CN104059873A - Non-tumorigenic MDCK cell line used for amplifying influenza viruses and screening method thereof - Google Patents
Non-tumorigenic MDCK cell line used for amplifying influenza viruses and screening method thereof Download PDFInfo
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Abstract
The invention discloses a non-tumorigenic MDCK cell line used for amplifying influenza viruses and a screening method thereof. The cell line is MDCK-2B2 with an accession number of CCTCC 201323. The invention provides the non-tumorigenic MDCK-2B2 cell line and a subclone cell line thereof; and the cell line can grow into adherent cells, and/or has an epithelioid form and/or can support duplication of a variety of viruses, and is capable of amplifying viruses with high titers. The invention also provides the screening method for the non-tumorigenic cell line. The growth speed and morphologic changes of cells can be used to assist in determination of tumorigenicity; i.e., simple-pore cells with a slow growth speed have substantially reduced tumorigenicity, and occurrence of giant cells enables tumorigenicity to be substantially increased; during large-scale screening of non-tumorigenic cell lines, such an auxiliary determination method can shorten time and save cost.
Description
Technical field
The present invention relates to MDCK derived cell system, more particularly, relate to a kind of system of the non-tumorigenic mdck cell for the influenza virus of increasing and screening method thereof.
Background technology
Annual influenza pandemic, no matter to developed country or developing country, all can bring larger impact.According to statistics, in influenza season prevailing, can there is upper respiratory tract infection in the people of the annual about 5-15% in the whole world, and approximately 250,000-500, can there is death [WHO. Influenza Fact Sheet www.who.int/mediacentre/factsheets/fs211/en/] in 000 people.Inoculation influenza vaccines are preventions and control one of major measure of influenza, can significantly reduce M & M, so provide effectively and quickly vaccine just to seem particularly important.At present, most of influenza vaccines still adopt chicken embryo to produce the strain that WHO announces, and due in influenza season prevailing, may have the shortage of chicken embryo supply, thereby cannot tackle fast the influenza of large-scale outbreak.Consider the uncertainty of chicken embryo supply and may have potential avian influenza virus, therefore in nineteen ninety-five, WHO suggestion adopts better virus amplification mode to replace chicken embryo to produce vaccine, and they first elect mammalian cell culture 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)].Deal with the influenza of flared with cells produce vaccine, large-scale production at short notice, the shortage of immune vaccine supply chain, also can solve the problem of a people to egg allergy.
In recent years, some successional clones are used to produce influenza vaccines gradually, wherein MDCK, Vero and PER.C6 [Alexander Doroshenko and Scott A Halperin. Trivalent MDCK Cell culture-derived influenza vaccine Optaflu (Novartis Vaccine).
expert Rev. Vaccines8 (6), 679-688 (2009)] titre of cell amplification influenza virus is higher, therefore become the main cell strain of producing influenza virus.The companies such as Su Wei pharmacy, Irving Baxter, DynPort, Crucell, Novartis, GlaxoSmithKline PLC and MedImmune have successively carried out with this wherein research of a kind of cell runoff yield in next life influenza vaccine.Wherein, research and development achievement with mdck cell is the plentifulest and substantial; adopted the influenza virus cracking deactivation vaccine that MDCK produces to obtain examination & approval in calendar year 2001 Su Wei pharmacy before this; afterwards because manufacturer's reason cannot be gone on the market in mass-producing; by 2007; the influenza vaccines Optaflu that Novartis produces based on mdck cell obtains the approval of European medicine evaluation office, successfully listing.The approval that the trivalent FluMist of MedImmune company research and development obtained FDA in 2003, but because this medicine adopts frozen preparation, be unfavorable for transport, therefore cannot go on the market.They changed formula afterwards, become refrigerated shipment, make this trivalent vaccine obtain the approval [MedImmune of FDA in January, 2007, Inc. http://pressroom.medimmune.com/press-releases/2007/01/08/fda-ap proves-medimmune-s-refrigerated-formulation-of-flumist-r/], MedImmune company continues to endeavour the research and development of this vaccine subsequently, obtain FDA approval in February, 2012, produce the influenza vaccines [MedImmune of first tetravalence, Inc. 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 to manufacture influenza virus vaccine, has following advantage: be first the advantage of viral growth, can amplify the influenza virus that titre is higher [Gaush & Smith, 1968 in mdck cell; Govorkova
et al., 1999]; Secondly, this technique is easily amplified fast; The 3rd is to review and to identify comprehensively this cell; The 4th this cell can be tamed into serum-free culture.Certainly, in addition, may also have some more advantages to cause manufacturer to be liked with this cell runoff yield in next life influenza vaccine.
For the source of Madin Darby dog kidney (MDCK) cell, multiple relatively independent mdck cell was once described.More consistent is to have set up these cells from the kidney of normal male Cocker (cocker spaniel) in 1958.ATCC has listed the MDCK(CCL-34 for S. Madin and N.B. Darby preservation) clone, but other many pedigrees of mdck cell also can be used.For example: 1963 MDCK-USD and 1961 MDCK-NIH [Gaush, PSEBM, 1996 122:931].
