Peristaltic pumps adapted for laminar flow experiments enhance in vitro modeling of vascular cell behavior

Abstract

Endothelial cells (ECs) are the primary cellular constituent of blood vessels that are in direct contact with hemodynamic forces over their lifetime. Throughout the body, vessels experience different blood flow patterns and rates that alter vascular architecture and cellular behavior. Because of the complexities of studying blood flow in an intact organism, particularly during development, the field has increasingly relied on in vitro modeling of blood flow as a powerful technique for studying hemodynamic-dependent signaling mechanisms in ECs. While commercial flow systems that recirculate fluids exist, many commercially available pumps are peristaltic and best model pulsatile flow conditions. However, there are many important situations in which ECs experience laminar flow conditions in vivo, such as along long straight stretches of the vasculature. To understand EC function under these contexts, it is important to be able to reproducibly model laminar flow conditions in vitro. Here, we outline a method to reliably adapt commercially available peristaltic pumps to study laminar flow conditions. Our proof-of-concept study focuses on 2D models but could be further adapted to 3D environments to better model in vivo scenarios, such as organ development. Our studies make significant inroads into solving technical challenges associated with flow modeling and allow us to conduct functional studies toward understanding the mechanistic role of shear forces on vascular architecture, cellular behavior, and remodeling in diverse physiological contexts.

Keywords: endothelial cells; in vitro modeling; laminar flow; pulsatile flow.

Figure 1

Adaptations to a peristaltic pump to deliver laminar flow.A, picture of the flow system setup, including the four head peristaltic pump, dampeners, reservoirs, and flow chamber. B, left panel, shows the schematic representation of a peristaltic pump' head including the positions of the dampeners. The right panel is a schematic representation of how to assemble the dampeners.

Figure 2

Quantifying the effect of dampeners in offsetting pump pulsation. A flow sensor was purchased to quantifiably measure the effects of dampeners in offsetting peristaltic pump pulsation. The flow sensor was placed directly prior to the flow chamber to ensure a representative measurement of flow forces felt by the endothelial cells (ECs) in the culture. A, schematic diagram of the flow circuit and placement of the flow sensor to measure flow forces generated by the original nonmodified peristaltic pump. B, pulse traces collected across 15 s of pump function, demonstrating marked pulsation of the fluid that is being flowed across the EC monolayer. C, average maximum and minimum flow forces generated by the endogenous function of the peristaltic pump at 1 and 24 h of culture. D, schematic diagram of the modified laminar flow circuit, including placement of our custom inlet/outlet dampeners and the flow sensor to measure forces generated by offsetting the pulsation coming out of the peristaltic pump. E, pulse traces collected across 15 s of pump function, demonstrating laminar flow (i.e., sixfold suppression of fluid pulsation) across the EC monolayer. F, average maximum and minimum flow forces generated by the modified laminar function of the peristaltic pump at 1 and 24 h of culture. G, schematic diagram of the modified laminar flow circuit, including placement of a commercial dampener and the flow sensor to measure forces generated by offsetting the pulsation coming out of the peristaltic pump. H, pulse traces collected across 15 s of pump function, demonstrating mild suppression of fluid pulsation across the EC monolayer. However, as shown, our dampeners suppressed pulsation to a much higher degree (commercial 1.4-fold from peristaltic; lab-built sixfold from peristaltic). I, average maximum and minimum flow forces generated after commercial dampener modifications of the peristaltic pump at 1 and 24 h of culture. The box plots are graphed showing the median versus the first and third quartiles of the data (the middle, top, and bottom lines of the box, respectively). The whiskers demonstrate the spread of data within 1.5 × above and below the interquartile range. All data points are shown as discrete dots (averages from each of the four individual pumps).

