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. (c) 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1071-1076, 2015