TECHNICAL NOTE Direct measurement of oxygen concentration inside cultured cartilage for relating to spatial growth of rabbit chondrocytes Masahiro Kino-oka, 1 Shogo Kagita, 2 Masrina Mohd Nadzir, 2 Hirofumi Inoue, 2 Katsura Sugawara, 3 and Masahito Taya 2, ⁎ Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita, Osaka 565-0871, Japan 1 Division of Chemical Engineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka 560- 8531, Japan 2 and Japan Tissue Engineering Co., Ltd., 6- 209- 1 Miyakita-dori, Gamagori, Aichi 443- 0022, Japan 3 Received 21 November 2009; accepted 19 March 2010 Available online 22 April 2010 In static culture of rabbit chondrocytes in collagen gel, the direct measurement of dissolved oxygen (DO) concentration revealed that the DO level at the top surface of gel decreased due to an increase in overall cell density with elapsed time. The local cell density at the top surface on day 21 was 5.7 × 10 7 cells/cm 3 , being 11 times that at the bottom of gel. This heterogeneity of cell distribution in the gel was considered to occur by limitation of oxygen supply into a deeper part of the gel. In shaking culture using a dish with gas-permeable film, the DO level was enhanced inside the gel and the overall cell density in the gel was achieved to be 2.9 times that in the static culture. © 2010, The Society for Biotechnology, Japan. All rights reserved. [Key words: Tissue engineering; Cultured cartilage; Chondrocytes; Dissolved oxygen measurement; Spatial cell distribution] Articular cartilage plays an important role in lubricating joint movement as an avascular and lymphatic tissue covering the surface of articular bone thinly, generally being less than 4 mm in thickness in vivo (1,2). Once the articular cartilage receives severe damage, it is hard to make spontaneous cure due to the lack of chondrocyte proliferative activity. Brittberg et al. (3) have established the autologous transplantation procedure using chondrocyte suspension through in vitro expansion, and made notable contribution to repairing defective parts. In this procedure, however, the chondrocyte expansion is conducted by two-dimensional (2-D) culture, causing low productivity of extracellular matrix (ECM). Ochi et al. (4) proposed the use of tissue-engineered construct prepared by culturing chondrocytes in collagen gel. The construct through the three-dimensional (3-D) culture leads to higher production of ECM (5). However, the 3-D environment is expected to be under the insufficient supply of nutrients to cells inside the construct, which results in the deterioration of cell growth (6). Dissolved oxygen (DO) is a key nutrient to govern the growth activity. Kellner et al. (7) indicated the distribution of DO in cylinderal cultured cartilage, concluding that insufficient DO supply inside cultured cartilage yielded an acellular region where the cell growth was severely suppressed. In previous work (8), we performed static culture of rabbit chondrocytes embedded in collagen gel according to the procedure reported by Ochi et al. (4), and the confocal laser scanning microscopy revealed the heterogeneous cell distribution with growth deterioration in a deeper region from top surface of gel, suggesting the lack of nutrients in this deeper region. In the present study, a direct measurement system to estimate DO level in the tissue- engineered construct was constructed using an optical fiber sensor, and the spatial distributions of cells and DO were analyzed. In addition, the culture condition was modified by use of a shaking vessel with gas-permeable bottom to examine the effect of improved oxygen supply on the distribution profile of the cells in the gel. For static culture of rabbit chondrocytes, the cells were embedded in 2.4% Atelocollagen ® gel (Koken Co., Tokyo) at seeding cell density of 2.0×10 6 cells/cm 3 . The gel placed on a 60 mm culture dish (Corning, Inc., Corning, NY, USA) was incubated statically at 37 °C under 5% CO 2 in air using medium as described elsewhere (8). Medium changes were conducted every two days. For shaking culture, the dish was put on a solid-geometrical rotary shaker (Mini shaker SHM-2001; LMS Co., Tokyo) at 20 rpm with an incline of 7°. A modified dish soled with gas-permeable film (Lumox TM dish; Greiner Bio-One, Frickenhausen, Germany) was employed for improving oxygen supply to the cells in the gel. For analyzing overall cell density in each gel (X), the triplicate samples were harvested, and the cells were suspended by the enzymatic digestion of gel, followed by direct cell counting on a hemocytometer (8). The local cell density in the gel (X ̂ ) was estimated using a confocal laser scanning microscope (FV300, Olympus, Tokyo) with a spatial cell distribution analyzer (SCDA) as shown previously (8). In brief, a gel specimen was applied to the double stainings for living cell cytoplasm by CellTracker TM Green CMFDA (Invitrogen, Carlsbad, CA, USA) and for nucleus by TO-PRO3 (Invitrogen). The stained specimen was subjected to the analysis of spatial cell distribution, and the custom-made software performed the quantitative determination of spatial cell positions in the Journal of Bioscience and Bioengineering VOL. 110 No. 3, 363 – 366, 2010 www.elsevier.com/locate/jbiosc ⁎ Corresponding author. Tel.: +81 06 6850 6251; fax: +81 06 6850 6254. E-mail address: taya@cheng.es.osaka-u.ac.jp (M. Taya). 1389-1723/$ - see front matter © 2010, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2010.03.009