Porous membranes enable the partitioning of cellular microenvironments in vitro, while still allowing physical and biochemical crosstalk between cells, a feature that is often necessary for recapitulating physiological functions. This article provides an overview of the different membranes used in tissue barrier and cellular co-culture models with a focus on experimental design and control of these systems. Specifically, we discuss how the structural, mechanical, chemical, and even the optical and transport properties of different membranes bestow specific advantages and disadvantages through the context of physiological relevance. This review also explores how membrane pore properties affect perfusion and solute permeability by developing an analytical framework to guide the design and use of tissue barrier or co-culture models. Ultimately, this review offers insight into the important aspects one must consider when using porous membranes in tissue barrier and lab-on-a-chip applications.