Pericytes require physiological oxygen tension to maintain phenotypic fidelity.

Pericytes function to maintain tissue homeostasis by regulating capillary blood flow and maintaining endothelial barrier function. Pericyte dysfunction is associated with various pathologies and has recently been found to aid cancer progression. Despite having critical functions in health and disease, pericytes remain an understudied population due to a lack of model systems which accurately reflect in vivo biology. In this study we developed a protocol to isolate and culture murine lung, brain, bone, and liver pericytes, that maintains their known phenotypes and functions. We demonstrate that pericytes, being inherently plastic, benefit from controlled oxygen tension culture conditions, aiding their expansion ex vivo. Primary pericytes grown in physiologically relevant oxygen tensions (10% O for lung; 5% O for brain, bone, and liver) also better retain pericyte phenotypes indicated by stable expression of characteristic transcriptional and protein markers. In functional tube formation assays, pericytes were observed to significantly associate with endothelial junctions. Importantly, we identified growth conditions that limit expression of the plasticity factor Klf4 to prevent spontaneous phenotypic switching in vitro. Additionally, we were able to induce pathological pericyte phenotypic switching in response to metastatic stimuli to accurately recapitulate in vivo biology. Here, we present a robust method for studying pericyte biology in both physiology and disease.