Species identification in cell culture: a two-pronged molecular approach

Cooper, Jason K.; Sykes, Greg; King, Steve; Cottrill, Karin; Ivanova, Natalia V.; Hanner, Robert; Ikonomi, Pranvera

doi:10.1007/s11626-007-9060-2

Cited by 174Identification and Quantification in FoodEnvironmental DNA in Biodiversity StudiesViral Infectious Diseases and Gene Expression in Insects

Species identification in cell culture: a two-pronged molecular approach

Cooper, Jason K., Sykes, Greg, King, Steve, Cottrill, Karin, Ivanova, Natalia V., Hanner, Robert, Ikonomi, Pranvera·2007·In Vitro Cellular & Developmental Biology - Animal

DOI 10.1007/s11626-007-9060-2

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Abstract

Species identification of cell lines and detection of cross-contamination are crucial for scientific research accuracy and reproducibility. Whereas short tandem repeat profiling offers a solution for a limited number of species, primarily human and mouse, the standard method for species identification of cell lines is enzyme polymorphism. Isoezymology, however, has its own drawbacks; it is cumbersome and the data interpretation is often difficult. Furthermore, the detection sensitivity for cross-contamination is low; it requires large amounts of the contaminant present and cross-contamination within closely related species may go undetected. In this paper, we describe a two-pronged molecular approach that addresses these issues by targeting the mitochondrial genome. First, we developed a multiplex PCR-based assay to rapidly identify the most common cell culture species and quickly detect cross-contaminations among these species. Second, for speciation and identification of a wider variety of cell lines, we amplified and sequenced a 648-bp region, often described as the "barcode region" by using a universal primer mix targeted at conserved sequences of the cytochrome C oxidase I gene (COI). This method was challenged with a panel of 67 cell lines from 45 diverse species. Implementation of these assays will accurately determine the species of cell lines and will reduce the problems of misidentification and cross-contamination that plague research efforts.

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HIBEC cells were originally from rat.
“confirmed that HIBEC cells were originally from rat and C201441 cells were from mouse”
From Investigation of Cross-Contamination and Misidentification of 278 Widely Used Tumor Cell Lines (2017) · cites this paper as [7]
C201441 cells were from mouse.
“confirmed that HIBEC cells were originally from rat and C201441 cells were from mouse”
From Investigation of Cross-Contamination and Misidentification of 278 Widely Used Tumor Cell Lines (2017) · cites this paper as [7]
Cooper and colleagues combined a multiplex PCR-based assay to identify common cell culture species and a protocol for sequencing a 648 bp region in the COI gene that accurately determines the species of cell lines.
“Cooper and colleagues implemented a two-pronged molecular approach in which they combined a multiplex PCR-based assay to identify the most common cell culture species and a protocol for sequencing of a 648 bp region located in the cytochrome C oxidase I (COI) gene, i.e., the “barcode region”, that accurately determines the species of cell lines [69].”
From Genetic Insights into the Economic Toll of Cell Line Misidentification: A Comprehensive Review (2026) · cites this paper as [69]

