Windows of operation as qualitative early-stage design tool for microfluidic (single-cell) cultivations

Microfluidic cultivation systems have transformed the study of cellular physiology by enabling high spatial and temporal analysis under precisely controlled environmental conditions. However, the successful application of these systems is hindered by technical challenges, including the lack of systematic characterization of key operational parameters and their interdependencies, which limits experimental reproducibility within a given setup and hampers the rational design of new systems. Here, we propose adapting ‘Windows of Operation’ — a framework originating from bioprocess engineering to visualize how different parameters define design limits — as a qualitative operational design tool for microfluidic cultivation, here further denoted as microfluidic window of operation (MWO). Through selected case studies, we demonstrate how defining MWOs can guide the identification and optimization of key experimental parameters. This provides a foundation for robust experimental design that links device function with operating parameters, thereby advancing feasibility, robustness, and comparability, while minimizing experimental bias.