery frozen in cell cultures, cell is not by bacterium, fungal contamination.
Three, cell growth curve is drawn
Method:
(1) suspension preparation
Get the cell that growth conditions to be measured is good, increase to and approach while converging, make cell suspension with ordinary method peptic cell, and count.
(2) inoculation
To the cell of inoculating respectively equal amts in 24 well culture plates, in 37 DEG C of CO
2in incubator, continue to cultivate.
(3) count detection
Count from inoculation time, the cell density in 24h counts 3 holes, calculates total cellular score with blood counting chamber, calculates mean value, gets the mean value in 3 holes, and 8 groups of so to the are finished.
(4) draw
Taking incubation time (d) as X-coordinate, cell density is as ordinate zou, and result is drawn on graph paper, obtains the growth curve of culturing cell.
Conclusion:
Prepare cell suspension by the bull testis cell to cultivated by ordinary method, counting, inoculate 24 well culture plates, every hole 1mL cell suspension, carry out continuous 7d timing numeration to being inoculated in the cell of 24 well culture plates, record cell density each time, drafting forms culturing cell growth curve as shown in Figure 7, after inoculation, 2d cell count starts to increase, 3d enters logarithmic phase, 6d Growth of Cells is slow, phase of cell growth general trend is all S-type, after cell inoculation, all there is the latent period of 24h, this phase is the laundering period of cell, reparation period after the damage that while being cell due to inoculation, trysinization causes, after this cell is exponential growth, it is exponential phase of growth, finally enter lag phase, Growth of Cells is slow, almost stop, visible have cell floating.And cell division index (MI maximum (cultivate 2d) occurs entering before logarithmic phase, and be maintained at a higher level in the time cultivating 2nd～4d, drop to subsequently initial level.The bull testis cell that the inventive method is cultivated
systempurity be 99.9%, be significantly increased compared with prior art culturing cell average purity 91%; And the PDT-35.9h that the cell colony doubling time (PDT) obtains with existing stationary culture with prior art collagenase digestion for 24h compares tool with 48h and increases significantly, and proves that cell purity is high, vitality is vigorous.
Four, Cell viability is measured
Method:
The survival rate that detects freeze-stored cell can adopt trypan blue exclusion test, will after cell recovery to be checked, make cell suspension, gets 10 μ L cell suspensions and adds 10 μ L1% trypan blue dye liquors, mixes.Blood counting chamber counting, healthy viable cell cell space is complete, and cell is transparent, not painted, is allly paintedly blue person for dead cell.Count 1000 cells, calculate cell survival rate.Add up two kinds of total cellular score, account for the per-cent reflection cell viability of total cellular score with viable cell.
Conclusion:
Before and after cell cryopreservation, bull testis cell is carried out to Cell viability mensuration.Result shows: frozen front Cell viability is between 96.8～98.7%, and frozen rear Cell viability is 92.8%～96.7%, lives and increases significantly compared with 78.6% with the frozen rear cell of existing cell culture technology.And the cell of recovery is compared with before frozen, and cellular form changes, but the speed of growth do not change, freeze-stored cell steady quality.
Five, the preparation of caryogram
Method:
1, add colchicine: the cell culture in the vegetative period of taking the logarithm, add colchicine in substratum, final concentration is 0.1～0.4g/mL.
2, in 37 DEG C of incubators, continue to cultivate 1～4h, make most cells in metaphase.
3, gather division phase cell: add 0.25% trypsin solution ordinary method and digest, then add substratum, stop the effect of trypsinase to cell.Proceed to 15mL centrifuge tube, with the centrifugal 8min of 1000rpm, collecting cell.
4, hypotonic: by sedimentation cell with being preheated to 37 DEG C, 0.075M KCl solution 2mL, resuspended, after piping and druming evenly, continue to add KC1 solution to l0mL, put into 37 DEG C of incubator incubation 15min.
5, pre-fix: in suspension, add fresh stationary liquid (acetic acid: methyl alcohol==1:3) 1mL, beat gently.
6, fixing: suspension, with the centrifugal 8min of 1000rpm, is removed to supernatant, add fresh stationary liquid 8mL, beat, room temperature leaves standstill 20min.
7, heavily fixing: repeat 2 times, remove part supernatant after centrifugal, residue 1～1.5mL, mixes.
8, drip sheet: drip 2～3 cell suspensions in 45.Inclination slide glass, is uniformly dispersed cell, dry under room temperature.
9, dyeing: with the Giemsa liquid dyeing l0min of 10 times of phosphate buffered saline buffer (pH6.8) dilutions, tap water rinses, dry air.
Claims (10)
1. a bull testis clone, its preserving number is: CCTCC C201399.
2. the application of bull testis clone claimed in claim 1.
3. application claimed in claim 2 refers in the application of producing in swine Fever Vaccine, sheep pox, sheep aphtha vaccine.
