Hollow Fiber Module Applied for Effective Proliferation and Harvest of Cultured Chondrocytes

Yoshiyuki Mori, Makoto Watanabe, Sakae Nakagawa, Yukiyo Asawa, Satoru Nishizawa, Kazumi Okubo, Hideto Saijo, Satoru Nagata, Yuko Fujihara, Tsuyoshi Takato, Kazuto Hoshi

Department of Cartilage & Bone Regeneration (Fujisoft), Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
Department of Cartilage & Bone Regeneration (Fujisoft), Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; Division of Tissue Engineering, The University of Tokyo Hospital, Tokyo, Japan.
Department of Sensory and Motor System Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
Department of Sensory and Motor System Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; Department of Cartilage & Bone Regeneration (Fujisoft), Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; Division of Tissue Engineering, The University of Tokyo Hospital, Tokyo, Japan.
Department of Sensory and Motor System Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; Division of Tissue Engineering, The University of Tokyo Hospital, Tokyo, Japan.
NAGATA Microtia and Reconstructive Plastic Surgery Clinic, Saitama, Japan.
Nomura Unison Co., Ltd, Nagano, Japan;.

DOI: 10.4236/msa.2013.48A008                

Abstract

Steady and useful culture for chondrocytes is essential for cartilage regenerative medicine. However, in conventional plate culture, the chondrocytes become dedifferentiated and lose their ability to make cartilage matrices. Three-dimensional culture mimicking the physiological environment in native chondrocytes is useful to maintain the chondrocyte properties during the proliferation culture. However, the three-dimensional culture is practically a hard task due to difficult harvest of the cells. Thus, we attempted to apply porous materials, hollow fibers for the three-dimensional culture, and developed their module to realize the effective harvest of the cells. Polyethersulfone-based hollow fibers, whose safety and cell affinity were confirmed by the experiment of the coculture with human chondrocytes, were collected to fabricate a module. The hollow fiber module was installed with screw ends, and enabled the easy removal of chondrocytes from the inner unit. Cultured human chondrocytes embedded within collagen hydrogel were put into the outer lumen of the hollow fiber module, while chondrocyte prolfieration medium was perfused through the inner lumen at 0 to 30 mL/min. After 2 weeks’ culture, the flow rate of 3 to 10 mL/min effectively supported the chondrocyte proliferation. Then, long-term culture using the hollow fiber module at flow rate of 5 mL/min was performed, revealing that the cell growth in this module at 3 weeks was approximately twice larger than that in static culture. The numbers of viable cells could be maintained by week 7. The hollow fiber module installed with screw ends can effectively culture and harvest the chondrocytes.

Keywords

Hollow Fiber Module; Proliferation; Cell Culture; Chondrocyte; Tissue Engineering

