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.

Astrocytes, a type of glial cell in the brain, are thought to be functionally and morphologically diverse cells that regulate brain homeostasis. Cell immortalization is a promising technique for the propagation of primary human astrocytes. The immortalized cells retain their astrocytic marker mRNA expression at lower levels than the primary cells. Therefore, improvement of the differentiation status is required. The use of a 3D formation technique to mimic structural tissue is a good strategy for reflecting physiological cell–cell interactions. Previously, we developed a spheroid formation method using highly viscous methyl cellulose (MC) medium. In this study, we applied this formation method to the well-established immortalized human astrocyte cell line HASTR/ci35. Stable HASTR/ci35 spheroids were successfully formed in MC medium, and laminin deposition was detected inside of the spheroids. Their functional markers were enhanced compared to conventional spheroids formed in U-bottom plates. The inflammatory response was moderately sensitive, and the ability to support neurite growth was confirmed. The HASTR/ci35 spheroid in the MC medium demonstrated the differentiation phenotype and could serve as a potent in vitro model for matured astrocytes.