Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Summary

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

Abstract

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system¹. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury^2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes⁴. This method focuses on neutrophil transmigration across human endothelial cells.

One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)⁵. Neutrophil isolation has been described visually elsewhere⁶; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-α results in increased E-selectin and IL-8 expression⁷. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature^7,8.

The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro^9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.

Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi μslide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

Protocol

1. Isolation and Maintenance of Human Vascular Endothelial Cells

1. Level 2 biosafety procedures must be used when working with human blood and tissue. Using scissors cut the cord from the placenta and then closely examine the cord for blood clots and damage caused by clamping the cord during delivery. Cut out and discard these portions of the cord.
2. Prepare fresh collagenase solution at 1 mg/ml by pouring 50 ml of warm cord buffer into a tube that contains 50 mg collagenase.
3. Change into sterile gloves and open an autoclaved instrument tray containing sterilized blunt cannulas, cord clamps, scissors, three-way stop-cocks and gauze in a lami....

Discussion

Researchers have routinely used endothelial cells from different vascular beds to study neutrophil recruitment and transmigration. Examples include, but are not limited to, dermal microvascular endothelial cells¹², liver sinusoidal endothelial cells¹³ and endothelial cells from human umbilical vein¹⁰. Among them HUVEC gained wide popularity because of their relative ease of isolation and availability. HUVEC are a powerful in vitro model system that mimics many endothelial process.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Collagenase Type 1	Worthington	4197
Cord buffer			Composition: 140 mM NaCl 4 mM KCl 10 mM D-glucose in 1mM NaH2PO4/Na2HPO4 buffer at pH 7.4
Endothelial Cell Media (ECM)			M199 with eagle salts supplemented with 16% human serum containing 100 units of penicillin 100 μg of streptomycin and 0.3 mg of L-glutamine/ml
M199	GIBCO	31100-035
Penicillin Streptomycin Glutamate (100X)	Invitrogen	10378-016
Ibidi chambers	Ibidi	80606
TNF-α	Prepro Tech	300-01A
Human Albumin 20% solution	Gemini Bioproducts	800120050
HBSS without Ca2+ and Mg2+	Sigma	H2487-10X
HBSS with Ca2+ and Mg2+	Sigma	H1387-10X