Match criteria for human cell line authentication: where do we draw the line?

Continuous human cell lines have been used extensively as models for biomedical research. In working with these cell lines, researchers are often unaware of the risk of cross-contamination and other causes of misidentification. To reduce this risk, there is a pressing need to authenticate cell lines, comparing the sample handled in the laboratory to a previously tested sample. The American Type Culture Collection Standards Development Organization Workgroup ASN-0002 has developed a Standard for human cell line authentication, recommending short tandem repeat (STR) profiling for authentication of human cell lines. However, there are known limitations to the technique when applied to cultured samples, including possible genetic drift with passage. In our study, a dataset of 2,279 STR profiles from four cell banks was used to assess the effectiveness of the match criteria recommended within the Standard. Of these 2,279 STR profiles, 1,157 were grouped into sets of related cell lines-duplicate holdings, legitimately related samples or misidentified cell lines. Eight core STR loci plus amelogenin were used to unequivocally authenticate 98% of these related sets. Two simple match algorithms each clearly discriminated between related and unrelated samples, with separation between related samples at ≥80% match and unrelated samples at <50% match. A small degree of overlap was noted at 50-79% match, mostly from cell lines known to display variable STR profiles. These match criteria are recommended as a simple and effective way to interpret results from STR profiling of human cell lines.

Development and characteristics of a human cell assay for screening agents for melanoma prevention.

This paper describes the development and initial evaluation of a human cell assay to identify potentially efficacious agents for preventing melanoma. Four human cell lines were used: normal melanocytes, a radial growth-phase-like melanoma cell line (WM3211), a vertical growth-phase-like melanoma cell line (Lu1205), and 83-2c, a cell strain cloned from metastatic melanoma. Four endpoints were evaluated in ultraviolet B-treated cells: annexin V, human leukocyte antigen-DR; E-cadherin, and N-cadherin. Annexin V was induced by nimesulide, 4-hydroxyphenylretinamide, and difluoromethylornithine in ultraviolet-B-treated radial growth-phase-like melanoma cells. None of the agents inhibited human leukocyte antigen-DR expression in ultraviolet-B-treated radial growth-phase-like melanoma cells, the only cells that strongly expressed human leukocyte antigen-DR. E-cadherin was overexpressed only in radial growth-phase-like melanoma cells relative to melanocytes, and ultraviolet B exposure dramatically reduced this expression. E-cadherin was only induced by difluoromethylornithine in ultraviolet-B-treated radial growth-phase-like melanoma cells. N-cadherin was over- expressed in all melanoma cell lines relative to melanocytes. In this study, all candidate preventive agents inhibited N-cadherin in ultraviolet B-treated radial growth-phase-like melanoma cells. Four agents inhibited N-cadherin in ultraviolet B-treated vertical growth-phase-like melanoma cells. The mean ratios of N-cadherin to E-cadherin levels and specific endpoint responses for both the radial growth-phase-like melanoma and vertical growth-phase-like melanoma cells were used to rank the agents. Agents were evaluated at clinically relevant concentrations. The rankings were difluoromethylornithine>4-hydroxyphenylretinamide>nimesulide>9-cis-retinoic acid>polyphenon E. Diphenylhydramine, D-mannitol, and nordihydroguaiaretic acid were inactive. The results of these initial studies suggest that ultraviolet-B-treated radial growth-phase-like melanoma cells are the most responsive to chemopreventive agents, and may be the cell line of choice for screening melanoma prevention agents.

Best practices in cell culture: an overview.

This overview describes a series of articles to provide an unmet need for information on best practices in animal cell culture. The target audience primarily consists of entry-level scientists with minimal experience in cell culture. It also include scientists, journalists, and educators with some experience in cell culture, but in need of a refresher in best practices. The articles will be published in this journal over a six-month period and will emphasize best practices in: (1) media selection; (2) use and evaluation of animal serum as a component of cell culture medium; (3) receipt of new cells into the laboratory; (4) naming cell lines; (5) authenticating cell line identity; (6) detecting and mitigating risk of cell culture contamination; (7) cryopreservation and thawing of cells; and (8) storing and shipping viable cells.

