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

Abstract

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.

Three-dimensional microscale hanging drop arrays with geometric control for drug screening and live tissue imaging

“…In comparison to the classical hanging drop culture ( 41 ), cell aggregation into multicellular spheroids is significantly faster in the miniaturized platform (1 day versus 4 in the classical assay), likely an effect of the smaller well size that affects the diffusion of soluble factors and enhanced cell-cell adhesion. Moreover, our technique can also be adapted to replenish the role of various macromolecules, including extracellular matrix components and polysaccharides, as reported by others ( 48 ).…”

Soluble ECM promotes organotypic formation in lung alveolar model

“…2c, 3a-b). [13][14][15][16] (which was not certified by peer review) is the author/funder. All rights reserved.…”

“…Previous studies show that the composite properties of ECM are capable of determining stem cell fate. [22] Additionally, soluble ECM enhances cell proliferation and survival in 2D mesenchymal cell cultures, and enhanced formation of tight junctions in hepatic spheroid cultures. [11] However, the effects of low, soluble ECM concentrations in organotypic self-patterning and growth have yet to be fully elucidated.…”

Enhancement and Maintenance of Hepatic Metabolic Functions by Controlling Three-Dimensional Aggregation of Cryo-Preserved Human iPS Cells Derived Hepatocyte-Like Cells

“…In addition, small molecule strategy is also reported to promote further hepatic differentiation from HLC 22,23 . Our MC spheroid formation method also has an advantage to form an "inner-manipulated" spheroid by adding biomolecules 24 or hydrogel beads 25,26 . For example, if we established HLC spheroid blended with hydrogel beads homogenously, differentiation of HLC sphere might be promoted by bringing out the differentiation factors effectively and evenly in the spheroid.…”