4. bull testis clone claimed in claim 2 application in testicular cell propagation research Pestivirus suis, capripox virus and sheep of virus.
5. bull testis clone claimed in claim 2 is separating or is identifying the application of capripox virus and development capripox virus vaccine.
6. the application of bull testis clone claimed in claim 2 in external source gene transfection, apoptosis and cytogamy research.
7. the establishment method of bull testis clone claimed in claim 1, is characterized in that, comprises the steps:
(1) former culture: gather nascent bovine testicle, be cut into 0.5～2.0mm
3tissue block, separate to obtain the bull testis cell of high vigor, cultivate, obtain primary bull testis cell;
(2) transfection and screening: extract, the purifying eukaryon expression plasmid pCI-neo-hTERT that contains hTERT gene, by extracting, purifying eukaryon expression plasmid pCI-neo-hTERT import the primary bull testis cell of step (1) gained and carry out transfection;
(3) screening and enlarged culturing: in the time that cell converges to 80%, reach in another 24 well culture plate after cell transfecting eukaryon expression plasmid pCI-neo-hTERT, carry out G418 screening with G418 screening liquid after 48h;
After the whole death of cell of control wells, the concentration reduction of G418 being screened to liquid maintains G418 screening, G418 is screened to positive clone cell and be inoculated in 96 well culture plates, then had digestive transfer culture to 24 well culture plate enlarged culturing, proceed G418 screening, obtain the positive cell of anti-G418;
(4) positive cell of the anti-G418 of step (3) gained is carried out to continuous passage culture in vitro in incubator, cultivate and exceed 60 generations, the bull testis clone of being immortalized; Described continuous passage culture in vitro adopts digestion method.
8. establishment method according to claim 7, is characterized in that, also comprises cell cryopreservation step, and this step is as follows in order:
Substratum in exchonge step (4) culturing bottle, continues to cultivate;
Use trysinization culturing cell, then add substratum termination reaction;
Counting;
Collect: centrifugal, remove supernatant liquor, add frozen storing liquid, mix, make cell resuspended, cultured cells is distributed into cryopreservation tube sealing;
Pre-freeze: 4 DEG C of pre-freeze cryopreservation tube 30～60min ,-20 DEG C of pre-freeze 1～2h ,-70 DEG C of pre-freeze 6～12h;
Frozen: the cryopreservation tube of-70 DEG C of pre-freezes is dropped into rapidly in liquid nitrogen cabinet;
The shared volume percent of each composition of described frozen storing liquid is: 10%DMSO, 60% foetal calf serum and 30%DMEM.
9. establishment method claimed in claim 8, is characterized in that, the bull testis cell cultures that the described cultivation of step (1) refers to high vigor is in DMEM substratum completely;
The described substratum of DMEM is completely that in liquid DMEM substratum, to add volume ratio be the DMEM substratum of 10% foetal calf serum.
10. establishment method according to claim 9, is characterized in that, described transfection comprises the steps:
(1) 24h before transfection, with being digested to individual cells, is inoculated into bull testis cell in 24 orifice plates, makes cell can reach 80%～90% fusion the same day in transfection;
(2) 4h before transfection, DMEM substratum is replaced with liquid DMEM substratum completely;
(3) with liquid DMEM substratum, the pCI-neo-hTERT plasmid of three part of 5 μ L is all diluted to 50 μ L, obtains the plasmid DNA of dilution;
(4) Lipofectamine2000 that dilutes respectively 6 μ L, 9 μ L, 12 μ L with liquid DMEM substratum, to 50 μ L, obtains the Lipofectamine2000 of dilution;
(5) plasmid DNA of dilution of difference mixing step (3) and the Lipofectamine2000 of the dilution of step (4), obtain mixture;
(6) substratum in 24 orifice plates of sucking-off step (1), with D-Hank ' s cleaning, adds the liquid DMEM substratum of 900 μ L;
(7) add the mixture of step (5) in different holes, mix; Set up blank simultaneously;
(8) be 5%CO in volume fraction
2in saturated humidity incubator, hatch, discard substratum, add DMEM substratum completely;
(9) observation of cell, and change in time liquid.
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| CN109735499B (en) * | 2019-01-15 | 2021-10-15 | 中国农业科学院兰州兽医研究所 | Newborn bovine testicular supporting cell immortalized cell line and establishment method and application thereof |
| CN111197028A (en) * | 2020-03-09 | 2020-05-26 | 崔立峰 | Human adipose-derived stem cell culture method |
| CN113355275A (en) * | 2021-06-11 | 2021-09-07 | 中国农业科学院兰州兽医研究所 | Lamb sheep testicular supporting cell, testicular supporting cell subclone, separation method and application thereof |
| CN113355275B (en) * | 2021-06-11 | 2024-03-05 | 中国农业科学院兰州兽医研究所 | Lamb testis support cell, testis support cell subcloning and separation method and application thereof |
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