Isoenzyme and molecular approach for authenticating and monitoring of animal cell lines.

Shirley B De Araújo,Maria Judite B Fernandes,Isabela C Simoni,Giancarlo F Patricio,Eliana B Rivas

doi:10.1590/0001-3765201920180487

Abstract

Authentication of cell lines is of paramount importance to validate the results from their use in biomedical research. Although isoenzyme polymorphism is the standard method, molecular methods based on mitochondrial DNA (mtDNA) have been developed to replace it. The aim of this study was the improvement of our isoenzyme electrophoretic analysis and the validation of one molecular technique targeted at mtDNA for the authentication of our animal cell lines. The combined method of cellular lysing through osmotic shock, followed by freezing-thawing in N2 to obtain isoenzyme extracts, and with 42 × 106 cells maintained the best efficiency. The superior electrophoretic conditions were PAGE run at 200 V. All cell lines had isoenzymatic mobility corresponding to their species to lactate dehydrogenase, malate-dehydrogenase, and glucose-6-phosphate dehydrogenase isoenzymes, and could be distinguished from each other. Two molecular techniques based on mtDNA were tested, one on the cytochrome b gene and other on cytochrome c oxidase I subunit gene. Due to difficulties in distinguishing all cell lines using only one these techniques, we merged the primers of two methods in such a way that there was a sufficient differentiation of all DNA fragments. The sequencing of these PCR products was also performed to validate these data.

Highlights

Several areas of basic and applied science demand cell lines, such as viral isolation and diagnosis; production of vaccines and biopharmaceuticals; toxicity testing of materials of synthetic or natural origin; and studies of complex physiological systems (Buttler 2005, Freshney 2005, CapesDavis et al 2010, Cree 2011)
Our initial isoenzyme studies were directed to the certification of cell lines using lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G-6-PHD) (Fernandes and Simoni 1995a)
It is noted that both isoenzymes do not distinguish several species of primates, and either isoenzyme distinguishes the human from chimpanzee cell lines (Halton et al 1983, Steube et al 1995)

Summary

Introduction

Several areas of basic and applied science demand cell lines, such as viral isolation and diagnosis; production of vaccines and biopharmaceuticals; toxicity testing of materials of synthetic or natural origin; and studies of complex physiological systems (Buttler 2005, Freshney 2005, CapesDavis et al 2010, Cree 2011). The certification or characterization of a cell line is based on a series of determinations, such as population growth, morphology, cytogenetics, absence of adventitious agents, viral susceptibility and, mainly, species identification (Hay 1992, Fernandes and Simoni 1995a, b, Freshney 2005, ATCC 2007). Our initial isoenzyme studies were directed to the certification of cell lines using lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G-6-PHD) (Fernandes and Simoni 1995a) These enzymes are sufficient for species identification, several authors recommend the use of three or more isoenzymes (Hay 1992, Nims et al 1998). We demonstrated a sensitivity of this technique for inter-species contamination monitoring of around 10%, observed by Nims et al (1998)

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Conclusion