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“KFE-5 cells at passage 28 were blotted onto FTA cards, DNA extracted, CO1 sequence amplified using PCR primer cocktail and procedures developed for teleost fish (Cooper et al., 2007; Ivanova et al., 2007).”
Method
Authentication of human cell-based products: the role of a new consensus standard
Kerrigan, L., & Nims, R. W. (2011) · Regenerative Medicine · doi:10.2217/rme.11.5 · Cited by 23 · see citing works
Structural features and phylogenetic implications of Cicadellidae subfamily and two new mitogenomes leafhoppers
Chen, X.-X., Yuan, Z.-W., Li, C., Dietrich, C., & Song, Y.-H. (2021) · PLoS ONE · doi:10.1371/journal.pone.0251207 · Cited by 22
“For a long time, gene cox1 has been considered as a universal barcode for identifying animal species [74], but in these 56 Cicadellidae species, it is the slowest evolving and least changing gene among 13 PCGs.”
Background
Restoration of Endogenous Retrovirus Infectivity Impacts Mouse Cancer Models
Ottina, E., Lévy, P., Eksmond, U., Merkenschlager, J., Young, G. R., Roels, J., Stoye, J. P., Tüting, T., Calado, D. P., & Kassiotis, G. (2018) · Cancer Immunology Research · doi:10.1158/2326-6066.cir-18-0038 · Cited by 22 · see citing works
“Species identification was carried out by the Cell Services facility at the Francis Crick Institute, using previously established multiplex PCR assays (20, 21).”
Method
Establishment and characterization of a piscean PCF cell line for toxicity and gene expression studies as in vitro model
Goswami, M., Sharma, B. S., Yadav, K., Bahuguna, S. N., & Lakra, W. S. (2014) · Tissue and Cell · doi:10.1016/j.tice.2014.04.004 · Cited by 23 · see citing works
Complete mitochondrial genomes of Bittacus strigosus and Panorpa debilis and genomic comparisons of Mecoptera.
Li, N., Hu, G.-L., & Hua, B.-Z. (2019) · International Journal of Biological Macromolecules · doi:10.1016/j.ijbiomac.2019.08.152 · Cited by 20
Host-Selection Patterns of Culex tarsalis (Diptera: Culicidae) Determine the Spatial Heterogeneity of West Nile Virus Enzootic Activity in Northern California
Campbell, R., Thiemann, T. C., Lemenager, D., & Reisen, W. K. (2013) · Journal of Medical Entomology · doi:10.1603/me13089 · Cited by 20 · see citing works
Development of cell culture system from the giant freshwater prawn Macrobrachium rosenbergii (de Man)
Goswami, M., Lakra, W., Rajaswaminathan, T., & Rathore, G. (2010) · Molecular Biology Reports · doi:10.1007/s11033-009-9659-3 · Cited by 20
Development and characterization of a skin cell line (SGA) from the mosquitofish Gambusia affinis and its susceptibility to fish Betanodavirus
Jyotsna, Vijayakumar, P., Ravi, M., Sudhakaran, R., Mekata, T., & Rajaswaminathan, T. (2020) · doi:10.1016/j.aquaculture.2019.734778 · Cited by 19
Establishment and characteristics of Iranian Sistani cattle fibroblast bank: a way to genetic conservation
Gorji, Z. E., Khaledi, K., Amoli, A. D., Ganjibakhsh, M., Nasimian, A., Gohari, N. S., Izadpanah, M., Vakhshiteh, F., Farghadan, M., Moghanjoghi, S. M., et al. (2017) · Conservation Genetics Resources · doi:10.1007/s12686-016-0640-x · Cited by 19
Optimization of cell lines as tumour models by integrating multi-omics data
Zhao, N., Liu, Y., Wei, Y., Yan, Z., Zhang, Q., Wu, C., Chang, Z., & Xu, Y. (2016) · Briefings in Bioinformatics · doi:10.1093/bib/bbw082 · Cited by 20 · see citing works
The necessity of identity assessment of animal intestinal cell lines: A case report
Steube, K. G., Koelz, A.-L., Uphoff, C. C., Drexler, H. G., Kluess, J., & Steinberg, P. (2011) · Cytotechnology · doi:10.1007/s10616-011-9420-3 · Cited by 18 · see citing works
Cell-Surface Integrins and CAR Are Both Essential for Adenovirus Type 5 Transduction of Canine Cells of Lymphocytic Origin
Agarwal, P., Gammon, E. A., Sajib, A. M., Sandey, M., & Smith, B. F. (2017) · PLoS ONE · doi:10.1371/journal.pone.0169532 · Cited by 18
Three-dimensional decellularized amnion membrane scaffold promotes the efficiency of male germ cells generation from human induced pluripotent stem cells
Ganjibakhsh, M., Mehraein, F., Koruji, M., Aflatoonian, R., & Farzaneh, P. (2019) · Experimental Cell Research · doi:10.1016/j.yexcr.2019.111544 · Cited by 18 · see citing works
Genetic characterization of Callosciurus (Rodentia: Sciuridae) Asiatic squirrels introduced in Argentina
