Aggregate culture of human induced pluripotent stem cells (hiPSCs) is a promising method to obtain high number of cells for cell therapy applications. This study quantitatively evaluated the effects of initial cell number and culture time on the growth of hiPSCs in the culture of single aggregate. Small size aggregates ((1.1 +/- 0.4) x 10(1) (2.8 +/- 0.5) x 10(1) cells/aggregate) showed a lower growth rate in comparison to medium size aggregates ((8.8 +/- 0.8) x 10(1) (6.8 +/- 1.1) x 10(2) cells/aggregate) during early-stage of culture (24-72 h). However, when small size aggregates were cultured in conditioned medium, their growth rate increased significantly. On the other hand, large size aggregates ((1.1 +/- 0.2) x 10(3) (3.5 +/- 1.1) x 10(3) cells/aggregate) showed a lower growth rate and lower expression level of proliferation marker (ki-67) in the center region of aggregate in comparison to medium size aggregate during early-stage of culture. Medium size aggregates showed the highest growth rate during early-stage of culture. Furthermore, hiPSCs proliferation was dependent on culture time because the growth rate decreased significantly during late-stage of culture (72-120 h) at which point collagen type I accumulated on the periphery of aggregate, suggesting blockage of diffusive transport of nutrients, oxygen and metabolites into and out of the aggregates. Consideration of initial cell number and culture time are important to maintain balance between autocrine factors secretion and extracellular matrix accumulation on the aggregate periphery to achieve optimal growth of hiPSCs in the culture of single aggregate. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.