But aspect the matrix of mdck cell, also existing some arguements, for example: the original clone of MDCK is all non-tumorigenic; Some have high tumorigenicity (likely 1-100 cell just may form tumour) derived from the clone of mdck cell; The cell matrix that the cell matrix of high tumorigenicity is also never used to produce virus vaccines and high tumorigenicity has brought significant supervision challenge [FDA. Use of MDCK Cells for Manufacture of Inactivated Influenza Vaccines:Introduction].
Each large production of vaccine is produced house the tumorigenicity of MDCK is also comparatively paid close attention to, after the nude mice of mdck cell strain injecting immune defect that Su Wei pharmacy adopts 4 to 5 weeks, can can form in injection site tubercle, in the tubercle of injection, there is the speciality of Madin-Darby canine kidney(cell line) and do not bring out the formation of tumour for the second time, therefore they think that this cell do not have tumorigenicity, and be safe [R. Brands, J. Visser for subunit vaccine production deactivation, highly purified
et al.influvac
tC: A Safe Madin Darby Canine Kidney (MDCK) Cell Culture-Based Influenza Vaccine].The subunit inactivated vaccine that Novartis produces has also been considered the problem of cell tumorigenicity, they think that the continuous cell line of aneuploid likely has tumorigenicity, therefore they have emphasized the inspection of residual intact cell in the process of quality inspection, in technological process, thoroughly remove complete mdck cell, and below the residual 10ng of being controlled at of the DNA that makes last every vaccinating agent, thereby stopped vaccine tumorigenicity [Alexander Doroshenko and Scott A Halperin. Trivalent MDCK cell culture-derived influenza vaccine Optaflu (Novartis Vaccine).
expert Rev. Vaccines8 (6), 679 – 688 (2009)].MedImmune company is attenuated live vaccine because of what produce, so they more pay close attention to the tumorigenicity of mdck cell, they adopt limiting dilution assay to filter out MDCK 9B9-1E4 cell strain, and they verify its non-tumorigenic, with 10
7mDCK 9B9-1E4 cell and 10
7the nude mice of the amount injecting immune defect of the cell lysate that cell is corresponding and 100 μ g/animal, the observation period is six months, found that and is observing the end of term, all there is extinction [Jonathan Liu, Sachin Mani in the knurl body of nude mice
et 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
Technical problem to be solved by this invention is to provide a kind of non-tumorigenic mdck cell for the influenza virus of increasing to be.
Second technical problem to be solved by this invention be, the screening method of the non-tumorigenic mdck cell system of the influenza virus that is provided for increasing.
The 3rd technical problem to be solved by this invention is to provide non-tumorigenic mdck cell to tie up to the application in amplification influenza virus.
In order to solve above-mentioned first problem, the invention provides a kind of non-tumorigenic mdck cell for the influenza virus of increasing system, described cell is MDCK-2B2, deposit number is CCTCC C201323.
In order to solve above-mentioned second technical problem, the invention provides the screening method for the non-tumorigenic mdck cell system of the influenza virus of increasing, it is characterized in that, comprise the following steps:
(1) improved limiting dilution assay, ATCC CCL-34 MDCK is gone down to posterity after preservation, adopt limiting dilution assay to carry out monoclonal screening, in order to ensure in each 96 orifice plates, cell count≤1, cell count when bed board is set as≤and 50, and after bed board, carry out immediately the inspection of single hole, determine that cell count is 1 single hole, respective markers on work, the single hole cell suitable fluid infusion good to mark;
(2) screening non-tumorigenic cell strain, observe the single hole cell that step (1) obtains, assist the judgement of tumorigenicity by the speed of growth of cell and the metamorphosis of cell, select growth phase to cell slowly, and form is normal, without the monoclonal cell strain of giant cell, the nude mice of SPF level is injected, filter out the cell strain that knurl body is withered away, carry out the further confirmation of pathological section for non-tumorigenic cell strain;
(3) set up cell bank.
In order to solve above-mentioned the 3rd technical problem, the invention provides the application that ties up to amplification influenza virus for the non-tumorigenic mdck cell of the influenza virus of increasing.
As a preferred version, described influenza virus is influenza A virus.
Clone Classification And Nomenclature of the present invention is: MDCK-2B2, deposit number is: CCTCC C201323, depositary institution is: Chinese Typical Representative culture collection center (preservation address: No. 16, Luo Jia Shan road, Wuchang District, Wuhan City, Hubei Province Wuhan University's Chinese Typical Representative culture collection center postcode 430072), preservation date is: on March 5th, 2013.
Clone of the present invention, derived from ATCC CCL-34 MDCK, can adopt method well known in the art derived from ATCC CCL-34 mdck cell.For example, CCL-34 mdck cell can, first containing (Minimum essential medium+10% foetal calf serum (FBS)+2mM glutamine) limited number of times that goes down to posterity in the substratum of serum, then be cloned each cell and identify each clone.Select the clone with good biology and physiological characteristic (including but not limited to doubling time, tumorigenicity situation and viral yield) and produce master cell bank (MCB).
Filter out each clone of mdck cell by limiting dilution assay, and each clone is amplified gradually, in the time that cell amplification arrives certain scale, carry out the mensuration of non-tumorigenic.The non-tumorigenic utilization nude mouse model of growing up is measured, and cell digest must accurate metering, placement on ice and inject nude mice after digestion in half an hour, and injection volume is 10
7cells/200 μ L/mice.
