The mechanics of the cellular microenvironment have critical impact on the behaviour of cells. However, how mechanical parameters 3D bioprinting such as shear forces or substrate stiffness affect cell stress and function is unclear. We investigate, how 3D bioprinting induced shear forces affect (i) plasma membrane integrity, using fluorescent membrane-dye incorporation , and (ii) long term cell stress, using lentiviral fluorescent reporters.
3D bioprinting of NIH/3T3 cells in alginate-based bioinks at various printing flow rates revealed an increased incorporation of FM 1-43 styryl-dye with increasing flow rates, indicating that higher shear stresses results in plasma membrane disruption. Moreover, high flow rates increased immediate and long term cell death, suggesting that shear forced induced plasmamembrane disruption can adversely affect cell performance. We correlate experimental data with mathematical modelling of the shear stresses experienced by cells during needle passage, and the cell distribution within the needle to determine the critical shear stress and time required for plasma membrane disruption. We also modulate additional parameters such as matrix stiffness, as well as needle diameter and length. We further correlate our results with data on long-term proliferation and apoptosis using lentiviral reporters. Our data suggest that FM 1-43 incorporation is an excellent tool to determine cell tolerance toward flow-induced shear stress, and to optimize 3D bioprinting processes. In addition, we expect our results to provide deeper insights into the relationship between the mechanical properties of the ECM and the behaviour of embedded cells in 3D bioprinting.