Rapid prototyping of concave microwells for the formation of 3D multicellular cancer aggregates for drug screening

Microwell technology has revolutionized many aspects of in vitro cellular studies from 2D traditional cultures to 3D in vivo-like functional assays. However, existing lithography-based approaches are often costly and time-consuming. This study presents a rapid, low-cost prototyping method of CO₂ laser ablation of a conventional untreated culture dish to create concave microwells used for generating multicellular aggregates, which can be readily available for general laboratories. Polymethylmethacrylate (PMMA), polydimethylsiloxane (PDMS), and polystyrene (PS) microwells are investigated, and each produces distinctive microwell features. Among these three materials, PS cell culture dishes produce the optimal surface smoothness and roundness. A549 lung cancer cells are grown to form cancer aggregates of controllable size from ≈40 to ≈80 μm in PS microwells. Functional assays of spheroids are performed to study migration on 2D substrates and in 3D hydrogel conditions as a step towards recapitulating the dissemination of cancer cells. Preclinical anti-cancer drug screening is investigated and reveals considerable differences between 2D and 3D conditions, indicating the importance of assay type as well as the utility of the present approach. A rapid, low-cost prototyping method of a conventional untreated culture dish to create concave microwells is demonstrated. A549 lung cancer cells are grown to form controllable size of cancer aggregates in microwells. Preclinical anti-cancer drug screening reveals considerable functional differences between 2D and 3D conditions, highlighting the importance of assay type as well as the utility of the present approach.