CN104059873B - For expanding non tumorigenic MDCK cell system and the screening technique thereof of influenza virus - Google Patents

Abstract

The invention discloses the non tumorigenic MDCK cell system for expanding influenza virus and screening technique thereof, described cell line is MDCK 2B2, and deposit number is CCTCC C201323.The invention provides cell line MDCK 2B2 and the subclonal cell line thereof of tool non-tumorigenic, it is grown to serve as attached cell and/or has Epithelial form and/or can support the duplication of various virus, can amplify relatively infectious titer；In addition the method that present invention finds screening non-tumorigenic cells system, the judgement of oncogenicity can be assisted by the metamorphosis of the speed of growth of cell and cell, i.e. grow relatively slow single hole cell, its oncogenicity can significantly reduce, the appearance of megabacterium can make oncogenicity dramatically increase, when Large-scale Screening non-tumorigenic cells system, such auxiliary judgment method can shorten time and cost-effective.

Description

For expanding non tumorigenic MDCK cell system and the screening technique thereof of influenza virus

Technical field

The present invention relates to MDCK derived cell system, systems a kind of non-tumorigenesis for expanding influenza virus Property mdck cell system and screening technique thereof.

Background technology

Annual influenza pandemic, no matter to developed country or developing country, all can bring and compare large effect.According to system Meter, in the season that influenza is prevailing, the people of the whole world the most about 5-15% can occur upper respiratory tract infection, and about 250,000-500,000 People death [WHO. Influenza Fact Sheet www.who.int/mediacentre/factsheets/ can occur fs211/en/].Inoculation influenza vaccines are one of prevention and the major measure controlling influenza, can significantly reduce sickness rate and death Rate, so providing vaccine to be just particularly important effectively and quickly.At present, most of influenza vaccines still use Embryo Gallus domesticus next life Produce the strain that WHO announces, due to the season prevailing in influenza, it is understood that there may be the shortage of Embryo Gallus domesticus supply, thus cannot quickly tackle The influenza of large-scale outbreak.In view of the uncertainty of Embryo Gallus domesticus supply with there may be potential bird flu virus, therefore 1995 Year, WHO be proposed with more preferable virus amplification mode to replace Embryo Gallus domesticus produce vaccine, they first elect mammalian cell cultivating system [WHO. Cell culture as a substrate for the production of influenza vaccines: memorandum from a WHO metting. Bull. World Helath Organ. 73(4), 431-435 (1995)].Produce vaccine with cell and tackle the influenza of suddenly outburst, can large-scale production at short notice, immune vaccine The shortage of supply chain, it is also possible to solve the people problem to egg allergy.

In recent years, some successional cell lines gradually be used to produce influenza vaccines, wherein MDCK, Vero and PER.C6 [Alexander Doroshenko and Scott A Halperin. Trivalent MDCK Cell culture-derived influenza vaccine Optaflu?? (Novartis Vaccine). Expert Rev. Vaccines 8 (6), 679-688 (2009)] titre of cell amplification influenza virus is higher, therefore become and produce influenza virus Main cell strain.The companies such as Su Wei pharmacy, Irving Baxter, DynPort, Crucell, Novartis, GlaxoSmithKline PLC and MedImmune Successively carry out with the research of a kind of cell runoff yield in next life influenza vaccine.Wherein, with the research and development achievement of mdck cell the most Plentiful and substantial, used the influenza virus cracking inactivated vaccine that produced of MDCK to obtain examination & approval in the pharmacy of calendar year 2001 Su Wei before this, later due to The reason of manufacturer cannot list in scale, and by 2007, the influenza vaccines Optaflu that Novartis produces based on mdck cell obtained To the approval of Europe drug assessment office, successfully list.Trivalent FluMist of MedImmune company research and development??Obtained in 2003 The approval of FDA, but owing to this medicine uses frozen preparation, be unfavorable for transport, therefore cannot list.Formula was carried out by they later Change, become Refrigerated Transport so that this trivalent vaccine obtains approval [MedImmune, the Inc. of FDA in January, 2007 http://pressroom.medimmune.com/press-releases/2007/01/08/fda-approves- Medimmune-s-refrigerated-formulation-of-flumist-r/], MedImmune company continues to cause subsequently The research and development of this vaccine of power, obtain FDA approval in February, 2012, produce influenza vaccines [MedImmune, the Inc. of first tetravalence http://pressroom.medimmune.com/press-releases/2012/02/29/

medimmune-announces-fda-approval-of-flumist%C2%AE-quadrivalent- influenza-vaccine-live-intranasal/]。

Mdck cell is used for manufacturing influenza virus vaccine, has following advantage: be first the advantage of viral growth, Mdck cell can amplify the higher influenza virus of titre [Gaush & Smith, 1968; Govorkovaet al., 1999]；Secondly, this technique is the most quickly amplified；3rd is can to review and this cell of comprehensive identification；4th this cell Serum-free culture can be tamed into.Certainly, in addition, some more advantages may be also had to cause manufacturer to like thin with this Born of the same parents' runoff yield in next life influenza vaccine.

