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Development and Characterization of Alginate-Based Bioinks incorporating Nicotiana tabacum Cells for Bioprinting
Kira Schnellbächer1, Adrian Rehn1, Robin Maatz1, Andreas Blaeser1
1 Technical University Darmstadt, IDD, Germany (schnellbaecher@idd.tu-darmstadt.de, blaeser@idd.tu-darmstadt.de)
Plant cells are valuable for producing various molecules due to their secondary metabolism [1]. Immobilization allows their use in continuous systems, improving product yield and simplifying downstream processing [2, 3]. Further enhancement can be achieved by printing a defined three-dimensional geometric shape from a hydrogel filled with plant cells, known as green 3D bioprinting [4]. This can improve the distribution of oxygen and nutrients and the transport of the final product. For successful printing, the hydrogel must be biocompatible and have appropriate rheological properties. A potentially suitable gelling agent is alginate, which is derived from plants and cross-links with CaCl2.
To assess the biocompatibility of alginate-based hydrogels, the components alginate and CaCl2, as well as the cross-linked gel, were tested with Nicotiana tabacum cells from a suspension culture. CaCl2 concentrations ranged from 10 to 450 mM and alginate concentrations from 0.5 to 1.5%. In the immobilization procedure, plant cells were mixed with the alginate media solution and added dropwise to a CaCl2 solution. The beads were analyzed by measuring fresh/dry weight and by staining with FDA/PI.
To estimate the suitability of the bioink for different printing processes, such as drop-on-demand bioprinting, the rheological properties were investigated. Therefore, the pure 1.5% alginate and the bioink with N. tabacum cells were investigated by measuring the viscosity at different shear rates. To determine the maximum tolerated stress for plant cells, shear rates up to 800 s-1 were applied and the cell viability assessed. After gelation, the mechanical stability of the hydrogel cell constructs was investigated using compression tests.
The addition of uncross-linked alginate to the medium has a strong effect on the viscosity and a strong growth inhibition was observed. CaCl2 has no or little effect up to a concentration of 20 mM. Based on these findings, the final gel used for immobilization consisted of 1.5% alginate cross-linked with 20 mM CaCl2. Here the tobacco cells showed growth with a sixfold increase in dry weight after 10 days and high viability with more than 85% viable cells after 17 days of culture, while the hydrogel remained stable. The bioink showed the desired shear-thinning behavior for bioprinting even stronger than pure alginate. No loss of cell viability was observed after shearing compared with the control.
In conclusion, 1.5% alginate hydrogel with 20 mM CaCl2 is a promising bioink-base for following 3D bioprinting approaches with N. tabacum, due to its biocompatibility, shear-thinning behavior and mechanical stability.
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[4] J. Seidel, T. Ahlfelf, M. Adolph, S. Kümmritz, J. Steingroewer, F. Krujatz, T. Bley, M. Gelinsky, A. Lode, Biofabrication (2017)
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