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Multicellular spheroids (spheroids) are expected to be a promising approach to mimic  in vivo  organ functions and cell microenvironments. However, conventional spheroids do not fully consider the existence of extracellular matrices (ECMs). In this study, we developed a tunable method for replenishing macromolecules, including ECM components and polysaccharides, into spheroids without compromising cell viability by injecting a microvolume cell suspension into a high density of methylcellulose dissolved in the culture medium. Adjusting the ECM concentration in the cell suspension enabled the generation of different three-dimensional microstructures, such as “ECM gel capsules”, which contained individually separated cells, and “ECM-loaded spheroids”, which had thin ECM layers between cells. ECM-loaded spheroids with a 30-fold dilution of Matrigel (0.3 mg/ml) showed significantly higher albumin secretion than control spheroids composed of Hep G2 or HuH-7 cells. Additionally, the expression levels of major CYP genes were decreased in ECM gel capsules with undiluted Matrigel (9 mg/ml) compared to those in control spheroids. However, 0.3 mg/ml Matrigel did not disrupt gene expression. Furthermore, cell polarity associated with tight junction proteins (ZO-1 and Claudin-1) and the transporter protein MRP2 was markedly induced by using 0.3 mg/ml Matrigel. Thus, high-performance three-dimensional tissues fabricated by this method are applicable to increasing the efficiency of drug screening and to regenerative medicine.

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Development of a tunable method to generate various three-dimensional microstructures by replenishing macromolecules such as extracellular matrix components and polysaccharides