In‐House Manufacturing of 3D ECM Culture Chips via Vacuum Thermoforming for Enhanced Imaging Applications

ABSTRACT Engineered 3D in vitro cancer models, particularly those that facilitate image‐based readouts capable of distinguishing the behavior of different cell populations, have become crucial tools in the discovery process. One such model, 96‐GLAnCE (Gels for Live Analysis of Compartmentalized Environments), allows for longitudinal imaging of tumor cell dynamics and therapy response. However, the widespread adoption of 96‐GLAnCE has been limited by the need for expensive, specialized fabrication equipment. To overcome this challenge, we have optimized a desktop vacuum thermoforming technique for the in‐house production of 96‐GLAnCE bottom chips using thin polystyrene films (0.192 mm). This optimization has led to the reliable and consistent fabrication of devices. Notably, using thin polystyrene films reduces the channel‐bottom thickness of the chips (from original 0.670 mm (±0.091) to 0.052 mm (±0.003)), enabling high‐magnification imaging for studying primary tumor cell phenotypes in 3D with single‐cell and subcellular resolution. Overall, the thermoformed 96‐GLAnCE chips provide an accessible fabrication approach for 96‐well plate‐compatible 3D ECM culture and enable longitudinal and high‐magnification image‐based analysis.