title: Potential neoplastic evolution of Vero cells: in vivo and in vitro characterization.
abstract: Vero cell lines are extensively employed in viral vaccine manufacturing. Similarly to all established cells, mutations can occur during Vero cells in vitro amplification which can result in adverse features compromising their biological safety. To evaluate the potential neoplastic evolution of these cells, in vitro transformation test, gene expression analysis and karyotyping were compared among low- (127 and 139 passages) and high-passage (passage 194) cell lines, as well as transformed colonies (TCs). In vivo tumorigenicity was also tested to confirm preliminary in vitro data obtained for low passage lines and TCs. Moreover, Vero cells cultivated in foetal bovine serum-free medium and derived from TCs were analysed to investigate the influence of cultivation methods on tumorigenic evolution. Low-passage Vero developed TCs in soft agar, without showing any tumorigenic evolution when inoculated in the animal model. Karyotyping showed a hypo-diploid modal chromosome number and rearrangements with no difference among Vero cell line passages and TCs. These abnormalities were reported also in serum-free cultivated Vero. Gene expression revealed that high-passage Vero cells had several under-expressed and a few over-expressed genes compared to low-passage ones. Gene ontology revealed no significant enrichment of pathways related to oncogenic risk. These findings suggest that in vitro high passage, and not culture conditions, induces Vero transformation correlated to karyotype and gene expression alterations. These data, together with previous investigations reporting tumour induction in high-passage Vero cells, suggest the use of low-passage Vero cells or cell lines other than Vero to increase the safety of vaccine manufacturing.
authors.affiliation: Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Via Bianchi 9, 25124, Brescia, Italy.
authors.forename: N A
authors.identifier: http://orcid.org/0000-0003-0330-9874
authors.initials: NA
authors.lastname: Andreani
authors.affiliation: Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Via Bianchi 9, 25124, Brescia, Italy.
authors.forename: S
authors.identifier: http://orcid.org/0000-0001-5863-2105
authors.initials: S
authors.lastname: Renzi
authors.affiliation: Department of Molecular and Translational Medicine, University of Brescia, V.le Europa 11, 25123, Brescia, Italy.
authors.forename: G
authors.identifier: http://orcid.org/0000-0003-0001-6435
authors.initials: G
authors.lastname: Piovani
authors.affiliation: Università Cattolica del Sacro Cuore di Piacenza, Piacenza, Italy.
authors.forename: P
authors.identifier: http://orcid.org/0000-0003-3165-4579
authors.initials: P
authors.lastname: Ajmone Marsan
authors.affiliation: Università Cattolica del Sacro Cuore di Piacenza, Piacenza, Italy.
authors.forename: L
authors.identifier: http://orcid.org/0000-0002-6290-8984
authors.initials: L
authors.lastname: Bomba
authors.affiliation: Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Via Bianchi 9, 25124, Brescia, Italy. riccardo.villa@izsler.it.
authors.forename: R
authors.identifier: http://orcid.org/0000-0001-8385-800X
authors.initials: R
authors.lastname: Villa
authors.affiliation: Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Via Bianchi 9, 25124, Brescia, Italy.
authors.forename: M
authors.identifier: http://orcid.org/0000-0001-7949-651X
authors.initials: M
authors.lastname: Ferrari
authors.affiliation: Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Via Bianchi 9, 25124, Brescia, Italy.
authors.forename: S
authors.identifier: http://orcid.org/0000-0002-2303-2027
authors.initials: S
authors.lastname: Dotti
keywords: In vitro test; In vivo test; RNAseq; Transformation; Tumorigenicity; Vero cells