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Cell cross-contamination in cell cultures: The silent and neglected danger

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Summary

Cell cross-contamination in cell cultures is a common problem during cell culturing and use. Contamination invalidates research results, compromises the comparison of results between laboratories, reduces reproducibility required in industrial production of cell lines, and may lead to unusable therapeutic products. The problem can be solved by increasing the awareness of its seriousness and by introducing regular quality control of cell cross-contamination in every laboratory where cells are grown and used.

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References

  • American Type Culture Collection catalog. Registry of animal cell lines certified by the cell culture collection committee. 1st ed. Rockville, MD: ATCC; 1964.

    Google Scholar 

  • American Type Culture Collection of cell lines and hybridomas. Hay, R.; Caputo, J.; Chen, T., et al., eds. Rockville, MD: ATCC; 1994.

    Google Scholar 

  • Appelbaum, F. Hematological disease. Acute leukemia. In: Wyngaarden, J.; Smith, L. H.; Bennett, J. C., eds. Cecil’s textbook of medicine. Philadelphia, PA: W. B. Saunders Co.; 1992:946–1947.

    Google Scholar 

  • Benedetti, A.; Pytel, B.; Baglioni, C. Loss of 2,5, oligoadenylate synthetase activity by production of antisense RNA results in lack of protection by interferon from viral infection. Proc. Natl. Acad. Sci. 84:658–662; 1987.

    Article  PubMed  Google Scholar 

  • Benedetti, A.; Pytel, B.; Baglioni, C. Retraction. Proc. Natl. Acad. Sci. 84:6740; 1987.

    Article  Google Scholar 

  • Brand, K. G.; Syverton, T. J. Immunology of cultivated mammalian cells. Species specificity determined by hemagglutination. J. Natl. Cancer Inst. 24:1007–1009; 1960.

    PubMed  CAS  Google Scholar 

  • Brunko, P.; Sauer, F. EEC legislation of medicinal products of biological origin. Dev. Biol. Stand. 75:21–25; 1991.

    PubMed  CAS  Google Scholar 

  • Chen, T. R. Reevaluation of HeLa, HeLa S3 and Hep-2 karyotypes. Cytogenet. Cell Genet. 48:19–24; 1988.

    PubMed  CAS  Google Scholar 

  • Christiansen, B.; Hansen, C.; Kieler, J., et al. Identity of non-malignant human uroepithelial cell lines classified as transformation grade I (TgrI) and II (TgrII). Anticancer Res. 13:2187–2192; 1993.

    Google Scholar 

  • Clonetics. Normal human cell systems catalog. San Diego, CA; 1995–1996.

  • Committee for proprietary medicinal products. Ad hock working party on biotechnology/pharmacy (CPMP), 1987. Guidelines on the production and quality control of medicinal products derived by recombinant DNA technology. TIB Tech. 5:G1–3.

  • Committee for proprietary medicinal products. Ad hock working party on biotechnology/pharmacy (CPMP), 1988. Guidelines in the production and quality control of monoclonal antibodies of murine origin intended for use in man. TIB Tech. G5–8.

  • Coons, A. H.; Kaplan, M. M. Localization of antigens in tissue cells. Improvement in methods for detection of antigen by means of fluorescent antibody. J. Exp. Med. 91:1–13; 1950.

    Article  PubMed  CAS  Google Scholar 

  • Dickinson, J. L.; Antalis, T. M. Response to letter to the editor. Leukemia 7:2079; 1993.

    Google Scholar 

  • Drexel, H. G.; Hane, B.; Hu, Z., et al. HeLa cross-contamination of a leukemia cell line. Leukemia 12:2077–2078; 1993.

    Google Scholar 

  • Embleton, M.; Gorochov, G.; Jones, P. T. In-cell PCR from mRNA: amplifying and linking the rearranged immunoglobulin heavy and light chain V-genes within single cells. Nucleic Acids Res. 20:3831–3837; 1992.

    Article  PubMed  CAS  Google Scholar 

  • Ferrone, S.; Pellegrino, M. A.; Reisfeld, R. A. A rapid method for direct HL-A typing of cultured lymphoid cells. J. Immunol. 107:613–615; 1971.