For the source of Madin Darby Testis et Pentis Canis (MDCK) cell, multiple relatively independent mdck cell was once described. To be that the kidney from normal male Cocker (cocker spaniel) in 1958 establishes these thin than more consistent Born of the same parents.ATCC lists as S. Madin and the MDCK(CCL-34 of N.B. Darby preservation) cell line, but mdck cell Other many pedigrees also can be used.Such as: 1963 MDCK-USD and 1961 MDCK-NIH [Gaush, PSEBM, 1,996 122: 931]。

But in terms of the substrate of mdck cell, there is also some arguements, such as: cell line original for MDCK is all non-cause Tumor；Some have high oncogenicity derived from the cell line of mdck cell, and (likely 1-100 cell is it is possible to formed swollen Tumor)；The cellular matrix of high oncogenicity is the most never used to produce the cellular matrix of viral vaccine and high oncogenicity and brings significantly Supervision challenge [FDA. Use of MDCK Cells for Manufacture of Inactivated Influenza Vaccines:Introduction]。

Each big production of vaccine is produced house and is the most more paid close attention to the oncogenicity of MDCK, the mdck cell strain injection that Su Wei pharmacy uses In 4 to 5 week after the nude mice of immunodeficiency, can form tuberosity in injection site, the tuberosity of injection has the speciality of Madin-Darby canine kidney(cell line) And do not induce the formation of second time tumor, therefore they think that this cell does not have oncogenicity, and for produce inactivation, highly purified Subunit vaccine be safe [R. Brands, J. Visser,et al. Influvac^TC: A Safe Madin Darby Canine Kidney (MDCK) Cell Culture-Based Influenza Vaccine].The Asia that Novartis produces Unit inactivated vaccine contemplates the problem of cellular neoplastic, and they think that the continuous cell line of aneuploid likely has Oncogenicity, therefore they highlight the inspection of the intact cell of residual during quality inspection, thoroughly remove complete in technical process Mdck cell, and make the DNA residual of last every vaccinating agent control at below 10ng, thus stopped the oncogenicity of vaccine [Alexander Doroshenko and Scott A Halperin. Trivalent MDCK cell culture- derived influenza vaccine Optaflu?? (Novartis Vaccine). Expert Rev. Vaccines 8(6), 679–688 (2009)].MedImmune company is then because produce is attenuated live vaccine, so they are thin to MDCK The oncogenicity of born of the same parents is more paid close attention to, and they use limiting dilution assay to filter out MDCK 9B9-1E4 cell strain, and they are to its non-tumorigenesis Property is verified, with 10⁷MDCK 9B9-1E4 cell and 10⁷Cell lysate that cell is corresponding and 100 μ g/animal The nude mice of amount injecting immune defect, the observation period is six months, found that observing the end of term, the tumor body of nude mice all there occurs and disappears Die [Jonathan Liu, Sachin Maniet al. Cloning and assessment of tumorigenicity and oncogenicity of a Madin-Darby canine kidney (MDCK) cell line for influenza vaccine production. Vaccine 28 (2010) 1285–1293.]。

Summary of the invention

The technical problem to be solved is to provide a kind of non tumorigenic MDCK cell for expanding influenza virus System.

The non-tumorigenic MDCK that second technical problem to be solved by this invention is to provide for expanding influenza virus is thin The screening technique of born of the same parents system.

3rd technical problem to be solved by this invention is to provide non tumorigenic MDCK cell and ties up to expand influenza virus In application.

In order to solve above-mentioned first problem, the invention provides a kind of non-tumorigenic MDCK for expanding influenza virus Cell line, described cell line is MDCK-2B2, and deposit number is CCTCC C201323.

In order to solve above-mentioned second technical problem, present invention provide for expanding the non-tumorigenic MDCK of influenza virus The screening technique of cell line, it is characterised in that comprise the following steps:

(1) limiting dilution assay improved, after ATCC CCL-34 MDCK is passed on preservation, uses limiting dilution assay to enter The monoclonal screening of row, in order to ensure in each 96 orifice plates, cell number≤1, cell number during bed board is set as≤50, and paving Carrying out the inspection of single hole after plate immediately, determine the single hole that cell number is 1, respective markers on work, the single hole good to labelling is thin The suitable fluid infusion of born of the same parents；

(2) screening non-tumorigenic cells strain, observes step (1) the single hole cell that obtains, by the speed of growth of cell and The metamorphosis of cell assists the judgement of oncogenicity, selects the relatively slow cell of growth, and form is normal, without giant cell Monoclonal cell strain the nude mice of SPF level is injected, filter out tumor body wither away cell strain, for non-tumorigenic cells strain Carry out further confirming that of pathological section；

(3) cell bank is set up.

In order to solve above-mentioned 3rd technical problem, present invention provide for expanding the non-tumorigenic MDCK of influenza virus Cell line application in amplification influenza virus.

As a preferred version, described influenza virus is influenza A virus.

Cell line Classification And Nomenclature of the present invention is: MDCK-2B2, and deposit number is: CCTCC C201323, and depositary institution is: China typical culture collection center (preservation address: Luo Jia Shan road, Wuchang District, Wuhan City, Hubei Province 16 China of Wuhan University Type Tissue Collection postcode 430072), preservation date is: on March 5th, 2013.

