Aiming for the clinical application of cartilage regeneration, a culture method for mesenchymal stem cells (MSCs) derived from human bone marrow to obtain scaffold-free cartilage-like disk-shaped sheet of uniform sizes without the shrinkage was investigated. A disk-shaped cell sheet having the same diameter as that of the membrane without the shrinkage was formed after the cultivation of MSCs (18.6 x 10(5) cells/well) for 3 weeks in a cell culture insert (CCI) containing a flat membrane whose porosity was 12%, while 6.2 and 31.0 x 10(5) MSCs/well, respectively, resulted in the shrinkage of the aggregate and the hole formation in the center part of the sheet. Cell aggregates shrunk also in a 96-well plate and CCIs having lower porosity. The disk-shaped cell sheet showed the comparable thickness (1.2 mm) and sulfated glycosaminoglycan (sGAG) density to those of the pellet formed in a pellet culture. The gene expression levels of aggrecan and type II collagen in the disk-shaped cell sheet were not lower than those in the pellet. In conclusion, the usage of CCI having 12% porosity and 18.6 x 10(5) MSCs/well could avoid the shrinkage from the formation of the scaffold-free cartilage-like disk-shaped cell sheet. (C) 2010, The Society for Biotechnology, Japan. All rights reserved.