Development of a Microwell System for Reproducible Formation of Homogeneous Cell Spheroids

<b>Background/Objectives:</b> Three-dimensional (3D) cell cultures are increasingly used because 3D cell aggregates better mimic tissue-level biological mechanisms and support studies of tissue physiology and drug screening. However, existing laboratory methods and commercial microwell platforms often yield inconsistent results and can be error-prone, time-consuming, or costly. The objective of this work was to develop a reproducible, high-yield, and cost-effective approach for generating homogeneous cell aggregates using custom 3D-printed microwell stamps. <b>Methods:</b> Custom conical and semi-spherical microwell stamps were fabricated using 3D printing. Stamp resolution was characterized by scanning electron microscopy (SEM). Negative imprints were cast in polydimethylsiloxane (PDMS), a biocompatible and hydrophobic polymer conducive to cell aggregation. These PDMS microwells were then used to generate pluripotent stem cell aggregates (embryoid bodies, EBs) and tumor spheroids from adherent cancer cell lines. <b>Results:</b> The 3D-printed stamps produced high-resolution conical and semi-spherical microwells in PDMS. Semi-spherical microwells enabled rapid, simple, and cost-effective formation of pluripotent stem cell aggregates that were homogeneous in size and shape. These aggregates outperformed those produced using commercial microwell plates and ultra-low attachment plates. The fabricated microwells also generated uniform tumor spheroids from adherent cancer cells, demonstrating their versatility. <b>Conclusions:</b> The in-house 3D-printed microwell stamps offer a reproducible, efficient, and economical platform for producing homogeneous cell aggregates. This system improves upon commercial alternatives and supports a broad range of applications, including pluripotent stem cell embryoid body formation and tumor spheroid generation.