An experimental workflow for bioprinting optimization: Application to a custom-made biomaterial ink

Abstract

Bioprinting is an emerging technology with significant potential in biomedical fields, enabling the creation of highly customized, cell-laden constructs. Despite the promise, achieving high-quality, reproducible prints remains challenging due to the lack of standardized protocols, which has hindered the widespread adoption of the technique. In this study, we present a systematic bioprinting protocol designed to optimize the performance of an in-house photo-curable biomaterial ink composed of gelatin methacryloyl and egg white protein. Printing quality was evaluated through the following three key assessments: extrusion, deposition, and printability. To facilitate accurate image analysis, we developed a custom three-dimensional (3D)- printed lens support specifically designed for a USB microscope. Additionally, we implemented a Python script to quantitatively assess bioprinting quality. Our results indicate that a pressure range of 70-80 kPa, combined with speeds between 300 and 900 mm/min, yields reliable extrusion flow, with 75 kPa and 600 mm/min emerging as optimal parameters for bioprinting 3D constructs. These findings underscore the importance of carefully tuning parameters—including pressure and speed—to achieve stable, high-resolution extrusions. Such optimization mitigates common printing issues, including tip clogging, filament dragging, and unintended merging of adjacent filaments, thereby enhancing structural accuracy. This work provides a comprehensive framework for evaluating and optimizing bioprinting parameters, offering a reproducible methodology to enhance print quality. It contributes to ongoing efforts to standardize bioprinting processes and advance their applications in tissue engineering and regenerative medicine.

Keywords

3D bioprinting protocol / Deposition / Egg white proteins / Extrusion / Gelatin methacryloyl / Photo-curable biomaterial ink / Printability

Funding

This work has been financially supported by the Spanish Ministry of Science and Innovation (grant no. PID2023-146072OB-I00). P.M.C was funded by the Spanish Government through “Plan de Recuperación, Transformación y Resiliencia” and by the European Union through “NextGenerationEU” (Programa Investigo 076-16). E.G.G was funded by the Ramón & Cajal Fellowship (RYC2021-033490-I, funded by MCIN/AE/10.13039/501100011033 and the EU “NextGenerationEU/PRTR”). The Bio X bioprinter was adquired through Contrato Programa Plan de Inversiones e Investigación from the Aragón Government (2022).

Conflict of interest

The authors declare they have no competing interests.