Shojiro Katoh1,2, Hiroshi Yoshioka3, Shoji Suzuki4, Hiroyuki Nakajima5, Masaru Iwasaki6, Rajappa Senthilkumar7, Senthilkumar Preethy7, Samuel Jk Abraham6,7,8,9,10. 1. Edogawa Evolutionary Lab of Science, Edogawa Hospital Campus, 2-24-18, Higashi Koiwa, Edogawa-Ku, Tokyo, 133-0052, Japan. 2. Department of Orthopaedic Surgery, Edogawa Hospital, 2-24-18, Higashi Koiwa, Edogawa-Ku, Tokyo, 133-0052, Japan. 3. Mebiol Inc., 1-25-8, Nakahara, Hiratsuka, 254-0075, Kanagawa, Japan. 4. Department of Clinical Education, University of Yamanashi -Faculty of Medicine, 1110, Shimokato, Chuo, Yamanashi, 409-3898, Japan. 5. II Department of Surgery, University of Yamanashi -Faculty of Medicine, 1110, Shimokato, Chuo, Yamanashi, 409-3898, Japan. 6. Centre for Advancing Clinical Research (CACR), University of Yamanashi -Faculty of Medicine, 1110, Shimokato, Chuo, Yamanashi, 409-3898, Japan. 7. The Fujio-Eiji Academic Terrain (FEAT), Nichi-In Centre for Regenerative Medicine (NCRM), PB 1262, Chennai, 600034, Tamil Nadu, India. 8. The Mary-Yoshio Translational Hexagon (MYTH), Nichi-In Centre for Regenerative Medicine (NCRM), PB 1262, Chennai, 600034, Tamil Nadu, India. 9. JBM Inc., 3-1-14, Higashi Koiwa, Edogawa-Ku, Tokyo, 133-0052, Japan. 10. Antony- Xavier Interdisciplinary Scholastics (AXIS), GN Corporation Co. Ltd., 3-8, Wakamatsu, Kofu, Yamanashi, 400-0866, Japan.
Abstract
BACKGROUND: Chondrocytes are used in cell-based therapies such as autologous chondrocyte implantation (ACI) and matrix-associated cartilage implantation (MACI). To transport the cartilage tissue to the laboratory for in vitro culturing, phosphate-buffered saline (PBS), Euro-Collins solution (ECS) and Dulbecco's Modified Eagle's Medium (DMEM) are commonly employed at 4-8 °C. METHODS: In this study, eight samples of human cartilage biopsy tissues from elderly patients with severe osteoarthritis undergoing arthroscopy, which would otherwise have been discarded, were used. The cartilage tissue samples were compared to assess the cell yield between two transportation groups: i) a thermo-reversible gelation polymer (TGP) based method without cool preservation (∼25 °C) and ii) ECS transport at 4 °C. These samples were subjected to in vitro culture in a two-dimensional (2D) monolayer for two weeks and subsequently in a three-dimensional (3D) TGP scaffold for six weeks. RESULTS: The cell count obtained from the tissues transported in TGP was higher (0.2 million cells) than those transported in ECS (0.08 million cells) both after initial processing and after in vitro culturing for 2 weeks in 2D (18 million cells compared with 10 million cells). In addition, mRNA quantification demonstrated significantly higher expression of Col2a1 and SOX-9 in 3D-TGP cultured cells and lower expression of COL1a1 in RT-PCR, characteristic of the hyaline cartilage phenotype, than in 2D culture. CONCLUSION: This study confirms that the TGP cocktail is suitable for both the transport of human cartilage tissue and for in vitro culturing to yield better-quality cells for use in regenerative therapies.
BACKGROUND: Chondrocytes are used in cell-based therapies such as autologous chondrocyte implantation (ACI) and matrix-associated cartilage implantation (MACI). To transport the cartilage tissue to the laboratory for in vitro culturing, phosphate-buffered saline (PBS), Euro-Collins solution (ECS) and Dulbecco's Modified Eagle's Medium (DMEM) are commonly employed at 4-8 °C. METHODS: In this study, eight samples of human cartilage biopsy tissues from elderly patients with severe osteoarthritis undergoing arthroscopy, which would otherwise have been discarded, were used. The cartilage tissue samples were compared to assess the cell yield between two transportation groups: i) a thermo-reversible gelation polymer (TGP) based method without cool preservation (∼25 °C) and ii) ECS transport at 4 °C. These samples were subjected to in vitro culture in a two-dimensional (2D) monolayer for two weeks and subsequently in a three-dimensional (3D) TGP scaffold for six weeks. RESULTS: The cell count obtained from the tissues transported in TGP was higher (0.2 million cells) than those transported in ECS (0.08 million cells) both after initial processing and after in vitro culturing for 2 weeks in 2D (18 million cells compared with 10 million cells). In addition, mRNA quantification demonstrated significantly higher expression of Col2a1 and SOX-9 in 3D-TGP cultured cells and lower expression of COL1a1 in RT-PCR, characteristic of the hyaline cartilage phenotype, than in 2D culture. CONCLUSION: This study confirms that the TGP cocktail is suitable for both the transport of human cartilage tissue and for in vitro culturing to yield better-quality cells for use in regenerative therapies.
Authors: Ozan S Kumru; Sangeeta B Joshi; Dawn E Smith; C Russell Middaugh; Ted Prusik; David B Volkin Journal: Biologicals Date: 2014-07-01 Impact factor: 1.856