Resolving drug uptake heterogeneity in 3D organ on chip models via live single-cell microsampling and ion mobility...

Three-dimensional (3D) cancer cell models, such as spheroids, organoids, and organ-on-chip systems, provide more physiologically relevant conditions by preserving native cell-cell and cell-matrix interactions. However, analytical techniques that maintain and assess the intrinsic heterogeneity of these systems have lagged behind their biological complexity. Current approaches either rely on live imaging, which lacks biochemical depth, or destructive, high-content assays like RNA sequencing and mass spectrometry imaging, which require either fixative agents or freezing the 3D model before analysis. The key problem addressed here is the lack of methods that integrate live imaging with high-resolution biochemical analysis at the single-cell level.

We developed an integrated platform that bridges this gap by combining live imaging of a 3D cancer model with microsampling and mass spectrometry-based whole single-cell analysis. Using a vascularized organ-on-chip system cultured with HepG2 hepatocellular carcinoma cells under a drug gradient, we demonstrated the platform's ability to capture heterogeneous uptake of the drug Tamoxifen. By modifying the physical barriers of a commercial organ-on-a-chip platform for precise capillary-based microsampling, introducing a single-cell preparation technique to mitigate matrix interference from Matrigel, and incorporating High-field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS), we reduced matrix contamination and enhanced signal-to-noise ratios. This workflow enables non-destructive linkage between dynamic imaging data and single-cell biochemical profiles, preserving the spatial and temporal integrity of 3D tumor models.

This platform enables capillary-based single-cell isolation and metabolomic analysis of individual cells from live 3D tumor microenvironments, overcoming limitations of current imaging and molecular methods. By integrating live microscopy with sensitive single-cell mass spectrometry, it offers a new route to investigate cellular heterogeneity, specifically with respect to drug distribution and therapeutic responses in realistic cancer models, advancing translational and preclinical cancer research.