Effect of dose-rate on induction of neoplastic transformation in vitro by low doses of 232 MeV protons.

PURPOSE: To determine the effect of dose-rate on induction of neoplastic transformation in vitro by low doses of 232 MeV protons. MATERIALS AND METHODS: The experimental system used was the human hybrid cell assay. The dose-rates examined were 50 cGy/min and 20 cGy/h. The dose-rate 20 cGy/h was chosen as this is in the range of the maximum dose-rate that can be experienced in an unshielded space environment following a solar flare. At low dose-rate (LDR), doses from 0.5-100 cGy were studied. At high dose rate (HDR), the dose range was 0.5-200 cGy. RESULTS: The data indicated no significant differences between the two dose-rates at doses up to 100 cGy. CONCLUSION: For the endpoint of neoplastic transformation in vitro, high dose-rate data may be sufficient to estimate low dose-rate effects (20 cGy/h) in the dose range up to 100 cGy from 232 MeV protons. The data are of relevance to risk estimation for space travel.

Short tandem repeat profiling: part of an overall strategy for reducing the frequency of cell misidentification.

The role of cell authentication in biomedical science has received considerable attention, especially within the past decade. This quality control attribute is now beginning to be given the emphasis it deserves by granting agencies and by scientific journals. Short tandem repeat (STR) profiling, one of a few DNA profiling technologies now available, is being proposed for routine identification (authentication) of human cell lines, stem cells, and tissues. The advantage of this technique over methods such as isoenzyme analysis, karyotyping, human leukocyte antigen typing, etc., is that STR profiling can establish identity to the individual level, provided that the appropriate number and types of loci are evaluated. To best employ this technology, a standardized protocol and a data-driven, quality-controlled, and publically searchable database will be necessary. This public STR database (currently under development) will enable investigators to rapidly authenticate human-based cultures to the individual from whom the cells were sourced. Use of similar approaches for non-human animal cells will require developing other suitable loci sets. While implementing STR analysis on a more routine basis should significantly reduce the frequency of cell misidentification, additional technologies may be needed as part of an overall authentication paradigm. For instance, isoenzyme analysis, PCR-based DNA amplification, and sequence-based barcoding methods enable rapid confirmation of a cell line's species of origin while screening against cross-contaminations, especially when the cells present are not recognized by the species-specific STR method. Karyotyping may also be needed as a supporting tool during establishment of an STR database. Finally, good cell culture practices must always remain a major component of any effort to reduce the frequency of cell misidentification.

Recommendation of short tandem repeat profiling for authenticating human cell lines, stem cells, and tissues.

Cell misidentification and cross-contamination have plagued biomedical research for as long as cells have been employed as research tools. Examples of misidentified cell lines continue to surface to this day. Efforts to eradicate the problem by raising awareness of the issue and by asking scientists voluntarily to take appropriate actions have not been successful. Unambiguous cell authentication is an essential step in the scientific process and should be an inherent consideration during peer review of papers submitted for publication or during review of grants submitted for funding. In order to facilitate proper identity testing, accurate, reliable, inexpensive, and standardized methods for authentication of cells and cell lines must be made available. To this end, an international team of scientists is, at this time, preparing a consensus standard on the authentication of human cells using short tandem repeat (STR) profiling. This standard, which will be submitted for review and approval as an American National Standard by the American National Standards Institute, will provide investigators guidance on the use of STR profiling for authenticating human cell lines. Such guidance will include methodological detail on the preparation of the DNA sample, the appropriate numbers and types of loci to be evaluated, and the interpretation and quality control of the results. Associated with the standard itself will be the establishment and maintenance of a public STR profile database under the auspices of the National Center for Biotechnology Information. The consensus standard is anticipated to be adopted by granting agencies and scientific journals as appropriate methodology for authenticating human cell lines, stem cells, and tissues.

Radiation-induced neoplastic transformation in vitro, hormesis and risk assessment.

