Chamber-Design-Associated In Situ Formation of HepG2 Spheroids in a Perfusable Organ-on-a-Chip

Perfused spheroid platforms enable continuous mass transport, but the role of chamber architecture in in situ spheroid assembly remains insufficiently understood. Here, we developed a perfusable organ-on-a-chip platform for the in situ formation and culture of HepG2 spheroids and compared rectangle-, rhombus-, and round-chamber designs under matched key channel dimensions. Two-dimensional (2D) simulations revealed geometry-associated differences in intragel velocity and shear-rate distributions. Experimentally, chamber design was associated with differences in spheroid morphology, qualified spheroid proportion, and viability, with the round chamber showing the best overall performance under standardized conditions. Rho-associated coiled-coil-containing protein kinase (ROCK) inhibition impaired early spheroid compaction without a significant geometry-dependent treatment effect, whereas Matrigel supplementation improved spheroid morphology, hepatic functional readouts, and viability. Under nominal APAP and TBHP exposure conditions, chip and ultra-low-attachment plate cultures exhibited distinct concentration- and time-dependent injury responses. Together, these findings identify chamber architecture and extracellular matrix (ECM) composition as important design variables in perfused spheroid culture and provide a comparative platform for studying liver spheroid development and drug-induced injury under perfusion.