Abstract
More and more plant cell suspension cultures are regarded as an attractive alternative to mammalian cells as host organism for production of complex recombinant proteins. The most important advantages of the production platform are low costs, easy scalability and enhanced safety by complete lack of animal components in the cultivation media. In order to characterize, understand and control such systems accurately, it is important to determine the cell-specific productivity (Qp) of plant cell-based production platforms. Compared to many microbial and mammalian cells the morphology of plant cells is nonhomogeneous and the cells tend to form aggregates, therefore commercial cell counting systems are too unreliable to determine cell numbers in plant suspension cultures. We addressed this limitation by developing a novel cell counting method based on a combination of cell-staining and automated confocal fluorescence microscopy. This method allowed us, for the first time, to determine the cell-specific productivity of transgenic tobacco (Nicotiana tabacum cv. Bright Yellow-2) cell suspension cultures producing the human antibody M12. In the future this method will be a useful tool in the development of optimized plant cell-based production processes.
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References
An GH (1985) High-efficiency transformation of cultured tobacco cells. Plant Physiol 79(2):568–570. doi:10.1104/Pp.79.2.568
Boehm R (2007) Bioproduction of therapeutic proteins in the 21st century and the role of plants and plant cells as production platforms. Ann NY Acad Sci 1102:121–134. doi:10.1196/annals.1408.009
Chartrain M, Chu L (2008) Development and production of commercial therapeutic monoclonal antibodies in Mammalian cell expression systems: an overview of the current upstream technologies. Curr Pharm Biotechnol 9(6):447–467
de Gunst MCM, Harkes PAA, Val J, van Zwet WR, Libbenga KR (1990) Modelling the growth of a batch culture of plant cells: a corpuscular approach. Enzyme Microb Tech 12(1):61–71. doi:10.1016/0141-0229(90)90182-P
Fischer R, Stoger E, Schillberg S, Christou P, Twyman RM (2004) Plant-based production of biopharmaceuticals. Curr Opin Plant Biol 7(2):152–158. doi:10.1016/j.pbi.2004.01.007
Holland T, Sack M, Rademacher T, Schmale K, Altmann F, Stadlmann J, Fischer R, Hellwig S (2010) Optimal nitrogen supply as a key to increased and sustained production of a monoclonal full-size antibody in BY-2 suspension culture. Biotechnol Bioeng 107(2):278–289
Holland T, Blessing D, Hellwig S, Sack M (2013) The in-line measurement of plant cell biomass using radio frequency impedance spectroscopy as a component of process analytical technology. Biotechnol J 8(10):1231–1240. doi:10.1002/biot.201300125
Jeffers P, Raposo S, Lima-Costa ME, Connolly P, Glennon B, Kieran PM (2003) Focussed beam reflectance measurement (FBRM) monitoring of particle size and morphology in suspension cultures of Morinda citrifolia and Centaurea calcitrapa. Biotechnol Lett 25(23):2023–2028
Jiang Z, Huang Y, Sharfstein ST (2006) Regulation of recombinant monoclonal antibody production in Chinese hamster ovary cells: a comparative study of gene copy number, mRNA level, and protein expression. Biotechnol Progr 22(1):313–318. doi:10.1021/Bp0501524
Kieran PM (2001) Bioreactor design for plant cell suspension cultures. In: Cabral JMS, Mota M, Tramper J (eds) Multiphase bioreactor design. Taylor & Francis, London, pp 391–426
Kirchhoff J (2012) Generation of highly productive polyclonal and monoclonal tobacco suspension lines from a heterogeneous transgenic BY-2 culture through flow cytometric sorting. Dissertation, RWTH Aachen University
Kirchhoff J, Raven N, Boes A, Roberts JL, Russell S, Treffenfeldt W, Fischer R, Schinkel H, Schiermeyer A, Schillberg S (2012) Monoclonal tobacco cell lines with enhanced recombinant protein yields can be generated from heterogeneous cell suspension cultures by flow sorting. Plant Biotechnol J 10(8):936–944. doi:10.1111/j.1467-7652.2012.00722.x
