Cell lines are among the most used in vitro models representing normal and disease tissue in basic research and pharmaceutical research and development. The implementation of short tandem repeat (STR) analysis, cytochrome 1 oxidase (CO1) barcoding, and other measures as part of routine authentication procedures has led to the discovery that some cell lines have been misidentified.
STR analysis and CO1 barcoding is critical to verify the identity of human cell lines. These assays are routinely performed at the time of accessioning a new cell line and at the replenishment of each distribution stock; the timing of inter- and intra-species authentication is critical to avoid distributing misidentified cell lines to the scientific community. Additionally, the results of the authentication tests of each replenished stock are verified by comparing to the baseline profile of the token stock derived from the depositor. Below is a list of cell lines found to be misidentified, along with the appropriate action taken by ATCC to correct the issue of misidentification.
Inappropriate Y – DNA profiling at ATCC for amelogenin, a sex-chromosome-specific PCR assay that can distinguish X chromosome-specific products from Y chromosome-specific products revealed the presence of Y chromosomes in six human cell lines of putative female origin. Confirmation of the general findings was accomplished by QM staining, C-banding, and FISH, with a whole chromosome paint probe to the human Y chromosome.
The cell lines include:
QM, C, FISH
QM, C, FISH
QM, C, FISH
QM, C, FISH
ATCC discontinued distribution of these lines following an investigation which uncovered no compelling reasons not to do so.
HeLa Derivatives – ATCC strongly advises against the use of these cell lines as models for original source material. Published data from researchers and testing performed during ATCC accessioning have established that these cell lines are contaminated with HeLa cells. These findings are based on isoenzyme analysis, HeLa marker chromosomes and DNA fingerprinting. HeLa contaminated cell lines should not be chosen for study when the specific organ or tissue of presumptive origin is of importance to the validity of the research as results can be compromised.
These cell lines include:
Identities in Question:
MDA-MB-435S (ATCC HTB-129) – ATCC advises against the use of this cell line as a model for original source material. Recent studies have generated questions about the origin of the parental cell line, MDA-MB-435, and by extension MDA-MB-435S and its derivatives listed below. Gene expression analysis of the cells produced microarrays in which MDA-MB-435 clustered with cell lines of melanoma origin instead of breast (PubMed IDs: 10700174, 1510101, 15679052).
Additional studies have since corroborated a melanocyte origin of MDA-MB-435, to which ATCC has responded by pursuing its own investigation into the identity of this cell line. The cell line to which MDA-MB-435 is reported to have been cross-contaminated with is the M14 melanoma line (PubMed IDs: 12354931, 17004106).
Derivatives of MDA-MB-435S with identifies in question are:
M4A4 LM3-2 GFP
M4Af LM3-4 CL16 GFP
OLGA-PH-J/92 [OL-J/92] (ATCC CRL-2576) – This cell line was originally deposited as a crayfish cerebral ganglion cell line. However, cytochrome c oxidase subunit I (COI) testing at ATCC cannot confirm the crayfish origin. Based on this analysis, the distribution of ATCC CRL-2576 has been discontinued.
EPC (ATCC CRL-2872) – Cytochrome c oxidase subunit I (COI) testing at ATCC revealed that EPC, originally deposited as a Carp cell line (Cyprinus carpio), was in fact a Fathead Minnow cell line (Pimephales promelas). Since the time of deposit, isoenzymology testing has correctly and consistently identified EPC (ATCC CRL-2872) as a fish cell line. However, isoenzymology does not allow for speciation between genus, and information regarding the species of fish was previously only provided by the depositor. These observations were confirmed via COI testing of the original stock available to ATCC.
MPanc-96 (ATCC CRL-2380) – STR analysis at ATCC revealed that MPanc-96, a human pancreatic cell line, has an STR pattern identical to that of another human pancreatic cell line, AsPC-1 (ATCC CRL-1682). These observations were confirmed with the original stock available to ATCC. The distribution of ATCC CRL-2380 has been discontinued.
SNB-19 (ATCC CRL-2219) and U-373 MG (ATCC HTB-17) – STR analysis at ATCC revealed that SNB-19, a human glioblastoma cell line has a STR pattern identical to that for U-373 MG (ATCC HTB-17). SNB-19 and U-373 MG also share derivative chromosomes. These observations were confirmed with the original stock available to ATCC. Since then distribution of SNB-19 was discontinued.
U-373 MG (ATCC HTB-17) – As a result of sequencing, the authenticity of ATCC HTB-17 has been questioned by R.F. Petersson in Stockholm and collaborator E.G. Van Meir in Atlanta (personal communication and see Ishii, N., et al. Brain Pathol 9: 469-79, 1999). They report similarities between U-373 MG (ATCC HTB-17) and another glioblastoma, U-251. The cell line U-373 MG, obtained from the original lab in Uppsala has differing genetic properties from the ATCC HTB-17 (U-373 MG). Following further investigations, ATCC stopped distribution of this cell line.
U-118 MG (ATCC HTB-15) AND U-138 MG (ATCC HTB-16) – Two human glioblastoma cell lines reportedly from different individuals have identical variable number of tandem repeats (VNTR) and similar STR patterns and karyotype. U-118 MG and U-138 MG are very similar cytogenetically and share at least six derivative marker chromosomes. ATCC will continue distribution with an alert that the two lines have similar origin.
ECV-304, a presumptive endothelial cell line (ATCC CRL-1998) and human bladder cell line T24 (ATCC HTB-4) – STR analysis at ATCC suggested that ECV-304 was a derivative of T24. In addition, karyotype analysis of the two cell lines showed that they shared-marker chromosomes. Combined, these results confirm that ECV-304 is a derivative of T24, a line that was developed years earlier.
It is important to emphasize that all stocks of ECV show similar properties, not just those from ATCC. It is clear that the contamination took place before the cell banks independently received initial stocks. ATCC has written to each recipient of ATCC CRL-1998 (ECV-304) to inform them of these findings.
HT-2 clone A5E (ATCC CRL-1841) – This HT-2 clone A5E cell line was originally deposited as derived from the BALB/c strain of mice. Recent testing at ATCC and by the NIST Mouse STR Consortium using mouse STR technology has shown that the STR profile has more alleles in common with a C57BL mouse strain rather than BALB/c. Based on this analysis, ATCC CRL-1841 has been put on the misidentified cell lines list.
If additional misidentified cell lines are discovered ATCC scientists will report these general findings on this page after the depositors and other cell resource banks have been informed.
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Cell Line Authentication Test Recommendations
Authentication of a cell line is the sum of the process by which a line’s identity is verified and shown to be free of contamination from other cell lines and microbes. Download this technical bulletin to learn how to achieve reliable and reproducible experimental data.Download the technical bulletin