Abstract
<jats:p>Three-dimensional (3D) cell culture models, particularly multicellular tumor spheroids, have become essential tools for studying cancer biology, drug screening, and preclinical testing due to their ability to mimic physiological tumor microenvironments. However, traditional invasion assays, such as Boyden-chamber- or Transwell-based systems, often suffer from variability introduced by spheroid handling and transfer, compromising data reproducibility. Here, we present a novel, 3D-printed migration and invasion platform, the MQm-sert, designed to standardize and streamline spheroid-based invasion assays while maintaining spheroid integrity. Fabricated via fused filament fabrication using biobased polylactic acid, the MQm-sert integrates a hanging-drop spheroid culture system (MQm-sert) with a membrane-based invasion chamber (M-sert), eliminating the need for disruptive spheroid transfer steps. Using synthetic tumor environment mimics (STEMs) composed of breast cancer cells (MDA-MB-231 and MCF7), mesenchymal stromal cells (MSCs), and human pulmonary microvascular endothelial cells (HPMECs), we quantified invasion dynamics and cellular interactions. This innovation significantly reduces experimental variability, as demonstrated by lower variance in invasive cell mass dimensions and cell density compared to conventional workflows. Beyond biological insights, the platform aligns with sustainability goals by leveraging cost-effective, open-source 3D printing, reducing reliance on commercial labware, and addressing key challenges in 3D cell culture standardization.</jats:p>