| Influenza,also known as pandemic influenza,is an acute infectious zoonotic disease of the upper respiratory tract caused by the influenza virus.It is primarily transmitted via droplets and respiratory secretions and has caused four worldwide pandemics.According to the World Health Organization(WHO),seasonal influenza epidemics worldwide are estimated to cause 3-5 million severe cases and 300,000-650,000 deaths annually.Vaccination is the most effective strategy for preventing and controlling infectious diseases and their associated complications.Because influenza viruses are characterized by rapid variability,seasonal influenza vaccines must be prepared and administered annually to match circulating strains.Currently,there are three types of influenza vaccines globally:split inactivated vaccine,live attenuated vaccine,and recombinant ha vaccine,which are approved for use in different countries.The production of influenza vaccines using chicken embryos as a matrix has been in use around the world for almost 70 years.However,this platform has a series of problems,such as difficulty in cleaning the chicken embryo supply,long production cycle,scale-up issues,cumbersome operation,and contamination.Additionally,containing a trace amount of ovalbumin may lead to allergic reactions,and it is difficult to respond to avian influenza outbreaks.Considering these issues,the WHO proposed cell culture as an alternative to chicken embryo cultured influenza vaccine production matrices as early as 1995.MDCK(Madin-Darby canine kidney)cells were established in 1958 by Madin and Darby and have since been subcultured to create an immortalized adherent epithelial cell line.These cells are susceptible to a variety of viruses and are widely used in viral research,including research on influenza virus,adenovirus,canine parvovirus,and others.MDCK cells are currently the most used cell line for influenza virus infection research,and they are also the preferred cell line for isolating,amplifying,and purifying influenza virus.Additionally,they have been used for influenza surveillance worldwide.In the mid-1990s,the World Health Organization(WHO)recommended using MDCK cells for the production of seasonal influenza vaccines instead of chicken embryos.In 2012,the FDA approved the use of MDCK cells for the production of cell culture-based influenza vaccines.Several MDCK-produced influenza vaccines are currently available worldwide.However,in China,the influenza vaccine market is mainly based on the traditional chicken embryo production platform and uses a split inactivated vaccine.Due to issues with the supply of chicken embryos,developing a scaled-up cell matrix for influenza production is considered the best solution.There are two main types of scaled cell culture:planarized adherent culture based on multi-surface and stacked surface,and single cell suspension culture.Single cell suspension culture offers several advantages,such as high cell density,easy scale-up,stable culture conditions,and easy control.Researchers have also successfully adapted some adherent-dependent cells,such as MDCK cells,BHK-21 cells,CHO cells,etc.,for suspension culture.The traditional culture method for MDCK cells is anchorage-dependent,but the bioproducts industry is focused on increasing productivity while reducing costs.Suspension culture offers higher cell density and easier process scaleup at reduced costs,which means a cheaper product for low-and middle-income countries(LMICs).Therefore,it is important to establish high-quality MDCK suspension cell lines and animal cell suspension culture-based influenza vaccine manufacturing processes.This paper focuses on establishing MDCK cell suspension cell lines using MDCK cells and influenza virus as the primary research objects.The aim is to develop a process for producing influenza virus vaccine suspensions using MDCK cells as the cell matrix.Firstly,In this article,the soft agar cloning method was used to purify the introduced MDCK cells,resulting in 23 cell clones.Among them,the clone formation rates of MDCKR11 and MDCK-R18 were the lowest,at 1.13%and 1.18%,respectively,while the formation rates of the other cell clones were higher than 1.20%.Subsequently,MDCKR11,with the lowest clone formation rate,was selected and successfully adapted to suspension culture using the "shaker culture adaptation method",supporting serum-free single-cell suspension culture and named MDCK-S.The entire domestication process took approximately 18 days,and the methods and processes used for suspension domestication are described in detail in the text.The doubling time of MDCK-S cells in shake flasks during suspension culture was approximately 27.16 hours,and the cell density reached a maximum of 1×107 cells/mL.Additionally,we confirmed the identity of MDCK-S cells through 18S sequencing and species-specific PCR,which showed that the 18S sequence was consistent with the MDCK(NBL-2)sequence and the specific PCR confirmed that MDCK-S was dog-derived cells,indicating that there was no contamination by other cell lines.Secondly,a comprehensive MDCK-S assay was performed,including morphology,growth kinetics,biochemical metabolic analysis,species identification,contamination examination,tumorigenicity and oncogenicity examination,chromosomal variability analysis,passage stability,transmission electron microscopy(TEM),and cell cycle analysis and so on.Our results show that MDCK-S cells are stable from passage to passage,cell state,cell cycle and metabolism during growth;No contamination with other cell lines,bacteria,fungi,viruses,or mycoplasmas;The soft agar cloning experiment showed that the clone formation rates of MDCK-S at passages 29,33,36,45,and 50 were 2.41%,2.27%,2.40%,2.17%,and 2.15%,respectively,with no significant differences between generations.The clone formation rate of MDCK-S was not significantly different from that of Hela cells(1.84%)and the adherent control group(P24)(2.1 1%).Chromosome number analysis showed that the chromosome number of MDCK-S cells was stable from passages 29 to 50,and 78±2 chromosomes was over 86%.Furthermore,tumorigenicity tests showed that the MDCK-S cells from the 50th passage were non-tumorigenic.Finally,we tested the sensitivity of MDCK-S cells to influenza viruses H3N2,H1N1,B-V,and B-Y and found that the sensitivity of MDCK-S cells to the four subtypes was comparable to that of MDCK(NBL-2)cells.Based on this,the optimal culture conditions for H3N2 in shake flask suspension culture of MDCK-S cells were determined and optimized using DoE design study and high throughput "shake tube" model,which were as follows:pH=7.88～8.00,MOI=0.001,TPCK final concentration 1.70μg/ml～6.0μg/ml and cell density 55.70～77.28×105cells/ml,the virus maintenance medium was Xene-S001 medium,the culture temperature was 34.5℃,the incubation time was 72h,the shaker speed was 100rpm/min,the CO2 content was 3%,the titer of influenza virus H3N2 under this condition 6.5 1gTCID50/ml.Here,we established a purification process for inactivated influenza virus vaccine prepared by MDCK-S suspension culture of influenza virus H3N2 through small-scale suspension culture of influenza virus H3N2,and evaluated its immunogenicity.The purification process involved the use of a 0.65μm filter,300KDa ultrafiltration concentration,Benzonase nucleic acid enzyme treatment,Capto Core 700,and Capto Q steps.Capto Core 700 and Capto Q each step yielding a lectin recovery rate of 55.03±2.01%,and 66.27±3.67%.The entire process for producing inactivated vaccine met the requirements of the pharmacopeia,with a total protein removal rate of 97.94±0.09%,host protein removal rate of 99.46±0.03%,and residual host cell DNA(rcDNA)removal rate of 99.99±0.01%.The use of nucleic acid enzyme degradation of rcDNA and two-step column purification in the purification process improved the safety of MDCK-S cell preparation of influenza virus.Inactivated virus was immunized in BALB/c mice,and the hemagglutination inhibition test was used to measure the hemagglutination inhibition antibodies in serum.There was no significant difference in the hemagglutination inhibition antibody titers between the influenza virus H3N2 prepared by this process and the inactivated virus cultivated in chicken embryos,indicating good immunogenicity and safety.In summary,this study established a method for domestication of MDCK cell suspension culture.The suspension cultured MDCK cells were sensitive to influenza virus and demonstrated good safety and immunogenicity.This method provides a new approach and strategy for large-scale production of influenza virus. |
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