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...

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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.