Figure 3

Phenotypic characterization of pulsatile versus laminar flow conditions.A, a cartoon of the cell alignment expected under pulsatile and laminar flow conditions (i.e., with and without dampeners). B, microscopy images show HUVEC alignment under pulsatile versus laminar flow conditions at 0 and 24 h as imaged utilizing our stage top incubated microscope system. C and D, distribution angles of HUVECs, showing the orientation of the cell’s longest axis relative to the direction of flow at 0 h (gray) and after 24 h (orange/blue) of pulsatile (C) versus laminar (D) flow. The alignment of the cells was carried out using the ImageJ plugin Directionality analysis, version 2.3.0, created by Jean-Yves Tinevez (https://imagej.net/plugins/directionality) implementing the method of Fourier components. n = 8 images, all cells within the image were included in the analysis. Data plotted using a bin size of 22.5^°. The radial axis represents the proportion of cells that fall into each bin. E, representative cell tracks of HUVECs across the 24-h period of pulsatile (top) or laminar (bottom) flow. Arrowheads represent the direction of cellular movement, and circles represent the starting point of individual cells. F–H, average total distance traveled (F), average distance from origin (G, calculated based off of the linear distance between the starting point and ending point of individual cells), and linearity of movement (H, calculated as the ratio of G to F) shown for pulsatile versus laminar flow over the 24 h period of treatment. For H, linearity of movement: “1” is a movement path that is completely linear, whereas “0” is a movement path that is completely nonlinear, that is, curved. The box plots are graphed showing the median versus the first and third quartiles of the data (the middle, top, and bottom lines of the box, respectively). The whiskers demonstrate the spread of data within 1.5 × above and below the interquartile range. All data points (individual cells; n > 8) are shown as discrete dots, with outliers shown above or below the whiskers. Data are representative of three independent replicates. HUVEC, human umbilical vein endothelial cell.

Figure 4

Immunostaining the EC junctional marker, VE-cadherin, under varying flow conditions.A, representative microscopy images of cells immunostained for VE-cadherin (grey) and nuclei (black) under no flow, pulsatile flow, or laminar flow conditions. B–D, zoomed in imaged of the white boxes shown in A, showing changes in localization and intensity of VE-cadherin staining depending on flow forces applied to the EC monolayer. E, quantification of VE-cadherin staining intensity at the EC junction. Black bar shows the no flow condition, orange bar shows staining intensity following exposure to pulsatile flow (original pump function), and blue bar shows staining intensity following exposure to laminar flow (condition with dampeners). F and G, histogram (F) and radial histograms (G) depicting orientation angles of the VE-cadherin positive junctional “fingers” relative to the direction of flow (flow applied from left to right in images). No flow (gray), pulsatile flow (orange), and laminar flow (blue). The distribution of data shows that under no flow conditions, VE-cadherin fingers, if present, are randomly distributed. Under pulsatile flow conditions, these fingers skew to have a slightly more perpendicular orientation, when considering junctions parallel to the direction of flow (i.e., “top” and “bottom” of the cells). Under laminar flow conditions, the fingers have a parallel orientation, when considering the junctions parallel to the direction of flow (i.e., “top” and “bottom” of the cells). See Fig. S3 for schematic description. The alignment of the VE-cadherin fingers was carried out using the module for quantitative orientation measurement of the ImageJ plugin OrientationJ created by Daniel Sage (http://bigwww.epfl.ch/demo/orientation/). n = 45 cells, five images. Data plotted using a bin size of 45^° and are representative of two independent experiments. F and G, represent varying presentation of the same dataset to demonstrate different visualization techniques, (F) the y-axis represents the proportion of cells that fall into each bin; (G) the radial axis represents the proportion of cells that fall into each bin. The box plots are graphed showing the median versus the first and third quartiles of the data (the middle, top, and bottom lines of the box, respectively). The whiskers demonstrate the spread of data within 1.5 × above and below the interquartile range. All data points (average intensity from individual images; n > 8) are shown as individual dots. p values are indicated above statistically significant datasets and were generated using one-way ANOVA. Data are representative of three independent replicates. EC, endothelial cell; VE, vascular endothelial.

Figure 5

Immunostaining of F-actin filaments with phalloidin-555 under pulsatile and laminar flow. A, images of F-actin filaments (white) and nuclei (blue) under no flow (top panel), pulsatile flow (middle panel), and laminar flow (lower panel). B, orientation angle of the actin filaments relative to the direction of the flow (flow applied from left to right). No flow, (gray), pulsatile flow (orange), and laminar flow (blue). Under pulsatile flow conditions, the actin filament angle of orientation is randomly distributed, though skewed in orientation toward the direction that flow is coming from, while under laminar flow, for the actin filaments align more narrowly to be parallel to the direction of flow. The alignment of the F-actin filaments was carried out using the ImageJ plugin Directionality analysis, version 2.3.0, created by Jean-Yves Tinevez (https://imagej.net/plugins/directionality) implementing the method of Fourier components. n = 8 individual images, all cells within the image were included in the analysis. Data plotted using a bin size of 22.5^°. The radial axis represents the proportion of cells that fall into each bin. Data are representative of three independent replicates.