Gabrielli, M., Cardoso, Y., Benitez, V., Gozzi, A. C., Guichón, M., & Lizarralde, M. (2014) · doi:10.1080/11250003.2014.940006 · Cited by 16
Epidermal growth factor enhances the malignant phenotype in canine mammary carcinoma cell lines.
Kennedy, K., Qurollo, B., Rose, B., & Thamm, D. H. (2011) · Veterinary and Comparative Oncology · doi:10.1111/j.1476-5829.2010.00248.x · Cited by 16 · see citing works
Best practices for authenticating cell lines
Nims, R. W., & Reid, Y. (2017) · In Vitro Cellular & Developmental Biology - Animal · doi:10.1007/s11626-017-0212-8 · Cited by 21 · see citing works
“Technologies with the capability of detecting interspecies mixed cultures include karyotyping (determination of modal chromosome number) and multiplex DNA barcode assays designed to detect the most common species of cultured cells (e.g., Cooper et al. 2007).”
BackgroundMethod
Development and characterization of five novel cell lines from snubnose pompano, Trachinotus blochii (Lacepede, 1801), and their application in gene expression and virological studies
Ahmed, A. N., Majeed, S. A., Taju, G., Vimal, S., Suryakodi, S., Badhusha, A., kuthoos, A. N. A., Palsamy, R. K., Paknikar, K. M., Rajwade, J. M., et al. (2021) · Journal of Fish Diseases · doi:10.1111/jfd.13542 · Cited by 14 · see citing works
Temporal activation of LRH‐1 and RAR‐γ in human pluripotent stem cells induces a functional naïve‐like state
Taei, A., Kiani, T., Taghizadeh, Z., Moradi, S., Samadian, A., Mollamohammadi, S., Sharifi-Zarchi, A., Guenther, S., Akhlaghpour, A., Abibeiglou, B. A., et al. (2020) · EMBO Reports · doi:10.15252/embr.201847533 · Cited by 15 · see citing works
“The contribution of 2a2iL-hPSCs in chimeric embryos was analyzed via immunohistochemistry and PCR for human-specific mitochondrial sequences (Table 1) (Cooper et al , 2007).”
An alternative method to isoenzyme profile for cell line identification and interspecies cross-contaminations: cytochrome b PCR-RLFP analysis
Losi, C. G., Ferrari, S., Sossi, E., Villa, R., & Ferrari, M. (2008) · In Vitro Cellular & Developmental Biology - Animal · doi:10.1007/s11626-008-9125-x · Cited by 13 · see citing works
Authentication of cell lines: ignore at your peril!
Freshney, R. I. (2008) · Expert Review of Anticancer Therapy · doi:10.1586/14737140.8.3.311 · Cited by 13 · see citing works
Anti‐angiogenic efficacy of aflibercept and bevacizumab in primary oral squamous cell carcinoma cells
Ganjibakhsh, M., Monshizadeh, R., Nasimian, A., Aminishakib, P., Farzaneh, P., Shiraji, S. T., Garajei, A., Rahrotaban, S., Baghaei, F., & Gohari, N. S. (2018) · Journal of Oral Pathology and Medicine · doi:10.1111/jop.12717 · Cited by 16 · see citing works
Development and characterization of a new gill cell line from the striped catfish, Pangasianodon hypophthalmus (Sauvage, 1878)
Sathiyanarayanan, A., Goswami, M., Nagpure, N. S., Gireesh Babu, P., & Das, D. K. (2022) · Fish Physiology and Biochemistry · doi:10.1007/s10695-022-01053-9 · Cited by 15 · see citing works
Establishment and characterization of a testicular Sertoli cell line from olive flounder Paralichthys olivaceus
Peng, L., Zheng, Y., You, F., Wu, Z.-H., Zou, Y.-X., & Zhang, P.-J. (2016) · Chinese Journal of Oceanology and Limnology · doi:10.1007/s00343-016-5091-4 · Cited by 13
“In addition, mitochondrial DNA, particularly COI gene sequence alignment, has been frequently used as a reliable molecular method to accurately identify the origin of established cell lines (Cooper et al., 2007; Swaminathan et al., 2012).”
Method
Host Selection Patterns ofCulex tarsalis(Diptera: Culicidae) at Wetlands Near the Salton Sea, Coachella Valley, California, 1998–2002
Reisen, W. K., Lothrop, H. D., & Thiemann, T. C. (2013) · Journal of Medical Entomology · doi:10.1603/me13078 · Cited by 11 · see citing works
A SRCF cell line from snowtrout, Schizothorax richardsonii: Development and characterization
Goswami, M., Sharma, B. S., Bahuguna, S. N., Nagpure, N. S., & Lakra, W. S. (2013) · Tissue and Cell · doi:10.1016/j.tice.2013.02.002 · Cited by 14 · see citing works
Comment to: “Spontaneous transformation of adult mesenchymal stem cells from cynomolgus macaques in vitro” by Z. Ren et al. Exp. Cell Res. 317 (2011) 2950-2957
Torsvik, A., Røsland, G. V., & Bjerkvig, R. (2011) · Experimental Cell Research · doi:10.1016/j.yexcr.2011.12.009 · Cited by 12 · see citing works
“As an alternative, species-specific primers [21] can be used to identify interspecies contamination.”
