Systematic standardization of organ-on-a-chip under controlled flow conditions: Use case with Caki-1 and A549 cell lines.

New in vitro models are urgently needed to improve the preclinical development of new drugs. Current standardised cellular models are mainly based on 2D cell culture, which lacks physiological flow conditions and often involves limited or simplified co-culture settings. • Organ-on-a-chip (OoC) systems use microchips incorporating extracellular matrices, surface coatings and cell cultures to mimic physiological conditions, enabling the formation of 3D structures, co-culture systems and dynamic flow. However, to perform reproducible and controlled experiments using OoC systems, it is necessary to systematically standardise cell culture conditions within the microfluidic channels. • The selection of the extracellular matrix (ECM) coating of the chip must be tailored and optimised for each specific cell type used in OoC systems. It is essential to establish the appropriate biological microenvironment following cell adhesion to the chip, including the time required for cell adhesion and maturation, as well as the appropriate flow conditions. • This protocol standardises flow and ECM coating conditions in OoC with kidney (Caki-1) and lung (A549) cell lines, facilitating reproducible experiments and enabling broader application across different OoC models.