The use of mild trypsinization conditions in the detachment of endothelial cells to promote subsequent endothelialization on synthetic surfaces

Abstract

A necessary condition for endothelialization of small diameter grafts is rapid and firm adhesion of endothelial cells upon exposure to flow. To retain integrins on the cell surface, we assessed the effects of trypsin concentration, the duration of trypsin incubation, and trypsin neutralization methods on endothelial cell adhesion. Human umbilical vein endothelial cells which were detached using 0.025% trypsin for 5 min and seeded onto glass pretreated with fibronectin had close to 100% cell retention when shear stresses as high as 200 dyn/cm² were applied for 2 min. An equivalent level of cell retention was observed on fibronectin coated Teflon-AF™ for shear stresses up to 60 dyn/cm² applied for 4 h. Using 0.025% trypsin, initial cell spreading and cell surface α₅β₁ integrins were increased relative to cells treated with 0.5% trypsin. After 1 h of attachment, focal adhesions formed when low trypsin concentrations were used but were less evident with high trypsin concentrations. These results showed that low trypsin concentrations produced faster spreading, a higher number of intact integrins, and rapid focal adhesion formation.

Introduction

There is considerable interest in promoting rapid and firm adhesion of endothelial cells (ECs) for a number of clinical applications, including seeding of vascular grafts and vascular tissue engineering [1]. ECs perform many vital functions: regulate platelet activation, adhesion, and aggregation; limit leukocyte adhesion; regulate smooth muscle cell migration and proliferation; and control blood flow and vessel tone [2], [3], [4], [5].

EC adhesion to materials is mediated by adsorbed cell adhesion proteins, primarily fibronectin and/or vitronectin [6], [7]. Cell adhesion proteins bind to cell surface integrins, a family of heterodimeric transmembrane proteins consisting of α and β subunits. Integrins α₅β₁ and α_Vβ₃ both play a major role in EC adhesion. Binding to extracellular matrix proteins activates integrins to associate with the actin cytoskeleton and eventually leads to integrin clustering into focal adhesions [8]. The formation of focal adhesions, which contain actin-associated proteins such as talin, vinculin, paxillin, and α-actinin, strengthens the adhesion of cells to the substrate [9].

Critical shear stress experiments are a common method used to quantify cell's adhesion strength to specific substrates. Prior to seeding a synthetic surface, ECs are usually treated with the proteolytic enzyme, trypsin, which cleaves proteins at the carboxyl side of the basic amino acids lysine and arginine, in order to remove cells from their culture substrate and hence be used for further applications [10]. Both α₅β₁ and α_Vβ₃ integrins are sensitive to trypsin [11].

Of those studies that do report the trypsin concentrations and incubation times to detach cells, the trypsin concentrations to detach cells range from 0.05% to 0.5% [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24] and incubation times range from 5 to 10 min [12], [13], [14] at either room (25 °C) or physiological temperature (37 °C). Taking the steps to neutralize the trypsin may also be critical for later cell attachment and spreading. The cell suspension is either centrifuged directly and resuspended in culture medium or a neutralizing solution containing serum or calcium ions is added to inhibit the trypsin before centrifugation.

While previous articles have reported relatively low critical shear stress values of 12–37 dyn/cm² after 15 min to 1 h of cell attachment [5], [6], [9], [14], [15], [16], [17], no direct assessment is reported in the literature on the effect of trypsin on the critical shear stress needed to detach 50% of the cells from the surface.

In a clinical setting there is limited time for either cultured or freshly isolated ECs to strongly attach and spread on graft surfaces. Therefore, it is important to develop methods to promote rapid and strong cell adhesion. We have optimized in vitro conditions to yield very high adhesion strength after short periods of attachment to glass and Teflon-AF™. Differences in cell adhesion were characterized by the effect of trypsin on initial cell attachment, cell spreading, the number of intact integrins (α₅β₁ and α_Vβ₃) present, and the localization of vinculin at 1 h post attachment.