Background
Establishment and characterization of Caspian horse fibroblast cell bank in Iran
Amoli, A. D., Mohebali, N., Farzaneh, P., Fazeli, S. A. S., Nikfarjam, L., Movasagh, S. A., Moradmand, Z., Ganjibakhsh, M., Nasimian, A., Izadpanah, M., et al. (2016) · In vitro Cellular & Developmental Biology-Animal · doi:10.1007/s11626-016-0120-3 · Cited by 12
Development and characterization of a cell line WAF from freshwater shark Wallago attu
Dubey, A., Goswami, M., Yadav, K., & Sharma, B. S. (2013) · Molecular Biology Reports · doi:10.1007/s11033-013-2936-1 · Cited by 13 · see citing works
“[38] used COI region for identification of 67 cell lines used for barcode analysis.”
Method
Establishment and characterization of a new fibroblast-like cell line from the skin of a vertebrate model, zebrafish (Danio rerio)
Sathiyanarayanan, A., Yashwanth, B. S., Pinto, N., Thakuria, D., Chaudhari, A., Babu, P. G., & Goswami, M. (2022) · Molecular Biology Reports · doi:10.1007/s11033-022-08009-5 · Cited by 12 · see citing works
Mosquito blood-feeding patterns and nesting behavior of American crows, an amplifying host of West Nile virus
Wheeler, S. S., Taff, C. C., Reisen, W. K., & Townsend, A. K. (2021) · Parasites & Vectors · doi:10.1186/s13071-021-04827-x · Cited by 12 · see citing works
Development and characterization of two cell lines PDF and PDH from Puntius denisonii (Day 1865)
Lakra, W., Goswami, M., Yadav, K., Gopalakrishnan, A., Patiyal, R., & Singh, M. (2011) · In vitro Cellular & Developmental Biology-Animal · doi:10.1007/s11626-010-9374-3 · Cited by 11
Evidence for a Previously Unrecognized Mycobacterial Endosymbiont in Acanthamoeba castellanii Strain Ma (ATCC®50370™)
Glaser, K. C., Hetrick, N. D., & Molestina, R. (2011) · Journal of Eukaryotic Microbiology · doi:10.1111/j.1550-7408.2010.00513.x · Cited by 11
“Procedures for DNA sequencing were followed as described previously (Cooper et al. 2007).”
Method
DNA Barcoding of Black Flies (Diptera: Simuliidae) in Slovakia and Its Utility for Species Identification
Kúdelová, T., Krčmárik, S., Lužáková, I., Bujačková, B., Matická, K., & Kúdela, M. (2023) · Diversity · doi:10.3390/d15050661 · Cited by 10
Assessing off-target cytotoxicity of the field lampricide 3-trifluoromethyl-4-nitrophenol using novel lake sturgeon cell lines
Vo, N. T. K., Moore, L. C., Spiteri, K. W., Hanner, R., Wilkie, M. P., & DeWitte‐Orr, S. J. (2018) · Ecotoxicology and Environmental Safety · doi:10.1016/j.ecoenv.2018.07.017 · Cited by 10 · see citing works
Self-citation
Linking large‐scale genetic structure of three Argynnini butterfly species to geography and environment
Polic, D., Yıldırım, Y., Lee, K., Franzén, M., Mutanen, M., Vilà, R., & Forsman, A. (2022) · Molecular Ecology · doi:10.1111/mec.16594 · Cited by 9
“2.7 | Sequencing of the mitochondrial COI gene The barcode region of mitochondrial cytochrome c oxidase 1 (COI) was amplified using the primers LepF1 and LepR1 (Cooper et al., 2007), and the PCR (polymerase chain reaction) amplification conditions as stated in Dincă et al. (2019).”
MethodInfluential
Establishment and characterization of novel cell lines derived from six lepidopteran insects collected in the field
Watanabe, K., Kobayashi, I., Hatakeyama, M., Kayukawa, T., & Akiduki, G. (2020) · In vitro Cellular & Developmental Biology-Animal · doi:10.1007/s11626-020-00438-5 · Cited by 9
Use of PCR-based assays for the detection of the adventitious agent porcine circovirus type 1 (PCV1) in vaccines, and for confirming the identity of cell substrates and viruses used in vaccine production
Kumar, D., Beach, N. M., Meng, X., & Hegde, N. R. (2011) · Journal of Virological Methods · doi:10.1016/j.jviromet.2011.10.017 · Cited by 12 · see citing works
CELLULAR, MOLECULAR, GENOMICS, AND BIOMEDICAL APPROACHES | Culture of Fish Cell Lines
Bols, N. C., Kawano, A., & Lee, L. E. J. (2011) · Elsevier eBooks · doi:10.1016/b978-0-12-374553-8.00253-7 · Cited by 7 · see citing works
The history, landscape, and outlook of human cell line authentication and security
Harbut, E., Makris, Y., Pertsemlidis, A., & Bleris, L. (2024) · SLAS Discovery · doi:10.1016/j.slasd.2024.100194 · Cited by 8 · see citing works
Comparative Mitogenomics of Flesh Flies: Implications for Phylogeny
Shang, J.-X., Xu, W.-T., Huang, X., Zhang, D., Yan, L.-P., & Pape, T. (2022) · Insects · doi:10.3390/insects13080718 · Cited by 8