Cell line of the present invention, derived from ATCC CCL-34 MDCK, can use method well known in the art derived from ATCC CCL-34 mdck cell.Such as, CCL-34 mdck cell can first (Minimum in the culture medium containing serum Essential medium+10% hyclone (FBS)+2mM glutamine) pass on finite number of time, then clone each cell And identify each clone.Select and there is excellent biology and physiological feature (includes but not limited to doubling time, oncogenicity situation and disease Poison yield) clone produce master cell bank (MCB).

Filtered out each clone of mdck cell by limiting dilution assay, and each clone is gradually amplified, when cell amplification arrives During certain scale, carry out the mensuration of non-tumorigenic.Non-tumorigenic utilizes nude mouse model of growing up to be measured, the cell digested Injection nude mice in necessary accurate metering, on ice placement and after digestion half an hour, injection volume is 10⁷cells/200μL/mice。

The routine observation injection monoclonal nude mice of mdck cell, can produce tuberosity at the position of injection, fixed with slide gauge Phase measures the size of tuberosity (with reference to normal tumor volume computing formula: general tumor volume=length × width x thickness (mm³), owing to being Long term monitoring, does not peel off tumor body, therefore uses length × wide value to represent).The tuberosity that some cells (example: MDCK-2B2) are formed Wither away in a short period of time；The time that the plastidogenetic tuberosity having is withered away is the longest；Most of cell is being seen At the end of examining the phase (6 months), tuberosity is not withered away, and the most persistently increases, i.e. induction of differentiation and the transfer of tumor body.

The passage withered away by tumor body within the observation period several times, again inject adult nude mice with identical amount, send out by result The cell of the time younger generation that existing tuberosity is withered away is compared, hence it is evident that extending, substantial portion of cell even occurs in the observation period The situation that interior tumor body cannot be withered away.MDCK-2B2 cell is verified through the oncogenicity of four times, finds that this cell was tied within the observation period Joint is withered away, but along with the raising of generation, the time that tuberosity is withered away is obviously prolonged.

The stand density of MDCK-2B2 cell line is determined by the present invention, finds that its high density grown is with MDCK Quite.

The production of HA titre after MDCK-2B2 infection of cell line influenza H1N1 is determined, find result with MDCK is suitable.

MDCK-2B2 cell has been carried out further clone, multiple clones of filtering out (example: MDCK-2B2-2G5 and MDCK-2B2-2G6 etc.), hereinafter referred to as MDCK-2B2 sub-clone, utilize nude mouse model of growing up to carry out non-tumorigenic mensuration, knot Fruit finds that the tuberosity that injection nude mice is formed can be withered away in the short period of time.

MDCK-2B2 sub-clone has been carried out the mensuration of stand density, found that high density and the MDCK-of its growth 2B2 is suitable.

MDCK-2B2 sub-clone infects the production of HA titre after influenza H1N1 be determined, find result with MDCK-2B2 is suitable.

Therefore it is good and suitable to think that MDCK-2B2 and MDCK-2B2 subclonal cell line is satisfied by non-tumorigenic, growing state The duplication of virus.

MDCK-2B2 and MDCK-2B2 subcloned cells can be grown to attached cell in the culture medium containing serum.

Every generation of MDCK-2B2 and MDCK-2B2 subcloned cells and pass on record every time should be enough detailed so that Each cell line has complete pedigree to use.

Additionally, the present invention also provides for screening the method detailed of non-tumorigenic cells strain, described cell has multiple concrete spy Levy, include but not limited to: non-tumorigenic cells (such as, formed in nude mouse after brief summary automatically wither away) and/or be grown to serve as Attached cell and/or there is Epithelial form and/or can support that various virus (including but not limited to influenza virus) replicates.

The present invention screens the method for non-tumorigenic cells strain and is different from traditional limiting dilution assay screening monoclonal, and cell spreads out It is conigenous ATCC CCL-34 MDCK, after carrying out passing on preservation for ATCC CCL-34 MDCK, uses limiting dilution assay sieve to carry out Monoclonal screening.In order to ensure in each 96 orifice plates, cell number≤1, cell number during bed board is set as≤50, and at bed board After carry out the inspection of single hole immediately, determine the single hole that cell number is 1, respective markers on work.

Select to be marked immediately after bed board, the phenomenon should not observed after individual cells sedimentation can be avoided.

The single hole cell suitable fluid infusion good to labelling, it has been observed that the growing state of cell exists significant difference after one week, Some single hole apoptosis, the single hole cell of survival also has bigger difference in the speed of growth.

Finding after to the single hole cell infusion amplified, grow relatively slow single hole cell, its oncogenicity can be notable Reduce.

The cell that after injection for the first time, tuberosity is withered away within the observation period is added up, finds along with the rising of generation, carefully The conformation of rules of born of the same parents has part and changes, it may appear that megabacterium, along with the appearance of these irregular bodies, the cause of cell Tumor can dramatically increase.