Koncz C, Schell J (1986) The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector. Molec Gen Genet 204(3):383–396. doi:10.1007/bf00331014
Lamboursain L, Jolicoeur M (2005) Determination of cell concentration in a plant cell suspension using a fluorescence microplate reader. Plant Cell Rep 23(10–11):665–672. doi:10.1007/s00299-004-0899-3
McDonald KA, Jackman AP, Hurst S (2001) Characterization of plant suspension cultures using the focused beam reflectance technique. Biotechnol Lett 23(4):317–324. doi:10.1023/A:1005646826204
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plantarum 15(3):473–497. doi:10.1111/j.1399-3054.1962.tb08052.x
Nagata T, Nemoto Y, Hasezawa S (1992) Tobacco BY-2 cell-line as the Hela-cell in the cell biology of higher-plants. Int Rev Cytol 132:1–30
Naill MC, Roberts SC (2004) Preparation of single cells from aggregated Taxus suspension cultures for population analysis. Biotechnol Bioeng 86(7):817–826. doi:10.1002/Bit.20083
Nicoloso FT, Val J, Vanderkeur M, Vaniren F, Kijne JW (1994) Flow-cytometric cell counting and DNA estimation for the study of plant-cell population-dynamics. Plant Cell Tiss Org 39(3):251–259. doi:10.1007/Bf00035978
Raven N, Schillberg S, Kirchhoff J, Brändli J, Imseng N, Eibl R (2010) Growth of BY-2 suspension cells and plantibody production in single-use bioreactors. In: Single-use technology in biopharmaceutical manufacture. Wiley, New York, pp 251–261. doi:10.1002/9780470909997.ch21
Schillberg S, Raven N, Fischer R, Twyman RM, Schiermeyer A (2013) Molecular farming of pharmaceutical proteins using plant suspension cell and tissue cultures. Curr Pharm Des 19(31):5531–5542
Tucker KG, Chalder S, al-Rubeai M, Thomas CR (1994) Measurement of hybridoma cell number, viability, and morphology using fully automated image analysis. Enzyme Microb Technol 16(1):29–35
Ullisch DA, Muller CA, Maibaum S, Kirchhoff J, Schiermeyer A, Schillberg S, Roberts JL, Treffenfeldt W, Buchs J (2012) Comprehensive characterization of two different Nicotiana tabacum cell lines leads to doubled GFP and HA protein production by media optimization. J Biosci Bioeng 113(2):242–248. doi:10.1016/j.jbiosc.2011.09.022
van Dussen L, Zimran A, Akkerman EM, Aerts JM, Petakov M, Elstein D, Rosenbaum H, Aviezer D, Brill-Almon E, Chertkoff R, Maas M, Hollak CE (2013) Taliglucerase alfa leads to favorable bone marrow responses in patients with type I Gaucher disease. Blood Cells Mol Dis 50(3):206–211. doi:10.1016/j.bcmd.2012.11.001
Vasilev N, Grömping U, Lipperts A, Raven N, Fischer R, Schillberg S (2013) Optimization of BY-2 cell suspension culture medium for the production of a human antibody using a combination of fractional factorial designs and the response surface method. Plant Biotechnol J 11(7):867–874. doi:10.1111/pbi.12079
Xu J, Ge X, Dolan MC (2011) Towards high-yield production of pharmaceutical proteins with plant cell suspension cultures. Biotechnol Adv 29(3):278–299. doi:10.1016/j.biotechadv.2011.01.002
Acknowledgments
The authors wish to thank Dr. Flora Schuster for her skilled technical assistance with the preparation of plant cell cultures and Dr. Richard M Twyman and Holger Spiegel for critical reading of the manuscript. This research was funded by the European Union Seventh Framework Programme under Grant Agreement No. 227420 CoMoFarm.
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Havenith, H., Raven, N., Di Fiore, S. et al. Image-based analysis of cell-specific productivity for plant cell suspension cultures. Plant Cell Tiss Organ Cult 117, 393–399 (2014). https://doi.org/10.1007/s11240-014-0448-x
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DOI: https://doi.org/10.1007/s11240-014-0448-x