Introduction

The history of the HeLa cell line is long and somewhat controversial (1). Despite its pivotal role as the first continuous human cancer cell line, the identity of the HeLa cell line has been accompanied by uncertainty. Although originally reported in 1952 as a cervical epithelial carcinoma from a female African-American individual, subsequent analysis of tumor pathology and clinical phenotype clearly identified the primary tumor as a rare adenocarcinoma (1). As the first human cell line to be widely cultured, HeLa has undergone strong adaptation to culture conditions. While karyotypically highly abnormal (4n=82) and carrying cytologically distinct marker chromosomes (2), it is phenotypically epithelial in appearance, grows rapidly (doubling time is ∼24 h), and shows a lack of contact inhibition (3). These characteristics and its ubiquity in cell culture laboratories have led to frequent cross-contamination. It has been estimated that 18% of cell lines submitted to repositories are contaminated with other cell lines, with HeLa alone being responsible for 25% of these cross-contamination events (2). In many cases, the originally deposited line has been completely replaced, resulting in the loss of potentially unique research resources in the very facilities designed to ensure their maintenance. There is also little doubt that cell line contamination in general—and HeLa contamination, in particular—has led to the publication of erroneous research data (4), with HeLa sublines masquerading variously as amnion cells (WISH), embryonic lung cells (L132) and liver cells (Chang liver) (5).

The advent of robust and fairly inexpensive short tandem repeat (STR)–based DNA (STR DNA) fingerprinting technologies (6) has enabled the development of international reference standards for cell line identification. These standards, in principle, should eliminate cell line cross-contamination as a source of error in research using established cell lines, and should allow validation of provenance for newly derived human cell lines. The major cell line repositories (Coriell, ATCC, ECACC, DSMZ, JCRB) routinely use STR DNA fingerprinting techniques to monitor the status of their cell line stocks. However, part of the problem with the use of contaminated cell lines is the requirement for the regular and routine assay of cell line identity. It has been suggested that mandatory monitoring of cell line identity, as a condition for funding and/or publication (7), could eliminate cell line contamination. Meanwhile, in the absence of such requirements, the expense of STR-based monitoring to research laboratories is clearly a barrier to self-regulation. Given that the HeLa cell line is disproportionately responsible for cross contamination events, a specific assay for this contamination could be included in cell culture practice, in much the same way as mycoplasma contamination can be routinely monitored by PCR (8). Here we present an assay that may remove some of these barriers.

L1 retrotransposons are active, autonomous mobile elements that are very common in the human genome [comprising 17% of the human genome sequence (9)]. Human-specific L1s (L1Hs) have been inserting into our DNA since the origin of the species and thus are readily employed as population (10) and individual specific (11) genetic markers with a number of useful characteristics. They are large (up to 7 kb) and stable insertions that undergo precise reversion only very rarely (12). During ongoing studies of human-specific, full-length, and potentially active L1 elements, we identified [using genome-wide transposon display methods (11)] a full-length L1 element that appeared to be HeLa-specific. Initial screening experiments showed that this insertion was present in a panel of widely geographically distributed HeLa isolates obtained from cell culture repositories in Europe and the United States, and absent from a large panel of human DNA samples. To fully realize the potential of this insertion as a HeLa-specific molecular diagnostic, we designed a single PCR-based genotyping assay that can be utilized in laboratories with access to standard molecular biology reagents and equipment. The assay is internally controlled, is sensitive to even low levels of HeLa contamination, and can be performed on unpurified cell pellets. We believe it will be of high utility in routine culture hygiene in cell culture laboratories and in tissue culture repositories.

Materials and methods

Tissue culture and DNA sources

The majority of HeLa and non-HeLa cell lines were obtained as gifts from collaborators (see Table 1). Where necessary, lines were revived from frozen storage and cultured at 37°C under 7% CO₂ in low glucose DMEM (Gibco/Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (Gibco/Invitrogen, Carlsbad, CA, USA) and 1× penicillin/streptomycin/glutamate (Gibco/Invitrogen).