The monoclonal nude mice of routine observation injection mdck cell, can produce tubercle at the position of injection, by size (the normal tumor volume calculation formula of reference: general tumor volume=length × wide × thick (mm of vernier callipers periodic measurement tubercle
3), owing to being long term monitoring, knurl body is not peeled off, therefore adopt length × wide value representation).The tubercle that some cells (example: MDCK-2B2) form is withered away in a short period of time; The time that the plastidogenetic tubercle having is withered away is longer; Most cells are in the time that the observation period (6 months) finishes, and tubercle is not withered away, and continue on the contrary to increase, and induced differentiation and the transfer of knurl body.
By knurl body within the observation period wither away passage several times, with identical amount again inject grow up nude mice, the cell that found that the younger generation of time of tubercle extinction is compared, obviously extend, the cell of considerable part even appears at the situation that in the observation period, knurl body cannot be withered away.MDCK-2B2 cell is through the tumorigenicity checking of four times, finds that this cell tubercle within the observation period withers away, but along with the raising of generation, the time that tubercle is withered away obviously extends.
The present invention measures the stand density of MDCK-2B2 clone, finds that the high-density of its growth is suitable with MDCK.
Production to HA titre after MDCK-2B2 infection of cell line influenza H1N1 is measured, and finds that result is suitable with MDCK.
MDCK-2B2 cell has been carried out to further clone, the multiple clones (example: MDCK-2B2-2G5 and MDCK-2B2-2G6 etc.) that filter out, hereinafter to be referred as MDCK-2B2 subclone, utilize the nude mouse model of growing up to carry out non-tumorigenic mensuration, found that the tubercle of injection nude mice formation can be withered away in the short period of time.
MDCK-2B2 subclone is carried out to the mensuration of stand density, found that the high-density of its growth is suitable with MDCK-2B2.
Production to HA titre after MDCK-2B2 subclone infection influenza H1N1 is measured, and finds that result is suitable with MDCK-2B2.
Therefore think that MDCK-2B2 and MDCK-2B2 subclonal cell line all meet copying of the good and suitable virus of non-tumorigenic, growing state.
MDCK-2B2 and MDCK-2B2 subclone cell can be containing being grown to attached cell in the substratum of serum.
Every generation of MDCK-2B2 and MDCK-2B2 subclone cell and go down to posterity at every turn record should be enough detailed, thereby make each clone have complete pedigree to use.
In addition, the present invention also provides the method detailed of screening non-tumorigenic cell strain, described cell has multiple specific features, include but not limited to: non-tumorigenic cell (for example, form in nude mouse after brief summary and automatically wither away) and/or be grown to serve as attached cell and/or there is Epithelial form and/or can support various virus (including but not limited to influenza virus) to copy.
The method that the present invention screens non-tumorigenic cell strain is different from traditional limiting dilution assay screening mono-clonal, cell-derived from ATCC CCL-34 MDCK, goes down to posterity after preservation for ATCC CCL-34 MDCK, adopts limiting dilution assay sieve to carry out monoclonal screening.In order to ensure in each 96 orifice plates, cell count≤1, cell count when bed board is set as≤and 50, and after bed board, carry out immediately the inspection of single hole, determine that cell count is 1 single hole, respective markers on work.
After being chosen in bed board, carry out immediately mark, can avoid the phenomenon that should not observe after individual cells sedimentation.
The single hole cell suitable fluid infusion good to mark, observed and finds after one week, and the growing state of cell exists significant difference, some single hole cell generation apoptosis, and the single hole cell of survival also has larger difference in the speed of growth.
After the single hole cell injection to amplifying, find, growth phase is to single hole cell slowly, and its tumorigenicity can significantly reduce.
The cell that after injection for the first time, tubercle is withered away within the observation period is added up, find the rising along with generation, the conformation of rules of cell has part and changes, and there will be megabacterium, be accompanied by the appearance of these irregular bodies, the tumorigenicity of cell can significantly increase.
The invention has the advantages that, the invention provides clone 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 various virus (including but not limited to influenza virus) to copy, and can amplify compared with infectious titer; In addition the present invention has found the method for screening non-tumorigenic clone, can assist by the metamorphosis of the speed of growth of cell and cell the judgement of tumorigenicity, be growth phase to single hole cell slowly, its tumorigenicity can significantly reduce, the appearance of megabacterium can make tumorigenicity significantly increase, in the time of Large-scale Screening non-tumorigenic clone, such auxiliary judgment method energy shortening time and saving cost.
Brief description of the drawings
Fig. 1 has summarized the technological line for derivative MDCK-2B2.Embodiment 1 has described the method in detail.
Fig. 2 shows that MDCK-2B2 cell has the photo of Epithelial form.This photograph taking was from latter 3 days of inoculation.
Fig. 3 is that the MDCK(that buys of MDCK-2B2 and ATCC is hereinafter to be referred as MDCK) growth curve in 10%FBS MEM substratum.MDCK-2B2 cell has the latent period of approximately 2 days, is then exponential phase of growth, after inoculation, within the 4th day, enters stationary phase, within the 4th day, reaches approximately 22 × 10
5the high-density of individual cell, lived through after plateau, entered the paracme in the 7th day; Mdck cell has the latent period of approximately 1 day, is then exponential phase of growth, after inoculation, within the 3rd day, enters stationary phase, within the 6th day, reaches approximately 24.7 × 10
5the high-density of individual cell, lived through after plateau, entered the paracme in the 7th day.