    PubMed  CAS  Google Scholar 

  • Fogh, J. Contamination in tissue culture. New York: New York Academy Press; 1973.

    Google Scholar 

  • Franks, D.; Daniel, M.; Gurner, B. V., et al. Variation in the forssman antigen in cell cultures. Exp. Cell Res. 36:310–324; 1963.

    Article  Google Scholar 

  • Freshney, R. I. Culture of animal cells. New York: John Wiley & Sons; 1994:243–252.

    Google Scholar 

  • Gartler, S. M. Genetic markers as tracers in cell culture. Natl. Cancer Inst. Monogr. 26:167–195; 1967.

    PubMed  CAS  Google Scholar 

  • Gartler, S. M. Apparent HeLa cell contamination of human heteroploid cell lines. Nature 217:750–751; 1968.

    Article  PubMed  CAS  Google Scholar 

  • Gignac, S. M.; Steube, K.; Schleithoff, L., et al. Multiparameter approach in the identification of cross-contaminated leukemia cell lines. Leuk. & Lymphoma 10:359–368; 1993.

    Article  CAS  Google Scholar 

  • Gilbert, D. A.; Reid, Y.; Gail, M., et al. Application of DNA fingerprints for cell line individualization. Am. J. Hum. Genet. 47:499–514; 1990.

    PubMed  CAS  Google Scholar 

  • Gold, M. A. Conspiracy of cells: one women legacy and the medical scandal it causes. Albany, NY: State University of NY Press; 1986.

    Google Scholar 

  • Grachev, V. P. Viral safety of biological products in WHO policy. Dev. Biol. Stand. 75:241–246; 1991.

    PubMed  CAS  Google Scholar 

  • Hampe, J.; Nurnberg, P.; Epplen, C., et al. Oligonucleotide fingerprinting as a means to identify and survey long term cultured B cell hybridomas and T cell lines. Hum. Antib. Hybrid. 3:186; 1992.

    CAS  Google Scholar 

  • Harris, H. Enzyme polymorphism in man. Proc. R. Soc. Lond. Ser. Biol. Sci. 164:298–301; 1966.

    CAS  Google Scholar 

  • Harris, N. L.; Gang, D. L.; Quay, S. C., et al. Contamination of Hodgkin’s disease cell cultures. Nature 289:228–230; 1981.

    Article  PubMed  CAS  Google Scholar 

  • Hay, R. J. Operator-induced contamination in cell culture systems. Dev. Biol. Stand. 75:193–204; 1991.

    PubMed  CAS  Google Scholar 

  • Hay, R. J. Methods for authenticating cell lines. Dev. Biol. Stand. 76:25–37; 1992.

    PubMed  CAS  Google Scholar 

  • Hay, R. J.; Caputo, J.; Macy, M. L. ATCC quality control methods for cell lines. Rockville, MD: ATCC; 1992:49–79.

    Google Scholar 

  • Hay, R. J.; Reid, Y. A.; McClintock, P. R., et al. Cell banks and their role in cancer research. J. Cell. Biochem. Suppl. 24:107–130; 1996.

    Article  PubMed  CAS  Google Scholar 

  • Honma, M.; Kataoka, E.; Ohnishi, K., et al. A new DNA profiling system for cell clone identification for use in cell banks in Japan. In Vitro Cell. Dev. Biol. 28A:24–28; 1992.

    Article  PubMed  CAS  Google Scholar 

  • Horaud, F. Absence of viral sequences in the WHO-Vero cell bank. A collaborative study. Dev. Biol. Stand. 76:43–46; 1992.

    PubMed  CAS  Google Scholar 

  • Hsu, T. C.; Benirschke, A. An atlas of mammalian chromosomes. New York: Springer-Verlag; 1967–1975.

    Google Scholar 

  • Hukku, B.; Halton, D.; Mally, M., et al. Cell characterization by use of multiple genetic markers. In: Acton, R. T.; Lynn, D. J., eds. Eukaryotic cell cultures. New York: Plenum Press; 1984:13–31.

    Google Scholar 

  • Innovative Chemistry, Inc., Marshfield, MA 02050.

