Optimizing Antimicrobial Treatment to Cryopreserve and Establish Oral Squamous Cell Carcinoma In-Vitro Models Derived from Habitual Tobacco Chewers.

Abstract

Objectives: Cryopreservation and establishment of reliable in vitro models of oral squamous cell carcinoma (OSCC) are frequently compromised by microbial contamination arising from the oral microbiota of habitual tobacco users. This study aimed to optimize an antimicrobial strategy prior to biobanking to eliminate resistant microbial contaminants and improve the reliability of OSCC primary cultures.

Methods: OSCC tissues obtained from habitual tobacco chewers were subjected to either conventional penicillin-streptomycin (Pen-Strep) treatment or a stepwise antimicrobial treatment before cryopreservation. The optimized protocol employed a combination of cell culture-grade antibiotics to selectively eliminate Pen-Strep-resistant microbial taxa. Post-thaw tissue viability and epithelial outgrowth were assessed. Microbial profiling of conventionally treated tissues was performed using 16S and 18S rRNA gene sequencing, and antibiotic susceptibility testing was carried out to determine resistance profiles.

Results: Conventional Pen-Strep treatment alone was insufficient to eliminate microbial contamination, whereas the stepwise antimicrobial treatment effectively eradicated resistant microorganisms, enabling sterile tissue preservation. Post-thaw assessments demonstrated epithelial-like outgrowth from day 3 onward, confirming preserved tissue viability and integrity. Microbial profiling identified contaminants including Neisseria elongata, Streptococcus gallolyticus, and Candida albicans. Antibiotic susceptibility testing highlighted the necessity of tailored combinatorial antimicrobial strategies to overcome resistance.

Conclusion: The refined biobanking protocol integrating a stepwise antimicrobial strategy prior to cryopreservation effectively mitigates microbial contamination while preserving OSCC tissue integrity. This approach enhances the reliability of OSCC primary cultures derived from tobacco chewers and provides a robust platform for translational oncology research and personalized medicine.