Homogeneous and Reproducible Mixing of Highly Viscous Biomaterial Inks and Cell Suspensions to Create Bioinks

Abstract

Highly viscous bioinks offer great advantages for the three-dimensional fabrication of cell-laden constructs by microextrusion printing. However, no standardised method of mixing a high viscosity biomaterial ink and a cell suspension has been established so far, leading to non-reproducible printing results. A novel method for the homogeneous and reproducible mixing of the two components using a mixing unit connecting two syringes is developed and investigated. Several static mixing units, based on established mixing designs, were adapted and their functionality was determined by analysing specific features of the resulting bioink. As a model system, we selected a highly viscous ink consisting of fresh frozen human blood plasma, alginate, and methylcellulose, and a cell suspension containing immortalized human mesenchymal stem cells. This bioink is crosslinked after fabrication. A pre-crosslinked gellan gum-based bioink providing a different extrusion behaviour was introduced to validate the conclusions drawn from the model system. For characterisation, bioink from different zones within the mixing device was analysed by measurement of its viscosity, shape fidelity after printing and visual homogeneity. When taking all three parameters into account, a comprehensive and reliable comparison of the mixing quality was possible. In comparison to the established method of manual mixing inside a beaker using a spatula, a significantly higher proportion of viable cells was detected directly after mixing and plotting for both bioinks when the mixing unit was used. A screw-like mixing unit, termed “HighVisc”, was found to result in a homogenous bioink after a low number of mixing cycles while achieving high cell viability rates.

Scaffolds for Cultured Meat on the Basis of Polysaccharide Hydrogels Enriched with Plant-Based Proteins

“…Higher temperatures of the hydrogel solutions can solve this problem, but also increase cell death [ 102 ]. Since this is not desirable, other mixing techniques can also be helpful to improve cellular distribution within high viscous gels [ 103 ]. For example, in the mixing technique used in this study, gellan with and without protein is the best candidate to encapsulate cells and to build up a 3D cell environment.…”

Analytics in Extrusion-Based Bioprinting: Standardized Methods Improving Quantification and Comparability of the Performance of Bioinks

“…This process is not reproducible, and air bubbles lead to inhomogeneity within the cartridge, resulting in fluctuations of the filament width. Air bubbles entrapped after the mixing steps have been addressed in the literature [15,20,26,35]. The mentioned studies implement strategies by either mixing, using a spatula or centrifuge, the cartridges or syringes to remove the entrapped air.…”

“…Furthermore, methodologies for the evaluation of printed structures are lacking standards as well. Methods in that field consist of the manual extraction of the metrics of the produced structures [16][17][18][19][20]. Such methods are prone to observer-dependent errors and are not reproducible.…”

Virtual Reality as Tool for Bioprinting Quality Inspection: A Proof of Principle

“…In literature, the use of single-use static mixers or reciprocating movements between two vessels with a bottleneck in between are state of the art ( Puertas-Bartolomé et al, 2020 ; Silva et al, 2020 ). Some complex solutions are under investigation, for example, active mixers ( Ortega et al, 2019 ; Wenger et al, 2020 ; Dani et al, 2021 ).…”