It is an advantage of the current invention that cell line MDCK-2B2 and the subcloned cells thereof that the invention provides tool non-tumorigenic System, it is grown to serve as attached cell and/or has Epithelial form and/or can support that various virus (includes but not limited to influenza Virus) replicate, relatively infectious titer can be amplified；In addition the method that present invention finds screening non-tumorigenic cells system is permissible Assisted the judgement of oncogenicity by the speed of growth of cell and the metamorphosis of cell, i.e. grow relatively slow single hole thin Born of the same parents, its oncogenicity can significantly reduce, and the appearance of megabacterium can make oncogenicity dramatically increase, at Large-scale Screening non-tumorigenic During cell line, such auxiliary judgment method can shorten time and cost-effective.

Accompanying drawing explanation

Fig. 1 outlines the technology path for deriving MDCK-2B2.Embodiment 1 details the method.

Fig. 2 is to show the photo that MDCK-2B2 cell has Epithelial form.This photograph taking was from latter 3 days of inoculation.

Fig. 3 is the MDCK(hereinafter referred to as MDCK that MDCK-2B2 and ATCC buys) growth in 10%FBS MEM culture medium Curve.MDCK-2B2 cell has the incubation period of about 2 days, followed by exponential phase of growth, within after inoculation the 4th day, enters stable phase, the Within four days, reach about 22 × 10⁵The high density of individual cell, after living through plateau, entered phase of decline in the 7th day；Mdck cell There is the incubation period of about 1 day, followed by exponential phase of growth, within after inoculation the 3rd day, enter stable phase, within the 6th day, reach about 24.7 × 10⁵The high density of individual cell, after living through plateau, entered phase of decline in the 7th day.

Fig. 4 is MDCK and MDCK-2B2 cell glucose consumption in 10%FBS MEM culture medium and the figure of lactic acid generation Sheet.Glucose consumes completely when detection in the 3rd day, and lactic acid started at second day to produce.

Fig. 5 is that MDCK and MDCK-2B2 cell consumes and glutamic acid and ammonia at 10%FBS MEM culture medium GLN The picture produced.

Fig. 6 is the H1N1 virus of display MDCK-2B2 cell inoculation different titers (m.o.i is respectively 0.01 and 0.001) After, the detection case of HA titre.Embodiment 2 details the method.

Fig. 7 is the time that the tuberosity formed after the MDCK-2B2 cell infusion nude mice of different generation is withered away in vivo.Implement Example 3 details the method.

Fig. 8 is the MDCK-2B2(P102 of higher generation time) tuberosity (12 specimen) peeled off after one week of cell infusion nude mice The paraffin enclosed mass done and slice map.

Fig. 9 is that MDCK-2B2 cell sets up the detection case of exogenous factor behind master library and work storehouse.Embodiment 4 details this Method.

Figure 10 is sub-clone inoculation different titers (m.o.i is respectively 0.01 and 0.001) showing MDCK-2B2 cell After H1N1 virus, the detection case of HA titre.

Figure 11 is the growing state of the sub-clone showing MDCK-2B2 cell.

Detailed description of the invention

Below in conjunction with specific embodiment, the present invention is expanded on further.Experimental technique used in following embodiment such as without Specified otherwise, is conventional method.Material used in following embodiment, reagent etc., if no special instructions, all can be from business way Footpath obtains.Should be understood that these embodiments are merely to illustrate the present invention rather than limit the scope of the present invention.In the following example The experimental technique of unreceipted actual conditions, generally according to normal condition, such as Sambrook et al., molecular cloning: laboratory hands Condition described in volume (New York:Cold Spring Harbor Laboratory Press, 1989), or according to manufacture Condition proposed by manufacturer.

Embodiment 1, there is the acquisition of non-tumorigenic MDCK-2B2 cell line

One, the acquisition of MDCK monoclonal cell

1, the digestion of cell and numeration

By ATCC CCL-34 MDCK P55 in the culture medium containing serum (Minimum essential medium+ 10% hyclone (FBS)+2mM glutamine) pass on finite number of time, MDCK P63, for cell, chooses one bottle of T-25 cell and enters Row digestion and numeration.

Take mono-bottle of T-25 cell of MDCK P63, abandon culture supernatant, use 1mL pancreatin (purchased from Gibco company) to wash two Time, it is subsequently adding 1mL pancreatin, puts into CO₂Incubator is hatched, Microscopic observation cell rounding after 15min, adds 9mL culture medium eventually Only digestion, mixes cell suspension, takes 10 μ L and drip in cell counting count board, count.

2, the adjustment of cell density and bed board

In order to ensure without the polyclonal existence of single hole in 96 porocyte plates, during bed board, reduce total cellular score, every piece of 96 orifice plates Total cellular score be 50, the liquid volume added in every hole is 200 μ L or 100 μ L, through comparing, adds the amount of liquid of 100 μ L, is more easy to observe Cell in 96 orifice plates, i.e. cell density are adjusted to 50cells/100 μ L × 96 ≈ 5 cells/mL.The cell plates that will complete At Microscopic observation, (this point is particularly important, cannot observe the situation in cell plate hole, thus cannot determine after cell settlement in time The later stage cell of amplification is derived from monoclonal or polyclone), labelling is carried out in the hole for individual cells, puts into CO₂Cultivate Case is hatched, and in order to reduce the volatilization of liquid, puts into wet box or to the 96 regular fluid infusion of porocyte plate simultaneously.