Fig. 4 is MDCK and the glucose consumption of MDCK-2B2 cell in 10%FBS MEM substratum and the picture of lactic acid generation.Glucose consumes completely in the time within the 3rd day, detecting, and lactic acid started at second day to produce.
Fig. 5 is the picture that MDCK and MDCK-2B2 cell glutamine consumption and L-glutamic acid and ammonia in 10%FBS MEM substratum produce.
Fig. 6 shows after the H1N1 virus of MDCK-2B2 cell inoculation different titers (m.o.i is respectively 0.01 and 0.001), the detection case of HA titre.Embodiment 2 has described the method in detail.
Fig. 7 is the time that the tubercle that forms after the MDCK-2B2 cell injection nude mice of different generations is withered away in vivo.Embodiment 3 has described the method in detail.
Fig. 8 is the MDCK-2B2(P102 of higher generation time) tubercle (12 samples) peeled off after one week of cell injection nude mice the paraffin enclosed mass and the slice map that do.
Fig. 9 is that MDCK-2B2 cell is set up the detection case of exogenous factor behind master library and work storehouse.Embodiment 4 has described the method in detail.
Figure 10 shows after the H1N1 virus of subclone inoculation different titers (m.o.i is respectively 0.01 and 0.001) of MDCK-2B2 cell, the detection case of HA titre.
Figure 11 is the growing state that shows the subclone of MDCK-2B2 cell.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.The experimental technique using in following embodiment if no special instructions, is ordinary method.Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, the people such as such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1, there is the acquisition of non-tumorigenic MDCK-2B2 clone
One, the acquisition of MDCK monoclonal cell
1, the digestion of cell and numeration
By ATCC CCL-34 MDCK P55 containing (Minimum essential medium+10% foetal calf serum (FBS)+2mM glutamine) in the substratum of serum, limited number of times goes down to posterity, MDCK P63, for cell, chooses one bottle of T-25 cell and digests and numeration.
Get mono-bottle of T-25 cell of MDCK P63, abandon culture supernatant, adopt 1mL pancreatin (purchased from Gibco company) washing twice, then add 1mL pancreatin, put into CO
2incubator is hatched, and Microscopic observation cell rounding after 15min adds 9mL substratum to stop digestion, mixes cell suspension, gets 10 μ L and drips in cell counting count board, counts.
2, the adjustment of cell density and bed board
In order to ensure in 96 porocyte plates without the polyclonal existence of single hole, when bed board, reduce total cellular score, the total cellular score of every 96 orifice plates is 50, the liquid volume added in every hole is 200 μ L or 100 μ L, through comparing, add the amount of liquid of 100 μ L, more easily observe the cell in 96 orifice plates, cell density is adjusted into 50cells/100 μ L × 96 ≈ 5 cells/mL.By the cell plate of completing, at Microscopic observation, (this point pole is important in time, after cell settlement, cannot observe the situation in cell plate hole, thereby cannot determine that the later stage cell of amplification stems from mono-clonal or polyclone), carry out mark for the hole of individual cells, put into CO
2incubator is hatched, and in order to reduce the volatilization of liquid, puts into wet box or to the regular fluid infusion of 96 porocyte plate simultaneously.
3, the screening of monoclonal cell
The cell plate that routine observation is completed, after one week, there is significant difference in the growth conditions of cell, some generation apoptosis, survival mono-clonal 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 is to cell continued growth on 96 porocyte plates slowly.
Two, the initial growth speed of MDCK monoclonal cell, cellular form are relevant to cell tumorigenicity
1, initial growth speed and tumorigenicity are negative correlation
Adopt the method for " acquisition of embodiment 1 MDCK monoclonal cell ", injection nude mice after the polyclonal cellular strain amplification that screening is obtained, from the experimental result of tumor after being inoculated into nude mice, only have grow in 96 porocyte plates cell energy comparatively slowly of only a few to disappear within a short period of time and to take off the tubercle that injection cell forms, Fig. 3 has also verified this point, and the initial growth speed of MDCK-2B2 is slow compared with MDCK.
2, the form of cell is relevant to tumorigenicity
Launch experiment for polyclonal cellular, from the experimental result of tumor after being inoculated into nude mice, identical cell strain can disappear and take off the tubercle that injection cell forms within a short period of time in the time that generation is lower, and after going down to posterity several times, within the observation period, cannot eliminate the tubercle of formation, some tubercles even continue to become large, occur to shift is the formation of secondary induced tumor.Examine identical cell strain generation when lower, form rule, without the existence of megabacterium form, has the existence of megabacterium after going down to posterity several times, and these irregular cell paste develop into tumour cell.
In conventional screening methods, obtain after monoclonal cell strain by limiting dilution assay, by after cell enlarged culturing, injection nude mice, part nude mice tubercle carries out slice analysis before disappearing and taking off, and remainder nude mice continues to observe, and what in 6 months, knurl body was withered away is non-tumorigenic cell strain.
