Generic method of printing window adjustment for extrusion-based 3D-bioprinting to maintain high viability of mesenchymal stem cells in an alginate-gelatin hydrogel

Abstract

Over the last decade, bioprinting of artificial tissues has been developed into a significant field of research. With an increasing number of printing technologies and bioinks used in bioprinting, its complexity increases as both the printing technology and the properties of the bioink influence the cell biological functionality and printing accuracy of the printed tissue. Therefore, optimization of bioprinting processes often remains a challenge, which could be solved by a smart fine-tuning of the process parameters. We present a novel method to adjust the printing window for extrusion-based bioprinting on the basis of a two-step assessment to determine process parameters such as nozzle size, extrusion flow rate, and printing temperature. First, a suitable printing temperature is deduced from the bioink properties and second nozzle size and extrusion flow rate is selected in a way that the immediate cell damage after printing is reduced. For both steps only basic rheological properties of the bioinks need to be known as well as detailed knowledge of the cell survival in the bioink for different shear stresses.This method is applied to an exemplary alginate-gelatin hydrogel to show how the printing temperature affects the achievable printing accuracy. For this bioink, viability of immortalized mesenchymal stem cells (iMSC) decreases with about 4% per thousand Pascal increase in maximum shear stress. For different combinations of flow rate, nozzle size and nozzle shape it is shown, that only the maximum shear stress experienced by the iMSCs influences average cell viability. Factors like flow rate, nozzle size and shape only play an indirect role by influencing the maximum shear stress and individually have no significant influence on cell viability.The experimental results allow a direct adjustment of printing parameters for the presented combination of hydrogel and cell type but are not limited to it. For other bioinks, the described generic method can be easily used to systematically adjust the printing parameters. For this purpose, only the basic rheological properties and the influence of shear stress on cell survival need to be known and process parameters can be set concerning the respective application.