A modular and reconfigurable microfluidic device for culturing spheroids under continuous perfusion

3D cell spheroids have become crucial in vitro models for biomedical research, yet maintaining their growth and viability remains challenging due to diffusion limitations. We developed a versatile microfluidic modular device with a reconfigurable channel design that is customizable by altering the channel configuration in the adhesive layer. The resealable adhesive layer also enables open access to the wells for loading cells, continuous perfusion after closing, and facile retrieval of spheroids for downstream analysis and imaging after culturing. We evaluated three channel configurations using Mouse Embryonic Fibroblasts (MEFs), human induced Pluripotent Stem Cells (hiPSCs), and MDA-MB-231 breast cancer cells. The device significantly improved spheroid growth in MEFs and hiPSCs, increasing up to 139.9% over controls in 14 days. In contrast, MDA-MB-231 spheroids exhibited slower growth, highlighting the need for balancing nutrient delivery with autocrine factor retention. Sphericity was maintained in MEF and MDA-MB-231 spheroids, while hiPSC spheroids experienced budding. In situ optical coherence tomography (OCT) provided noninvasive 3D viability assessments of the spheroids. Our findings demonstrate that this modular microfluidic device, combined with OCT analysis, offers a powerful platform for advancing spheroid culture techniques and opens up new opportunities in applications such as drug testing, studying spheroid-spheroid interactions, and collecting spheroid secretions.