This article describes the preparation of cell-enclosing hyaluronic acid (HA) microparticles with solid core and microcapsules with liquid core through cell-friendly horseradish peroxidase (HRP)-catalyzed hydrogelation. The spherical vehicles were made from HA derivative possessing phenolic hydroxyl moieties (HA-Ph) cross-linkable through the enzymatic reaction by extruding cellsuspending HA-Ph aqueous solution containing HRP from a needle of 180 lm in inner diameter into the ambient coaxial flow of liquid paraffin containing H 2 O 2 in a microtubule of 600 lm in diameter. By altering the flow rate of liquid paraffin, the diameters of gelatin and HA-Ph microparticles were varied in the range of 120-220 lm and 100-300 lm, respectively. The viability of the enclosed human hepatoma HepG2 cells in the HA-Ph microparticles of 180 lm in diameter was 94.2 6 2.3%. The growth of the enclosed HepG2 cells was enhanced by decreasing the HRP concentration. The microcapsules of 200 lm in diameter were obtained by extruding HA-Ph aqueous solution containing thermally liquefiable cell-enclosing gelatin microparticles of 150 lm in diameter using the same microfluidic system. The enclosed cells grew and filled the cavity within 10 days. Spherical tissues covered with a heterogeneous cell layer were obtained by degrading the microcapsule membrane using hyaluronidase after covering the surface with a heterogeneous cell layer.

We employed an ozone‐induced grafting process to prepare membrane‐hydrogel hybrid biomaterials. Exposure of isotactic polypropylene to ozone resulted in surface oxidation, as detected by ESCA, and the formation of peroxides and hydroperoxides. The amount of oxygen‐bearing moieties, as detected by FT–IR, was increased when a microporous membrane with a large surface area was used. Ozonation for an extended period of time, 1–2 h, resulted in a degradation of microporous PP, observed with SEM as an enlargement of pores and brittle characteristics of the material. The molecular weight of PP was dramatically reduced after as little as 5 min of ozonation. Exposure to ozone for longer periods of time contributed to further reductions of the molecular weight and gradual modification of chemical composition of polypropylene, restricted, however, to the surface or intercrystalline amorphous regions. It was possible to graft 2‐hydroxyethylmethacrylate (HEMA) to the ozonated samples, such that the graft copolymer acted as continiuous matrix consequently linked to and reinforced by the PP crystals. © 1997 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 35: 1461–1467, 1997

The usefulness of cell-enclosing microcapsules in biomedical and biopharmaceutical fields is widely recognized. In this study, we developed a method enabling the preparation of microcapsules with a liquid core in one step using two enzymatic reactions, both of which consume H2 O2 competitively. The microcapsule membrane prepared in this study is composed of the hydrogel obtained from an alginate derivative possessing phenolic hydroxyl moieties (Alg-Ph). The cell-enclosing microcapsules with a hollow core were obtained by extruding an aqueous solution of Alg-Ph containing horseradish peroxidase (HRP), catalase, and cells into a co-flowing stream of liquid paraffin containing H2 O2 . Formation of the microcapsule membrane progressed from the surface of the droplets through HRP-catalyzed cross-linking of Ph moieties by consuming H2 O2 supplied from the ambient liquid paraffin. A hollow core structure was induced by catalase-catalyzed decomposition of H2 O2 resulting in the center region being at an insufficient level of H2 O2 . The viability of HeLa cells was 93.1% immediately after encapsulation in the microcapsules with about 250 µm diameter obtained from an aqueous solution of 2.5% (w/v) Alg-Ph, 100 units mL(-1) HRP, 9.1 × 10(4) units mL(-1) catalase. The enclosed cells grew much faster than those in the microparticles with a solid core. In addition, the thickness of microcapsule membrane could be controlled by changing the concentrations of HRP and catalase in the range of 13-48 µm. The proposed method could be versatile for preparing the microcapsules from the other polymer derivatives of carboxymetylcellulose and gelatin.

We report a method for preparation of mammalian cell-enclosing hydrogel particles through horseradish peroxidase (HRP)-catalysed hydrogelation by dropping cell-suspending aqueous solution into an aqueous coagulation solution. An aqueous solution of 10% (w/v) gelatin derivative possessing phenolic hydroxyl (Ph) moieties (Gelatin-Ph), HepG2 cells and 10 U/mL HRP was dropped into an aqueous coagulation solution containing 1 mM H2O2. The resultant hydrogel formed through the HRP-catalysed reaction consuming H2O2 had a spherical shape. The sphericity decreased with decreasing concentrations of Gelatin-Ph, HRP and H2O2. The thickness of the hydrogel membrane layer of the hydrogel particles could be controlled by altering incubation time in the H2O2 solution. The cells encapsulated in the particles with a thinner hydrogel membrane grew faster. These results demonstrate that we successfully established the method of cell-encapsulation in hydrogel particles based on dropping aqueous polymer solution into aqueous coagulation solution through HRP-catalysed reaction.

Cancer stem-like cells (CSCs) are rare subpopulations of cancer cells. The development of three-dimensional tissues abundant in CSCs is important to both the understanding and establishment of novel therapeutics targeting them. Here, we describe the fabrication of multicellular tumor spheroids (MTSs) abundant in CSCs by employing alginate microcapsules with spherical cavities templated by cell-enclosing gelatin microparticles. Encapsulated human pancreatic cancer cell line PANC-1 cells grew for 14 days until they filled the cavities. The percentage of cells expressing reported CSC markers CD24, CD44, and epithelial-specific antigen (ESA), increased during this growth period. The percentage at 24 days of incubation, 22%, was 1.6 times higher than that of MTSs formed on a nonadherent surface in the same period of incubation. The MTSs in microcapsules could be cryopreserved in liquid nitrogen using a conventional method. No significant difference in the content of CSC marker-expressing cells was detected at 3 days of incubation when thawed after cryopreservation for 2 weeks, compared with cells incubated without prior cryopreservation.

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Cell encapsulation has been investigated as a bioproduction system in the biomedical and pharmaceutical fields. We encaps-ulated human induced pluripotent stem (hiPS) cells in duplex microcapsules prepared from an alginate derivative possessing phenolic hydroxyl moieties, in a single-step procedure based on two competing enzymatic reactions catalyzed by horseradish peroxidase (HRP) and catalase. The encapsulated cells maintained 91.4% viability and proliferated to fill the microcapsules following 19 days of culture. Encapsulated hiPS cells showed pluripotency comparable to that of unencapsulated cells during the cultures, as demonstrated by the expression of the SSEA-4 marker.

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