Kousuke Moriyama

Enzymatic preparation of a redox-responsive hydrogel for encapsulating and releasing living cells

Horseradish peroxidase-mediated oxidative cross-linking of a thiolated poly(ethylene glycol) is promoted in the absence of exogenous hydrogen peroxide, by adding a small amount of a phenolic compound under physiological conditions. The prepared hydrogel can encapsulate and release living mammalian cells.

Control of cellular adhesiveness in an alginate-based hydrogel by varying peroxidase and H2O2 concentrations during gelation

An aqueous solution of alginate possessing phenolic hydroxyl (Alg-Ph) groups is gellable via a horseradish peroxidase (HRP)-catalyzed oxidative crosslinking reaction between Ph groups, consuming H(2)O(2) as an electron acceptor. This study evaluates the effect of H(2)O(2) and HRP concentrations on cellular adhesiveness and proliferation on the resultant enzymatically crosslinked Alg-Ph gels. After 4h of seeding, 81.1% of L929 fibroblast cells adhere to an Alg-Ph hydrogel prepared with 1 U ml(-1) HRP and 1mM H(2)O(2). Increasing the concentration of H(2)O(2) to 15 mM decreases the percentage of adhering cells to 28.4%. The cellular adhesion at this H(2)O(2) concentration is increased to 82.6% by increasing the HRP concentration to 10 U ml(-1). The cells adhering to the Alg-Ph hydrogels with higher cellular adhesiveness establish a confluent monolayer during 168 h of culture. A cell sheet can then be harvested within 5 min of immersion in a medium containing alginate lyase at 1.0 mg ml(-1). The harvested cell sheet re-adhere, and the cells contained in the sheet proliferate after being transferred to another cell culture dish.

Immobilization of alkaline phosphatase on magnetic particles by site-specific and covalent cross-linking catalyzed by microbial transglutaminase

Bacterial alkaline phosphatase (BAP) was site-specifically and covalently immobilized on magnetic particles (MPs) using the enzymatic reaction of microbial transglutaminase (MTG). Immobilization efficiency was affected by the chemical surface treatment of MPs and immobilized BAP exhibited more than 90% of the initial activity after 10 rounds of recycling.

Hematin is an Alternative Catalyst to Horseradish Peroxidase for In Situ Hydrogelation of Polymers with Phenolic Hydroxyl Groups In Vivo

Hematin, an iron-containing porphyrin used in the management of porphyria attacks, was evaluated as an alternative catalyst to horseradish peroxidase (HRP) for in situ gelation of polymers with phenolic hydroxyl (Ph) moieties in vivo. An aqueous solution of gelatin derivative with Ph moieties was gellable in the presence of both hematin and H2O2. A total of 98.6% adhesion of L929 fibroblast cells 4 h after seeding and their similar morphology to those on substrate coated with unmodified gelatin indicated no obvious substrate cytotoxicity. High cytocompatibility of the gelation process under conditions inducing gelation within 20 s was demonstrated by 95.0% viability of enclosed cells in vitro. Furthermore, no adverse effects of hematin were found compared with HRP by histological observation of cutaneous tissue surrounding in situ formed gels. The versatility of hematin for gelation of a variety of polymers possessing Ph groups was demonstrated by the gelation of a carboxymethyl cellulose derivative.

Enzymatically prepared redox-responsive hydrogels as potent matrices for hepatocellular carcinoma cell spheroid formation

Cellular spheroids have been received much attention in the biological and biomedical fields, especially as a base material for drug assays, regenerative medicine, and tissue engineering. Hydrogels have potential for scalable preparation of spheroids because they provide a spatial environment suitable for three‐dimensional cell cultivation. Herein, the potential use of a redox‐responsive hydrogel as a scaffold for preparation and recovery of spheroids is reported. A hydrogel composed of poly(ethylene glycol) (PEG), which can be degraded using cysteine as a reducing agent under mild conditions, is prepared by mixing an octa‐thiolated PEG derivative (8‐arm PEG‐SH), horseradish peroxidase and a small phenolic compound (Glycyl‐L‐tyrosine). Human hepatocellular carcinoma cells (HepG2) are encapsulated in the hydrogel and cellular spheroids formed by proliferation within the scaffolds. After seven days of cultivation, the size of the HepG2 spheroids reached a diameter between ≈40 and 60 μm, depending on the 8‐arm PEG‐SH concentration. Liver‐specific functions of the HepG2 spheroids such as albumin secretion and urea production are retained at higher levels than those of cells prepared from traditional two‐dimensional mono layers. These results suggest that the system presented here has potential for preparation of cellular spheroids for tissue engineering applications.

