Fabrication of microchannel networks in multicellular spheroids

Multicellular spheroids are indispensable in cell biology as three-dimensional environments, in tissue engineering for achieving higher functions, and in drug validation by pharmaceutical companies. However, spheroids have limited nutrient exchange because they lack microvasculature. In this study, we established a method for fabricating microchannel networks in multicellular spheroids to overcome this limitation. Alginate hydrogel beads with almost the same size as animal cells were prepared by discharging alginate solution using an inkjet system. Using these beads, we produced “heterospheroids” that comprised the same number of cells and hydrogel beads based on the rapid cell/particle aggregation method. The hydrogel beads occupied the branched, connected spaces in the heterospheroids, which resembled microvasculature. The connectivity of the microchannel networks was confirmed using confocal laser microscopy and staining cells with 1-μm polystyrene beads after digesting the hydrogel beads using alginate lyase solution. The microchannel networks improved the albumin secretion rate and suppressed the expression of hypoxia-inducible factor-1α in Hep G2 cells. Experiments with rat primary hepatocytes demonstrated that the branched luminal-like structures overcame the limitations of albumin secretion and ammonia clearance. These findings suggest that it is possible to fabricate microchannel networks that can effectively maintain cellular functions by enhancing material exchange.