Abstract
Cell line authentication is critical for preventing the use of mixed or misidentified cell lines in research. Current efforts include short tandem repeat (STR) analysis and PCR-based assays to detect mixed species cultures. Using PCR analysis with mouse-specific primers, we identified contaminating mouse DNA in growth factor conditioned medium (CM) derived from the L-WRN cell line (L-WRN CM), as well as in human organoid cultures maintained in the L-WRN CM. DNA isolated from L-WRN CM matched the L-WRN cell signature by STR analysis. Organoid lines that were positive for murine DNA by PCR were further analyzed via bulk RNA-sequencing and transcripts were aligned to the human and mouse genomes. RNA analysis failed to detect mouse-specific gene expression above background levels, suggesting no viable murine cells were present in the organoid cultures. We interpret our data to show conclusive evidence that mouse cell-derived CM can be a source of contaminating murine DNA detected in human organoid cultures, even though live, transcriptionally-active murine cells are not present. Together, our findings suggest that multiple methods may be required to authenticate human organoid or cell lines and urges cautious interpretation of DNA-based PCR cell line authentication results.
Highlights
As the use of primary human 3-dimensional (3D) organoids in research increases, so does the need for stringent cell line authentication measures for these cultures
To confirm the finding that murine DNA was detected in L-WRN-cultured organoids only, we identified V1rh10 amplification in samples of cultured colonoids and enteroids; the matched primary tissue samples did not amplify this marker (Figure 1)
The short tandem repeat (STR) profile of the cell-free DNA (cfDNA) from the L-WRN conditioned medium (CM) contained 8 loci with intact alleles out of 18 potential loci, of which 100% of the alleles identified in the CM matched the L-cell and L-WRN cell profiles
Summary
As the use of primary human 3-dimensional (3D) organoids in research increases, so does the need for stringent cell line authentication measures for these cultures. The use of misidentified or contaminated cell lines has been a concern for more than 70 years and has called into question experimental conclusions, lead to poor reproducibility, wasted research dollars, and led to manuscript retractions (American Type Culture Collection Standards Development Organization Workgroup ASN-0002, 2010). To address these issues, guidelines have been submitted by many organizations to ensure proper cell. Despite the development of guidelines that suggest regular testing (Barallon et al, 2010), many contaminated lines continue to be used for in vitro research. Testing for cell line contamination has become a priority for researchers, and efforts have centered around detecting mixed or misidentified cell lines (Babic et al, 2019)
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