  • International Workshop on Current Issues—continuous cell lines. Dev. Biol. Stand. 76:3–37; 1991.

    Google Scholar 

  • Jeffreys, A. J.; Wilson, V.; Thein, S. L. Hypervariable minisatellite regions of human DNA. Nature 314:67–73; 1985a.

    Article  PubMed  CAS  Google Scholar 

  • Jeffreys, A. J.; Wilson, V.; Thein, S. L. Invalid species “fingerprints” of human DNA. Nature 316:76–79; 1985b.

    Article  PubMed  CAS  Google Scholar 

  • Kawamura, A. Fluorescent antibody techniques and their application. Tokyo, Japan: Tokyo University Press; 1969.

    Google Scholar 

  • Kozak, R. W. Introduction to the issues: recently developed methods for characterizing cell lines. Dev. Biol. Stand. 76:21–24; 1992.

    PubMed  CAS  Google Scholar 

  • King, B.; Lichtenstein, A.; Berenson, J., et al. Polymerase chain reaction-based microsatellite typing assay used for tumor cell line identification. Am. J. Pathol. 144:486–491; 1994.

    PubMed  CAS  Google Scholar 

  • Lavappa, K. S. Survey of ATTC stocks of human cell lines for HeLa contamination. In Vitro 14:469–475; 1978.

    Article  PubMed  CAS  Google Scholar 

  • Leibovitz, A.; Wright, S.; Pathak, M., et al. Detection and analysis of G6PD phenotype B cell line contamination. J. Natl. Cancer Inst. 63:635; 1979.

    Google Scholar 

  • Lichter, P.; Chang Tang, C.; Call, K. High resolution mapping of human chromosomes by in situ hybridization with cosmid clones. Science 274:64–69; 1990.

    Article  Google Scholar 

  • Lukens, J. Classification and differentiation of acute leukemias. In: Lee, R.; Bithell, T.; Foerster, J., et al., eds. Wintrobe’s clinical hematology. Philadelphia, PA: Lea & Febiger; 1993: 1873–1887.

    Google Scholar 

  • Macville, M.; Zimonjic, D.; Popesku, N., et al. Spectral karyotyping of cervical carcinoma cell lines. J. Histochem. Cytochem. 44:782; 1996.

    Google Scholar 

  • Macy, M. L. Identification of cell line species by isoenzyme analysis. TCA Man. 4:833–3; 1978.

    Article  Google Scholar 

  • Magrath, D. I. Safety of vaccines produced in continuous cell lines. Dev. Biol. Stand. 75:17–20; 1991.

    PubMed  CAS  Google Scholar 

  • Mann, D.; O’Brien, S.; Gilbert, D., et al. Origin of the HIV-susceptible human CD4+ cell line H9. Aids Res. Hum. Retroviruses 5:253–255; 1989.

    Article  PubMed  CAS  Google Scholar 

  • Markovic, O. Kinetics of peroxidase activity in normal polymorphonuclear leukocytes. University in Belgrade, Yugoslovia; 1977a. Doctorate Thesis.

  • Markovic, O. Cell culture purity assessment based on classic cytochemical principles. J. Histochem. Cytochem. 44:789; 1996a.

    Google Scholar 

  • Markovic, O. New application of image analysis based cytochemistry and immunocytochemistry in cell cultures. In Vitro Cell. Dev. Biol. 32A:58; 1996b.

    Google Scholar 

  • Markovic, O.; Hay, R. J.; Steenbergen, K. Rapid screening and detection of minimal cell-cross contamination in cell cultures. J. Histochem. Cytochem. 44:75–76; 1996c.

    PubMed  CAS  Google Scholar 

  • Markovic, N.; Lipkin, L.; Markovic, O., et al. Device for quantitative cytochemistry: a computerized microdensitometer grain counter. J. Histochem. Cytochem. 26:792–802; 1978.

    PubMed  CAS  Google Scholar 

  • Markovic, O.; Markovic, N.; Rakic, L. J. Quantitative cytochemistry of enzymes. Belgrade, Yugoslavia: Univ Press; 1986a.

