Multiplexed Fluid Flow Device to Study Cellular Response to Tunable Shear Stress Gradients

Abstract

Endothelial cells (ECs) line the interior of blood and lymphatic vessels and experience spatially varying wall shear stress (WSS) as an intrinsic part of their physiological function. How ECs, and mammalian cells generally, sense spatially varying WSS remains poorly understood, due in part to a lack of convenient tools for exposing cells to spatially varying flow patterns. We built a multiplexed device, termed a 6-well impinging flow chamber, that imparts controlled WSS gradients to a six-well tissue culture plate. Using this device, we investigated the migratory response of lymphatic microvascular ECs, umbilical vein ECs, primary fibroblasts, and epithelial cells to WSS gradients on hours to days timescales. We observed that lymphatic microvascular ECs migrate upstream, against the direction of flow, a response that was unique among all the cells types investigated here. Time-lapse, live cell imaging revealed that the microtubule organizing center relocated to the upstream side of the nucleus in response to the applied WSS gradient. To further demonstrate the utility of our device, we screened for the involvement of canonical signaling pathways in mediating this upstream migratory response. These data highlight the importance of WSS magnitude and WSS spatial gradients in dictating the cellular response to fluid flow.

Sphingosine 1-phosphate receptor 1 regulates the directional migration of lymphatic endothelial cells in response to fluid shear stress

“…Previous work suggests that PI3K is activated downstream of S1P, S1PR1 and Gai in ECs [56]. Consistent with this understanding, we found that chemical inhibition of PI3K prevented HLMVEC migration against the flow direction in response to impinging flow [36]. In addition, the requirement of S1P could imply an involvement of additional S1P binding receptors such as S1PR2, which has been previously shown to play a role in lymphatic vessel contraction [57].…”

Perpendicular alignment of lymphatic endothelial cells in response to spatial gradients in wall shear stress

“…4). We have previously observed that HLMVECs migrate against the direction of flow 24,30,31 . Here, we observed that, in the presence of flow, HLMVECs moved towards the constriction site (x-direction) but did not show migration in the direction of flow (y-direction) ( Supplementary Fig.…”

“…Determination of cell migration: brightfield movies were processed in the same way as our previous publications 24,30 . Movies were processed with Fiji software and analyzed using custom Matlab routines.…”

The Combined Contribution of Vascular Endothelial Cell Migration and Adhesion to Stent Re-endothelialization

“…Factors including stent deployment depth, the scale of endothelial denudation, hemodynamic changes, and the structure and material properties of the stent potentially regulate re-endothelialization in stented vessels ( Conway and Schwartz, 2015 ; Kang et al, 2015 ; Liang et al, 2016 ; Ostrowski et al, 2016 ). Due to the lack of effective in vivo and in vitro models, the cell sources of endothelial repair after stent implantation have not been fully understood.…”