Guidelines for Cell Line Authentication by STR profiling

Are the newly purchased cells and the cells that have been passed down for generations in the laboratory, or the cells stored in the ultra low temperature refrigerator for several years contaminated? Are the identifications correct? Do they affect the results of experiments and researches? If the contaminated cells are used without identification, a large number of experiments are conducted, a paper is written, but when the conclusion is then overthrown and the paper recalled, it means a lot of time, material and manpower are wasted, and everything has to start from scratch.

1.Necessity of cells STR identification

Misidentification and cross contamination of mammalian cells used in biomedical research have been a widespread problem all the time. According to statistics, about 20% of cell lines in foreign laboratories have been misidentified and cross-contaminated. National Institutes of Health (NIH) and American type culture collection (ATCC) have appealed researchers in the past two years to identification of cells before use.

In 2008, molecular biologist Winand Dinjens of the Josephine Nefkens Institute and his colleagues found that esophageal adenocarcinoma cells had a similar genotype to the esophageal squamous cell carcinoma cell line. These results prompted them to start to pay attention to cell identity issues. And then Dinjens began to validate these cell lines, performed genotype comparison with the original tissue, and found that these cell lines already had different genotypes. Among the 13 esophageal adenocarcinoma cell lines, three of them including SEG-1, BIC-1 and SK-GT-5 were contaminated by the mix of other cancer cells. But these three had been used in two clinical trials in several laboratories, more than 100 articles had been published in SCI journals and applied for 11 American patents. Also, these results were published in the latest issue of the Journal of the National Cancer Institute. (JNCI).

In 2007, NIH issued a notice which strongly recommended to perform authentication in cells culture. And at the end of 2008, experts suggested that ATCC  should be committed to the identification of human cell lines and develop standard procedures for identification.

In recent years, a large number of studies have shown that STR genotyping is one of the most effective and accurate methods for cell lines cross contamination and characteristic identification. And STR genotyping for cell identification has also been strongly recommended by some institutions such as ATCC. Furthermore, ATCC cell bank in the United States, DSMZ cell bank in Germany, and JCRB cell bank in Japan provide various data for cell strains to alignment.

In early 2011, ATCC successfully developed the STR identification standard for human cell lines to limit the expansion of invalid scientific data due to cell line cross contamination and misidentification. More importantly, the precise identification of cell lines plays a crucial role in the development of cell-based medical products which can avoid the risk of exposing human cells to misidentified cells.

2.Cells STR identification methods

It is required to provide indicators of general and specific biological traits of cells. General characteristics include cell morphology, specific structure, cell growth curve, division index, doubling time, vaccination rate, chromosome analysis, isozyme examination, DNA fingerprinting, etc. And specificity indicators are often identified based on the specificity of the cells type and function. For instance, if it is the glandular cell, it’s generally identified special products including secreted proteins or hormones. If tumor cells, it also need to prove that they are indeed derived from tumor tissue rather than others, which still retain their original characteristics, and therefore, it is usually necessary to conduct colony formation experiments, nude mice tumorigenic experiments and invasion experiments on normal tissues.

(1) Identification of normal cell lines

The identification of normal cell lines mainly focuses on the following four aspects: (1) Identification of the germline sources of cells: Commonly used methods include chromosome analysis, isozyme analysis, DNA fingerprinting and other techniques. (2) Identification of tissue sources of cells: By means of morphology detection and tissue-specific antigens detection, etc. (3) Whether the cells undergo transformation and malignant transformation: Mainly identified by karyotype analysis, cell growth behavior observation (whether the cells lose the contact inhibition), nude mice tumor formation experiment, etc. (4) Identification of cells cross contamination: Mainly identified by isozyme and DNA fingerprinting techniques.

(2)Identification of tumor cell lines

The identification of tumor cell lines mainly focuses on its malignant development, and the direction of tumor cell identification includes the characteristics such as chromosome abnormalities, changes in growth characteristics of contact inhibition and density dependent growth, colony forming ability, tumor formation in nude mice, invasive growth in animals, and that of some genes and moleculars.

3.Technology Application

(1) As a clinical diagnostic tool

The most important technology application is the quantitative detection of the ratio of antigenic specificity in peripheral blood and tissues, and the analysis of  phenotype and function. Altman et al have detected the antigenic specificity in a large number of asymptomatic AIDS patients with a detection rate of up to 2%. Zerbini et al. have applied tetramer technology and immunohistochemical method to detect peptide-specific CD8 in tumor tissues of hepatocellular carcinoma patients for the first time, which provides broad prospects for the application of immunotherapy to MAGE antigen in HCC. Moreover, in situ staining of tetramers in autoimmune diseases was first used in TCR transgenic mice. And through the quantitative detection, phenotypic and functional analysis of antigenic specificity, it lays the foundation for elucidation of the pathogenesis of viral infectious diseases, tumors and autoimmune diseases.

(2) Immunotherapy for adoptive tumors

Mei-denbauer et al intravenously infused a large number of Melan-A antigenic specificity (42.1%) into 8 patients with refractory malignant melanoma. The results showed that the ratio of antigenic specificity in peripheral blood mononuclear cells rose from 0.01-0.07% to 2% before and after infusion, and these cells could survive in vivo for several weeks, had the function of secreting INF-7, and preferentially aggregated to the tumors to play a role.

(3) Monitoring of vaccine efficacy

In recent years, the use of vaccines pulsed treatment of antigenic peptides to immunize the body to induce an effective CTL response is becoming a research hotspot for its significant benefits. And moreover, tetramers constructed with homologous peptides can be used to monitor the efficacy of the vaccine.

4.The significance of cells STR identification

Due to the importance of cell culture systems in the development of biopharmaceutical research and technology, appropriate cell line identification process has become the most interesting point for every researcher. However, the cross contamination is still a widespread problem. And with the increasingly wide use of new cell lines in different laboratories around the world, the basic principles of quality control in cell line identification have also changed dramatically.

What’s more, cross contamination affects every area of scientific research from laboratory experiment to clinical medicine, it is also reflected from these published research papers that have led to suspicious results using the "wrong" cell lines to stem cell lines and others used in clinical practice. And there are going to have to be some drastic changes in the handling process of cell culture, otherwise, cross contamination will be a more serious problem.

Cross contamination poses a serious problem for experimental research. If the results of studies are questioned by using the wrong cell lines, the previous experiments may have to be re-done again, therefore, it is necessary to identify the cell lines before use.