The speed of growth of cell of the present invention and the metamorphosis of cell are assisted the judgement of tumorigenicity, the cell plate that routine observation is completed, after one week, there is significant difference in the growth conditions of cell, some generation apoptosis, survival mono-clonal 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 is to cell continued growth on 96 porocyte plates slowly.Experimental result through multiple injection nude mice gathers discovery, and remaining growth phase significantly reduces cell tumorigenicity slowly, in the time of injection nude mice, can select poky cell, and this has reduced time and the cost of screening non-tumorigenic cell strain undoubtedly.The non-tumorigenic cell strain filtering out is after going down to posterity, along with the increase of generation, the probability of tumorigenicity can increase, after what the non-tumorigenic cell strain that we filter out had went down to posterity 25 generations, still keep non-tumorigenic, after 2 generations of going down to posterity that have, just have non-tumorigenic, in mitotic stability test process, we find, after going down to posterity, the more cell strain tumorigenicity of megabacterium can significantly increase, and this non-tumorigenic cell strain stable for we have judged whether to screen has been saved the time.
The growthhabit of embodiment 2, MDCK-2B2 cell and biochemical indicator
One, MDCK-2B2 cell has Epithelial form
Get P85 MDCK-2B2 and carry out (40 ×) observation under mirror, find to be the fusiformis of regular edges, attached cell, tool Epithelial form.
Two, MDCK-2B2 has higher stand density
Inoculate identical its beginning density (2.5 × 10
5cells/5mL) MDCK-2B2 and MDCK are in the Tissue Culture Flask of T-25, and each 7 bottles, interval 24 hours is each to be extracted wherein one bottle and carry out cell counting, continuous counter 7 days, and the total cellular score of MDCK-2B2 in 7 days is respectively: 1.8 × 10
5cells, 2.9 × 10
5cells, 11.6 × 10
5cells, 22 × 10
5cells, 20 × 10
5cells, 20.8 × 10
5cells and 10.5 × 10
5cells; The total cellular score of MDCK in 7 days is respectively: 2.3 × 10
5cells, 4.8 × 10
5cells, 19.1 × 10
5cells, 21 × 10
5cells, 23.5 × 10
5cells, 24.87 × 10
5cells and 14.5 × 10
5cells.As shown in Figure 3, MDCK-2B2 cell has the latent period of approximately 2 days, is then exponential phase of growth, after inoculation, within the 4th day, enters stationary phase, within the 4th day, reaches approximately 22 × 10
5the high-density of individual cell, lived through after plateau, entered the paracme in the 7th day; Mdck cell has the latent period of approximately 1 day, is then exponential phase of growth, after inoculation, within the 3rd day, enters stationary phase, within the 6th day, reaches approximately 24.7 × 10
5the high-density of individual cell, lived through after plateau, entered the paracme in the 7th day.
From latent period, MDCK-2B2 experience enters exponential phase of growth after the latent period of 2 days, and MDCK just enters exponential phase of growth after 1 day latent period of experience, and this has also verified in embodiment 1 that initial growth speed and tumorigenicity are negative correlation.
The high-density that MDCK-2B2 reaches on the 4th day is 22 × 10
5, then maintain the plateau of three days; MDCK enters plateau on the 3rd day, and density is 19.1 × 10
5cells, maintains the plateau of four days, within the 6th day, reaches approximately 24.7 × 10
5the high-density of individual cell.The cell density of contrast plateau, MDCK-2B2 is a little less than MDCK.
Three, MDCK-2B2 cellular biochemical index
Inoculate identical initial density (2.0 × 10
6cells/40mL) MDCK-2B2 and MDCK are in the Tissue Culture Flask of T-225, each 1 bottle, interval is drawn 1mL cell conditioned medium for 24 hours in EP pipe, put into Nova BioProfile 400 Biochemical Analyzers and analyze, record glucose (Gluc) consumption, lactic acid (Lac) generation; Glutamine (Gln) consumes and L-glutamic acid (Glu) and ammonia (NH4
+) produce situation.
The qualification of embodiment 3, MDCK-2B2 cell chromosome
1, harvested cell, 0.85% NaCl solution rinses cell walls twice;
2,0.25% Trypsin-EDTA digestion 10-15 minute, until cell face occur naked eyes wrinkle as seen shape while changing with the lower cell 453g(1500rpm/min of suction pipe piping and druming, eppendorf 5810R) centrifugal 10 minutes of room temperature, remove supernatant, approximately stay 0.5ml;
3, hypotonic: to add 37 DEG C of 0.075M KCl 4-6ml, suction pipe piping and druming, 37 DEG C of water-bath 3-5 minute
4, pre-fix: add 1.5ml ± (methyl alcohol: Glacial acetic acid=3:1) fixing agent, gently mix 5 minutes 453g(1500rpm/min of 37 DEG C of water-baths, eppendorf 5810R) centrifugal 10 minutes of room temperature.Remove the honest and upright and thrifty 0.5ml of staying;
5, fixing: add fixing agent 8ml ±, gently mix 37 DEG C of water-baths 10 minutes, 453g(1500rpm/min, eppendorf 5810R) centrifugal 10 minutes of room temperature.Remove supernatant, approximately stay 0.5ml;
6, repeat to fix: the same.453g(1500rpm/min, eppendorf 5810R) centrifugal 10 minutes of room temperature, remove supernatant, add suitably several fixing agents depending on pipe floor cells amount;
7, drip sheet: every 1-2 drips;
8, roasting sheet: 75 DEG C of baking boxs toast 3 hours;
9, row dyeing in second day is processed, and karyomit(e) is counted.
