Intracellular calcium dynamics of lymphatic endothelial and muscle cells co-cultured in a Lymphangion-Chip under pulsatile flow

Abstract

The lymphatic vascular function is regulated by pulsatile shear stresses through signaling mediated by intracellular calcium [Ca²⁺]_i. Further, the intracellular calcium dynamics mediates signaling between lymphatic endothelial cells (LECs) and muscle cells (LMCs), including the lymphatic tone and contractility. Although calcium signaling has been characterized on LEC monolayers under uniform or step changes in shear stress, these dynamics have not been revealed in LMCs under physiologically-relevant co-culture conditions with LECs or under pulsatile flow. In this study, a cylindrical organ-on-chip platform of the lymphatic vessel (Lymphangion-Chip) consisting of a lumen formed with axially-aligned LECs co-cultured with transversally wrapped layers of LMCs was exposed to step changes or pulsatile shear stress, as often experienced in vivo physiologically or pathologically. Through real-time analysis of intracellular calcium [Ca²⁺]_i release, the device reveals the pulsatile shear-dependent biological coupling between LECs and LMCs. Upon step shear, both cell types undergo a relatively rapid rise in [Ca²⁺]_i followed by a gradual decay. Importantly, under pulsatile flow, analysis of the calcium signal also reveals a secondary sinusoid within the LECs and LMCs that is very close to the flow frequency. Finally, LMCs directly influence the LEC calcium dynamics both under step changes in shear and under pulsatile flow, demonstrating a coupling of LEC–LMC signaling. In conclusion, the Lymphangion-Chip is able to illustrate that intracellular calcium [Ca²⁺]_i in lymphatic vascular cells is dependent on pulsatile shear rate and therefore, serves as an analytical biomarker of mechanotransduction within LECs and LMCs, and functional consequences.