Comprehension of terminal differentiation and dedifferentiation of chondrocytes during passage cultures Masrina Mohd Nadzir, 1 Masahiro Kino-oka, 2 Nao Maruyama, 1 Yasuaki Sato, 1 Mee-Hae Kim, 2 Katsura Sugawara, 3 and Masahito Taya 1, ⁎ Division of Chemical Engineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka 560-8531, Japan, 1 Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita, Osaka 565-0871, Japan, 2 and Japan Tissue Engineering Co., Ltd., 6-209-1 Miyakita-dori, Gamagori, Aichi 443-0022, Japan 3 Received 1 December 2010; accepted 16 June 2011 Available online 21 July 2011 A high density collagen type I coated substrate (CL substrate) was used to evaluate the chondrocyte phenotypes in passaged cultures. With increasing age of cell population (population doubling (PD)=0–14.5), the frequency of non-dividing spindle shaped cells without ALP activity increased, accompanied with an increase in gene expression of collagen type I, meaning the senescence of dedifferentiated cells. At the middle age of cell population (PD = 5.1 and 6.6), the high frequency of polygonal shaped cells with ALP activity existed on the CL substrate together with up-regulated expressions of collagen types II and X, indicating the terminal differentiation of chondrocytes. When the chondrocytes passaged up to the middle age were embedded in collagen gel, the high frequency of single hypertrophic cells with collagen type II formation was recognized, which supports the thought that the high gene expression of collagen type II was attributed to terminal differentiation rather than redifferentiation. These results show that the CL substrate can draw out the potential of terminal differentiation in chondrocytes, which is unattainable by a polystyrene surface, and that the CL substrate can be a tool to evaluate cell quality in three-dimensional culture with the collagen gel. © 2011, The Society for Biotechnology, Japan. All rights reserved. [Key words: Tissue engineering; Rabbit chondrocytes; Terminal differentiation; Passage cultures; Collagen-coated substrate; Cultured cartilage] In reconstructive surgery for repairing articular cartilage defects, serial monolayer cultures of isolated chondrocytes are performed for expanding cells so as to be sufficient for subsequent tissue cultures. One of the drawbacks in this approach is the partial or complete loss of proliferative ability with increasing passage number of subculture (1), which leads to hindering spatial growth in a scaffold typically employed for tissue reconstruction. The decrease in proliferating vitality of chondrocytes in vitro has been associated with cellular senescence due to aging toward terminal differentiation. The progression of chondrocytes toward terminal differentiation is characterized by prolonged state of cell cycle arrest with significant increase in apparent cellular volume as well as with enhancement of collagen type X synthesis and alkaline phosphatase activity (2–4). However, the transition toward terminal differentiation in serial monolayer cultures for cell expansion of chondrocytes is still unclear and the effort to study its progression in three-dimensional (3-D) cultures has been hampered by lack of a suitable in vitro model. An alternative is to use a two-dimensional (2-D) culture system for the evaluation of cell behavior. However, it is well-known that chondrocytes dedifferentiate when grown in vitro on a traditional tissue culture polystyrene (PS) surface in a way of monolayer manner, acquiring a fibroblastic-like morphology, and that instead of the cartilage-specific collagen (collagen type II), they synthesize the collagen type I (5,6). Despite the fact that chondrocytes in suspension and pellet cultures have potential to undergo terminal differentiation (7–9), the limitation for single cell analysis in these systems prevents thorough understanding of the cell phenotypes and the heterogeneity in the cell population. The previous findings have resolved this dilemma by modifying a conventional PS surface by coating with high-density collagen type I (CL substrate). The CL substrate provides a 3-D mimicking environ- ment to chondrocytes, and enabled to evaluate the cell states for dedifferentiation in a quantitative manner (10). Recent studies have demonstrated that the cell morphology indicates the states of rabbit chondrocytes, and on the CL substrate the morphological change from round to stretch shape was observed during serial subcultures with higher mRNA expression of collagen type I, suggesting that the cell morphology can offer an indicator for chondrogenic potency during dedifferentiation process (11). The time-lapse observation of each single cell yields a wealth of quantifiable data on cellular properties such as changes in cell morphology, adhesion, migration and cell division pattern. Another feature for time-lapse experiment allows the movie to be rewound, thus one can survey the morphological and behavioral properties of the cells which were targeted at the end of culture, in a backward Journal of Bioscience and Bioengineering VOL. 112 No. 4, 395 – 401, 2011 www.elsevier.com/locate/jbiosc ⁎ Corresponding author. Tel.: +81 (0)6 6850 6251; fax: +81 (0)6 6850 6254. E-mail address: taya@cheng.es.osaka-u.ac.jp (M. Taya). 1389-1723/$ - see front matter © 2011, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2011.06.005