Centrifugal casting allows rapid biofabrication of tubular tissue constructs by suspending living cells in an in situ cross-linkable hydrogel. We hypothesize that introduction of laser-machined micropores into a decellularized natural scaffold will facilitate cell seeding by centrifugal casting and increase hydrogel retention, without compromising the biomechanical properties of the scaffold. Micropores with diameters of 50, 100, and 200 m were machined at different linear densities in decellularized small intestine submucosa (SIS) planar sheets and tubular SIS scaffolds using an argon laser. The ultimate stress and ultimate strain values for SIS sheets with laser-machined micropores with diameter 50 m and distance between holes as low as 714 m were not significantly different from unmachined control SIS specimens. Centrifugal casting of GFP-labeled cells suspended in an in situ cross-linkable hyaluronan-based hydrogel resulted in scaffold recellularization with a high density of viable cells inside the laser-machined micropores. Perfusion tests demonstrated the retention of the cells encapsulated within the HA hydrogel in the microholes. Thus, an SIS scaffold with appropriately sized microholes can be loaded with hydrogel encapsulated cells by centrifugal casting to give a mechanically robust construct that retains the cell-seeded hydrogel, permitting rapid biofabrication of tubular tissue construct in a “bioreactor-free” fashion.