Rapid screening and detection of cellular cross-contamination in cell cultures: a new application of cytochemistry.

Abstract

Cellular cross-contamination is a serious problem that can potentially affect all who use cell culture systems in research and production work. Reports from national testing services indicate that frequencies as high as 17-36% of the lines examined may be incorrectly identified by source laboratories, and intra/interspecies contamination was identified in 11-25% of cell cultures examined (1). National cell banks employ a variety of techniques to attempt cell line authentication, i.e., fluorescent antibody staining, isoenzymology, cytogenetics, and, more recently, DNA fingerprinting via Southern blotting and PCR methodologies (2). Each of these procedures has advantages and disadvantages, and none is capable on a routine basis of detecting very low levels, e.g., 0.5% or less, of cross-contamination of one cell line with another from the same species. In March of 1994, a histiocyte lymphoma cell line U-937 (AKC CRL 1593) was found to be cross-contaminated with a myeloid cell line K-562 (AKC CCL243). An extraordinary, cumbersome, and expensive effort, including PCR and cytogenetics studies, was required to solve this problem. Initial stocks, after deposit, contained as little as 0.4% of the offending K-562 line (3). After proliferation for 4 weeks and many passages, the &nding cell line emerged more apparently. The U-937 line was established by C. Sundstrom and K. Nilsson in 1976 and has been extensively used to study biology of human lymphoma cells and hematological malignancies in vitro (4). In search of a rapid, reliable, and inexpensive method to screen the U-937 cell line routinely for purity and to detect early andlor minor contamination with K-562, we applied a series of cytochemical and immunocytochemical methods. We found that esterase cytochemistry is very helpful in this case and will serve in quality control for purity of large production batches. Alpha-naphthyl acetate esterase and naphthol AS-D chloroacetate esterase were present in both cell types in different amounts, as assessed by image analysis. However, alpha-naphthyl butyrate esterase was positive in U-937 and negative in K-562 cell line cells. A clear distinction on slides can be made between green-bluish (counterstain, methylene blue) K-562 cells and brown-red (esterase activity deposit) U-937 cells (Figure 1). This technique allows detection of cross-contamination with K-562 cells below 0.1%. Morphological criteria, based on Wright-Giemsa modified staining, were used in addition. The entire procedure (preparation of cells and reagents, fixation, cytochemical visualization of enzyme activity, and counterstaining) can be performed within 2 hr (5.6). We recommend the alpha-naphthyl butyrate esterase cytochemical assay as a screening procedure for detecting K-562 cell cross-contamination in U-937 cell line. This test has been applied at ATCC to a stock of U-937 now available as CRL1593.2(4). Contaminant K-562 cells have not been detected in this population. A battery of cytochemical tests can be applied on many cell lines, and specific identification characteristics can bc identified. Once such identification cards are created, cytochemistry can be used as quality control in every cell culture laboratory. This type of assessment of cell cross-contamination will save researchers from possible disappointments and will increase their confidence in the purity of cell lines. However, this application has not yet been sufficiently exploited. We hope that our letter will motivate researches to explore further applications of cytochemistry in cell culture studies.