Elsevier

Biologicals

Volume 68, November 2020, Pages 112-121
Biologicals

Establishment of a low-tumorigenic MDCK cell line and study of differential molecular networks

https://doi.org/10.1016/j.biologicals.2020.07.003Get rights and content

Abstract

Influenza is an acute respiratory infection caused by the influenza virus, and vaccination against influenza is considered the best way to prevent the onset and spread. MDCK (Madin-Darby canine kidney) cells are typically used to isolate the influenza virus, however, their high tumorigenicity is the main controversy in the production of influenza vaccines. Here, MDCK-C09 and MDCK-C35 monoclonal cell lines were established, which were proven to be low in tumorigenicity. RNA-seq of MDCK-C09, MDCK-C35, and MDCK-W73 cells was performed to investigate the putative tumorigenicity mechanisms. Tumor-related molecular interaction analysis of the differentially expressed genes indicates that hub genes, such as CUL3 and EGFR, may play essential roles in tumorigenicity differences between MDCK-C (MDCK-C09 and MDCK-C35) and MDCK-W (MDCK-W73) cells. Moreover, the analysis of cell proliferation regulation-associated molecular interaction shows that downregulated JUN and MYC, for instance, mediate increased proliferation of these cells. The present study provides a new low-tumorigenic MDCK cell line and describes the potential molecular mechanism for the low tumorigenicity and high proliferation rate.

Introduction

Influenza is a respiratory disease with high morbidity and mortality rates. The influenza virus exists as three types: A, B, and C. Among these, the influenza A virus easily mutates due to its antigenicity, and therefore is highly contagious and causes death of humans and livestock [1,2]. The influenza B virus mainly infects humans and often causes local outbreaks. To date, no ideal treatment has been found for either flu or bird flu, and vaccination remains the most effective means to prevent disease occurrence and epidemics. Traditional influenza vaccines are produced by a method using chicken embryos; however, this method is limited by many factors, and more rapid and effective vaccine production techniques are needed to meet market demand. Due to easy culture, rapid proliferation, and high efficiency of influenza virus infection, MDCK (Madin-Darby canine kidney) cells are one of the most important cell lines in influenza vaccine production. MDCK cells were established from the kidney tissue of a Cocker Spaniel in the United States by Madin and Darby in 1958 [1]. Currently, MDCK cells are used to isolate the influenza virus since the antigenicity and gene characteristics of the isolated cell lines are relatively stable and closer to the original specimens [2]. Therefore, the development of influenza vaccines by MDCK cell culture has been the focus of domestic and foreign experts. At present, several companies worldwide have been approved for the use of MDCK cells in the production of influenza vaccines: Flucelvax® inactivated vaccine (influenza A and B) was approved by the US FDA in November 2012 for influenza vaccination in adults over 18 years of age; and Flumist®, the MedImmune four-valent live attenuated vaccine by AstraZeneca, was approved by the US FDA in 2010 for vaccination of individuals aged 2–45 years.

The main controversy in the use of MDCK cells in influenza vaccine production is their high tumorigenicity. A research report by Novartis showed that serum-free suspension-cultured MDCK cells are very tumorigenic, with as little as 10 cells being capable of forming tumors in nude mice. However, it has been shown that the company's MDCK cell line, 33016-PF, obtained by domestication, does not form tumors in nude mice. On the other hand, to produce a high-titer virus vaccine, increasing the cell proliferation rate for the achievement of a high cell density vaccine production process is also an important research focus. It takes approximately 12 weeks to produce a flu vaccine using a reactor for cell culture [3]. When a flu epidemic occurs, frozen cells in the cell bank are resuscitated, and a cell line with rapid proliferation can quickly enter the large-scale vaccine production process. Direct application of wild-type epidemic strains for vaccine production in a tertiary biosafety environment is an important reason why the proliferation rate of MDCK cells used to produce influenza vaccines needs to be increased.

