Molecular detection and characterization of sustainable intracellular contaminants in commercially used cell cultures for pre-clinical studies

Abstract

Microbial contamination in cell and tissue culture is a constant problem, which can compromise development and applications of cell lines. An immediate consequence of cell culture contamination is loss of researcher time, money and effort spent developing cultures and setting up experiments. There are adverse effects detected on cultures suffering from undetected biological contamination. This hidden contamination can potentially achieve high densities altering the growth and characteristics of the cultures. The objective of this study was to assess the molecular detection and characterization of sustainable intracellular contaminants in commercially used cell cultures use for regulatory pre-clinical studies for developing new chemical entities. This study was prompted by a series of observations by multiple researchers that cell lines were harbouring visible black particulate contaminants, capable of intracellular mobility with secondary impacts on cell adherence and lyses. This was initially limited to human liver cells, HepG2, C3A and CaCO₂ cell lines, but recently similar evidence has been found in a series of other cell lines as well. Giemsa staining did show many spore-like entities in the cytoplasm mainly of 0.5-1 μ diameter, rounded and transparent in colour. Electron microscope examination of C3A infected cell line revealed the presence of numerous intracellular bacteria located in vacuoles or free in the host cytoplasm. In addition, the interaction of this bacterium with epithelial cells was associated with the elongation of micro-villar extension that extruded from the host cell membranes and engulfed the bacteria. This internalization mechanism strongly resembles Salmonella- or Shigella-induced macro-pinocytosis. The strain was characterized using the 16S RNA sequence that amplified the gene from many genera. The closest phylogenetic relative was |HQ877772.1| Escherichia sp. A94 with 88% 16S ribosomal RNA gene sequence similarity. It was proposed that unidentified strain be assigned as type strain of species of the bacteria origin based on the 16S rRNA gene sequence search in Ribosomal Database project, small subunit rRNA and large subunit rRNA database together with the phylogenetic tree analysis.

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Keywords: Intracellular contaminants, cell cultures, bacteria culture, pre-clinical studies