Authentication of SNCA Knockout Cell Lines for Reliable Results

Dr. Shapiro: Good afternoon, Pamela, thank you for meeting today. In my previous discussions with Dr. Birgitt Schüle, she discussed the significance of the cell lines that were made available through the collaboration between MJFF and ATCC and went into detail on their development and potential application in Parkinson’s disease research. After she deposited these cell lines at ATCC, they were authenticated. Can you tell us about the authentication process that was performed at ATCC? 

Ms. Wood:  Cell line authentication was performed on each MJFF cell line at multiple stages of our accessioning process—this includes the original material deposited at ATCC, the low-passage Master Cell Bank produced and sequestered at ATCC, and on each batch of vials made available to the research community. Authentication of the MJFF cell lines includes the standard set of identification and purity tests conducted on all ATCC human cell lines as well as a set of tests specific to these special induced pluripotent stem cells (iPSCs). 

Standard authentication tests include species determination using a cytochrome oxidase 1 (CO1) PCR assay. This test not only confirms the human identity of our cell lines but also provides assurance that the cultures are not cross-contaminated with cells of another animal species. Identity and purity are further authenticated by STR profiling, which is a DNA barcoding assay that uses short DNA sequences in the human genome to establish a DNA fingerprint for every human cell line. The DNA profile of each sample is run against ATCC’s extensive database to authenticate identity and provide confidence that these cell lines are of unique identity and pure. Additionally, because of the related nature of the MJFF cell lines, a special genotyping assay was developed to further differentiate cell clones, which have identical or nearly identical STR profiles as a result of their derivation from a single human individual. Here, we used a targeted allele-specific PCR assay (fragment analysis with fluorescently labeled primers and capillary electrophoresis) designed to distinguish the various frameshift mutations in these clones.

In addition to identity testing, customized viability and growth testing is performed on every batch of MJFF iPSCs. Highly skilled laboratory scientists thaw sample vials of each batch and then count and seed according to the specialized growth protocols required by each of these cell lines. MJFF iPSCs require routine culturing, specialized flasks, surface coatings, media, and reagents all aimed at maintaining the cells in their undifferentiated state. In QC testing, each batch is assessed for viable cell recovery by performing colony counts within the first week. These cells are further cultured for karyology and other specialized iPSC testing.

One of the specialized characterization tests performed on MJFF iPSCs includes the assessment of the cells’ ability to self-perpetuate and differentiate (i.e., pluripotency). The PluriTest Assay uses bioinformatics to compare microarray gene expression data from a test sample to a reference set of known expression profiles in the stem cell database. The pluripotency score indicates how strongly a reference set of pluripotency signatures are expressed in the sample. The novelty score indicates the general model fit for the sample. 

Another iPSC characterization assay uses flow cytometry to evaluate surface antigen expression of stem cell markers SSEA1, SSEA4, and Tra-1-60. SSEA1 is expressed on differentiated human iPSCs and therefore should have low expression levels. SSEA4 and Tra-1-60 are expressed in undifferentiated human iPSCs and should have high expression levels. 

In addition to the authentication and characterization tests mentioned above, there are some standard microbial tests conducted on all ATCC cell cultures that also apply to iPSCs. These include (1) sterility testing using the BacT/ALERT® 3D instrument to screen for bacterial and fungal contaminants under aerobic and anaerobic conditions, (2) mycoplasma testing via the direct culture method and ATCC’s PCR-based method, and (3) human pathogenic virus testing for HIV, HepB, HPV, EBV, and CMV using a PCR-based assay.

At ATCC, quality control testing is performed under ISO 9001–certified and ISO/IEC 17025–accredited processes. The manufacturing procedures, safe storage with 24/7 monitoring, cold-chain support, and quality management system at ATCC work together to provide the highest quality material to our Parkinson’s disease researchers. By purchasing fully authenticated cultures directly from ATCC, the researcher is a step ahead at meeting the guidelines required for publication and funding.

Dr. Shapiro: Is the cell authentication and characterization data you mentioned available to the customer?

Ms. Wood: Cell line–specific data is available within the catalog description of each MJFF cell line found on the ATCC website. This includes handling information, quality control specifications, and images of each cell line in culture. 

Additional information be found on the certificates of analysis. These certificates can be downloaded through the ATCC website using the batch number on the vial received. They provide batch-specific results that are more comprehensive than the catalog description.

These SNCA knockout isogenic cell lines provide powerful tools for screening drug candidates that target the SNCA mutant or rescue its phenotype. If you are interested in learning more about these cell models or would like to explore our growing portfolio, please visit our MJFF collection webpage.

Did you know?

ATCC offers cell authentication services for human cell STR profiling, mouse cell STR profiling, and mycoplasma testing.