Establishment of a Novel Fetal Ovine Heart Cell Line by Spontaneous Cell Fusion: Experimental Study

Abstract

Background The culture of immortal cell lines has become an indispensable tool in the field of modern biotechnology and has been used in the production of human and viral veterinary vaccines, therapeutic recombinant proteins, interferons, and monoclonal antibodies. Several approaches are used to immortalize cells in culture, such as transduction of cells with viral oncogenes, induced expression of telomerase reverse transcriptase, and spontaneous immortalization by serial passage of primary cell lines. Objective This study aimed to establish an immortal cell line by serial passage of fetal ovine heart cells that could be used to produce veterinary viral vaccines. Methods We serially passaged primary heart cells prepared from a fetal ovine heart till passage 140. We studied the events that led to the transformation and immortalization of the cell line under light and phase contrast microscopy. DNA samples of the cell line at passages 22 (before transformation) and 47 (after transformation) were genotyped according to single nucleotide polymorphisms (SNPs) using the OvineSNP50 BeadChip (Illumina). We sequenced the cytochrome b gene, control region, and tRNA-Phe and 12S rRNA genes of the mitochondrial genome of the cell line at passages 26 and 59 by Sanger sequencing. The susceptibility of the cell line to sheep pox, Peste des petits ruminants (PPR), lumpy skin disease (LSD), Rift Valley fever (RVF), and camel pox viruses was investigated. Results We established a unique immortal cell line called fetal ovine heart–Saudi Arabia (FOH-SA) by serial passage of fetal ovine heart cells. We demonstrated that the transformation or immortalization of the cell line resulted from spontaneous cellular and nuclear fusion of 2 morphologically distinct cardiocytes at passage 29. Fused cells at passage 29 gave rise to progeny cells, which grew into multicellular filaments that persisted at passages 30, 31, and 32. Trypsinization of the filamentous multicellular growth at passage 32 gave an epithelial-type immortal heart cell line. SNP genotyping revealed 65% and 96% homozygosity in SNP genotypes of the cell line at passages 22 and 47, respectively. Partial sequencing of the mitochondrial genome of the cell line revealed mutational events in the control region and the tRNA-Phe and 12S rRNA genes of the mitochondrial genome of passage 59 cells. The cell line was found to be permissive to sheep pox, PPR, LSD, RVF, and camel pox viruses. Conclusions We established an immortal cell line by serial passage of primary fetal ovine heart cells, which was permissive to many animal viruses. It could be used in animal virus isolation, vaccine production, and biotechnology. The study reported spontaneous cell fusion of cardiocytes as a method of cell immortalization. The findings of this study might help address the mystery of how the VERO cell line evolved.

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Identifiers

DOI

10.2196/53721

Journal

JMIRx Bio

Year

2024