Transformation and Tumorigenicity Testing of Simian Cell Lines and Evaluation of Poliovirus Replication

RESEARCH ARTICLE Transformation and Tumorigenicity Testing of Simian Cell Lines and Evaluation of Poliovirus Replication Silvia Dotti*, Tina Lombardo, Riccardo Villa, Andrea Cacciamali, Cinzia Zanotti, Nadia Andrea Andreani, Stefano Cinotti, Maura Ferrari Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna, Brescia, Italy * Abstract a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Dotti S, Lombardo T, Villa R, Cacciamali A, Zanotti C, Andreani NA, et al. (2017) Transformation and Tumorigenicity Testing of Simian Cell Lines and Evaluation of Poliovirus Replication. PLoS ONE 12(1): e0169391. doi:10.1371/journal.pone.0169391 The key role of cell cultures in different scientific fields is worldwide recognized, both as in vitro research models alternative to laboratory animals and substrates for biological production. However, many safety concerns rise from the use of animal/human cell lines that may be tumorigenic, leading to potential adverse contaminations in cell-derived biologicals. In order to evaluate the suitability of 13 different cell lines for Poliovirus vaccine production, safety and quality, in vitro/in vivo tumorigenicity and Poliovirus propagation properties were evaluated. Our results revealed that non-human primate cell lines CYNOM-K1, FRhK-4, 4MBr-5 and 4647 are free of tumorigenic features and represent highly susceptible substrates for attenuated Sabin Poliovirus strains. In particular, FRhK-4 and 4647 cell lines are characterized by a higher in vitro replication, resulting indicated for the use in large-scale production field. Editor: Ilya Ulasov, Swedish Neuroscience Institute, UNITED STATES Received: January 14, 2016 Accepted: December 16, 2016 Introduction Published: January 3, 2017 Poliomyelitis is a highly contagious disease caused by a virus of the Enterovirus genus, belonging to the Picornaviridae family, known as Poliovirus and composed by a 7,500 nucleotides (+) single stranded RNA molecule [1,2]. Three different serotypes of wild Poliovirus were identified and classified as type 1, type 2 and type 3 [3]. No specific therapy is available against the virus, but effective inactivated and attenuated vaccines are essential to prevent the disease. Since the development of the first vaccines by Salk in 1955 and Sabin in 1960 [4,5], Poliovirus study greatly improved, taking advantage of cell cultures to isolate the virus from infected people [6,7], microcarrier technology [8,9] and simian cell lines for large-scale production of infected cells for vaccine manufacture [10–14]. Immortalization of animal and human cells, derived from primary cell cultures, is a phenomenon mainly due to genetic mutations or infections by oncogenic viruses, which can result in the appearance of transformed features and tumorigenic properties. Furthermore, cells can undergo several modifications during in vitro cultivation, resulting in the appearance of novel Copyright: © 2017 Dotti et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: This work was supported by the Ministry of Health project, MINSALPOLIOVIRUS. Competing Interests: The authors have declared that no competing interests exist. PLOS ONE | DOI:10.1371/journal.pone.0169391 January 3, 2017 1 / 13 Poliovirus Replication in Simian Cell Lines biochemical, biological and genetic characteristics that differ from primary or diploid cell ones. This represents an important issue in order to establish the biosafety of the cell lines used as substrates and to monitor the possible transmission of animal pathogens to human recipients [15]. Among continuous cell lines, the human HeLa cell line, naturally contaminated by human Papillomavirus, revolutionized the study of Poliovirus biology. On the other hand, Vero cells, widely used in Poliovirus vaccine manufacturing, became immortalized through a spontaneous, unknown process and they acquired tumorigenic properties with increasing in vitro passage levels [16–18]. Moreover, recent studies have demonstrated that the in vitro establishment of two African green monkey kidney derived cell lines, named BS-C-1 and CV-1, gave rise to transformed colonies and tumor formation in the rat model [18–20]. The aim of this research was to identify cell lines free of any transformation ability and tumorigenicity, suitable for Poliovirus vaccine production. In this respect, thirteen simian cell lines have been screened in vitro and in vivo for transformation and tumorigenicity features and their permissiveness to Poliovirus infection investigated, in comparison with other wellestablished substrates. Materials and Methods Cell lines All the investigated simian cell lines reported in Table 1 were stored at the Italian Biobank of Veterinary Resources of IZSLER, the OIE Collaborating Centre for Veterinary Biological Biobank (Brescia, Italy; www.ibvr.org) and are available upon request. All these are continuous, spontaneously immortalized cell lines, exception made for CYNOM-K1, CV-1 (finite cell lines) and 4MBr-5 (EFG-dependent line). The investigations were performed at the passages indicated. Moreover, seven cell lines used as controls or as substrates are reported in a separate section of Table 1. MRC-5, LLC-MK2 and RK13.6 were used as substrates in adventitious agents investigation, while HEp2 and 3T3BALB/c as positive and negative controls in tumorigenicity assays. LCP were infected with Maedi-Visna virus (VIR RE RSCIC 312) and used as retrovirus positive sample. Furthermore, MRC-5 and LLC-MK2 cell lines were selected to prepare “master” batches of three types of Poliovirus (see Poliovirus propagation section). Cells were cryopreserved in vapor phase nitrogen until use. After thawing at 37˚C, they were diluted in MEM culture medium (Sigma-Aldrich, Milan, Italy), free of antibiotics, supplemented with 4mM L-glutamine (Sigma-Aldrich) and centrifuged at 125 g for 5 minutes at 20˚C, in order to remove the dimethyl sulfoxide cryoprotectant agent. Cells were stained with Trypan Blue (Sigma-Aldrich), counted and checked for viability by a Cellometer1 Automated Cell Counter (Nexcelom Bioscience, USA). Finally, 1x105 viable cells of each cell line were seeded in a 75 cm2-flask and incubated at 37˚C in 5% CO2 in the below reported culture media, enriched with 10% (v/v) of Fetal Bovine Serum (FBS; Euroclone, Milan, Italy). BGMK, BS-C-1, CYNOM-K1, HeLa, HEp2, LCP, LLC-MK2, MA-104, MARC-145, RK13.6 and Vero cell lines were amplified in MEM, while FRhK-4, FrP3, RC 37 and 4647 cell lines in D-MEM (Sigma-Aldrich). NCTC cl 3526 cell line was maintained in NCTC 135 medium (Thermo Fisher Scientific) and CV-1 in Eagle’s basal medium in Hanks’ BSS with amino acids and vitamins (...truncated)