Abstract
Contaminations of cell cultures with microbiological organisms are well documented and can be managed in cell culture laboratories applying reliable detection, elimination and prevention strategies. However, the presence of viral contaminations in cell cultures is still a matter of debate and cannot be determined with general detection methods. In the present study we screened 577 human cell lines for the presence of murine leukemia viruses (MLV). Nineteen cell lines were found to be contaminated with MLV, including 22RV1 which is contaminated with the xenotropic murine leukemia virus-related virus variant of MLV. Of these, 17 cell lines were shown to produce active retroviruses determined by product enhanced reverse transcriptase PCR assay for reverse transcriptase activity. The contaminated cell lines derive from various solid tumor types as well as from leukemia and lymphoma types. A contamination of primary human cells from healthy volunteers could not be substantiated. Sequence analyses of 17 MLV PCR products and five complete MLV genomes of different infected cell lines revealed at least three groups of related MLV genotypes. The viruses harvested from the supernatants of infected cell cultures were infectious to uninfected cell cultures. In the course of the study we found that contamination of human genomic DNA preparations with murine DNA can lead to false-positive results. Presumably, xenotransplantations of the human tumor cells into immune-deficient mice to determine the tumorigenicity of the cells are mainly responsible for the MLV contaminations. Furthermore, the use of murine feeder layer cells during the establishment of human cell lines and a cross-contamination with MLV from infected cultures might be sources of infection. A screening of cell cultures for MLV contamination is recommended given a contamination rate of 3.3%.
Highlights
Human and animal cell cultures are highly susceptible to a multitude of contaminations
Either the donor organism or the cells are screened for potential virus infections, such as Epstein-Barr virus (EBV), hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), human T-cell lymphotropic virus (HTLV) or other suspected human pathogenic viruses
A BLAST search comparing the primer sequences with sequences from the public databases showed for the outer primer for more than 220 entries and for the inner primer for more than 130 entries complete concordance with murine leukemia viruses, with XMLV and xenotropic murine leukemia virus related virus (XMRV) as well as with mouse chromosomal sequences attributed to endogenous MLV
Summary
Human and animal cell cultures are highly susceptible to a multitude of contaminations. Special attention should be paid to infections caused by mycoplasmas and mycobacteria among the bacterial contaminations. These organisms are growing very slowly and cannot be detected during routine cultivation of the cells [2]. Cell lines are commonly used for the production of viruses and for the investigation of virus infections, only sporadic reports address the possible problem of unintended viral contamination of cell cultures. One reason for this dearth of attention may be the generally assumed species- and tissue-specificity of the viruses. Animal cells are rarely screened for contaminating viruses because animal viruses are usually not pathogenic to man (with the exception of monkey or bat cell cultures that are known to be reservoirs for human pathogenic viruses)
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