3, the screening of monoclonal cell

The cell plates that routine observation is completed, after one week, there is significant difference in the growth conditions of cell, some generation apoptosis, Survival monoclonal majority converges rate and reaches more than 80%, proceeds to 24 porocyte culture plates and cultivate after digestion.The growth phase of remaining only a few To the continued growth on 96 porocyte plates of cell slowly.

Two, the initial growth speed of MDCK monoclonal cell, cellular morphology are relevant to cellular neoplastic

1, initial growth speed and oncogenicity are negative correlation

The method using " acquisition of embodiment 1 MDCK monoclonal cell ", after polyclonal cellular strain screening obtained amplification Injection nude mice, from the point of view of the experimental result of tumor after being inoculated into nude mice, only only a few grows the thinnest in 96 porocyte plates The tuberosity that born of the same parents' energy fading within a short period of time injection cell is formed, Fig. 3 also demonstrates this point, the initial growth of MDCK-2B2 Speed is slow compared with MDCK.

2, the form of cell is relevant to oncogenicity

Launching experiment for polyclonal cellular, from the point of view of the experimental result of tumor after being inoculated into nude mice, identical cell strain is in generation relatively The tuberosity that time low, energy fading within a short period of time injection cell is formed, and after passing on several times, shape cannot be eliminated within the observation period The tuberosity become, some tuberositys the most persistently become greatly, the transfer i.e. formation of secondary induced tumor occur.Examine identical cell When strain generation is relatively low, form rule, without the existence of megabacterium form, after passing on several times, then there is the existence of megabacterium, this A little irregular cytons develop into tumor cell.

In conventional screening methods, after obtaining monoclonal cell strain by limiting dilution assay, after cell expansion is cultivated, injection Nude mice, carries out slice analysis before part nude mice tuberosity fading, remainder nude mice continues to observe, and what in 6 months, tumor body was withered away is Non-tumorigenic cells strain.

The speed of growth of cell of the present invention and the metamorphosis of cell assist the judgement of oncogenicity, and routine observation is completed Cell plates, after one week, there is significant difference, some generation apoptosis in the growth conditions of cell, and survival monoclonal majority converges rate and reaches More than 80%, proceed to 24 porocyte culture plates after digestion and cultivate.The relatively slow cell of growth of remaining only a few is at 96 porocytes Continued growth on plate.Experimental result through multiple injection nude mice collects discovery, remaining growth relatively slow cell tumorigenesis Property significantly reduce, inject nude mice time can select poky cell, this undoubtedly reduce screening non-tumorigenic cells strain Time and cost.The non-tumorigenic cells strain filtered out is after passing on, and along with the increase of generation, the probability of oncogenicity can increase Adding, be also to maintain non-tumorigenic after what the non-tumorigenic cells strain that we filter out had passed on for 25 generations, have passes on after 2 generations just tool Have non-tumorigenic, in mitotic stability test process it was found that pass on after, the cell strain oncogenicity meeting that megabacterium is more Dramatically increase, for us, this judges whether that having screened stable non-tumorigenic cells strain saves the time.

Embodiment 2, the growthform of MDCK-2B2 cell and biochemical indicator

One, MDCK-2B2 cell has Epithelial form

Take P85 MDCK-2B2 and carry out (40 ×) observation under mirror, find the fusiformis in regular edges, attached cell, have epithelium Sample form.

Two, MDCK-2B2 has higher stand density

Inoculate its beginning density (2.5 × 10 identical⁵Cells/5mL) MDCK-2B2 and MDCK is in the Tissue Culture Flask of T-25 In, each 7 bottles, be spaced 24 hours respectively extraction wherein one bottle carry out cell counting, continuous counter 7 days, MDCK-2B2 is in 7 days Total cellular score is respectively as follows: 1.8 × 10⁵Cells, 2.9 × 10⁵Cells, 11.6 × 10⁵Cells, 22 × 10⁵Cells, 20 × 10⁵Cells, 20.8 × 10⁵Cells and 10.5 × 10⁵cells；MDCK total cellular score in 7 days is respectively as follows: 2.3 × 10⁵Cells, 4.8 × 10⁵Cells, 19.1 × 10⁵Cells, 21 × 10⁵Cells, 23.5 × 10⁵Cells, 24.87 × 10⁵Cells and 14.5 × 10⁵cells.As it is shown on figure 3, MDCK-2B2 cell has the incubation period of about 2 days, followed by index life For a long time, within after inoculation the 4th day, enter stable phase, within the 4th day, reach about 22 × 10⁵The high density of individual cell, lives through plateau After, entered phase of decline in the 7th day；Mdck cell has an incubation period of about 1 day, followed by exponential phase of growth, after inoculation the 3rd day Enter stable phase, within the 6th day, reach about 24.7 × 10⁵The high density of individual cell, after living through plateau, entered in the 7th day Phase of decline.

From the point of view of incubation period, entering exponential phase of growth after the incubation period that MDCK-2B2 experiences 2 days, MDCK is then in experience 1 Being put into exponential phase of growth after it incubation period, this also demonstrates initial growth speed and oncogenicity in embodiment 1 is negative correlation.

