Low-Level Mouse DNA in Conditioned Medium Generates False Positive Cross-Species Contamination Results in Human Organoid Cultures

• Published in: Frontiers in cell and developmental biology 2020-11, Vol.8, p.587107-587107
• Main Authors: Bohm, Margaret S., Dame, Michael K., Boyd, Joseph, Su, Kevin, Wu, Angeline, Attili, Durga, Chu, Vi, Colacino, Justin A., Spence, Jason R.
• Format: Article
• Language: English
• Subjects: Cell and Developmental Biology; cell-free DNA; cfDNA; colonoid; conditioned medium; enteroid; murine contamination
• ISSN: 2296-634X; 2296-634X
• DOI: 10.3389/fcell.2020.587107

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.