Embodiment 4, MDCK-2B2 cell produce the calibrating of malicious ability
1, cell counting
Go down to posterity each three bottles of MDCK, MDCK-2B2 T-25, cell density and the volume of three bottles are in full accord, and after 2 days, observation of cell covers with substantially, takes out the each one bottle of digestion of MDCK, MDCK-2B2 T-25, calculates total cellular score.
2, virus inoculation
Four bottles of remaining cells are washed twice with PBS respectively, add the MEM basic medium of serum-free (in * bovine serum, to contain the nonspecific inhibitor of influenza virus, can affect copying of influenza virus) (it is in order to improve the replication of influenza virus in cell that * adds appropriate pancreatin object to+TPCK-pancreatin, the working concentration of pancreatin is 2.5 μ g/mL)+virus (be respectively 1/100 and 1/1000 according to m.o.i value, calculate viral inoculum size).
3, receipts poison and the hemagglutination test (HA test) of virus
Start collecting cell supernatant liquor from virus infected cell 24h hour and carry out red cell agglutination experiment, in 96 hole blood clotting plate holes, add 50 μ L physiological saline, then add 50 μ L cell conditioned medium liquid, each sample does two multiple holes, mix, after doubling dilution, every hole adds 1% red cell suspension 50 μ L, observes blood clotting result after 30min.Every sampling in 24h hour once, respectively in 24h, 48h, 72h and 96h sampling.As shown in Figure 6, in the time that 24h samples, the titre of HA is 0, and at 48h, 72h and 96h, m.o.i value is 1/100 o'clock, and the HA titre that the HA titre that MDCK produces produces than MDCK-2B2 is slightly high; M.o.i value is 1/1000 o'clock, and the HA titre that when 48h, MDCK produces is significantly higher than the HA titre producing with MDCK-2B2, and the HA titre that when 72h, both produce is suitable, and the HA titre that when 96h, MDCK produces is slightly high.
4, the preparation of 1% red cell suspension
By physiological saline washing for chicken blood 3 times, centrifugal 5 min of the first two times 1 500r/min, last 1 synchronized centrifugal 10min, abandons supernatant, is made into 1% suspension by stroke-physiological saline solution, put 4 DEG C stand-by.Generally put 4 DEG C and can preserve about 7 days, once haemolysis should discard.
The calibrating of embodiment 5, MDCK-2B2 cell non-tumorigenic
Digestion MDCK-2B2 cell, counting, by the not MEM substratum suspension containing FBS for cell, is adjusted into 5 × 10 by cell density
7cells/mL, the adult nude mice of injection 3 ~ 4weeks SPF level, every nude mice is injected 200 μ L cell suspending liquids from subcutaneous shoulder blade, and the cell injection volume of every nude mice is 10
7cells.
One, the calibrating of the non-tumorigenic of MDCK-2B2 continuous passage
For the MDCK clone of amplification, according to 10
7injection volume injection nude mice, the formation of periodic measurement tubercle and the situation of taking off that disappears of cells/200 μ L/mice.Observation period is set as 6 months, and the formation of each cell strain tubercle exists significant difference with the situation of taking off that disappears.Most cells strain tubercle within the observation period becomes large, brings out secondary tumour, the transfer that even has tumour kitchen range having; Small part cell is in the time of 2weeks, and the tubercle maximum of formation, disappears and take off gradually within the observation period with posterior tubercle.Find through injection polyclonal cellular, the in the situation that of identical generation, after injection MDCK-2B2, disappear and take off the required shortest time of tubercle.After continuous passage, most cells have the non-tumorigenic of lower generation to change the tumorigenicity of higher generation time into, and MDCK-2B2 still disappears and takes off tubercle in the time of higher generation time within the observation period of six months by a definite date, can keep non-tumorigenic.As shown in Figure 7, the tubercle forming in injection site after MDCK-2B2 P77 injection nude mice, disappeared and takes off at the 23rd day, and MDCK-2B2 P79 disappears and takes off at the 31st day tubercle of injection, P89 disappears and takes off tumorigenicity at the 109th day tubercle of injection, and P100 disappears and takes off at the 153rd day tubercle of injection.
Two, the calibrating of the non-tumorigenic of MDCK-2B2 higher generation time
To MDCK-2B2 continuous passage to P102, according to 10
7the adult nude mice of injection volume injection 3 ~ 4weeks of cells/200 μ L/mice, inject after one week tubercle is peeled off, be kept in 4% formalin solution (10 times of dilutions of formaldehyde solution of commercially available 37%), send the qualification of cutting into slices of department of pathology of shanghai Medicine institute of Fudan University.
Pathological section result shows (as shown in Figure 8), the existence of negative for tumor cells in tubercle, still think that MDCK-2B2 P102 is for without tumorigenicity.