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	S. MacNeil, “Progress and Opportunities for Tissue-Engineered Skin,” Nature, Vol. 445, 2007, pp. 874-880. doi:10.1038/nature05664
[2]	Y. Z. Jiang, S. F. Zhang, Y. Y. Qi, L. L. Wang and H. W. Ouyang, “Cell Transplantation for Articular Cartilage Defects: Principles of Past, Present, and Future Practice,” Cell Transplant, Vol. 20, 2011, pp. 593-607. doi:10.3727/096368910X532738
[3]	K. von der Mark, V. Gauss, H. von der Mark and P. Muller, “Relationship between Cell Shape and Type of Collagen Synthesised as Chondrocytes Lose Their Cartilage Phenotype in Culture,” Nature, Vol. 267, 1977, pp. 531-532. doi:10.1038/267531a0
[4]	G. Kretzmer, “Industrial Processes with Animal Cells,” Applied Microbiology and Biotechnology, Vol. 59, No. 3, 2002, pp. 135-142. doi:10.1007/s00253-002-0991-y
[5]	L. Gillberger, “Emerging Trends in Plasma-Free Manufacturing of Recombinant Protein Therapeutics Expressed in Mammalian Cells,” Biotechnology Journal, Vol. 4, No. 2, 2009, pp. 186-201. doi:10.1002/biot.200800241
[6]	M. Ochi, Y. Uchio, K. Kawasaki, S. Wakitani and J. Iwasa, “Transplantation of Cartilage-Like Tissue Made by Tissue Engineering in the Treatment of Cartilage Defects of the Knee,” The Journal of Bone & Joint Surgery British, Vol. 84, No. 4, 2002, pp. 571-578. doi:10.1302/0301-620X.84B4.11947
[7]	T. Takahashi, T. Ogasawara, Y. Asawa, Y. Mori, E. Uchinuma, T. Takato and K. Hoshi, “Three-Dimensional Microenvironments Retain Chondrocyte Phenotypes during Proliferation Culture,” Tissue Engineering, Vol. 13, No. 7, 2007, pp. 1583-1592. doi:10.1089/ten.2006.0322
[8]	K. Yonenaga, S. Nishizawa, Y. Fujihara, Y. Asawa, S. Kanazawa, S. Nagata, T. Takato and K. Hoshi, “The Optimal Conditions of Chondrocyte Isolation and Its Seeding in the Preparation for Cartilage Tissue Engineering,” Tissue Eng Part C Methods, Vol. 16, No. 6, 2010, pp. 1461-1469. doi:10.1089/ten.tec.2009.0597
[9]	Y. Tanaka, T. Ogasawara, Y. Asawa, H. Yamaoka, S. Nishizawa, Y. Mori, T. Takato and K. Hoshi, “Growth Factor Contents of Autologous Human Sera Prepared by Different Production Methods and Their Biological Effects on Chondrocytes,” Cell Biology International, Vol. 32, No. 5, 2008, pp. 505-514. doi:10.1016/j.cellbi.2007.12.012
[10]	T. Takahashi, T. Ogasawara, J. Kishimoto, G. Liu, H. Asato, T. Nakatsuka, E. Uchinuma, K. Nakamura, H. Kawaguchi, T. Takato and K. Hoshi, “Synergistic Effects of FGF-2 with Insulin or IGF-I on the Proliferation of Human Auricular Chondrocytes,” Cell Transplant, Vol. 14, No. 9, 2005, pp. 683-693. doi:10.3727/000000005783982675
[11]	K. Yonenaga, S. Nishizawa, M. Akizawa, Y. Asawa, Y. Fujihara, T. Takato and K. Hoshi, “Utility of NucleoCounter for the Chondrocyte Count in the Collagenase Digest of Human Native Cartilage,” Cytotechnology, Vol. 62, No. 6, 2010, pp. 539-545. doi:10.1007/s10616-010-9304-y
[12]	S. Satoh, J. Kobayashi, J. Mizoguchi, M. Nogawa and M. Otani, “Serum-Free Cultivation of Anchorage-Dependent Cells on Microcarrier: Effective Production of Human Macrophage Colony-Stimulating Factor,” Cytotechnology, Vol. 5, No. 2, 1991, pp. 95-114. doi:10.1007/BF00573882
[13]	J. Wu, “Mechanisms of Animal Cell Damage Associated with Gas Bubbles and Cell Protection by Medium Additives,” Journal of Biotechnology, Vol. 43, No. 2, 1995, pp. 81-94. doi:10.1016/0168-1656(95)00133-7
[14]	B. G. Boedeker, “Production Processes of Licensed Recombinant Factor VIII Preparations,” Seminars in Thrombosis and Hemostasis, Vol. 27, No. 4, 2001, pp. 385-394. doi:10.1055/s-2001-16891
[15]	D. Wang, W. Liu, B. Han and R. Xu, “The Bioreactor: A Powerful Tool for Large-Scale Culture of Animal Cells,” Current Pharmaceutical Biotechnology, Vol. 6, No. 5, 2005, pp. 397-403. doi:10.2174/138920105774370580
[16]	M. Takagi, K. Shiwaku, T. Ionue, Y. Shirakawa, Y. Sawa, H. Matsuda and T. Yoshida, “Hydrodynamically Stable Adhesion of Endothelial Cells onto a Polypropylene Hollow Fiber Membrane by Modification with Adhesive Protein,” Journal of Artificial Organs, Vol. 6, No. 3, 2003, pp. 222-226. doi:10.1007/s10047-003-0218-8
[17]	W. C. Sanford, “A New Method for Dispersing Strongly Adhesive Cells in Tissue Culture,” In Vitro, Vol. 10, 1974, pp. 281-283. doi:10.1007/BF02615309
[18]	H. Yamaoka, H. Asato, T. Ogasawara, S. Nishizawa, T. Takahashi, T. Nakatsuka, I. Koshima, K. Nakamura, H. Kawaguchi, U. I. Chung, T. Takato and K. Hoshi, “Cartilage Tissue Engineering Using Human Auricular Chondrocytes Embedded in Different Hydrogel Materials,” Journal of Biomedical Materials Research Part A, Vol. 78, No. 1, 2006, pp. 1-11. doi:10.1002/jbm.a.30655
[19]	H. Yamaoka, Y. Tanaka, S. Nishizawa, Y. Asawa, T. Takato and K. Hoshi, “The Application of Atelocollagen Gel in Combination with Porous Scaffolds for Cartilage Tissue Engineering and Its Suitable Conditions,” Journal of Biomedical Materials Research Part A, Vol. 93, 2010, pp. 123-132.
[20]	Y. Tanaka, H. Yamaoka, S. Nishizawa, S. Nagata, T. Ogasawara, Y. Asawa, Y. Fujihara, T. Takato and K. Hoshi, “The Optimization of Porous Polymeric Scaffolds for Chondrocyte/Atelocollagen Based Tissue-Engineered Cartilage,” Biomaterials, Vol. 31, No. 16, 2010, pp. 4506-4516. doi:10.1016/j.biomaterials.2010.02.028