Development of cell-selective films for layered co-culturing of vascular progenitor cells

Abstract

Cell-sheet assemblies are currently being studied for tissue engineering. However, tissues engineered from completely biological cell sheets lack substrate cues and possess poor mechanical strength. Recent studies demonstrate the use of synthetic bioresorbable films as scaffolds that may address these issues. Here, we describe the application of a micro-thin, biaxially-stretched polycaprolactone (μXPCL) with surface modifications for layered tissue engineering, and present the results of biphasic cell-sheet constructs using surfaces optimised for specific cell types. Polyacrylic acid (PAAc) was grafted onto μXPCL film surfaces by low-pressure plasma immobilisation. This provided a surface suitable for perivascular cells, forming the medial compartment. Subsequently, endothelial progenitor cell (EPC)-selective CD34 antibody was conjugated onto the reverse surface (intimal compartment) to select and anchor EPCs for improved adhesion and proliferation. Using the blood vessel as a model, a biphasic culture system was then setup to represent a tunica intima (endothelial cells) and tunica media (smooth muscle cells). When suitable cell types were cultured in the corresponding compartments, confluent layers of the respective populations were achieved distinctively from each other. These results demonstrate the use of μXPCL films with cell-selective modifications for layered co-cultures towards the generation of stratified tissue.

Introduction

Structure and organisation are critical in native tissue/organ systems to maintain proper physiological function [1], [2]. However, existing three dimensional tissue-engineering scaffolds are isotropic, difficult to modify and consequently, offer little control over tissue organisation. To address these deficiencies, recent approaches have exploited the use of cell sheets for the assembly of layered tissue constructs [1], [3], [4], [5], [6]. For example, Okano's group has demonstrated the use of temperature responsive materials to generate aligned cell sheets, which can subsequently be stacked to form organised blood vessels [1]. Bringing this a step further, Sarkar et al. used microporous films as scaffolds to generate cell-sheet constructs, which could be stacked to form stratified tissue. The planar film surfaces allow for spatial control and amelioration of cellular responses [7]. The film also functions as scaffold to provide mechanical support, and as a physical barrier between different cell types, to prevent overgrowth of one cell type over another, maintaining tissue organisation. Taking advantage of the film geometry, different cellular requirements for attachment and proliferation on a planar surface can be catered for on either side of the film. For example, endothelial progenitor cells (EPC), which are required for seeding of the intima surface, have poor adhesive qualities on synthetic surfaces; this can be addressed by the placement of CD34 antibodies, as demonstrated in coronary stents [8], [9].

Polycaprolactone (PCL) films are being developed for tissue engineering applications, and are receiving much interest due to low toxicity and unique prolonged degradation rates [10]. We have previously demonstrated the fabrication of biaxially-stretched PCL films with 10–20 μm thickness and superior mechanical properties arising from the drawn and aligned polymer chains (μXPCL films) [11]. The use of a micro-thin geometry as a cell-sheet scaffold will minimise the amount of synthetic material within the construct. To achieve suitable surface modification without compromising the bulk properties of the film, we investigated the use of low-pressure plasma-based techniques, whereby surface reactions are limited to a depth of 10 nm [12], [13]. Here, we plasma-immobilised a hydrogel layer [14], [15] onto μXPCL films and subsequently conjugated cell-selective CD34 antibodies to promote attachment of endothelial progenitor cells. Based on these techniques, we developed polymeric films with specific modifications to achieve compartmentalisation. Using the blood vessel as a model, we demonstrate the utility of surface modified μXPCL films for layered co-culture of vascular progenitor cells, recapitulating the tunica intima (endothelial) and tunica media (smooth muscle) components of native blood vessels.