The foundation in embodiment 6, MDCK-2B2 cell master library and the storehouse of working
Set up master library and work storehouse for MDCK-2B2.Master library is carried out to Identification cell lines is fixed, tumorigenicity detects and carry out as shown in Figure 9 exogenous factor detection; The detection of Identification cell lines and exogenous factor is carried out in work storehouse.
The identification experiment of cell comprises cellular form and chromosome analysis, and to observation of cell form after cell continuous passage, cellular form is normal, without noticeable change.Cell for the different generations of MDCK-2B2 carries out chromosome analysis, finds that chromosome number is consistent with the mdck cell that ATCC buys.
The detection of exogenous factor, comprises that sterility test, cell method detect viral exogenous factor and detect virokine with animal and chicken embryo.
One, sterility test (sterility test comprises bacterium, fungi and mycoplasma)
1, bacterium, fungi detect
A, by MDCK-2B2 cell suspension inoculation THIOGLYCOLLIC ACID salt broth, improvement Martin's substratum and each two of nutrient agar medium inclined-plane (inoculating MEM substratum in contrast) simultaneously; B. postvaccinal substratum is divided into two parts, portion is put 30 DEG C～35 DEG C and is cultivated 14 days; Another part is put 20 DEG C～25 DEG C and is cultivated 14 days, should observe day by day and record whether have bacteria growing between incubation period, and result shows without bacterium and fungal contamination.
2, mycoplasma inspection
Mycoplasma semi-fluid substratum should boil 10～15 minutes before use, is cooled to 56 DEG C of left and right, then adds death of monks or nuns energy new-born calf serum (substratum and serum volume ratio are 8:2), fully shakes up.Mycoplasma broth culture is without boiling, but also should add equally new-born calf serum before using.Inoculation trial-product (MDCK-2B2 cell suspension), respectively inoculates 4, puts 36 ± 1 DEG C and cultivates 21 days.From 4, got 2 in postvaccinal the 7th day and carry out time culture, each 2 of every 1 substratum transferred species mycoplasma semi-fluid substratum and mycoplasma broth culture, put 36 ± 1 DEG C and cultivate 21 days, observed 1 time every 3 days, and result shows without mycoplasma contamination.
Two, cell method detects viral exogenous factor
1, cell culture method is directly observed and hemadsorption test
Get MDCK master library cytomixis bottle cell sample and inoculate 6 of little square vases, in flakes rear observation two weeks.Observe latter stage get 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, hemabsorption result is negative.
2, different passages are cultivated and are checked
Get mdck cell (every bottle of cell inoculation 10 of MDCK master library cell culture supernatant biased sample inoculation MRC-5, VERO and different generations
7cells MDCK-2B2), observe latter stage get 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; Cultivating the 7th day time, get respectively each one bottle of the monolayer cell supernatant liquor of every kind of inoculation, be inoculated in respectively on the corresponding monolayer cell of fresh preparation, continue to cultivate 7 days, observation of cell pathology is also carried out cellular form observation and hemabsorption.Must not there is cytopathy in the monolayer cell of every kind of inoculation, hemabsorption should be all negative.
Three, detect virokine with animal and chicken embryo
1, injection suckling mouse (work storehouse does not need to detect)
Digestion MDCK-2B2 cell, is adjusted into 10 by cell density
7cells/mL, the suckling mouse of injection in 24h, intracerebral injection brood, inoculum size is 0.01 mL/only, routine observation, cumulative observation 21 days, finds all survivals; Abdominal injection brood, inoculum size is 0.1 mL/, routine observation, cumulative observation 21 days, finds all survivals.
2, be injected into mouse (work storehouse does not need to detect)
Digestion MDCK-2B2 cell, is adjusted into 2 × 10 by cell density
6cells/mL, the one-tenth mouse of injection 3 ~ 4 weeks, intracerebral injection brood, inoculum size is 0.03 mL/only, routine observation, cumulative observation 21 days, finds all survivals; Abdominal injection brood, inoculum size is 0.5 mL/, routine observation, cumulative observation 21 days, finds all survivals.
3, injection chicken embryo (allantoic cavity)
Hatch chicken embryo to 11 age in days, irradiate at dark indoor candler, observe air chamber, the position that label vascular is sparse, with punch tool punching, along air chamber vertical line to allantoic cavity, injection density is 5 × 10
6mDCK-2B2 0.2 mL/only, inject altogether 10 chicken embryos, put hatching box and hatch, after 4 days, chicken embryo is taken out, put 4 DEG C and spend the night (preventing from getting allantoic fluid is to poke blood vessel), draw the allantoic fluid 100 μ L of clarification in the injection from each chicken embryo in second day.
On 96 hole blood-coagulation-boards, every hole adds 50 μ L physiological saline, the allantoic fluid that corresponding aperture adds 50 μ L to draw, and after doubling dilution, every hole adds 50 μ L red cell suspensions, and result shows: urine hemagglutination test is negative.
4, injection chicken embryo (yolk sac)
Hatch chicken embryo to 6 age in days, irradiate at dark indoor candler, observe air chamber, the position that label vascular is sparse, with punch tool punching, is 2 × 10 along air chamber vertical line to the contrary position injection density of allantoic cavity
6only (after injection, back suction finds that there is yolk liquid, shows inject successfully) of MDCK-2B2 0.5 mL/, inject altogether 10 chicken embryos, after 5 days, irradiate observation with candler, discovery chicken embryo is all survived.
