Redox-responsive functionalized hydrogel marble for the generation of cellular spheroids

https://doi.org/10.1016/j.jbiosc.2020.05.010Get rights and content

Liquid marbles (LMs) have recently shown a great promise as microbioreactors to construct self-supported aqueous compartments for chemical and biological reactions. However, the evaporation of the inner aqueous liquid core has limited their application, especially in studying cellular functions. Hydrogels are promising scaffolds that provide a spatial environment suitable for three-dimensional cell culture. Here, we describe the fabrication of redox-responsive hydrogel marbles (HMs) as a three-dimensional cell culture platform. The HMs are prepared by introducing an aqueous mixture of a tetra-thiolated polyethylene glycol (PEG) derivative, thiolated gelatin (Gela-SH), horseradish peroxidase, a small phenolic compound, and human hepatocellular carcinoma cells (HepG2) to the inner aqueous phase of LMs. Eventually, HepG2 cells are encapsulated in the HMs then immersed in culture media, where they proliferate and form cellular spheroids. Experimental results show that the Gela-SH concentration strongly influences the physicochemical and microstructure properties of the HMs. After 6 days in culture, the spheroids were recovered from the HMs by degrading the scaffold, and examination showed that they had reached up to about 180 μm in diameter depending on the Gela-SH concentration, compared with 60 μm in conventional HMs without Gela-SH. After long-term culture (over 12 days), the liver-specific functions (secretion of albumin and urea) and DNA contents of the spheroids cultured in the HMs were elevated compared with those cultured in LMs. These results suggest that the developed HMs can be useful in designing a variety of microbioreactors for tissue engineering applications.

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Materials

PTE-200 SH (Sunbright) (4-arm PEG-((CH2)2-SH)4, Mw 20 kDa) was purchased by the NOF Corporation (Tokyo, Japan). Gelatin type A and poly(tetrafluoroethylene) (PTFE; particle size: 1 μm) were supplied by Sigma–Aldrich (St. Louis, MO, USA). Horseradish peroxidase (HRP; activity: 100 unit/mg) was supplied by Wako Pure Chemical Industries (Osaka, Japan). Glycyl-l-tyrosine hydrate (Gly–Tyr), l-cysteine (Cys), 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC), and squalene oil were acquired by

Physicochemical properties of hydrogel marbles prepared by HRP catalysis under various Gela-SH concentrations

The physicochemical properties of HMs depend on the intrinsic properties of the polymer backbone. The HMs were prepared by incorporating four components (4-arm PEG-SH, Gela-SH, HRP, and Gly–Tyr) for hydrogelation and covering a droplet of this solution in a superhydrophobic powder (Fig. 1A and B, i). The HMs can be produced with any desired volume between 10 and 50 μL (Fig. 1B, ii), which results in HMs with diameters around 2.5–4.5 mm. Drops with large volumes (50 μL) of aqueous polymer tended

Discussion

Many approaches have been proposed for the fabrication of 3D cellular spheroids (25, 26, 27, 28, 29). Among them, hydrogels have been the most widely studied as 3D cell culture in the past decade as they enable biomimetic extracellular matrix (30). Normally, the hydrogelation process requires hydrogen peroxide (H2O2) for HRP-catalyzed gelation. However, too much H2O2 may negatively impact the biological components in the hydrogel system (21). Therefore, in previous studies, we developed a novel

Acknowledgments

This work was supported by the Japan Society for the Promotion of Science, JSPS KAKENHI (grant numbers JP17K19016 and JP19H00841 to N.K. and JP16H06369 to M.G.). The authors would like to thank Mr. Yusei Hamada for his essential help and technical support in the initial stage of this research. We also thank Stephanie Knowlton, PhD, from Liwen Bianji, Edanz Editing China, for editing the English text of a draft of this manuscript.

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