Takahashi Tsuguharu, Ogasawara Toru, Kishimoto Junji, Liu Guangyao, Asato Hirotaka, Nakatsuka Takashi, Uchinuma Eijyu, Nakamura Kozo, Kawaguchi Hiroshi, Takato Tsuyoshi, Hoshi Kazuto
Department of MENICON Cartilage & Bone Regeneration, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-Ku, Tokyo 113-0033, Japan.
Division of Tissue Engineering, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-Ku, Tokyo 113-0033, Japan.
Cell Transplant. 2005 Oct;14(9):683-693. doi: 10.3727/000000005783982675.
Chondrocyte preparation with the safety and efficiency is the first step in cartilage regenerative medicine. To prepare a chondrocyte proliferation medium that does not contain fetal bovine serum (FBS) and that provides more than a 1000-fold increase in cell numbers within approximately 1 month, we attempted to use the medium containing 5% human serum (HS), but it exerted no more than twofold increase in 2 weeks. To compensate for the limited proliferation ability in HS, we investigated the combinational effects of 12 factors [i.e., fibroblast growth factor(FGF)-2, insulin-like growth factor(IGF)-I, insulin, bone morphogenetic protein-2, parathyroid hormone, growth hormone, dexamethasone, 1α25-dihydroxy vitamin D, L-3,3',5'-triodothyronine, interleukine-1 receptor antagonist, 17β-estradiol, and testosterone] on the proliferation of human auricular chondrocytes by analysis of variance in fractional factorial design. As a result, FGF-2, dexamethasone, insulin, and IGF-I possessed promotional effects on proliferation, while the combination of FGF-2 with insulin or IGF-I synergistically enhanced the proliferation. Actually, the chondrocytes increased 7.5-fold in number in 2 weeks in a medium containing 5% HS with 10 ng/ml FGF-2, while the cell number synergistically gained a 10-12-fold increase with 5 μg/ml insulin or 100 ng/ml IGF-I in the same period. The proliferation effects were more enhanced at a concentration of 100 ng/ml for FGF-2, and especially for the combination of 100 ng/ml FGF-2 and 5 μg/ml insulin (approximately 16-fold within 2 weeks). In the long-term culture with repeated passaging, this combination provided more than 10,000-fold within 8 weeks (i.e., passage 4). Thus, we concluded that such a combination of FGF-2 with insulin or IGF-I may be useful for promotion of auricular chondrocyte proliferation in a clinical application for cartilage regeneration.
制备安全且高效的软骨细胞是软骨再生医学的第一步。为了制备一种不含胎牛血清（FBS）且能在约1个月内使细胞数量增加1000倍以上的软骨细胞增殖培养基，我们尝试使用含有5%人血清（HS）的培养基，但在2周内其细胞数量增加不超过两倍。为了弥补HS中有限的增殖能力，我们通过析因设计的方差分析研究了12种因子[即成纤维细胞生长因子（FGF）-2、胰岛素样生长因子（IGF）-I、胰岛素、骨形态发生蛋白-2、甲状旁腺激素、生长激素、地塞米松、1α,25-二羟基维生素D、L-3,3',5'-三碘甲状腺原氨酸、白细胞介素-1受体拮抗剂、17β-雌二醇和睾酮]对人耳软骨细胞增殖的联合作用。结果，FGF-2、地塞米松、胰岛素和IGF-I对增殖具有促进作用，而FGF-2与胰岛素或IGF-I的组合协同增强了增殖。实际上，在含有5%HS和10 ng/ml FGF-2的培养基中，软骨细胞在2周内数量增加了7.5倍，而在同一时期，加入5 μg/ml胰岛素或100 ng/ml IGF-I时，细胞数量协同增加了10 - 12倍。FGF-2浓度为100 ng/ml时增殖作用更强，特别是100 ng/ml FGF-2与5 μg/ml胰岛素的组合（2周内约增加16倍）。在长期传代培养中，这种组合在8周内（即第4代）使细胞数量增加超过10000倍。因此，我们得出结论，FGF-2与胰岛素或IGF-I的这种组合可能有助于在软骨再生的临床应用中促进耳软骨细胞增殖。
