Biologically active artificial scaffolds for cell seeding are developed by mimicking extracellular matrices using synthetic materials. Here, we propose a feasible approach employing biocatalysis to integrate natural components, that is, gelatin and heparin, into a synthetic scaffold, namely a polyethylene glycol (PEG)-based hydrogel. Initiation of horseradish peroxidase-mediated redox reaction enabled both hydrogel formation of tetra-thiolated PEG via disulfide linkage and incorporation of chemically thiolated gelatin (Gela-SH) and heparin (Hepa-SH) into the polymeric network. We found that the compatibility of the type of gelatin with heparin was crucial for the hydrogelation process. Alkaline-treated gelatin exhibited superior performance over acid-treated gelatin to generate dual functionality in the resultant hydrogel originating from the two natural biopolymers. The Gela-SH/Hepa-SH dual functionalized PEG-based hydrogel supported both cellular attachment and binding of basic fibroblast growth factor (bFGF) under cell culture conditions, which increased the proliferation and phenotype transformation of NIH3T3 cells cultured on the hydrogel. Inclusion of bFGF and a commercial growth factor cocktail in hydrogel matrices effectively enhanced cell spreading and confluency of both NIH3T3 cells and HUVECs, respectively, suggesting a potential method to design artificial scaffolds containing active growth factors.

In this report, a strategy for constructing three-dimensional (3D) cellular architectures comprising viable cells is presented. the strategy uses a redox-responsive hydrogel that degrades under mild reductive conditions, and a confluent monolayer of cells (i.e., cell sheet) cultured on the hydrogel surface peels off and self-folds to wrap other cells. As a proof-of-concept, the self-folding of fibroblast cell sheet was triggered by immersion in aqueous cysteine, and this folding process was controlled by the cysteine concentration. Such folding enabled the wrapping of human hepatocellular carcinoma (HepG2) spheroids, human umbilical vein endothelial cells and collagen beads, and this process improved cell viability, the secretion of metabolites and the proliferation rate of the HepG2 cells when compared with a two-dimensional culture under the same conditions. A key concept of this study is the ability to interact with other neighbouring cells, providing a new, simple and fast method to generate higherorder cellular aggregates wherein different types of cellular components are added. We designated the method of using a cell sheet to wrap another cellular aggregate the 'cellular furoshiki'. the simple selfwrapping furoshiki technique provides an alternative approach to co-culture cells by microplate-based systems, especially for constructing heterogeneous 3D cellular microstructures.

Hydrogels possessing the ability to control cell functions have great potential as artificial substrates for cell culture. Herein, we report dual-functionalizable protein–polymer hybrid hydrogels prepared by thiol oxidation catalyzed by horseradish peroxidase and a phenolic molecule. A chimera protein of streptavidin (SA) and the SpyCatcher protein, with a cysteine residue at its N-terminus, (C-SA-SC) was constructed and co-cross-linked with thiol-functionalized four-arm polyethylene glycol (PEG-SH) to obtain hydrogels possessing two orthogonal conjugation moieties. Hydrogel formation using C-SA-SC conjugated with biotinylated or SpyTagged functional molecules (premodification strategy) resulted in the formation of hydrogels with a uniform distribution of the functional molecules. Postmodification of the functional molecules of the C-SA-SC hydrogel with biotin or SpyTag could alter the three-dimensional (3D) spatial distribution of the functional molecules within the hydrogels depending on the mode of conjugation (SA/biotin or SpyCatcher/SpyTag), the size of the functional molecules, and the length of time of the modification. NIH-3T3 cells cultured on a C-SA-SC hydrogel, dual-functionalized with a biotinylated-Arg-Gly-Asp-Ser (RGDS) peptide and a basic fibroblast growth factor (bFGF) with SpyTag, showed cell adhesion to the PEG-SH-based hydrogels and cell morphological changes in response to the immobilized RGDS peptide and the bFGF. Moreover, the cells showed higher proliferation on the dual-functionalized C-SA-SC hydrogel than the cells cultured on hydrogels without either the RGDS peptide or the bFGF, demonstrating the benefits of dual-functionalizable hydrogels. The C-SA-SC hydrogel presented in this study is capable of being orthogonally functionalized by two different functional molecules with different 3D distributions of each molecule within the hydrogel and thus has the potential for use as a cell culturing scaffold for creating artificial cellular microstructures.

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