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.

Culture systems for three-dimensional tissues, such as multicellular spheroids, are indispensable for high-throughput screening of primary or patient-derived xenograft (PDX)-expanded cancer tissues. Oxygen supply to the center of such spheroids is particularly critical for maintaining cellular functions as well as avoiding the development of a necrotic core. In this study, we evaluated two methods to enhance oxygen supply: (1) using a culture plate with a gas-permeable polydimethylsiloxane (PDMS) membrane on the bottom, and; (2) embedding hydrogel beads in the spheroids. Culturing spheroids on PDMS increased cell growth and affected glucose/lactate metabolism and CYP3A4 mRNA expression and subsequent enzyme activity. The spheroids, comprised of 5000 Hep G2 cells and 5000 20 µm-diameter hydrogel beads, did not develop a necrotic core for nine days when cultured on a gas-permeable sheet. In contrast, central necrosis in spheroids lacking hydrogel beads was observed after day 3 of culture, even when using PDMS. These results indicate that the combination of gas-permeable culture equipment and embedded hydrogel beads improves culture 3D spheroids produced from primary or PDX-expanded tumor cells.

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