The qualification of embodiment 7, MDCK-2B2 subclone cell
MDCK-2B2 continuous passage is to P102, and without tumorigenicity, we to its bed board, select more excellent cell strain with it with reference to the method for embodiment mono-.
One, the calibrating of the non-tumorigenic of MDCK-2B2 subclone cell
Carry out tumorigenicity calibrating for the mono-clonal forming after bed board MDCK-2B2 (being designated hereinafter simply as MDCK-2B2 subclone).Tumorigenicity is examined and determine with reference to embodiment 5.
MDCK-2B2-2G5 P91 latter 43 days in injection, tubercle disappears and takes off; MDCK-2B2-2G6 P92 latter 34 days in injection, tubercle disappears and takes off; MDCK-2B2 P89 disappears and takes off at the 109th day tubercle of injection.Comparatively speaking,, in the situation that generation is close, MDCK-2B2 subclone time of taking off tubercle that disappears obviously shortens.
Two, the calibrating of the product of MDCK-2B2 subclone cell poison ability
Carry out the calibrating of the product poison ability of MDCK-2B2 subclone cell with reference to embodiment 4, as shown in figure 10, the product poison ability of MDCK-2B2-2G6 is close with MDCK-2B2.
Three, the monitoring of the stand density of MDCK-2B2 subclone cell
With reference to the second section of embodiment 2, cell is counted, as shown in Figure 11, the high-density of MDCK-2B2-2G5 and MDCK-2B2-2G6 is close with MDCK-2B2.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (4)
1. the system of the non-tumorigenic mdck cell for the influenza virus of increasing, is characterized in that, described cell is MDCK-2B2, and deposit number is CCTCC C201323.
2. the screening method that described in claim 1 for the non-tumorigenic mdck cell of the influenza virus of increasing is, is characterized in that, comprises the following steps:
(1) improved limiting dilution assay, ATCC CCL-34 MDCK is gone down to posterity after preservation, adopt limiting dilution assay to carry out monoclonal screening, in order to ensure in each 96 orifice plates, cell count≤1, cell count when bed board is set as≤and 50, and after bed board, carry out immediately the inspection of single hole, determine that cell count is 1 single hole, respective markers on work, the single hole cell suitable fluid infusion good to mark;
(2) screening non-tumorigenic cell strain, observe the single hole cell that step (1) obtains, assist the judgement of tumorigenicity by the speed of growth of cell and the metamorphosis of cell, select growth phase to cell slowly, and form is normal, without the monoclonal cell strain of giant cell, the nude mice of SPF level is injected, filter out the cell strain that knurl body is withered away, carry out the further confirmation of pathological section for non-tumorigenic cell strain;
(3) set up cell bank.
3. described in claim 1, tie up to the application of amplification influenza virus for the non-tumorigenic mdck cell of the influenza virus of increasing.
4. the application in amplification influenza virus according to claim 3, is characterized in that, described influenza virus is influenza A virus.
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| CN106801031A (en) * | 2017-01-22 | 2017-06-06 | 西北民族大学 | Without Tumor formation mdck cell clone strain |
| CN106884003A (en) * | 2017-01-22 | 2017-06-23 | 西北民族大学 | Without Tumor formation mdck cell clone strain |
| CN112195148A (en) * | 2020-08-17 | 2021-01-08 | 上海生物制品研究所有限责任公司 | Serum-free suspension-adapted MDCK cell and application thereof in preparation of influenza virus vaccine |
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| CN101189326A (en) * | 2004-12-23 | 2008-05-28 | 米迪缪尼疫苗股份有限公司 | Non-tumorigenic MDCK cell line for propagating viruses |
| CN101511385A (en) * | 2006-09-11 | 2009-08-19 | 诺华有限公司 | Making influenza virus vaccines without using eggs |
| WO2012033328A2 (en) * | 2010-09-06 | 2012-03-15 | Sk Chemicals Co., Ltd. | Mdck-derived cell lines adapted to serum-free culture and suspension culture and method for preparing vaccine virus using the cells |
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| CN101189326A (en) * | 2004-12-23 | 2008-05-28 | 米迪缪尼疫苗股份有限公司 | Non-tumorigenic MDCK cell line for propagating viruses |
| CN101511385A (en) * | 2006-09-11 | 2009-08-19 | 诺华有限公司 | Making influenza virus vaccines without using eggs |
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| CN106801031A (en) * | 2017-01-22 | 2017-06-06 | 西北民族大学 | Without Tumor formation mdck cell clone strain |
| CN106884003A (en) * | 2017-01-22 | 2017-06-23 | 西北民族大学 | Without Tumor formation mdck cell clone strain |
| CN112195148A (en) * | 2020-08-17 | 2021-01-08 | 上海生物制品研究所有限责任公司 | Serum-free suspension-adapted MDCK cell and application thereof in preparation of influenza virus vaccine |
| CN112195148B (en) * | 2020-08-17 | 2023-01-03 | 上海生物制品研究所有限责任公司 | Serum-free suspension-adapted MDCK cell and application thereof in preparation of influenza virus vaccine |
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