<scp>3D</scp> ‐Printed Structures Versus Drilled Cavities: A Comparison of Microconfinement Methods for Rheological Characterisation of Multicellular Aggregates

Three-dimensional (3D) cell culture systems are increasingly recognised as more physiologically relevant models than traditional two-dimensional cultures, as they better mimic the native tumour microenvironment and enable the study of complex cellular behaviour. However, applying atomic force microscopy (AFM) to these models is challenging due to the inherent instability of multicellular aggregates, which complicates reproducible mechanical property measurements. To address this, we developed and evaluated two distinct strategies for confining multicellular aggregates for AFM analysis. In the first approach, aggregates were encapsulated in porous 3D-printed truncated conical microstructures fabricated by two-photon polymerisation. These structures were designed to allow medium perfusion, which is hypothesised to improve nutrient and metabolite exchange by enhancing fluid accessibility within the confined environment. In the second, cylindrical cavities were microfabricated into the base of PolyHEMA-coated Petri dishes to provide a simple yet robust platform for aggregate retention. Both methods successfully confined aggregates without compromising cellular integrity, enabling reproducible and reliable measurements of stiffness and viscoelasticity. Human pancreatic cancer cells (PANC-1) were used both as single cells and as monotypic aggregates. Aggregates confined in either system were consistently softer than substrate-attached single cells, indicating reduced cytoskeletal tension and altered remodelling in 3D environments. Among the two approaches, cylindrical cavities provided more reliable aggregate retention, whereas the 3D-printed truncated conical structures may possibly facilitate improved medium access through their porous architecture. Together, these microconfinement strategies enable reproducible mechanical characterisation of multicellular aggregates and extend the applicability of AFM to tumour mechanobiology and the assessment of anticancer therapies.