The high density that MDCK-2B2 reaches on the 4th day is 22 × 10⁵, then maintain the plateau of three days；MDCK the 3rd day Entering plateau, density is 19.1 × 10⁵Cells, maintains the plateau of four days, within the 6th day, reaches about 24.7 × 10⁵Individual cell High density.The cell density of contrast plateau, MDCK-2B2 is slightly below MDCK.

Three, MDCK-2B2 cellular biochemical index

Inoculate identical initial density (2.0 × 10⁶Cells/40mL) MDCK-2B2 and MDCK cultivates in the cell of T-225 In Ping, each 1 bottle, be spaced 24 hours draw 1mL cell conditioned medium in EP pipe, put into Nova BioProfile 400 biochemical analysis Instrument is analyzed, and record glucose (Gluc) consumes, lactic acid (Lac) produces；Glutamine (Gln) consumes and glutamic acid (Glu) And ammonia (NH4⁺) situation about producing.

Embodiment 3, the qualification of MDCK-2B2 cell chromosome

1, harvesting, 0.85% NaCl solution rinses cell wall twice；

2,0.25% Trypsin-EDTA digests 10-15 minute, occurs that naked eyes visible wrinkle shape is used when changing until cell face Lower cell 453g(1500rpm/min, the eppendorf 5810R of suction pipe piping and druming) room temperature is centrifuged 10 minutes, removes supernatant, about stay 0.5ml；

3, hypotonic: to add 37 DEG C of 0.075M KCl 4-6ml, suction pipe is blown and beaten, 37 DEG C of water-baths 3-5 minute

4, pre-fix: add 1.5ml ± (methanol: glacial acetic acid=3:1) fixative, light mixing 5 minutes 453g of 37 DEG C of water-baths (1500rpm/min, eppendorf 5810R) room temperature is centrifuged 10 minutes.Go honest and upright and thrifty to stay 0.5ml；

5, fixing: add fixative 8ml ±, gently mix, 37 DEG C of water-baths 10 minutes, 453g(1500rpm/min, Eppendorf 5810R) room temperature is centrifuged 10 minutes.Remove supernatant, about stay 0.5ml；

6, repeat to fix: ibid.453g(1500rpm/min, eppendorf 5810R) room temperature is centrifuged 10 minutes, goes Clearly, suitably several fixatives are added depending on pipe floor cells amount；

7, sheet is dripped: every 1-2 drips；

8, roasting sheet: 75 DEG C of oven cooking cycle 3 hours；

9, row dyeing in second day processes, and counts chromosome.

Embodiment 4, the calibrating of MDCK-2B2 cell Toxin producing C

1, cell counting

Passing on each three bottles of MDCK, MDCK-2B2 T-25, the cell density of three bottles is completely the same with volume, after 2 days, observes Cell covers with the most, takes out each one bottle of digestion of MDCK, MDCK-2B2 T-25, calculates total cellular score.

2, virus inoculation

Remaining four bottles of cells are washed twice with PBS respectively, adds the MEM basal medium of serum-free (in * Ox blood serum Containing the nonspecific inhibitor of influenza virus, the duplication of influenza virus can be affected) (* adds appropriate pancreatin mesh to+TPCK-pancreatin Be to improve influenza virus replication capacity in cell, the working concentration of pancreatin is 2.5 μ g/mL)+virus (according to M.o.i value is respectively 1/100 and 1/1000, calculates the inoculum concentration of virus).

3, viral receipts poison and hemagglutination test (HA test)

Within 24h hour, start to collect cell supernatant from virus infected cell and carry out red cell agglutination experiment, to 96 hole blood clottings Adding 50 μ L normal saline in plate hole, be subsequently adding 50 μ L cell supernatants, each sample does two multiple holes, and mixing, multiple proportions is dilute After releasing, every hole adds 1% red cell suspension 50 μ L, observes blood clotting result after 30min.Every sampling in 24h hour once, i.e. distinguish Sample at 24h, 48h, 72h and 96h.As shown in Figure 6, when 24h samples, the titre of HA is 0, at 48h, 72h and 96h, When m.o.i value is 1/100, the HA titre that MDCK produces is more slightly higher than the HA titre that MDCK-2B2 produces；M.o.i value is 1/1000 Time, the HA titre that during 48h, MDCK produces is significantly higher than the HA titre produced with MDCK-2B2, the HA titre produced both during 72h Quite, the HA titre that during 96h, MDCK produces is slightly higher.

4, the preparation of 1% red cell suspension

By Sanguis Gallus domesticus brine 3 times, the first two times 1 500r/min is centrifuged 5 min, last 1 time synchronized centrifugal 10min, abandons supernatant, is made into 1% suspension with physiological saline solution, put 4 DEG C stand-by.Typically put 4 DEG C can preserve about 7 days, the most molten Blood should discard.

