"Cell Line Authentication Methods". In ... - Wiley Online Library

Cell Line Authentication Methods Yvonne A Reid, American Type Culture Collection, Manassas, VA, USA A Scott Durkin, American Type Culture Collection, Manassas, VA, USA Kate Steenbergen, American Type Culture Collection, Manassas, VA, USA Robert J Hay, American Type Culture Collection, Manassas, VA, USA

Secondary article Article Contents . Introduction . Microbial Contaminant Detection . Isoenzymology . Cytogenetics . DNA Fingerprinting . Summary

In vitro cell culture systems are crucial research tools for discerning the complex mechanisms and pathways that regulate cell growth, differentiation and development. The validity of conclusions drawn from this research demands unequivocal verification of cell line identity.

Introduction Cell line authentication is a crucial, under-appreciated task facing the research scientist. Cell lines are used in diverse disciplines such as basic cell biology, genetic mapping, gene expression and gene therapy. The validity of conclusions drawn from this research demands the unequivocal verification of cell line identity. Kaplan and Hukku (1998) have found that 7% of cell lines examined are cross-contaminated by cells of the same or different species. Accurate cell line authentication requires a comprehensive strategy employing multiple complementary techniques that identify microbial contaminants as well as interspecific and intraspecific cell line contaminants. Intraspecific identification, e.g. differentiating one human liver cell line from another human liver cell line, has always been problematic. However, the advent of molecular biology has produced a reliable set of tools in this area. An overview of the currently available cell line authentication techniques will be presented.

Microbial Contaminant Detection Microbial contaminants of cell cultures include bacteria, yeasts, fungi, moulds and mycoplasmas. Bacteria, yeasts, moulds and fungi are usually obvious during routine microscopic examination of the cells, although slowgrowing organisms may be difficult to detect. Most common species can be detected by routine inoculation into a series of broths and agar (Hay, 1992b). Mycoplasma contamination cannot easily be detected and so continues to be widespread (at least 20% of cultures tested worldwide; Rottem and Barile, 1993; Hay, 1998). Mycoplasma contamination affects virtually every parameter of cell biology (McGarrity et al., 1985), thus compromising research if not rendering it invalid. Detection methods include direct cultivation (Hay, 1992b)

followed by staining with the DNA fluorescent stain Hoechst 33258. The stain binds with mycoplasma DNA, allowing for easy detection on the surface of the cell. Other indirect methods include DNA hybridization, ELISA (enzyme-linked immunosorbent assay), immunofluorescence, autoradiography and specific biochemical assays. More recently the PCR (polymerase chain reaction) technique has been used for detection (Harasawa et al., 1993; Hu and Buck, 1993). It has high sensitivity, is able to detect all the common species, allows for speciation of the contaminating organism and avoids the difficulties sometimes associated with interpretation of Hoechst results by providing clear, easy-to-read results in one day.

Isoenzymology Isozyme analysis determines the species of origin of a given cell line and detects cellular cross-contamination. This technique identifies enzymatically active proteins catalysing the same reactions and occurring in the same species but differing by their multiple molecular forms. These isoenzymes or isozymes are separated chromatographically or electrophoretically and the distribution patterns of the enzymes (zymograms) are characteristic of the species or tissue. O’Brien et al. (1980), and later Halton et al. (1983) have described useful isozyme markers for cell line verification. The technique examines the electrophoretic mobility of several different isozymes in a cell homogenate. Zymograms are visualized by staining with chromogenic substrates. The number of bands and their respective electrophoretic mobilities are species specific. When tested in combination glucose-6-phosphate dehydrogenase (G6PD), lactic dehydrogenase (LDH), and nucleoside phosphorylase (NP) can be used to verify species of origin. Additional enzymes such as adenosine deaminase (ADA), esterase D (ESD), peptidase D (PEP-D), phosphogluco-

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Cell Line Authentication Methods

mutases 1 and 3 (PGM1 and PGM3), 6-phosphogluconate dehydrogenase (PGD), acid phosphatase (ACP1), glyoxylase 1 (GLO1), malate dehydrogenase (ME2), a-fucosidase (FUCA) and adenylate kinase (AK1) are useful in detecting intraspecific cross-contamination in human cell lines (Hay, 1992a).

Cytogenetics Cytogenetics establishes interspecific and intraspecific identifications. This technique involves culturing the cells in the presence of the antimicrotubule drug colcemid to synchronize the cell division cycle, harvesting the cells in a hypotonic solution, then fixing and staining for microscopic examination. Kaplan and Hukku (1998) review in detail the utility of analysing Giemsa-stained spreads of metaphase chromosomes in differentiating normal cell lines from various species. They also review the value of identifying unique marker chromosomes in tumour cell lines to detect intraspecific cell line contamination.

