Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

Abstract

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses 1 .Our flow chamber design and flow circuit ( Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs 2,3 . This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts.The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (> 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry ( Fig. 11E), or scanning electron microscopy 5 . The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12) . Video LinkThe video component of this article can be found at https://www.jove.com/video/3349/ Protocol 1. Endothelial progenitor cell isolation 1. Prior to any collection of peripheral human blood, submit your research protocol to your Institutional Review Board (IRB), and after its approval, obtain the volunteer donors' informed consent (peripheral blood collection and EPC isolation had been approved by the Duke University IRB and is in full compliance with U.S. regulatory requirements related to the protection of human research participants). 2. When working with animal-derived EPCs, have your research protocol approved by your Institutional Animal Care and Use Committee (IACUC). All our porcine experiments had been approved by the Duke University IACUC and were conducted in accordance with the highest standards of humane care. 3. For isolation of endothelial progenitor cells, collect 50 ml of peripheral blood via standard phlebotomy technique from a consented volunteer donor into blood collection bags filled with the anticoagulant citrate phosphate dextrose and dilute the solution 1:1 with Hank's buffered salt solution (without CaCl 2 , MgCl 2 , MgSO 4 ) and layer on equal volumes of Histopaque to create well-defined layers. 4. Centrifuge (30 min, 740 g, low break setting) and colle...

Maximizing fibroblast adhesion on protein-coated surfaces using microfluidic cell printing

“…While various applications of surface micro-patterning are limited by the real-time analysis of the printing process 33–35 , the microfluidic printing technology described herein utilizes a microscope objective aligned with the print surface, allowing real-time analysis of the process. A parallel plate flow chamber is an often used and validated model for determining cell adhesion 32 ; however, cellular interactions constrained by two material surfaces may not be an appropriate model for many FBR cell adhesion measurements in which cells directly experience unrestrained differential flow based on their relative location to the device surface. The ability to interrogate cellular interactions under continuous flow over time represents a distinct advantage of the described vertical microfluidic printing device, which is able to create a reversible seal on a wet or dry surface 19 .…”

Increased Yield of Endothelial Cells from Peripheral Blood for Cell Therapies and Tissue Engineering

“…Following, one of the two glass slides was designated as a static control and transferred to a quadriPERM cell culture vessel (Greiner Bio-One, Frickenhausen, Germany, in 10 ml of EC medium), and incubated. The other glass slide was inserted into a parallel plate flow chamber [19] in a flow circuit consisting of a 125 ml reservoir (Cole Parmer, Pyrex, IL, USA), peristaltic pump (L/S variable-speed economy drive and Easy Load Pump Head; Masterflex, IL, USA), and pulse dampener (Masterflex) in EC medium (total circuit volume 100 ml). pBD-ECs in our parallel plate flow chamber were exposed to a shear stress of 25 dynes/cm 2 calculated according to $$\mathrm{normal\tau }=6\mathrm{normal\mu }\mathrm{normalQ}/\mathrm{normalw}{\mathrm{h}}^2$$ where τ denotes the wall shear stress (dynes/cm 2 ), μ the medium viscosity at 37°C (0.01 g/cm/s), w the channel width (2.5 cm), h the channel height (0.04 cm), Q the volumetric flow rate (cm 3 /s) [19].…”

A multi-component parallel-plate flow chamber system for studying the effect of exercise-induced wall shear stress on endothelial cells

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