## 4 Conclusions  
In this study, we aimed to optimize an SA-G bioink formulation to match the properties of the native photoreceptor layer and to be successfully extruded and printed with good resolution. To assess its suitability as a substrate for photoreceptors in in vitro culture, we conducted a comprehensive characterization of an SA-G bioink. Our results showed that the stiffness of the bioink closely mimics the in vivo photoreceptor stiffness. This parameter plays a crucial role in the development of a functional photoreceptor layer, as it influences the cell behavior guiding the cell phenotype and genotype. In addition, through rheological and printability tests, we demonstrated the bioink's ability to fabricate multilayered structures characterized by high shape fidelity. This feature is crucial for obtaining a microstructure able to drive photoreceptor polarization, thus replicating their highly organized in vivo configuration. Ultimately, the viability of fibroblasts, both in contact with material extracts and embedded within the bioink, proved that the SA-G material is non-cytotoxic. Taken together, these findings suggest that the proposed SA-G bioink may be used as a tissue mimetic to produce a cellularized construct with the specific pore size and geometry capable of promoting photoreceptor development, alignment, and functional culture in vitro. Future studies should focus on incorporating stem cells into the SA-G bioink to form a functional photoreceptor layer in vitro. This step will require the selection of the appropriate culture medium and growth factors and the execution of several tests to determine the optimal cell density for mimicking the native high packing density of photoreceptors. Different printing patterns will then be explored to determine  
Figure 8: Fluorescence microscopy images of the LIVE/DEAD assay. Green and red indicate live and dead cells, respectively: (**a**) Image of a printed filament 1 day after printing; (**b**) Image of a corner within the printed grid 1 day after printing; (**c**) View of a pore of the printed grid and a zoomed-in view of a filament 5 days after printing. Scale bar = 200 \(\upmu\)m.  
which microstructure best supports native-like spatial cell organization. The formation of a functional photoreceptor layer will be evaluated by conducting a thorough biological analysis, including of cell viability and proliferation and the expression of specific markers. Finally, in vivo studies need to be performed to assess the functional integration and stability of the engineered photoreceptor layer.  
The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app14166980/s1](https://www.mdpi.com/article/10.3390/app14166980/s1)]([https://www.mdpi.com/article/10.3390/app14166980/s1](https://www.mdpi.com/article/10.3390/app14166980/s1)), Figure S1: Average line width at different printing speeds using a 25G conical nozzle.  
Conceptualization, B.B., S.M. and F.B.; methodology, B.B. and F.I.; investigation, B.B. and F.I.; data curation, B.B. and F.P.; writing--original draft preparation, B.B.; writing--review and editing, S.M., F.I., F.P., F.B. and M.L. All authors have read and agreed to the published version of the manuscript.  
This research received no external funding.  
The raw data supporting the conclusions of this article will be made available by the authors on request.  
The authors declare no conflict of interest.