Low Levels of Physiological Interstitial Flow Eliminate Morphogen Gradients and Guide

Supplementary Figures

Low levels of physiological interstitial flow eliminate morphogen gradients and guide angiogenesis

Venktesh S. Shirurea, Andrew Leziaa, Arnold Taoa, Luis F. Alonzob, and Steven C. Georgea, c ,#

aDepartment of Biomedical Engineering, Washington University in St. Louis, St. Louis, MO 63130.

bDepartment of Biomedical Engineering, University of California, Irvine, CA 92697.

cDepartment of Energy, Environment, and Chemical Engineering, Washington University in St. Louis, St. Louis, MO 63130.

# Corresponding Author:

Steven C. George, M.D., Ph.D.

Department of Biomedical Engineering, Washington University in St. Louis

Email:

Figure S1. The vessels developed in the microfluidic device have circular cross sectional morphology. The vessels (green) were developed for 8 days under interstitial flow conditions and z-stacks of the images were obtained by confocal microscopy. A) The top view (white border line) and cross sectional views (at yellow lines)of the central chamber of the device were obtained. The arrows indicate circular cross sectional view of vessels. B) A vessel sprouted into the upstream chamber of the device was imaged. The top view (white border line)and cross sectional views (at yellow lines) of a vessel in upstream chamber were obtained. The arrow indicate circular cross sectional view of the vessel and arrow head shows lumen along length of the vessel.

FigureS2. Fibroblast migration is not biased by interstitial flow. A) The vascular tissues were created using GFP-transduced endothelial cells (green) and azurite transduced normal human lung fibroblasts (blue). The tissue was exposed to interstitial flow, created by using ΔP =10 mm H2O from day 2, and the image was acquired on day 8. B) The fluorescent area of fibroblast was measured by using imageJ in the upstream (U) and downstream (D) chamber on day 8, and presented as an index of fibroblast migration.

FigureS3. The concentration gradients for short time at the beginning does not impact vessel guidance. A) The unsteady state concentration gradient in a tissue could exist for a short time (<1 hr). The temporal concentration profiles of morphogens in a fibrin tissue were calculated by the mathematical model. An interstitial flow (6 µm/s) of VEGF containing fluid was created from left to right in the tissue that had no VEGF at t = 0. The concentrations were determined along a horizontal line running at the vertical center of the tissue at indicated time. B) A vascular tissue developed for two days and exposed with various conditions. The interstitial flow (6 µm/s) was created in the absence (Only IF) or presence (IF and GF) of concentration gradients of VEGF. The concentration gradient and non-gradient conditions were created by perfusing VEGF-media or non-VEGF-media (2 ng/ml) in the low pressure fluidic line, while maintaining the high pressure fluidic line with VEGF-media (2 ng/ml). The scale bar shows 100 µm C) The data of vessel density was quantified for top chamber.

FigureS4. The barrier function of vessel for abluminal to luminal transport. The vessels were developed using mixture of ECFC-ECs (GFP; green) and NHLF in a fibrin gel in the central tissue chamber for 6 days. TritClabeled-dextran of 70 KDa (red) was added to one fluidic line, and fluid flow was created by applying ΔP = 10 mm H2O. The images were taken after 30 min. The lumen had minimal to no dextran (arrow) compared to the surrounding stroma (arrow head). The scale bar indicates 100 µm.

FigureS5. The interstitial flow of fluid mainly passes around the vessel. A) A hypothetical vessel of diameter 50 µm (blue) was placed in the microfluidic device filled with fibrin (gray). The interstitial flow with ΔP =10 mm H2O in the direction of the arrow was applied. B) The velocity profile found by COMSOL showing high velocities in non-vessel (red) and low velocity in the vessel (blue). C) The fluid flow rate passing through unit length of the device (ECM) and the vessel wall were presented as percentages with respect to flow rate per unit length through the device.

Figure S6. The stiffness of matrix affects directional bias of angiogenesis created by interstitial flow. Vascular tissues was created over the course of two days using 2 mg/ml (soft ECM) and 10 mg/ml (stiff ECM) of fibrinogen. The vascular tissues were then exposed to ΔP = 5 mm (6 μm/s) for 4 days using VEGF containing medium. The scale bar shows 100 µm. A) Vessel sprouting is enhanced in the upstream chambers of both soft and stiff ECM. The image analysis was performed to quantify the vessel length (B) and total number of endpoints (C) in upstream and downstream chambers, indicated by U and D. ** significantly different from stiff matrix conditions and the downstream chamber of soft ECM condition;$significantly different from downstream chamber.

FigureS7. Non-physiological level of capillary flow is required to create interstitial concentration gradient under interstitial flow. The steady state concentration profiles of morphogens in a fibrin tissue at various capillary flow velocities (Uc) were calculated by the mathematical model. An interstitial flow of fluid with morphogen was created from left to right in the tissue. The tissue compartment initially (t=0) had no morphogen. The concentrations were determined along a horizontal line running at the vertical center of the tissue. The Pe number was varied by increasing interstitial fluid velocity of 0.2-10 um/s and the diffusion coefficient was 10-11 m2/s.