DNA Fingerprinting Molecular biology provides techniques that allow investigators to discriminate between two samples that differ in their genetic code by a single base pair, a level of discrimination that readily lends it to intraspecific identification. These techniques take advantage of informative DNA regions that are dispersed throughout the genome. This polymorphic DNA is arranged in VNTR (variable numbers tandem repeat) DNA. The basic repetitive element of a VNTR is typically 10–20 bp. Elucidation of these regions is called DNA fingerprinting (Jeffreys et al., 1985). This is a process that takes several days. It entails extracting DNA from the cell line, cutting it into precise pieces with restriction endonucleases, size-fractionating the mixture using agarose gel electrophoresis, transferring the resulting restriction fragment pattern onto a solid support, then visualizing the polymorphic bands by probing with DNA labelled with a reporter molecule. Gilbert et al. (1990) used the highly informative multilocus probe 33.6 (Jeffreys et al., 1985) to develop baseline DNA fingerprints for human cell lines. This probe yielded a median composite DNA fingerprint frequency of 2.9  10 2 17. Although this probe was highly informative, scoring the resulting complicated banding patterns proved too difficult for routine screening. A strategy utilizing a combination of single locus hypervariable VNTR probes developed by Nakamura et al. (1987) yields an average DNA fingerprint frequency of 5.2  10 2 7 (Durkin and Reid, 1998). Although less informative than the multilocus probe, this strategy can be 2

employed as a routine screening tool that detects intraspecific contaminants at the 5% level. By incorporating the PCR technique, DNA fingerprints can be generated in as little as a single day. This process requires preparation of a crude cell extract, amplification of the target loci by PCR, size fractionation of the PCR products using polyacrylamide gel electrophoresis and subsequent visualization of the PCR products. The loci examined with PCR are of a different class of repetitive DNA known as short tandem repeats (STRs). The small size of an STR locus (the basic motif is 2–5 bp) makes it more amenable to PCR than the larger-sized VNTR loci. Edwards et al. (1992) demonstrated the utility of STRs for differentiating humans at the DNA level. Examination of eight STR loci can produce a DNA fingerprint frequency of approximately 1.0  10 2 9. This technique is generally used for intraspecific contaminant detection because of the specificity of the PCR primers; the level of routine crosscontaminant detection is 10%.

Summary Many laboratories employ rigorous interspecific and intraspecific cross-contamination monitoring in the cell line authentication process. This requires testing for microbial contaminants such as bacteria, yeasts, fungi, moulds and mycoplasmas by routine inoculation into a series of broths and agar. Cell line cross-contamination, both interspecific and intraspecific, is examined by generating isozyme profiles, preparing karyotypes and producing DNA fingerprints. The logical evolution of the authentication process will be the creation of a centralized authority. This authority should disseminate the standards of authentication agreed upon by the community. It should acquire, maintain and curate the data generated by collaborating laboratories as a community reference database against which a cell line can be authenticated.

References Durkin AS and Reid YA (1998) Short tandem repeat loci utilized in human cell line identification. ATCC Connection 18: 1–7. Edwards A, Hammond HA, Jin L, Caskey CT and Chakraborty R (1992) Genetic variation at five trimeric and tetrameric tandem repeat loci in four human population groups. Genomics 12: 241–253. Gilbert DA, Reid YA, Gail MH et al. (1990) Application of DNA fingerprints for cell-line individualization. American Journal of Human Genetics 47: 499–514. Halton DM, Peterson WD and Hukku B (1983) Cell culture quality control by rapid isoenzyme characterization. In Vitro 19: 16–24. Harasawa R, Uemori T and Asada K (1993) Sensitive detection of mycoplasmas in cell cultures by using two-step polymerase chain reaction. In: Kahane I and Adoni A (eds) Rapid Diagnosis of Mycoplasmas, pp. 227–232. New York: Plenum Press.

Cell Line Authentication Methods

Hay RJ (1992a) Cell line preservation and characterization. In: Freshney RI (ed.) Animal Cell Culture: A Practical Approach, pp. 95–148. Oxford: IRL. Hay RJ (1992b) Testing for microbial contaminants – bacteria and fungi. In: Hay RJ, Caputo J and Macy ML (eds) ATCC Quality Control Methods for Cell Lines, 2nd edn, pp. 19–33. Rockville, MD: American Type Culture Collection. Hay RJ (1998) Cell banking and authentication. In: Brown F, Griffiths E, Horaud JC and Petricciani JC (eds) Safety of Biological Products Prepared from Mammalian Cell Culture, Developments in Biological Standardization, 93, pp. 15–19. Basel: Karger. Hu M and Buck C (1993) Application of polymerase reaction technique for detection of mycoplasma contamination. Journal of Tissue Culture Methods 15: 155–160. Jeffreys AJ, Wilson V and Thein SL (1985) Hypervariable ‘minisatellite’ regions in human DNA. Nature 314: 67–73. Kaplan J and Hukku B (1998) Cell characterization and authentication. Methods in Cell Biology 57: 203–216. McGarrity GJ, Sarama J and Vanaman V (1985) Cell culture techniques. ASM News 51: 170–183. Nakamura Y, Leppert M, O’Connell P et al. (1987) Variable number of tandem repeat (VNTR) markers for human gene mapping. Science 235: 1616–1622.

O’Brien SJ, Shannon JE and Gail HE (1980) A molecular approach to the identification and individualization of human and animal cells in culture: isozyme and allozyme genetic signatures. In Vitro 16: 119–134. Rottem S and Barile MF (1993) Beware of mycoplasmas. Trends in Biotechnology 11: 143–150.

Further Reading Chen TR (1977) In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Experimental Cell Research 104: 255–262. McGarrity GJ (1976) Spread and control of mycoplasmal infection of cell cultures. In Vitro 12: 643–648. Lincoln CK and Gabridge MA (1998) Cell culture contamination: sources, consequences, prevention and elimination. In: Mather JP and Barnes (eds) Animal Cell Culture Methods, pp. 50–64. New York: Academic Press. Nelson-Rees WA, Flandermeyer RR and Hawthorne PK (1975) Distinctive banded marker chromosomes of human tumor cell lines. International Journal of Cancer 16: 74–82. Peterson WD, Simpson WF and Hukku B (1979) Cell culture characterization: monitoring for cell identification. Methods in Enzymology 58: 164–178.

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