Dose-response curves for various low-LET radiation sources have consistently been demonstrated to be J-shaped for the cancer-relevant endpoint of neoplastic transformation in vitro. Most of these studies have been performed where the radiation has been delivered at intermediate to high dose-rates (30-3000 mGy/min), where the threshold dose for induction of neoplastic transformation is around 100-200 mGy. Below these doses, the transformation frequency is less than that seen spontaneously, indicative of a hormetic effect. More recently, data have been obtained for low dose rates (<0.5 mGy/min) of low-LET radiation, and again hormetic effects are apparent but with threshold doses now being >1000 mGy. Similar trends have been reported in animal experiments as well as in human epidemiologic studies. Indeed, the relative risks for induction of neoplastic transformation in vitro in the dose range 1 to 1000 mGy agree well with those for incidence of radiation-induced breast cancer and leukemia in humans. These findings support the notion that the endpoint of neoplastic transformation in vitro is a plausible endpoint to not only study mechanisms involved in response to low doses of radiation, but also to provide information of potential importance to risk assessment.

Correlation of in vitro chemopreventive efficacy data from the human epidermal cell assay with animal efficacy data and clinical trial plasma levels.

The human epidermal cell (HEC) assay, which uses carcinogen exposed normal skin keratinocytes to screen for cancer prevention efficacy, was used to screen possible preventive agents. The endpoints measured were inhibition of carcinogen-induced growth and induction of involucrin, an early marker of differentiation. Sixteen of twenty agents (apigenin, apomine, budesonide, N-(2-carboxyphenyl)retinamide, ellagic acid, ibuprofen, indomethacin, melatonin, (-)-2-oxo-4-thiazolidine carboxylic acid, polyphenon E, resveratrol, beta-sitosterol, sulfasalazine, vitamin E acetate, and zileuton) were positive in at least one of the two assay endpoints. Four agents (4-methoxyphenol, naringenin, palmitoylcarnitine chloride, and silymarin) were negative in the assay. Nine of the sixteen agents were positive for both endpoints. Agents that showed the greatest response included: ellagic acid > budesonide, ibuprofen > apigenin, and quinicrine dihydrochloride. Fifty-eight of sixty-five agents that have been evaluated in the HEC assay have also been evaluated in one or more rodent bioassays for cancer prevention and several are in clinical trials for cancer prevention. The assay has an overall predictive accuracy of approximately 91.4% for efficacy in rodent cancer prevention irrespective of the species used, the tissue model, or the carcinogen used. Comparison of the efficacious concentrations in vitro to plasma levels in clinical trials show that concentrations that produced efficacy in the HEC assay were achieved in clinical studies for 31 of 33 agents for which plasma levels and/or C(max) levels were available. For two agents, 9-cis-retinoic acid (RA) and dehydroepiandrosterone (DHEA), the plasma levels greatly exceeded the highest concentration (HC) found to have efficacy in vitro. Thus, the HEC assay has an excellent predictive potential for animal efficacy and is responsive at clinically achievable concentrations.

The human epithelial cell cytotoxicity assay for determining tissue specific toxicity: method modifications.

The Human Epithelial Cell Cytotoxicity (HECC) Assay (Meth Cell Sci, 22: 17-24, 2000) has been modified to include three additional cell lines and to allow protocol adjustments for slow growing cell lines. This manuscript presents methods using human epithelial cells from ten different normal human tissues including: skin, mammary, prostate, renal, bronchial, lung, oral, ecto-cervix, colon, and liver. The HECC Assay can also be used to evaluate other types of drugs, personal care products, environmental chemicals, and potential toxicants. Human epithelial cells at an early passage are seeded into multi-well dishes. The cells are exposed to multiple concentrations of each test agent. A preliminary assay using an exposure of five days at 1 mM (if soluble) and four log dilutions is used to determine the highest concentration for the HECC Assay. In the HECC Assay, cultures are exposed for three to four days. Following the exposure period, endpoint measurements for inhibition of growth, mitochondrial function, and PCNA (proliferating cell nuclear antigen) expression or albumin synthesis (hepatocytes) are made. Data are analyzed to determine the concentration that inhibited an endpoint by 50 percent (TC(50)) for each agent in each target epithelial cell line or culture and the data are compared to determine the relative sensitivity of each epithelial cell line to the test agent.