Lewicki, J. | bioRxiv Science

bioRxiv2019

Optimization of 3D bioprinting of human neuroblastoma cells using sodium alginate hydrogel

There are many parameters in extrusion-based three-dimensional (3D) bioprinting of different materials that require fine-tuning to obtain the optimal print resolution and cell viability. To standardize this process, methods such as parameter optimization index (POI) have been introduced. The POI aims at pinpointing the optimal printing speed and pressure to achieve the highest accuracy keeping theoretical shear stress low. Here we applied the POI to optimize the process of 3D bioprinting human neuroblastoma cell-laden 2% sodium alginate (SA) hydrogel using freeform reversible embedding of suspended hydrogels (FRESH). Our results demonstrate a notable difference between optimal parameters for printing 2% SA with and without cells in the hydrogel. We also detected a significant influence of long-term cell culture on the printed constructs. This observation suggests that the POI has to be evaluated in the perspective of the final application. When taking these conditions into consideration, we could define a set of parameters that resulted in good quality prints maintaining high neuroblastoma cell viability (83% viable cells) during 7 days of cell culture using 2% SA and FRESH bioprinting. These results can be further used to manufacture neuroblastoma in vitro 3D culture systems to be used for cancer research.

bioengineering↗

bioRxiv2019

Recombinant spider silk protein matrices facilitate multi-analysis of calcium-signaling in neural stem cell-derived AMPA-responsive neurons

Neural progenitors or stem cells (NSCs) show great promise in drug discovery and clinical application. Yet few efforts have been made to optimize biocompatible materials for such cells to be expanded and used in clinical conditions. We have previously demonstrated that NSCs are readily cultured on substrates of certain recombinant spider silk protein without addition of animal- or human-derived components. The question remains however whether this material allows differentiation into functional neurons and glia, and whether such differentiation can take place also when the NSCs are cultured within the material in a pseudo-3D context. Here we demonstrate that \"foam\"-like structures generated from recombinant spider silk protein (4RepCT) provided excellent matrices for the generation and multicellular analysis of functional excitatory neurons from NSCs without addition of animal- or human-derived components. NSCs isolated from the cerebral cortices of rat embryos were cultured on either 4RepCT matrices shaped as foam-like structures without coating, or on conventional polystyrene plates coated with poly-L-ornithine and fibronectin. Upon treatment with recombinant proteins including the growth factor BMP4 or a combination of BMP4 and the signaling factor Wnt3a, the cortical NSCs cultured in 4RepCT foam-like structures differentiated efficiently into neurons that responded to glutamate receptor agonists, such as AMPA, to at least the same extent as control cultures. Matrices derived from recombinant spider silk proteins thus provide a functional microenvironment for neural stem cells without any animal- or human-derived components, and can be employed in the development of new strategies in stem cell research and tissue engineering.

bioengineering↗