Peptide Tag-Induced Horseradish Peroxidase-Mediated Preparation of a Streptavidin-Immobilized Redox-Sensitive Hydrogel

Several methods have recently been reported for the preparation of redox-sensitive hydrogels using enzymatic reactions, which are useful for encapsulating sensitive materials such as proteins and cells. However, most of the reported hydrogels is difficult to add further function efficiently, limiting the application of the redox-sensitive hydrogels. In this study, peptide sequences of HHHHHHC and GGGGY (Y-tag) were genetically fused to the N- and C-termini of streptavidin (C-SA-Y), respectively, and C-SA-Y was mixed with horseradish peroxidase and thiol-functionalized 4-arm polyethylene glycol to yield a redox-sensitive C-SA-Y immobilized hydrogel (C-SA-Y gel). The C-SA-Y immobilized in the hydrogel retained its affinity for biotin, allowing for the incorporation of proteins and small molecules to hydrogel via biotin. C-SA-Y gel was further prepared within a water-in-oil (w/o) emulsion system to yield a nanosized hydrogel, to which any intracellular and cytotoxic agent can be modified, making it a potential drug delivery carrier.

Enzyme-mediated preparation of hydrogels composed of poly(ethylene glycol) and gelatin as cell culture platforms

A redox-responsive hydrogel composed of poly(ethylene glycol) and gelatin was prepared via an enzymatic oxidative reaction. Fibroblast cells adhered on the hydrogel showed proliferation and they could be recovered as a cell sheet by degradation of the scaffold under mild reductive conditions, thus providing a novel platform for cell culture.

Controlling thermo-reversibility of gelatin gels through a peroxidase-catalyzed reaction under mild conditions for mammalian cells.

A variety of cross-linking methods is used for obtaining gelatin gels having a tolerance to thermo-reversible gel–sol transition at physiological temperature. In this paper, we investigated the applicability of horseradish peroxidase-catalyzed cross-linking of tyrosine residues originally contained in native gelatin molecules for preparing such gelatin gels. The gelatin gels obtained through exposure to the enzymatic reaction showed a higher resistance to thermo-reversibility at 37°C than gels obtained through a thermally-induced gelation alone. In addition, the resistance property to thermo-reversible gel–sol transition was tunable by controlling enzymatic reaction conditions: higher peroxidase concentration and thermally-induced pre-gelation accomplished by cooling the gelatin solution prior to the enzymatic reaction produced gels with higher resistance to thermo-reversibility. Fibroblast cells enclosed in the gelatin gels obtained through the enzymatic reaction with thermally-induced pre-gelation showed 93% viability. These results demonstrate the feasibility of peroxidase-catalyzed reaction for obtaining gelatin gels having a tolerance to thermo-reversible gel-to-sol transition at physiological temperature toward applications in biomedical and biopharmaceutical fields.

Persistent random deformation model of cells crawling on a gel surface

In general, cells move on a substrate through extension and contraction of the cell body. Though cell movement should be explained by taking into account the effect of such shape fluctuations, past approaches to formulate cell-crawling have not sufficiently quantified the relationship between cell movement (velocity and trajectory) and shape fluctuations based on experimental data regarding actual shaping dynamics. To clarify this relationship, we experimentally characterized cell-crawling in terms of shape fluctuations, especially extension and contraction, by using an elasticity-tunable gel substrate to modulate cell shape. As a result, an amoeboid swimmer-like relation was found to arise between the cell velocity and cell-shape dynamics. To formulate this experimentally-obtained relationship between cell movement and shaping dynamics, we established a persistent random deformation (PRD) model based on equations of a deformable self-propelled particle adopting an amoeboid swimmer-like velocity-shape relationship. The PRD model successfully explains the statistical properties of velocity, trajectory and shaping dynamics of the cells including back-and-forth motion, because the velocity equation exhibits time-reverse symmetry, which is essentially different from previous models. We discuss the possible application of this model to classify the phenotype of cell migration based on the characteristic relation between movement and shaping dynamics.

Dynamically controlled construction of microstructures based on photo-induced phase transition of a spirobenzopyran-modified polymer solution

Dynamically controlled construction of microstructures was demonstrated using a spirobenzopyran-containing poly(N-isopropylacrylamide). Under patterned light irradiation, the polymer accumulated at the irradiated area from a uniformly dissolved state and formed micro-patterned structures based on phase transition. Furthermore the microstructures were collapsed due to redissolution of the precipitated polymer when light irradiation was stopped.