Authors: Cooper, J. K., Sykes, G., King, S., Cottrill, K., Ivanova, N. V., Hanner, R., & Ikonomi, P.
Affiliations: American Type Culture Collection (US)
University of Guelph (CA)
Corresponding author: Ikonomi, Pranvera
ORCID: Hanner, Robert
Venue: In Vitro Cellular & Developmental Biology - Animal
Publisher: Springer Science and Business Media LLC
Published: 2007-10-13
Volume: 43
Issue: 10
Pages: 344-351
ISSN: 1071-2690 (ISSN-L)
1543-706X
DOI: 10.1007/s11626-007-9060-2
OpenAlex: W1907982143
Cited by: 174
References: 0
Citations by year: 2026: 7 · 2025: 7 · 2024: 7 · 2023: 11 · 2022: 10 · 2021: 10 · 2020: 8 · 2019: 10 · 2018: 10 · 2017: 13 · 2016: 8 · 2015: 7 · 2014: 11 · 2013: 14 · 2012: 4
FWCI: 2.64
Type: journal article
Language: en
Open access: closed
Licence: www.springer.com/tdm (tdm)
Locations: In Vitro Cellular & Developmental Biology - Animal (publishedVersion)
PubMed (publishedVersion)
BioOne Complete (BioOne) (submittedVersion)
Funders: Genome Canada
Natural Sciences and Engineering Research Council of Canada
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Identification and Quantification in Food
Physical Sciences › Environmental Science › Ecology › Environmental DNA in Biodiversity Studies
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
Keywords: cross-contamination detection, mitochondrial genome, cytochrome c oxidase I, short tandem repeat profiling, multiplex PCR, cell culture contamination, research reproducibility
MeSH terms: Animals, Electron Transport Complex IV, Humans, Phylogeny, Species Specificity, Cell Culture Techniques, K562 Cells, Protein Subunits

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Species identification in cell culture: a two-pronged molecular approach

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