Bottom-up approaches have emerged as a new philosophy in tissue engineering, enabling precise control over tissue morphogenesis at the cellular level. We previously prepared large bone-like tissues using cell-laden microbeads (microtissues) by following a modular approach to ensure cell viability. However, a long-term culture of such avascular macroscopic tissues (macrotissues) has not been evaluated. In the present study, microtissues were fabricated by cultivating human fibroblasts on Cytopore-2 microbeads in spinner flasks for 16 days. We then examined the long-term perfusion culture for macrotissues. Specifically, following assembly in a perfusion chamber for 15 days, cell death was found to be prominent at a depth of 500 µm from the surface of macrotissues towards the interior, suggesting that there was a new mass transfer limit leading to cell death instead of tissue maturation. Subsequently, we developed a strategy by incorporating microchannel structures in centimeter-sized tissue constructs to promote mass transport. By installing glass rods (1 mm diameter, 1 mm wall-to-wall spacing) in the perfusion chamber, stable microchannel architectures were introduced during the microtissue assembly process. Based on live/dead assay and scanning electron microscopy (SEM), these channelled macrotissues (length × diameter, 1.6 × 2.0 cm) demonstrated high cell viability and compact packing of microbeads. Comparative biochemical analysis further suggested a more homogeneous spatial distribution of cells and extracellular matrix (ECM) in the channelled macrotissues than in solid ones. Viable 3D large tissues can therefore be prepared by assembling cell-laden microbeads in conjunction with microchannel carving, meeting clinical needs in tissue repair. Copyright © 2012 John Wiley & Sons, Ltd.