Short tandem repeat profiling provides an international reference standard for human cell lines

In a pilot study, DNA derived from 33 human cancer cell lines was compared by using two STR profile multiplex systems: Second Generation Multiplex (United Kingdom Forensic Science Service) (25) and Powerplex 1 (Promega) (26). The information on cell line identification and assortment obtained was identical in the two systems, with the exception of a single pair of samples that showed identical SGM profiles but an additional allele in one sample's Powerplex 1 profile (at the D13S317 locus). On the basis of the pilot study, the SGM STR multiplex was chosen for an extended study.

For the extended study, 20 samples of DNA (1 μg/ml) or cells were requested from each of the major cell banks in the U.S. (American Type Culture Collection, Manassas, VA; Coriell Cell Repositories, Camden, NJ), Europe (European Collection of Animal Cell Cultures, Salisbury, U.K.; Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) and Asia [The Institute of Physical and Chemical Research (Japan) RIKEN, Tsukuba, Japan; Japanese Collection of Research Bioresources, Tokyo] and from five cancer research institutes carrying large stocks of human cancer cell lines. The purpose of the project was explained to the 11 contributors, and each was asked to provide 20 DNA or cell samples of their own choosing. The analysis was blind and confidential for both the contributing centers and the analytical center (LGC, Teddington, U.K.). No data on the claimed origin or the STR profile of any cell line were released before the study was complete. A small number of unexpected findings were made, and requests were made for additional samples to be analyzed, in every case with an identical result.

DNA was prepared from the cells, where necessary, by using a commercial silica-gel-based purification kit (Qiagen, Crawley, U.K.). The SGM kit comprises the six STR loci: tyrosine hydroxylase, HUMTH01, 11p15.5; von Willebrand factor (vWF), HUMVWFA31/A, 12p-12pter; D8S1179, chromosome 8; D21S11, 21q11.2–21q21; α Fibrinogen (FGA), HUMFIBRA, 4q28 and D18S51, 18q21.3 plus the sex chromosome marker amelogenin, HUMAMGX/Y, Xp22.1–22.3 and Yp11.2. SGM amplifications contained 2 ng of target DNA in a 50-μl reaction volume containing 1× PARR buffer (Cambio, Cambridge, U.K.), 1.25 units AmpliTaq Gold DNA Polymerase (Perkin–Elmer) and 200 μM of each dNTP (Amersham Pharmacia). Samples were amplified by using the following conditions: 95°C for 18 min, 30 cycles of 95°C for 30 seconds, 58°C for 75 seconds, 72°C for 15 seconds, and 72°C for 25 min. Primer concentrations were 45 nM AMG 1/2, 87.5 nM TH01 1/2, 125 nM vWF 1/2, 560 nM D8S1179 1/2, 210 nM D21S11 1/2, 100 nM FGA 1/2, and 100 nM D18S51 1/2. Samples that failed to give measurable peaks at all loci were reanalyzed by using a different concentration of DNA. A small proportion of samples failed twice but were not processed further, as the aim of this study was to determine the utility of the method under routine conditions.

All of the STRs used in this study have a tetranucleotide repeat sequence, although intermediate sized alleles have been observed for TH01, FGA, and D21S11. Multiplex PCR reactions were carried out by using fluorescent dye-linked primers. Labeled products were detected by electrophoretic size fractionation on a Perkin–Elmer–ABI Prism 377 Genetic Analyser and analyzed by using genescan and genotyper analysis software (Perkin–Elmer). The end result for each cell line was an electropherogram, with each STR allele represented as one or more peaks of the appropriate color (see Fig. 1).

The data were further analyzed to categorize peaks according to their size in relation to an internal standard run (GS500, Perkin–Elmer) in every lane in the gel. This analysis enabled every peak to be allocated a size corresponding to the number of repeat units present (e.g., TH01 9 has 9 AATG repeats and results in a peak at 170.5 ± 0.5 bp in SGM analysis). An algorithm was developed to compare the allelic profiles, with each profile (questioned profile) being checked against every other profile (reference profiles) in the database. For each comparison, the number of alleles present in both reference and questioned profiles was scored and expressed as a percentage of the total number of alleles in the questioned profile. The principle can be seen operating in Table 1, where a consensus profile is taken as the reference profile, and 16 examples of HeLa cross-contaminants taken as questioned profiles.

The term cross-contamination is used in this study to indicate misidentification of one cell line by another, rather than contamination by a microbiological organism. Other tissue culture terminology follows the internationally agreed nomenclature (27), as used in the recently published United Kingdom Coordinating Committee on Cancer Research guidelines for the use of cell lines in cancer research (28).

