Bioanalytic Hybrid System Merging 3-Dimensional Cell Culture and Chromatographic Precision for Unprecedented Preclinical Insights in Molecular Imaging

Verena Pichler; Verena Schwingenschlögl-Maisetschläger; Irem Duman; Xavier Monforte; Stefanie Ponti; Lukas Zimmermann; Elma Joldic; Monika Dumanic; Chrysoula Vraka; Marcus Hacker; Christian Kraule; Andreas Herbert Teuschl-Woller

doi:10.2967/jnumed.124.269133

Article Figures & Data

Figures

Additional Files

Download figure
Open in new tab
Download powerpoint
Download figure
Open in new tab
Download powerpoint
FIGURE 1.
Technical setup of silk sponge as stationary phase and column body. (A) Technical drawing of column body including flow direction (cranial to caudal). (B) Stepwise improvement by changing from noncompartmentalized to compartmentalized system. From top to bottom: final composition of column including silk, frits, and caps; PET/CT image of preliminary setup without compartments; CT image of final setup including compartments separated by frits. (C) Logistic setup of column preparation, radiotracer production, transfer to radioactive area, and measurement within 24-h time frame (created in bioRender). (D) Setup for automated administration of radiotracer to column and schematic representation of overall system. (E) Pressure limitation test of system for empty and filled columns. No additional pressure was introduced in system using silk scaffolds. HPLC = high-performance liquid chromatography; QC = quality control.
Download figure
Open in new tab
Download powerpoint
FIGURE 2.
Radiotracer interaction studies in dynamic 3D cell culture system. (A) Analysis of void volume and administration volume of system to ensure equal medium distribution for incubation. (B) Quantification of cellular accumulation of radiotracer [¹⁸F]FDG as marker for cell viability and proliferation in MDCK-WT and MDCK-Pgp cells (n = 3). Cell line–dependent accumulation was observed, with higher reproducibility for MDCK-WT than for MDCK-Pgp cells. (C) Cell viability assessment of seeded cells in dependency on cell count to estimate ideal cell seeding capacity to avoid overgrowth in HCT116 cells after 5 d (n > 3), and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay performed to analyze cell viability within column. (D) Dynamic scan to analyze fluidic behavior of administered radiotracer. (E) Comparison of behavior of HT29 cells in monolayer, multicellular tumor spheroid, and our dynamic system exposed to [¹⁸F]FDG (active transport), [¹⁸F]fluoromisonidazole (passive distribution), [¹⁸F]fluoroethylcholine (active transport), and [¹⁸F]NaF (passive distribution) at 37°C and 4°C. (F) Small-animal PET/CT scan of 600-μm-sized HT1080 spheroids incubated with [¹⁸F]FDG (n = 4, 1 representative dataset is shown to visualize direct readout), and analysis of cell viability by [¹⁸F]FDG. (G) Quantification of PET tracer with high specific binding and low nondisplaceable binding. Radiotracer interaction of [¹⁸F]FPSMA was investigated on PC3-PSMA–positive cells (increasing cell number in 5 × 10⁴ to 5 × 10⁶ cells per sponge) in comparison to PC3-PSMA–negative cells and silk without cells as background (n = 3, 1 representative dataset is shown). (H) Quantification of investigational PET tracer with high specific binding and high nondisplaceable binding. [¹¹C]DPMARE (investigational radiotracer toward muscarinic acetylcholine receptors) was investigated in CHO-M1, expressing human M1 subtype of muscarinic acetylcholine receptors, and CHO-K1 cells as negative control (n = 2, 1 representative dataset is shown). AD = applied dose; FEC = fluoroethylcholine; FMISO = fluoromisonidazole.