Microwell‐based flow culture increases viability and restores drug response in prostate cancer spheroids

3D cancer spheroids represent a highly promising model for study of cancer progression and therapeutic development. Wide‐scale adoption of cancer spheroids, however, remains a challenge due to the lack of control over hypoxic gradients that may cloud the assessment of cell morphology and drug response. Here, we present a Microwell Flow Device (MFD) that generates in‐well laminar flow around 3D tissues via repetitive tissue sedimentation. Using a prostate cancer cell line, we demonstrate the spheroids in the MFD exhibit improved cell growth, reduced necrotic core formation, enhanced structural integrity, and downregulated expression of cell stress genes. The flow‐cultured spheroids also exhibit an improved sensitivity to chemotherapy with greater transcriptional response. These results demonstrate how fluidic stimuli reveal the cellular phenotype previously masked by severe necrosis. Our platform advances 3D cellular models and enables study into hypoxia modulation, cancer metabolism, and drug screening within pathophysiological conditions. Graphical and Lay Summary In this study, the authors present the use of a novel Microwell Flow Device (MFD) for generating laminar flow around individual 3D in vitro cultures. Using the MFD, the authors demonstrate that flow increases the growth of prostate cancer spheroids while reducing central necrosis. This culture platform is instrumental in preserving spheroid viability for downstream analysis and drug response.