    Google Scholar 

  • Markovic, N.; Markovic, O. Esterases inside blasts and neutrophils indicate prognosis of acute leukemia. Clin. Res. 36:414; 1988.

    Google Scholar 

  • Markovic, O.; Markovic, N. Cytochemistry and immunocytochemistry in the classification of bone marrow and blood cells and in the diagnosis of hematological disorders. In: Ogawa, K.; Barka, T., eds. Electron microscopic cytochemistry and immunocytochemistry in biomedicine. Boca Raton, FL: CRC Press; 1992:611–638.

    Google Scholar 

  • Markovic, N.; Markovic, O.; Markovic, S. Image processing assisted measurement of intracellular effect of enzyme targeting drugs. Cell Vision 2:71–78; 1995.

    CAS  Google Scholar 

  • Markovic, N.; Markovic, O.; Roberts, J., et al. A new assay for intracellular measurement of inosine monophosphate dehydrogenase, a guide for better selection of patients for enzyme-targeted chemotherapy. J. Histochem. Cytochem. 40:895–902; 1992b.

    Google Scholar 

  • Markovic, O.; Shulman, N. R. Megakaryocyte maturation indicated by methanol inhibition of acid phosphatase shared by megakaryocytes and platelets. Blood 50:905–913; 1977b.

    PubMed  CAS  Google Scholar 

  • Markovic, O.; Young, D.; Markovic, N. Enzyme kinetics in single cells. Concept and model. Clin. Chem. 23:1472–1475; 1977c.

    CAS  Google Scholar 

  • Matsuba, I.; Lernmark, A.; Madsen, O., et al. Gene probes to detect cross-culture contamination in hormone producing cell lines. In Vitro Cell. Dev. Biol. 24:1970–1975; 1988.

    Article  Google Scholar 

  • McCulloch, E. A.; Parker, R. C. In: Begg, R. W., ed. Canadian cancer conference. Vol. 2. New York: Academic Press; 1957.

    Google Scholar 

  • McGarrity, G. J. Detection of mycoplasma infection in cell culture. Adv. Cell Cult. 2:99–131; 1982.

    Google Scholar 

  • Miele, M.; Bonatti Menickini, P. The fluorescence of amplified regions alters the stability of chromosomes in drug resistance Chinese hamster cells. Mutat. Res. 219:171–178; 1989.

    PubMed  CAS  Google Scholar 

  • Morita, T.; Shinohora, N.; Honma, M., et al. Establishment and characterization of a new cell line from a bladder cancer. Urol. Res. 23:143–149; 1995.

    Article  PubMed  CAS  Google Scholar 

  • Nelson-Rees, W. A. Inter and intraspecies contamination of human breast tumor cell line specificity. Original and natural history of cell lines. Prog. Clin. Biol. Res. 26:25; 1978.

    PubMed  CAS  Google Scholar 

  • Nelson-Rees, W. A. Double minutes in human carcinoma cell lines, with special reference to breast tumors. J. Natl. Cancer Inst. 63:537; 1979.

    PubMed  CAS  Google Scholar 

  • Nelson-Rees, W. A.; Daniels, D.; Flandermeyer, R. R. Cross contamination of cells in cell culture. Science 212:446–452; 1981.

    Article  PubMed  CAS  Google Scholar 

  • Nelson-Rees, W. A.; Flandermeyer, R. R. HeLa cultures defined. Science 191:96–97; 1976.

    Article  PubMed  CAS  Google Scholar 

  • Nelson-Rees, W. A.; Flandermeyer, R. R. Inter- and intraspecies contamination of human breast tumor cell lines HBC and Br Ca5 and other cell cultures. Science 195:1343–1344; 1977.

    Article  PubMed  CAS  Google Scholar 

  • Nelson-Rees, W. A.; Flandermeyer, R. R.; Daniels, D. T-1 cells are HeLa and not from normal human kidney origin. Science 209:719–720; 1980.

    Article  PubMed  CAS  Google Scholar 

  • Nelson-Rees, W. A.; Flandermeyer, R. R.; Hawthorne, P. K. Banded marker chromosomes as indicators of intraspecies cellular contamination. Science 184:1093–1096; 1974.

