Engineering tissues with a perfusable vessel-like network by assembling endothelialized alginate hydrogel fiber and spheroid-enclosing microcapsules

摘要

Development of the technique for constructing an internal perfusable vascular network is a challenging issue in the fabrication of dense three-dimensional tissues in vitro. In this study, we report a method for realizing it. Firstly, we prepared hydrogel fiber of about 200 mm in diameter and HepG2 cell-enclosing microcapsules of about 500 mm in diameter from alginate derivative possessing phenolic hydroxyl (Ph) moieties. The surfaces of the hydrogel constructs were covered with gelatin derivative possessing Ph moieties through horseradish peroxidase catalyzed cross-linking Ph moieties. After covering the surfaces of the hydrogel constructs with human umbilical vein endothelial cells (HUVECs) by incubating them in the medium containing the cells, we assembled them in collagen gel (Fig. 1). The endothelial cells on the hydrogel constructs sprouted and spontaneously formed a network connecting the hydrogel constructs with each other. We, then, degraded the hydrogel constructs by the treatment with alginate lyase. The formation of a perfusable vascular-tike network was confirmed by introducing solution containing tracer particles of 3 mm in diameter into the lumen templated by the hydrogel fiber (Fig. 2). The introduced solution flowed into the spontaneously formed capillary branches and passed around the individual spherical tissues. Fig. 1. (A) Schematic of the assembly of hydrogel microcapsules with enclosed spherical tissues and a hydrogel fiber in collagen gel, both covered with endothelial cells in advance. Microphotographs of (B) a hydrogel fiber covered with MUVECs, (C) microcapsules enclosing spherical tissues of HepG2 cells at 6 days of culture before and after covering with HUVECs, and (D) empty microbeads covered with HUVEs. Bars: 200 μm. Fig. 2. Transmitted light (left) and fluorescence (right) microphotographs of tissue constructs (A, B) with and (C, D) without the internal HUVEC (green in panel A) network (vascularized and avascular tissue constructs) (A, C) before and (B, D) during perfusion of 3 μm fluorescent microparBcles (red in panels B and D). Dotted lines indicate the tubular cavity formed after degradation of hydrogel fibers. Bars: 1 mm.