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.
Highlights
Multicellular spheroids are expected to be a promising approach to mimic in vivo organ functions and cell microenvironments
To examine whether biological extracellular matrices (ECMs) components can co-aggregate with cells, cells labelled with PKH26 were re-suspended with fluorescein isothiocyanate (FITC)-collagen and injected into MC medium, and the progression of aggregation was observed over time (Fig. 1a)
The amounts of FITC-labelled fibronectin per spheroid using 0.5 mg/ml, 0.1 mg/ml or 0.01 mg/ml were 7%, 3% and 0.6%, respectively. We defined such spheroids with diluted ECM as “ECM-loaded spheroids” to distinguish them from the “ECM gel capsules”. These results indicate that the structures of 3D spheroid tissue at the endpoint of aggregation vary depending on the concentrations of ECM
Summary
Multicellular spheroids (spheroids) are expected to be a promising approach to mimic in vivo organ functions and cell microenvironments. Spheroids of the hepatoma cell lines Hep G2 and HuH-7 were successfully generated in hanging drops or on low adhesion surfaces[6,7,8,9,10] These culture systems do not fully consider the influence of the ECM. A method of generating micrometre-sized ECM scaffolds on cell surfaces via the formation of type I collagen fibres has been reported[16] These techniques can precisely control the thickness of the ECM between cells, multi-step manipulation is required to fabricate a thick ECM layer. The available types of ECMs are restricted, because this technique depends on the interaction between two types of ECM components or between integrin receptors and ECM components Another approach is encapsulating cells into an ECM gel by water-in-oil emulsion methods[17,18]. We hypothesized that the MC medium method can aggregate cells and ECM molecules simultaneously
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