Innovative use of forensic STRs for authentication of human cell lines stored over 34 years

Aim: In forensic science, the authentication of biological samples is critical for accurate analysis and evidence validation. To evaluate the authenticity and genetic stability of 91 long-term preserved human cell line samples under cryogenic conditions over 34 years, and to explore the applicability of forensic-grade short tandem repeat (STR) profiling for long-term cell line authentication. Methods: We conducted STR-based authentication using 23 forensic STR markers. STR profiles were analyzed using the Tanabe and Masters algorithms for authentication. The study also examined the effects of prolonged passaging and genetic modification on STR stability, and investigated potential contamination events by comparing profiles with public databases. Results: All uniquely labeled human cell lines were successfully revived and yielded complete STR profiles, confirming the efficacy of long-term cryopreservation. One male cell line showed the Y-indel in the absence of a Y-allele at Amelogenin, indicating that the Y-indel alone is insufficient for accurate sex determination. Authentication using the Tanabe and Masters algorithms revealed that stringent criteria provided more definitive results than scoring-based methods. Instances of relatedness among unauthenticated cell lines to problematic references in existing databases suggest early-stage contamination rather than recent laboratory errors. STR profile stability persisted through routine cell line passages, and genetically edited cell lines retained profiles identical to their parental counterparts, indicating a need for additional differentiation markers for accurate derivation authentication. No significant differences in the number of altered alleles between normal and tumor cell lines, implying cell type alone did not dictate STR stability. Conclusion: This study represented one of the most extensive single-laboratory investigations into cell line preservation using forensic-grade tools, providing new reference alleles and valuable insights into cell line authentication. The findings supported the application of forensic STR kits beyond traditional forensic samples, offering a new perspective on their utility in genetic research and laboratory management.