No abstract

We report a method for preparing cell-laden hydrogel tubes. This method uses a coaxial double-orifice spinneret, simpler than triple-orifice spinnerets which have been used for preparing similar constructs. The intended application was to create a template for preparing filament-like structures composed of two heterogeneous living cell layers. An aqueous solution containing an alginate derivative possessing phenolic hydroxyl moieties (Alg-Ph), catalase and horseradish peroxidase (HRP) was extruded into an ambient flow of H(2)O(2) aqueous solution. This operation enabled the Alg-Ph solution to be gellable through a HRP-catalyzed reaction, cross-linking the Ph moieties together. By altering flow rates of the Alg-Ph and H(2)O(2) solutions along with the concentrations of catalase and H(2)O(2), the diameter and membrane thickness of the hydrogel tubes were controllable between 250-550 µm and 70-140 µm, respectively. The viability of the HeLa cells enclosed in the hydrogel tubes with a diameter of 300 µm and a membrane thickness of 80 µm was 95.4%. Subsequently, the enclosed HeLa cells grew and filled the hollow core. A filament-like structure of HeLa cells covered with a layer of fibroblast 10T1/2 cells was obtained when confluency of fibroblast 10T1/2 cells was reached and the hydrogel matrix was degraded with alginate lyase.

No abstract

A method for identifying each cell secreting reactive oxygen species (ROS) is highly desirable to advance the understanding of the physiological and pathological processes attributed to extracellular ROS. Here, we first report a method for realizing this. The individual cells secreting hydrogen peroxide (H2O2), a common ROS, could be coated by a hydrogel membrane through a horseradish peroxidase-catalyzed reaction consuming H2O2 secreted from the cells themselves. This hydrogel membrane coating was proved to be cytocompatible. In addition, the hydrogel membrane made from an alginate derivative could be removed on demand without causing damage to the enclosed cells. These results demonstrated the feasibility of the proposed method to be an effective tool in cellular ROS studies.

Multicellular tumor spheroids (MTS) are gaining increased recognition as valuable tools and key elements in anticancer drug discovery and tumor therapy test programs. However, the lack of reproducible and uniform MTS sizes is a major problem for pharmaceutical assays. Here, we show the usefulness of duplex microcapsules with a Ca-alginate gel membrane as a platform for producing MTS with a highly homogeneous size distribution. HeLa cells were enclosed with 86.9% viability within the microcapsules. The enclosed cells grew and formed MTS with the same size as the cavity of the microcapsules by arresting their growth with the microcapsule membrane. The cells in the resultant MTS had a higher proportion in G 0 ⁄ G 1 phase (71.2%) than 2-D cultured cells in the stationary phase (64.3%) or those in MTS formed on a nonadherent surface (65.3%) (P < 0.01). Furthermore, the cells in MTS formed within microcapsules showed higher tolerance to mitomycin C (1-1000 nM) and gemcitabine (4.5-4500 nM) than 2-D cultured cells (P < 0.01). In addition, the expression of MDR1, MCT1, HIF-1a, and GRP78 mRNA was 2.9-, 3.2-, 3.8-, and 5.5-fold higher, respectively, than those in 2-D cultured cells (P < 0.04). Cryopreserved encapsulated cells in the microcapsules showed 80.5% viability and formed MTS with a comparable tolerance of 100 and 1000 nM mitomycin C to those that were not cryopreserved (P > 0.09). These findings suggest the duplex microcapsule may be a promising tool for producing MTS for pharmaceutical applications. (Cancer Sci 2012; 103: 549-554) T hree-dimensional tumor cell constructs are increasingly recognized as valuable tools and key elements in anticancer drug discovery and tumor therapy test programs because their gene expression profiles reflect those of cancer cells in vivo more accurately than those of 2-D cultured cells.(1-3) The multicellular tumor spheroid (MTS) is a well-known 3-D tumor cell construct and various techniques have been reported for their preparation.(3) One conventional technique uses cultivation on dishes with non-adherent surfaces or hanging drop cultures.(1)One of the critical obstacles, however, is difficulty in preparing MTS with a well-defined size under conditions of sufficient medium renewals that are required for pharmaceutical assays with proper statistical analysis.(3) One of the solutions to this problem is to culture tumor cells in homogeneous microcapsules. (4,5) We recently reported an original duplex microcapsule suitable for mass production of spherical tissues with a narrow size distribution from a variety of cells.(6) The duplex microcapsules were prepared from gelatin and sodium alginate using a flow-focusing system. (6)(7)(8) The spherical tissues formed in the microcapsule can be harvested easily and safely when necessary for subsequent applications by liquefying the microcapsule membrane.The aim of this study was to evaluate the feasibility of duplex microcapsules as a platform for producing MTS for pharmaceutical assays. We used the HeLa human cervical cancer cell line an...