Embodiment 5, the calibrating of MDCK-2B2 cell non-tumorigenic

Digestion MDCK-2B2 cell, counting, the cell MEM culture medium without FBS is suspended, cell density is adjusted to 5×10⁷Cells/mL, injects 3 ~ 4weeks SPF level adult nude mice, and every nude mice injects 200 μ L cells at subcutaneous scapula The cell infusion amount of suspension, i.e. every nude mice is 10⁷cells。

One, the calibrating of the non-tumorigenic of MDCK-2B2 continuous passage

For the MDCK clone of amplification, according to 10⁷The injection volume injection nude mice of cells/200 μ L/mice, periodic measurement The formation of tuberosity and fading situation.Observation period is set as 6 months, and the formation of each cell strain tuberosity and fading situation exist significance difference Different.Most cells strain tuberosity within the observation period becomes big, i.e. induction secondary tumor, the transfer that even there is tumor stove having；Few Part cell is when 2weeks, and the tuberosity of formation is maximum, with posterior tubercle gradually fading within the observation period.Through injection polyclonal cellular Find, in the case of identical generation, after injection MDCK-2B2, the shortest time needed for fading tuberosity.After continuous passage, most Cell has the non-tumorigenic of relatively low generation to be changed into the oncogenicity of higher generation time, and MDCK-2B2 when higher generation time still for Fading tuberosity in the observation period of six months phases, can keep non-tumorigenic.As it is shown in fig. 7, after MDCK-2B2 P77 injection nude mice , there is fading at the 23rd day in the tuberosity formed in injection site, MDCK-2B2 P79 is in the 31st day tuberosity fading of injection, and P89 exists Injecting the 109th day tuberosity fading oncogenicity, P100 is in the 153rd day tuberosity fading of injection.

Two, the calibrating of the non-tumorigenic of MDCK-2B2 higher generation time

To MDCK-2B2 continuous passage to P102, according to 10⁷Cells/200 μ L/mice injection volume injection 3 ~ 4weeks adult nude mice, peels off tuberosity after injecting one week, is saved in the formalin solution (formalin of commercially available 37% of 4% 10 times of dilutions) in, send Shanghai medical college department of pathology of Fudan University to carry out section and identify.

Pathological section result shows (as shown in Figure 8), the existence of negative for tumor cells in tuberosity, therefore it is considered that MDCK-2B2 In P102 generation, is without oncogenicity.

Embodiment 6, MDCK-2B2 cell master library and the foundation in work storehouse

Master library and work storehouse are set up for MDCK-2B2.Master library is carried out cell and differentiates fixed, oncogenicity detection and such as Fig. 9 institute Show and carry out exogenous factor detection；Work storehouse then carries out cell and differentiates and the detection of exogenous factor.

The identification experiment of cell includes cellular morphology and chromosome analysis, to observation of cell form after cell continuous passage, Cellular morphology is normal, without notable change.Cell for MDCK-2B2 difference generation carries out chromosome analysis, finds Chromosome number The mdck cell that mesh is bought with ATCC is consistent.

The detection of exogenous factor, including sterility test, cell method detection virus exogenous factor and detects with animal and Embryo Gallus domesticus Virokine.

One, sterility test (sterility test includes antibacterial, fungus and mycoplasma)

1, antibacterial, fungal detection

A, by MDCK-2B2 cell suspension inoculation THIOGLYCOLLIC ACID salt broth, improvement Martin's culture medium and Nutrient agar Each two of inclined-plane (inoculation MEM culture medium is as comparison simultaneously)；B. postvaccinal culture medium being divided into two parts, portion puts 30 DEG C ～35 DEG C cultivate 14 days；Another part is put 20 DEG C～25 DEG C and is cultivated 14 days, should observe and record whether have bacterium day by day during cultivation Growth, result shows without antibacterial and fungal contamination.

2, mycoplasma inspection

Mycoplasma semifluid culture medium should be boiled 10～15 minutes before use, is cooled to about 56 DEG C, is subsequently adding and goes out Energy new-born calf serum (culture medium and serum volume ratio are 8:2), fully shakes up.Mycoplasma broth bouillon is without boiling, but makes Also new-born calf serum should be added equally with front.Inoculation test sample (MDCK-2B2 cell suspension), each inoculation 4, put 36 ± 1 DEG C of trainings Support 21 days.From 4, take 2 in postvaccinal 7th day carry out time culture, every 1 culture medium transferred species mycoplasma semifluid training Supporting base and each 2 of mycoplasma broth bouillon, put 36 ± 1 DEG C and cultivate 21 days, observed 1 time every 3 days, result shows without mycoplasma Pollute.

Two, cell method detection virus exogenous factor

1, cell culture method is directly observed and hemadsorption test

Take MDCK master library mixing with cells bottle cell sample inoculation little square vase 6, rear observation two weeks.Observe and take carefully latter stage Born of the same parents' supernatant, adds 0.5% chicken erythrocyte suspension, puts 2～8 DEG C 30 minutes, microscopy cell, then puts 20～25 DEG C 30 minutes, and blood is inhaled Adhesion test result is negative.