    Article  PubMed  CAS  Google Scholar 

  • Nicols, E. A.; Ruddle, F. H. Comparative sensitivity of electrophoretic assays for human enzymes. Biochem. Genet. 17:127–132; 1979.

    Article  Google Scholar 

  • Nuovo, G. Keys to successful in situ PCR. J. Histochem. Cytochem. 44:781; 1966.

    Google Scholar 

  • O’Brien, S. J.; Kleiner, G.; Olsen, R., et al. Enzyme polymorphism as genetic signatures in human cell cultures. Science 195:1345–1348; 1977.

    Article  PubMed  CAS  Google Scholar 

  • O’Brien, S. J.; Shannon, J. E.; Gail, M. H. Molecular approach to the identification and individualization of human and animal cells in culture: isozyme and allozyme genetic structure. In Vitro 16:119–135; 1980.

    PubMed  CAS  Google Scholar 

  • Office of Biologics Research Review (OBRR). Points to consider in the characterization of cell lines used to produce biologicals. Bethesda, MD: FDA; 1987.

    Google Scholar 

  • Peterson, W. D., Jr. Biochemical identification and standards. Natl. Cancer Inst. Monogr. 29:55; 1968.

    PubMed  Google Scholar 

  • Peterson, W. D., Jr.; Stulberg, C. S.; Swanborg, N. K., et al. G6PD isoenzymes in human cell cultures determined by sucrose agar gel and cellulose acetate zymograms. Proc. Soc. Exp. Biol. Med. 128:772–776; 1968.

    PubMed  CAS  Google Scholar 

  • Pearson, J. Changing issues of quality control: diploid and non-diploid cell lines. Dev. Biol. Stand. 76:13–17; 1992.

    PubMed  CAS  Google Scholar 

  • Petricciani, J. C. Regulatory philosophy and acceptability of cells for the modulation of biologicals. Dev. Biol. Stand. 75:9–15; 1991.

    PubMed  CAS  Google Scholar 

  • Petricciani, J. C. Cell line issue: historical and future prospective. Dev. Biol. Stand. 76:5–11; 1992.

    PubMed  CAS  Google Scholar 

  • Quinnan, G. J. Workshop introduction and overview. International Workshop on current issues—continuous cell lines. Dev. Biol. Stand. 76:3–4; 1992.

    Google Scholar 

  • Raap, T. Sensitive and high resolution fluorescent in situ hybridization. J. Histochem. Cytochem. 44:781; 1996.

    Google Scholar 

  • Reid, Y. A.; Gilbert, D. A.; O’Brien, S. J. The use of DNA hypervariable probes for human cells identification. ATCC Newsletter 10:1–3; 1990.

    Google Scholar 

  • Reid, Y. A.; O’Neill, K.; Chen, T. R., et al. Cell cross-contamination of U-937. J. Leukocyte Biol. 57:804; 1995.

    PubMed  CAS  Google Scholar 

  • Satoh, M.; Takeuchi, M. Cross-contamination of cell lines as revealed by DNA fingerprinting in the IFO animal cell bank. IFO Res. Commun. 16:18–23; 1993.

    Google Scholar 

  • Schmidt, B. F.; Chao, J.; Zhu, Z., et al. Signal amplification in the detection of single copy DNA and RNA by enzyme-catalyzed deposition (CARD) of the novel fluorescent reporter substrate, Cy 3.29-tyramide. J. Histochem. Cytochem. 44:781; 1996.

    Google Scholar 

  • Shimada, Y.; Imamura, M.; Wagata, T. Characterisation of 21 newly established esophageal cancer cell lines. Cancer 69:277–284; 1992.

    Article  PubMed  CAS  Google Scholar 

  • Siciliano, M.; Barker, P. E.; Cailleau, R. Mutually exclusive genetic signatures of human breast tumor cell lines with a common chromosomal marker. Cancer Res. 39:919–922; 1979.

    PubMed  CAS  Google Scholar 

  • Simpson, W. F.; Stulberg, C. S. Species identification of animal cell strains by immunofluorescence. Nature 189:616–617; 1963.

