Development of Semi-Solid Media for Cloning and Screening of Hybridoma Cell Line
Abstract
Establishment of the hybridoma cell line is an important step for monoclonal antibody production. The screenings of a candidate hybridoma cell clone by limiting dilution is a laborious method and require highly skilled personnel. Currently, a method of easier, faster and more convenient than limiting dilution is cultured in semi-solid culture media was developed. Although, the methylcellulose based semi-solid media kit have available, the culture media kit is very expensive. Moreover, we found that using media based methylcellulose alone unable to generate a 3D colony of the hybridoma cell suggests that there must be other substances in media that are not disclose. In this study, we aimed to develop in-house semi-solid based methylcellulose for generating of 3D colony of the hybridoma cell. The various viscosity substances such as cellulose, methylcellulose, type I collagen and gelatin is used to determine the optimal combination and proportion for semi-solid media to generate 3D colony of the hybridoma cell. The best recipe of the semi-solid media that allow hybridoma cell survival, proliferate and form 3D colonies that remain physically separated from other colonies was 1.5 % methylcellulose, 0.25%gelatin, 1% collagen, in DMEM supplemented with 10% fetal bovine serum. Keywords: 3D cell culture, semi-solid medium, hybridoma cell lineReferences
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tumorsphere culture system for the efficient and low-cost enrichment of cancer stem cells with natural polymers. Experimental and Therapeutic Medicine, 15(1), 85-92. doi:10.3892/etm.2017.5419
Brezski, R. J., Kinder, M., Grugan, K. D., Soring, K. L., Carton, J., Greenplate, A. R., Jordan, R. E. (2014). A monoclonal
antibody against hinge-cleaved IgG restores effector function to proteolytically-inactivated IgGs in vitro
and in vivo. Monoclonal Antibodies, 6(5), 1265-1273. doi:10.4161/mabs.29825
Gu, H., Ji, R., Zhang, X., Wang, M., Zhu, W., Qian, H., Xu, W. (2016). Exosomes derived from human mesenchymal
stem cells promote gastric cancer cell growth and migration via the activation of the Akt pathway. Molecular
Medicine Reports, 14(4), 3452-3458. doi:10.3892/mmr.2016.5625
Kelley, B., (2007). Very large scale monoclonal antibody purification: the case for conventional unit operations.
Biotechnology Progress. 23(5), 995-1008.
Kubow, K. E., Vukmirovic, R., Zhe, L., Klotzsch, E., Smith, M. L., Gourdon,D., Vogel, V. (2015). Mechanical forces
regulate the interactions of fibronectin and collagen I in extracellular matrix. Nature Communications,
6, 8026. doi :10.1038/ncomms9026
Kusnezow, W., Hoheisel, J. D., (2002). Antibody microarrays: promises and problems. Bio Techniques, Suppl 33(6),
S14-S23. doi.org/10.2144/dec02kusnezow
Laurino, J. P., Shi, Q., Ge, J., (1999). Monoclonal antibodies, antigens and molecular diagnostics: a practical overview.
Annals of Clinical & Laboratory Science, 29(3), 158-166.
Lertworapreecha, M., Sudthimusic S., Tontikapong, K. (2019). Establishment of hybridoma cell lines producing monoclonal antibodies against somatic (O) and flagella (H) antigens of Salmonella typhimurium and Salmonella weltevreden. Research report. 42 p. Thaksin University. (in Thai).
Li, X., Bian, H., Yu, S., Xiao, W., Shen, J., Lan, C., Tang, Y. (2018). A rapid method for antigen-specific hybridoma
clone Isolation. Analytical Chemistry, 90(3), 2224-2229. doi:10.1021/acs.analchem.7b04595
Lipman, N. S., Jackson, L. R., Trudel, L. J., Weis-Garcia, F., (2005). Monoclonal versus polyclonal antibodies:
distinguishing characteristics, applications, and information resources. Institute for Laboratory
Animal Research Journal, 46(3), 258-268.
Liu, Y., Wang, Y., Liu, J., Zuo, W., Hao, L., Zhang, L., & Zhen, B. (2014). High throughput monoclonal antibody
generation by immunizing multiple antigens. Science China Life Sciences, 57(7), 710-717.
doi:10.1007/s11427-014-4688-0
Mechetner, E. (2007) Development and characterization of mouse hybriodmas. In: Albitar M, (Ed.). Monoclonal antibodies: methods and protocols. (pp. 1-14). Totowa, N.J: Humana Press.
Zhu, Z. W., Chen, L., Liu, J. X., Huang, J. W., Wu, G., Zheng, Y. F., & Yao, K. T. (2018). A novel three-dimensional
tumorsphere culture system for the efficient and low-cost enrichment of cancer stem cells with natural polymers. Experimental and Therapeutic Medicine, 15(1), 85-92. doi:10.3892/etm.2017.5419
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2020-01-15
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บทความวิจัยจากการประชุมวิชาการระดับชาติ"วิทยาศาสตร์วิจัย"ครั้งที่ 11