DNA from 253 human cell lines, including 33 cell lines in a pilot study, was analyzed by STR profiling. Two further samples of Vero cells, which are of monkey origin, were sent by different centers and failed to amplify, as would be expected. One center was excluded, because the DNA was too dilute for routine analysis. Of the remaining 10 centers, 173/198 (87.4%) of samples gave complete results within two runs. Including the samples where partial data were obtained (up to two loci missing), the success rate increased to 188/198 (94.9%). Three centers sent cells rather than DNA, and the success rate for complete analysis was 46/59 (78.0%) and for partial analysis was 55/59 (93.2%).

STR profiling was evaluated as a simple method for cell line identification suitable for the establishment of reference genotypes. Two hundred and fifty-three cell lines from international cell banks and cancer research institutes worldwide were analyzed. It was demonstrated that this method can provide a universal reference standard for human cell lines. If this approach is applied internationally by using a common set of STR primers, cross-contamination of human cell lines will be readily detectable and such misrepresentation almost entirely eliminated, except as a result of deliberate fraud.

The methodology used deliberately did not set out to customize analysis for different sample or cell types, and so the results illustrate the success rate for obtaining complete profiles under routine circumstances at minimal cost. Samples with low DNA concentrations could have been processed further, as is standard for certain forensic samples, or another sample obtained. STR profiles should be readily obtainable for all human cell lines.

Where appropriate primers are available, DNA profiling can be applied to DNA samples from all species. In addition, human specific STR primers, such as those used in this study, could provide rapid confirmation that particular human chromosomes are present in somatic cell hybrids between human cells and cells of other species. Within an established cell line designation, analysis of additional STR loci can be used to characterize subline specific markers if required, as shown in the pilot study.

The STR profiles indicate that the HeLa sublines are not identical, and that there are both gains and losses of alleles, which may reflect the phenotypic differences observed in different laboratories. For the same strain of each cell line to be used worldwide, the international cell banks will need to agree to have a common stock.

Apart from simple cross-contamination, we checked whether viral transformation or long-term exposure to chemotherapeutic drugs would produce sublines that appeared to be distinct by STR profiling. However, the differences between the parental and derived cell lines were no greater than those between the HeLa sublines, and in all cases the sublines were identified as being closely related to the parental line.

Electropherograms provide information about the relative heights of peaks at heterozygous loci, which may reflect the number of copies of that allele present (Fig. 1). This information is lost in the simple numerical code. The code could be modified to indicate the relative peak height at each locus, for example by indicating the ratio of the two peaks as a bracketed suffix, e.g., TH01 7, 9 (1:1.8). Large differences in the height of two peaks at the same locus can be caused by a variety of reasons, including cell mixtures, differential amplification efficiency between heterozygous loci (for example because of a mutation in the primer binding site), or the presence of additional copies of one allele. Many of the samples in this study showed large differences in peak height at one or more loci. This finding is typical of cancer cells and reflects their relatively high genetic instability compared with normal cells.

For normal human DNA, STR profiling will show two alleles at most loci, as expected for highly polymorphic loci in a diploid genotype. Additional alleles at a locus are rarely seen in normal profiles but theoretically can occur by trisomy, gene duplication, or mixed populations of cells or hybrids. If most of the loci show more than two peaks, this is an indication of an hybrid or mixture of cells, whereas single loci with more than two peaks are more likely explained by trisomy or gene duplication events.

Because the loci were chosen on the basis of their high degree of heterozygosity in human populations, relatively few homozygous loci with single alleles would be expected. The significantly elevated levels of homozygosity observed in this study are consistent with the characteristic loss of heterozygosity, common in many cancer cells, particularly in the later stages of cancer progression from which almost all cancer cell lines are derived. At the TH01 locus, the incidence of homozygosity increased from about 20 to over 50%.

STRs are subject to mutation at a relatively high rate: in 10,844 parent/child allelic transfers at 9 loci, 23 mismatches were observed, 22 of which were because of a single-step mutation (29). It is generally accepted that replication slippage (30) is the major mechanism causing new mutations in microsatellites (29). STR loci have a higher frequency of polymorphism than single nucleotide polymorphisms and are the preferred option for forensic applications, but technological developments in the near future will provide rapid automated DNA profiling on arrays.

If DNA profiling of cell lines becomes accepted as normal practice, then editors of scientific journals could require authors to confirm that all cell lines used have been checked for authenticity during the period of the study. This analysis could become a prerequisite for publication, so that the problem of cell line cross-contamination can be reduced to a minimum in the future. This advance will be of great benefit to the scientific community, which can in future confidently develop or extend cell culture-based studies from other researchers without fear of false premises and without their own results being attributable to contamination.

This paper was submitted directly (Track II) to the PNAS office.

See commentary on page 7656.