No abstract

No abstract

No abstract

Short title: Neuronal differentiation of neural stem cells in 3D spider silk matrices Abbreviations: NSCs (neural stem cells), BMP4 (Bone Morphogenic protein 4), TuJ1 (Neuronal class III B-tubulin), P (poly-ornithine), F (fibronectin), ECM (extracellular matrix), 3D (three dimensional), BW (BMP4+Wnt3a) 2 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 animalor 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.

2627 Abbreviations: FRESH, freeform reversible embedding of suspended hydrogels; POI, 28 parameter optimization index; SA, sodium alginate; 2 29 Abstract 30 There are many parameters in extrusion-based three-dimensional (3D) bioprinting of 31 different materials that require fine-tuning to obtain the optimal print resolution and cell 32 viability. To standardize this process, methods such as parameter optimization index (POI) 33 have been introduced. The POI aims at pinpointing the optimal printing speed and pressure 34 to achieve the highest accuracy keeping theoretical shear stress low. Here we applied the 35 POI to optimize the process of 3D bioprinting human neuroblastoma cell-laden 2% sodium 36 alginate (SA) hydrogel using freeform reversible embedding of suspended hydrogels (FRESH).37 Our results demonstrate a notable difference between optimal parameters for printing 2% 38 SA with and without cells in the hydrogel. We also detected a significant influence of long-39 term cell culture on the printed constructs. This observation suggests that the POI has to be 40 evaluated in the perspective of the final application. When taking these conditions into 41 consideration, we could define a set of parameters that resulted in good quality prints 42 maintaining high neuroblastoma cell viability (83% viable cells) during 7 days of cell culture 43 using 2% SA and FRESH bioprinting. These results can be further used to manufacture 44 neuroblastoma in vitro 3D culture systems to be used for cancer research. 45 3 46 2. Introduction 4748 Stem cell and tumor biology allow for the generation of small organ-like or tumor-like 49 structures to be developed in vitro, and this holds great promise for significant improvement 50 of approaches in drug discovery and precision medicine. Bioprinting has emerged as an 51 important tool for improving the conditions and control of such cell culture rationale [1,2].52 However, to achieve optimal results, many parameters of the microenvironment must be 53 taken into account. We and others have demonstrated the significance of, e.g., oxygen levels 54 [3,4], substrate stiffness [5,6], substrate roughness [7], and biomaterial properties [8] for 55 progenitor cells to respond properly to external signaling factors such as growth factors, and 56 to execute the appropriate transcriptional programs. Yet, 2-dimensional cell culture is in 57 itself a limiting factor both in stem cell and tumor biology and it has been shown that, for 58 example, certain tumor cells grown in 2D conditions are more sensitive to chemotherapeutic 59 reagents than when grown in three dimensions [9], which may explain some of the lack of 60 progress in cancer research heavily debated during recent years [10,11]. 6162 Three-dimensional (3D) bioprinting was first reported to deposit viable cells by Smith et al in 63 2004 [12]. More than a decade later, 3D bioprinting is constantly being improved in terms of 64 hardware, software, biomaterials, and applications. Standard 3D extrusion-based 65 bioprinters, despite being ...