At present, the study of tumorigenesis in nude mice was a stable method to evaluate the characteristics of cell tumorigenesis. In 2012, tumorigenic MDCK cells were successfully generated following Hras and cMyc oncogene transfection of MDCK9B9-1E4 cloned cells. Through a series of experiments such as tumor formation in nude mice, it was found that p53 pathway plays an important role in tumor formation. In addition, with the application of sequencing technology, more and more genes related to function have been discovered [4]. In 2018, the regulatory capacity of transporter ABC increased after hypertonic stress through transcriptome technology [5]. In the present paper, we established a monoclonal MDCK cell line with low tumorigenicity. Subsequently, we obtained the differentially expressed genes (DEGs) using transcriptome sequencing to find the potential molecular mechanism of reduced tumorigenicity of the monoclonal cells.

Section snippets

Preparation of monoclonal cells

Monoclonal cells were prepared by limiting dilution. MDCK cells were derived directly from the ATCC stock (ATCC CCL-34) and cultured with adherent culture method in DMEM supplemented with 10% heat-inactivated fetal bovine serum (CellMax, Beijing, China). The cell culture bottle of T75 is 10 ml DMEM medium mixed with FBS. The monolayer was washed twice with 10 mL PBS, and the cells were detached with 5 mL 0.25% trypsin-EDTA (Gibco, Grand Island, USA) digesting at 37 °C for 5 min. Cells were

Biological characteristics of the monoclonal MDCK cell lines

To study the biological characteristics of MDCK cells, 10 dominant cell lines were selected to establish a primitive cell bank by single cell clone culture and screening. All were epithelioid cells, numbered as: MDCK-C01, MDCK-C04, MDCK-C09, MDCK-C19, MDCK-C20, MDCK-C21, MDCK-C23, MDCK-C25, MDCK-C26 and MDCK-C35. Fig. 1 shows the growth pattern of cells cultured for 24 h. Among these, MDCK-C23, MDCK-C25, MDCK-C26 and MDCK-C35 showed island growth (Fig. 1g–j) at 48 h before each generation

Discussion

In the present study, MDCK-C09 and MDCK-C35 cell lines with low tumorigenicity were screened by cell monoclonal technique (Fig. 1). More than 80% of the two cell lines contain n = 78 ± 2 chromosomes and are sensitive to influenza virus (Table 2). An in vivo tumor formation assay showed that these two MDCK cell lines are low in tumorigenicity, and MDCK-W73 is high in tumorigenicity. Subsequently, differential gene expression among MDCK-C09, MDCK-C35, and MDCK-W73 cell lines was analyzed (Fig. 3

Conclusion

MDCK cells, currently used for the production of influenza vaccines, are tumorigenic and pose biosafety issues. Here, we established two low-tumorigenic monoclonal MDCK cell lines with a high proliferation rate. RNA-Seq results showed that JUN, MYC, EED, YWHAB and APP might regulate the proliferation of MDCK cells. Cell migration and osteoblast differentiation occur in the process of tumor formation. According to the molecular interaction network, epidermal growth factor receptor (EGFR) forms a

Ethics approval and consent to participate

This study obtained ethics approval from Ethics Committee of the Experimental Animals of Northwest University for Nationalities (NO: 2014012).

Availability of data and material

Not applicable.

Funding

This work was financially supported by the Major National Special Funds for Science and Technology (2015ZX09102016), the Program for Changjiang Scholars and Innovative Research Team in the University (IRT_17R88), the Fundamental Research Funds for the Central Universities (31920190004 and 31920200069).

Authors’ contributions

GM and YK contributed to the design, analysis, and interpretation of data for the study. ZQ and JW drafted the work, interpreted the data and conducted the experiments. GM, DH, XX, FW, FW and SB conducted the experiments. SB and ZM collected data. JX and YH finally approved the version to be published.

Declaration of competing interest

The authors declare that they have no competing interest.

Acknowledgements

The authors would like to thank Dr. Natalie Ward (Medical College of Wisconsin, Wauwatosa, WI, United States) for editing this manuscript.

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