In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Summary

This protocol replicates physiological or pathological blood flow in vitro to aid in determining cell response in disease pathologies. By introducing a pressure damping chamber downstream of a blood pump, blood flow across the vasculature can be recapitulated and imposed on a monolayer of vascular endothelium or a mimetic co-culture.

Abstract

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.

Introduction

Morbidity rates for cardiovascular diseases are the largest in America, with many resulting from unhealthy vasculature. Healthy arteries consist of elastic tissue, with soft luminal surface coated with an endothelial cell (EC) monolayer. Arterial flow may be modeled as an oscillating wave function with positive mean flow rate. The pulsatility index (PI) is the quotient of oscillation magnitude and mean flow (PI = (Max. - Min.) / Mean),¹ and has been modeled in vitro with variable vessel elasticity.² Arterial elasticity is important in storage of flow energy from heart contractions, dilating under systolic pressure, and plays a s....

Protocol

1. Silanization and Biomolecule Functionalization of Slide or Polycarbonate Membrane

Note: Many of the chemicals and solutions within this protocol have high evaporation rates (ethanol (EtOH), acetone, etc.). Other steps entail long incubation times for low evaporation rates. Paraffin film is recommended to seal containers. Caution: Many of the chemicals (including: sulfuric acid, acetone, (3-aminopropyl)triethoxysilane, glutaraldehyde, EtOH) are considered hazardous, or volatile. Consult material safety data sheet (MSDS) of each material for proper storage, handling, and disposal before use.

1. Place new glass slide measu....

Results

Maintenance of flow conditions is reliant on correct assembly of the flow circuit (Figure 1). Tubing diameter is an important selection in assembly, with larger diameters reducing flow resistance and subsequent pressure drop before and after the culture chamber. To ensure intended pressure and flow velocity, assemble the system with flow meter before experiment with intended tubing. Alignment of culture chamber vacuum channel (Figure 2), with perforations on silicon gasket (not pictured).......

Discussion

This protocol describes a method of reproducing pulsatile flow in vitro, and may be instrumental first step in determining contribution of flow conditions to disease pathologies. Previous studies using this protocol have found flow conditions contribute to vascular inflammatory response.^1,10 Additionally, this protocol is intended for experienced laboratories. As such, neither in depth fluid mechanics, nor biochemical analysis is described herein. For more advanced fluid dynamics,¹

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acetone	Sigma-Aldrich	34850
Sulfuric Acid	Sigma-Aldrich	320501
(3-Aminopropyl)triethoxysilane	Sigma-Aldrich	440140
Glutaraldehyde Solution	Sigma-Aldrich	G5882
Ethanol	Sigma-Aldrich	459844
Glass Slide (70 mm x 50 mm)	Sigma-Aldrich	CLS294775X50
Polycarbonate Membrane	Millipore Corp.	HTTP09030
Silicone Gasket	Grace Bio-Labs	RD 475464
Fibronectin (25 μg/ml)	Sigma-Aldrich	F1141
Collagen Type-I	Sigma-Aldrich	C3867
NaHCO3	Fluka	36486
NaOH	Sigma-Aldrich	S5881
Damping Chamber			This chamber is custom made, and may be requested using the engineering drawing of Figure 3.
Blood Pump	Harvard Apparatus	529552
Poly-Vinyl Carbonate Tubing	US Plastic	65066, 65063, 65062	Various sizes may be required
Luer Connections	Nordson Medical	Various	Various sizes will be required, and a number of parts should be purchased for replacement use.
Culture Chamber	Machined in-house	Custom	Acrylic may be purchased in sheets and machined for intended use. The engineering drawing shown in Figure 2 may be used to recreate this chamber.
Square Petri Dish	Cole-Parmer	EW-14007-10
Glass Slide Holder	Capitol Scientific	WHE-900303
Fetal Bovine Serum	Mediatech, Inc.	35-010-CV
Dulbecco's Modified Eagle Medium	Mediatech, Inc.	10-013-CV
Flow Meter	Sonotec, GmbH	Sonoflow co.55/060
Sylgard Elastomer Kit	Sigma-Aldrich	761036-5EA
14 G Steel Cannula	General Laboratory Supply	S8365-1