A bioreactor for in vitro studies of lymphatic endothelial cells with simultaneous fluid shear stress and membrane strain

Abstract

Reproducing the in vivo physiologic conditions and biomechanical environment to stimulate natural growth and behavior of lymphatic endothelial cells (LECs) is critical in studying the lymphatic system and its response to stimuli. In vitro studies that deconstruct the biomechanical environment, e.g. independently incorporate flow-induced shear stress or membrane strain have demonstrated the significance of mechanotransduction in LECs (and vascular endothelial cells). Such studies have facilitated the investigation of intracellular signaling pathways stimulated by a particular mechanical cue but do not accurately reproduce natural physiologic behavior of in vivo LECs given the absence of other natural mechanical cues. In this study, we present a novel experimental device designed to reconstruct the in vivo biomechanical environment, i.e. a device that enables the simultaneous application of flow-induced shear stress and cyclic stretching of LECs in vitro. The device is uniquely capable of simulating physiologically-relevant conditions for lymphatic endothelial cells, such as low-flow, high-strain scenarios. Using this device, we observed that, like vascular ECs, LECs aligned in the direction of fluid shear stress when steady flow was applied. In our case the behavior was observed under conditions closer to the physiological mean flow in the lymphatic vessels than vascular levels of shear stress. When concurrent cyclic stretching was applied, the alignment in the direction of flow and perpendicular to the uniaxial stretch was detected in a substantially shortened timeframe. Additionally, the distribution of alignment angles was more closely clustered around 90° under the flow/stretch scenario after 6 h than the 24 h flow only scenario, perhaps indicating a greater sensitivity to cyclic stretching than to fluid shear stress in the morphological alignment response of LECs. We also observed alignment of cell nuclei and F-actin filaments in Human Dermal Lymphatic Endothelial Cells (HDLECs) after only 6 h of combined flow and stretch. These observations underscore the importance of including both sources of mechanical stress when studying the growth and behavior of LECs.