We present a simple but accurate algorithm to calculate the flow and shear rate profile of shear thinning fluids, as typically used in biofabrication applications, with an arbitrary viscosity-shear rate relationship in a cylindrical nozzle. By interpolating the viscosity with a set of power-law functions, we obtain a mathematically exact piecewise solution to the incompressible Navier-Stokes equation. The algorithm is validated with known solutions for a simplified Carreau-Yasuda fluid, full numerical simulations for a realistic chitosan hydrogel as well as experimental velocity profiles of alginate and chitosan solutions in a microfluidic channel. We implement the algorithm in an easy-to-use Python tool, included as Supplementary Material, to calculate the velocity and shear rate profile during the printing process, depending on the shear thinning behavior of the bioink and printing parameters such as pressure and nozzle size. We confirm that the shear stress varies in an exactly linear fashion, starting from zero at the nozzle center to the maximum shear stress at the wall, independent of the shear thinning properties of the bioink. Finally, we demonstrate how our method can be inverted to obtain rheological bioink parameters in-situ directly before or even during printing from experimentally measured flow rate versus pressure data.

Alginate dialdehyde–gelatin (ADA–GEL) hydrogels have been reported to be suitable matrices for cell encapsulation. In general, application of ADA–GEL as bioink has been limited to planar structures due to its low viscosity. In this work, ring shaped constructs of ADA–GEL hydrogel were fabricated by casting the hydrogel into sacrificial molds which were 3D printed from 9% methylcellulose and 5% gelatin. Dissolution of the supporting structure was observed during the 1st week of sample incubation. In addition, the effect of different crosslinkers (Ba2+ and Ca2+) on the physicochemical properties of ADA–GEL and on the behavior of encapsulated MG-63 cells was investigated. It was found that Ba2+ crosslinked network had more than twice higher storage modulus, and mass decrease to 70% during incubation compared to 42% in case of hydrogels crosslinked with Ca2+. In addition, faster increase in cell viability during incubation and earlier cell network formation were observed after Ba2+ crosslinking. No negative effects on cell activity due to the use of sacrificial materials were observed. The approach presented here could be further developed for cell-laden ADA–GEL bioink printing into complex 3D structures.

Numerous cell functions are accompanied by phenotypic changes in viscoelastic properties, and measuring them can help elucidate higher-level cellular functions in health and disease. We present a high-throughput, simple and low-cost microfluidic method for quantitatively measuring the elastic (storage) and viscous (loss) modulus of individual cells. Cells are suspended in a high-viscosity fluid and are pumped with high pressure through a 5.8 cm long and 200 μm wide microfluidic channel. The fluid shear stress induces large, near ellipsoidal cell deformations. In addition, the flow profile in the channel causes the cells to rotate in a tank-treading manner. From the cell deformation and tank treading frequency, we extract the frequency-dependent viscoelastic cell properties based on a theoretical framework developed by R. Roscoe that describes the deformation of a viscoelastic sphere in a viscous fluid under steady laminar flow. We confirm the accuracy of the method using atomic force microscopy-calibrated polyacrylamide beads and cells. Our measurements demonstrate that suspended cells exhibit power-law, soft glassy rheological behavior that is cell cycle-dependent and mediated by the physical interplay between the actin filament and intermediate filament networks.

Bioprinting offers the opportunity to fabricate precise 3D tumor models to study tumor pathophysiology and progression. However, the choice of the bioink used is important. In this study, cell behavior was studied in three mechanically and biologically different hydrogels (alginate, alginate dialdehyde crosslinked with gelatin (ADA–GEL), and thiol-modified hyaluronan (HA-SH crosslinked with PEGDA)) with cells from breast cancer (MDA-MB-231 and MCF-7) and melanoma (Mel Im and MV3), by analyzing survival, growth, and the amount of metabolically active, living cells via WST-8 labeling. Material characteristics were analyzed by dynamic mechanical analysis. Cell lines revealed significantly increased cell numbers in low-percentage alginate and HA-SH from day 1 to 14, while only Mel Im also revealed an increase in ADA–GEL. MCF-7 showed a preference for 1% alginate. Melanoma cells tended to proliferate better in ADA–GEL and HA-SH than mammary carcinoma cells. In 1% alginate, breast cancer cells showed equally good proliferation compared to melanoma cell lines. A smaller area was colonized in high-percentage alginate-based hydrogels. Moreover, 3% alginate was the stiffest material, and 2.5% ADA–GEL was the softest material. The other hydrogels were in the same range in between. Therefore, cellular responses were not only stiffness-dependent. With 1% alginate and HA-SH, we identified matrices that enable proliferation of all tested tumor cell lines while maintaining expected tumor heterogeneity. By adapting hydrogels, differences could be accentuated. This opens up the possibility of understanding and analyzing tumor heterogeneity by biofabrication.

Two-dimensional (2D) cancer models have been the standard for drug development over the past few years, but they frequently do not resemble in vivo properties adequately. 3D models are superior in many aspects and are, therefore, more similar to human pathophysiology. Over the past years, the emerging field of biofabrication has made significant advances, resulting in even more sophisticated 3D models. With this study, a hydrogel is created for biofabrication that is suitable for mimicking the tumor microenvironment in vitro and is further tested as a new vascularized melanoma model in vivo. The alginate/hyaluronic acid/gelatin bioink shows good shape-fidelity, high cell survival rates, and enables successful cultivation of melanoma cells and adipose-derived stem cells as well as cell differentiation in vitro. In vivo, in the arteriovenous loop model, it proves to be a unique method to study melanoma progression, tumor vascularization, and ultimately and reliably metastases in an isolated and controlled environment. These results show that this 3D model is very application-oriented for molecular research and therapy development.