JMIR Publications

ABSTRACT

Background:

Immortal cell lines became indispensible tool in the field of modern biotechnology and have been used in production of human and veterinary viral vaccines, therapeutic recombinant proteins, interferons, and monoclonal antibodies. Serial passaging of primary cell cultures is a common approach to spontaneously immortalize cell lines.

Objective:

The objective of the study was to establish an immortal cell line by serially passaging primary fetal ovine heart cells that could be used to produce veterinary vaccines instead of production in primary cell lines.

Methods:

We serially passaged primary heart cells prepared from a fetal ovine heart till passage 140. We studied the events that led to transformation and immortalization of the cell line under light and phase contract microscopy. The fetal ovine cell line was genotyped by nucleotide polymorphism (SNP) using the OvineSNP50 Beadchip. We sequenced two segments of the fetal ovine cell line mitochondrial genome using Sanger Sequencing. The fetal ovine cell line was authenticated by DNA barcoding. The sensitivity of the established fetal ovine heart cell line to animal viruses was tested.

Results:

We established a unique immortal cell line designated, FOH-SA, by serial passage of fetal ovine heart cells. In a novel phenomenon in cell biology, we demonstrated that the immortalization of the cell line supervened as a result of spontaneous cellular and nuclear fusion of two morphologically distinct cardiocytes at passage 29. Fused cells gave progeny cells which grew into multicellular filaments. Trypsinization of the filamentous multicellular growth gave the immortal heart cell line. Comparative SNP genotyping of the cell line before and after cell fusion revealed a large scale genetic conversion resulting in 96% homozygosity in SNPs genotypes in the progeny cells. Partial sequencing of the mitochondrial (mt) genome of the cell line revealed the occurrence of large mutational events in the control region, the tRNA-Phe and 12S rRNA genes of the mt genome of progeny cells. The cell line was found permissive to sheep pox, peste des petits ruminants (PPR), lumpy skin disease (LSD), rift valley fever (RVF), and camel pox viruses. This study would resolve the over half a century mystery of how the African Green Monkey cell line (VERO) had evolved. The cell line was authenticated at the European Collection of Authenticated Cell Cultures (ECACC) and we deposited a copy at the American Type Culture Collection (ATCC).

Conclusions:

We established an immortal cell line by serial passaging of primary fetal ovine heart cells and in a novel finding in cell biology, we demonstrated that the cell line evolved from spontaneous fusion of two distinct cardiocytes. The cell line was permissive to many animal viruses and it would be of importance in isolation of animal viruses, vaccine development, and other biotechnological fields.