    Article  Google Scholar 

  • Stacey, G. N.; Bolton, B.; Doyle, A. The quality control of cell banks using DNA fingerprinting. Experientia (Suppl.) 58:361–370; 1991.

    CAS  Google Scholar 

  • Stacey, G. N.; Bolton, B.; Doyle, A. The DNA fingerprinting, a valuable new technique for the characterization of cell lines. Cytotechnology 9:211–216; 1992a.

    Article  PubMed  CAS  Google Scholar 

  • Stacey, G. N.; Bolton, B.; Morgan, D., et al. Multilocus DNA fingerprint analysis of cell banks: stability studies and culture identification in human B-lymphoblastoid and mammalian cell stocks. Cytotechnology 8:13–20; 1992b.

    Article  PubMed  CAS  Google Scholar 

  • Steube, K. G.; Grunicke, D.; Drexler, H. Isoenzyme analysis as a rapid method for the examination of the species identity of cell cultures. In Vitro Cell. Dev. Biol. 31:115–119; 1995.

    CAS  Google Scholar 

  • Stevenson, R. Development of cell banking in the US 1960–1985: a strategic approach to quality control. Adv. Cell Cult. 51:267–288; 1987.

    Google Scholar 

  • Stulberg, C. S. Extrinsic cell contamination of tissue cultures. In: Fogh, J., ed. Contamination in tissue culture. New York: Academic Press; 1973:1–27.

    Google Scholar 

  • Stulberg, C. S.; Peterson, W. D., Jr. Problems and experience in the characterization of tissue culture cells. Rev. Biol. 41:124–130; 1966.

    Article  CAS  Google Scholar 

  • Stulberg, C. S.; Peterson, W. D.; Simpson, W. F. Identification of cells in culture. Am. J. Hematol. 1:237–242; 1976.

    Article  PubMed  CAS  Google Scholar 

  • Stulberg, C. S.; Simpson, W. F.; Berman, L. Species related antigens of mammalian cell strains as detected by immunofluorescence. Proc. Soc. Exp. Biol. Med. 108:434–439; 1961a.

    PubMed  CAS  Google Scholar 

  • Stulberg, C. S.; Simpson, W. F.; Peterson, W. D., Jr., et al. Determination of species antigens of cultured cells by immunofluorescence. Fed. Proc. Fed. Am. Soc. Exp. Biol. 20:150; 1961b.

    Google Scholar 

  • Thacker, J.; Webb, M.; Debenham, P. Fingerprinting cell lines: use of human hypervariable DNA probes to characterize mammalian cell cultures. Somatic Cell Mol. Genet. 14:519–525; 1988.

    Article  PubMed  CAS  Google Scholar 

  • van Helden, P. D.; Wiid, I.; Albrecht, C. F., et al. Cross-contamination of human esophageal squamous carcinoma cell lines detected by DNA fingerprint analysis. Cancer Res. 48:5660–5662; 1988.

    PubMed  Google Scholar 

  • Vilien, M.; Christiansen, H.; Wolf, H., et al. Comparative studies of normal, “spontaneously transformed” and malignant human uroepithelial cells in vitro. Eur. J. Cancer & Clin. Oncol. 19:775–789; 1983.

    Article  CAS  Google Scholar 

  • Webb, M. B.; Debenham, P. G. Cell line characterization by DNA fingerprinting: a review. Dev. Biol. Stand. 76:39–42; 1992.

    PubMed  CAS  Google Scholar 

  • Westwood, L. C.; Macpherson, I.; Titmuss, D. H. J. Transplantation of normal cells in tissue culture, its significance relating to malignancy and in vitro vaccine production. Br. J. Exp. Pathol. 38:138; 1957.

    PubMed  CAS  Google Scholar 

  • World Health Organization Study Group (WHOSG). Cells, Products safety. Dev. Biol. Stand. 68:1–81; 1987.

    Google Scholar 

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Markovic, O., Markovic, N. Cell cross-contamination in cell cultures: The silent and neglected danger. In Vitro Cell.Dev.Biol.-Animal 34, 1–8 (1998). https://doi.org/10.1007/s11626-998-0040-y

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