2, different passages are cultivated and are checked

Take MDCK master library cell culture supernatant biased sample inoculation MRC-5, VERO and the mdck cell of different generation (every bottle of cell inoculation 10⁷Cells MDCK-2B2), observe latter stage take cell conditioned medium, add 0.5% chicken erythrocyte suspension, put 2～ 8 DEG C 30 minutes, microscopy cell, then put 20～25 DEG C 30 minutes, microscopy cell；When cultivating the 7th day, take every kind of inoculation respectively Each one bottle of cell monolayer supernatant, be inoculated in respectively on the corresponding cell monolayer of fresh preparation, continue cultivate 7 days, observe Cytopathy also carries out cellular morphology observation and hemabsorption.Must not there is cytopathy, blood in the cell monolayer of every kind of inoculation Adsorption test should be feminine gender.

Three, with animal and Embryo Gallus domesticus detection virokine

1, injection neonatal rat (work storehouse is not required to detection)

Digestion MDCK-2B2 cell, is adjusted to 10 by cell density⁷Cells/mL, the neonatal rat in injection 24h, intracerebral injection Brood, inoculum concentration is 0.01 mL/only, routine observation, and cumulative observation 21 days finds all survivals；Lumbar injection is a brood of, inoculation Amount is 0.1 mL/, routine observation, cumulative observation 21 days, finds all survivals.

2, Mus (work storehouse is not required to detection) it is injected into

Digestion MDCK-2B2 cell, is adjusted to 2 × 10 by cell density⁶Cells/mL, the one-tenth Mus of injection 3 ~ 4 weeks, Intracerebral injection is a brood of, and inoculum concentration is 0.03 mL/only, routine observation, and cumulative observation 21 days finds all survivals；Lumbar injection Brood, inoculum concentration is 0.5mL/, routine observation, and cumulative observation 21 days finds all survivals.

3, injection Embryo Gallus domesticus (allantoic cavity)

Hatch Embryo Gallus domesticus to 11 ages in days, irradiate at dark indoor candler, observe air chamber, the position that label vascular is sparse, use Card punch punches, and is 5 × 10 along air chamber vertical alignment allantoic cavity injection density⁶MDCK-2B2 0.2 mL/only, inject 10 altogether Embryo Gallus domesticus, puts hatching box and hatches, and after 4 days, is taken out by Embryo Gallus domesticus, puts 4 DEG C overnight (preventing from taking allantoic fluid is to poke blood vessel), in second It draws the allantoic fluid 100 μ L of clarification at the injection of each Embryo Gallus domesticus.

On 96 hole blood-coagulation-boards, every hole adds 50 μ L normal saline, and corresponding aperture adds the allantoic fluid that 50 μ L draw, and multiple proportions is dilute After releasing, every hole adds 50 μ L red cell suspensions, and result shows: urine hemagglutination test is feminine gender.

4, injection Embryo Gallus domesticus (yolk sac)

Hatching Embryo Gallus domesticus to 6 ages in days, irradiate at dark indoor candler, observe air chamber, the position that label vascular is sparse, with beating Hole device punching, the injection location density contrary along air chamber vertical alignment allantoic cavity is 2 × 10⁶MDCK-2B2 0.5 mL/ only (after injection, back suction is found to have yolk liquid, shows to inject successfully), altogether 10 Embryo Gallus domesticus of injection, after 5 days, irradiate with candler and observe, Find that Embryo Gallus domesticus is all survived.

Embodiment 7, the qualification of MDCK-2B2 subcloned cells

MDCK-2B2 continuous passage is to P102, and without oncogenicity, we are with reference to the method for embodiment one, to its bed board, with it Select more excellent cell strain.

One, the calibrating of the non-tumorigenic of MDCK-2B2 subcloned cells

Monoclonal (hereinafter referred to as MDCK-2B2 sub-clone) for being formed after bed board MDCK-2B2 carries out oncogenicity inspection Fixed.Oncogenicity calibrating is with reference to embodiment 5.

MDCK-2B2-2G5 P91 43 days after injection, tuberosity fading；MDCK-2B2-2G6 P92 34 days after injection, knot Joint fading；MDCK-2B2 P89 is in the 109th day tuberosity fading of injection.Comparatively speaking, in the case of generation is close, MDCK-2B2 The time of sub-clone fading tuberosity substantially shortens.

Two, the calibrating of the Toxin producing C of MDCK-2B2 subcloned cells

The calibrating of the Toxin producing C of MDCK-2B2 subcloned cells, as shown in Figure 10, MDCK-is carried out with reference to embodiment 4 The Toxin producing C of 2B2-2G6 is close with MDCK-2B2.

Three, the monitoring of the stand density of MDCK-2B2 subcloned cells

With reference to the Part II of embodiment 2, cell is counted, as shown in Figure 11, MDCK-2B2-2G5 and MDCK- The high density of 2B2-2G6 is close with MDCK-2B2.

The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art Member, under the premise without departing from the principles of the invention, it is also possible to make some improvements and modifications, these improvements and modifications also should be regarded as Protection scope of the present invention.

Claims (3)

1. the non tumorigenic MDCK cell system being used for expanding influenza virus, it is characterised in that described cell line is MDCK- 2B2, deposit number is CCTCC C201323.

2. the non tumorigenic MDCK cell being used for expanding influenza virus described in claim 1 ties up to expand answering in influenza virus With.

Application in amplification influenza virus the most according to claim 2, it is characterised in that described influenza virus is A type Influenza virus.