Cell Species Identification: Safeguarding the Pure Lineage of Research Cells

Have you ever doubted the true reliability of the cells used in your experiments? Cell cross-contamination is a subtle yet common issue that can distort experimental data and even render results invalid. Cell species identification, as a powerful tool to identify cell origin, can effectively detect cross-contamination, ensuring the scientific integrity and reproducibility of experimental data. It is not only used for identifying species cross-contamination, but is also widely applied for confirming non-human or mouse-derived cells. In this article, we will delve into the applications and core technologies behind cell species identification to help you quickly master this scientific tool.

1. Applications of Cell Species Identification

1.1 Detecting Cross-Contamination between Different Species

According to the best practices recommended by the International Cell Line Authentication Committee (ICLAC), the identification of cell lines and tissue samples should involve four steps ^[1]:

•  Validate cell line identity using resources such as Cellosaurus and the ICLAC website before use.

•  Perform STR (Short Tandem Repeat) analysis.

•  Identify cross-contamination with non-human species cells in cultures.

•  Perform mycoplasma testing.

The third step specifically involves confirming whether cross-species cell contamination exists using species identification.

The "GB/T 40172-2021 General Guidelines for Detection Methods of Mammalian Cell Cross-Contamination" published in 2021 also defines inter-species cross-contamination and outlines detection methods, including cell morphology analysis, isoenzyme profiles, karyotype analysis, and others. Each method has its own sensitivity and advantages or limitations (see Table 1).

Table 1: Methods for Detecting Cell Cross-Contamination

1.2 Identifying Non-Human or Mouse-Derived Cells

STR (Short Tandem Repeat) analysis is the gold standard for identifying human and mouse-derived cells. For other species, such as canine cells and Vero cells, STR methods are still in the early stages of development and are not yet widely applied. These cells are typically identified through species identification technologies, followed by key phenotype tests to verify the accuracy of the results. This approach effectively fills the gap in non-human and mouse-derived cell identification.

2. Technical Analysis of Species Identification

2.1 CO1 Gene DNA Barcoding Technology

The Cytochrome C Oxidase Subunit 1 gene (CO1) located in mitochondria is highly conserved across species and has a moderate mutation rate, making it an ideal marker for species identification and genetic diversity analysis. For non-human cell identification, the preferred method recommended by ICLAC is CO1 gene-based DNA barcoding technology ^[2][3].

Figure 1. DNA Barcoding Results for B95-8 Cells

Challenges: DNA barcoding requires the design of specific primers for target DNA amplification, followed by comparison and analysis using a database. This process is relatively complex and thus is better suited for the identification of certain species.

2.2 Optimized PCR Method Based on CO1 Gene

Pricella® has developed a rapid and efficient CO1 gene PCR detection method for 10 commonly used species (including human, mouse, rat, Chinese hamster, monkey, rabbit, pig, cow, dog, and sheep). This method uses a mixture of specific primers for each species to amplify target DNA. Results are then analyzed using a genetic analyzer, providing clear and intuitive results.

For example, the species identification result for Vero cells is shown in Figure 3: the DNA amplification result is good, with the amplification peak for the internal reference gene (18S) normal. A specific peak appears at the monkey-specific primer location (2-AF), while no peaks appear for other species-specific primers, confirming that the Vero cells are derived from monkeys and there is no cross-contamination with other species.

Figure 2. Vero Cell Species Identification Results

Cell species identification is an indispensable tool in cell research, providing reliable evidence for identifying cross-contamination and effectively filling the gap in non-human and mouse-derived cell STR identification. With the increasing research on cross-species cell studies, rigorous and scientific species identification ensures the validity of research and promotes the standardized use of cell resources.

References

[1] Institutional Best Laboratory Practices for Cell Line and Tissue Sample Authentication to Ensure Valid, Reproducible and Robust Research. ICALC.

[2] Non-human Cell Lines Authentication. ICLAC.

[3] ANSI/ATCC ASN-0003-2015 (2015) Species-level identification of animal cells through mitochondrial cytochrome c oxidase subunit 1 (CO1) DNA barcodes. ANSI eStandards Store.