3D-Printed Stackable Air–Liquid Interface Platform for Isotopic Tracing of Cancer-Derived Volatiles

Abstract

Analysis of biogenic volatile organic compounds (BVOCs) offers a noninvasive window into cellular metabolism; however, conventional in vitro culture systems often exhibit low volatile recovery and poor physiological relevance, severely limiting trace-level BVOC profiling. In this study, we developed a 3D-printed, paper-based, stackable air–liquid interface (ALI) platform to enhance BVOC extraction from lung cancer cell lines. Using 3D printing with low-emissive materials, we fabricated a stackable architecture that incorporates multiple cell-culture layers within a single headspace, thereby increasing cell density relative to headspace volume. To mimic the high gas-exchange efficiency of the human lung, the platform integrates a paper-based scaffold to establish a dual air–liquid interface; this configuration enables continuous nutrient supply from the basolateral side while allowing direct emission of volatiles into the apical gas phase. This dual-interface structure eliminated the liquid partition barrier, increased detection sensitivity, and enabled identification of 242 BVOCs. To validate the biosynthetic origins of the detected BVOCs, we performed 13C isotopic tracing using [U–13C]glucose. Isotopic enrichment analysis revealed that 46 BVOCs exhibited increased M + n/M + 0 ratios of the precursor ion. Consistent mass shifts in both precursor and fragment ions further confirmed that at least five volatiles were isotopically labeled, directly linking their origins to glucose metabolism. Collectively, these findings demonstrate that the stackable ALI platform enhances in vitro volatile detection sensitivity and provides a robust analytical tool for elucidating BVOC biosynthetic pathways.