An integrated cell printing system for the construction of heterogeneous tissue models.

A new three-dimensional (3D) cell printing system was developed and investigated to organize multiple cells/biomaterials with a control precision within 100 μm. This system can be used for the in vitro construction of heterogeneous tissue models. The proposed printing system was achieved by the integration of extrusion printing and alternating viscous and inertial force jetting (AVIFJ) techniques using dual-nozzle switching. In this technique, hydrogels containing high cell densities were extruded using extrusion printing, while droplets containing single cells were precisely manipulated using AVIFJ. The droplets that contained single cells were at the scale of pico-liters and could be accurately positioned at the micron scale. Stable hydrogel structures with adjustable diameters were also printed, with cell viabilities exceeding 90% after printing. A heterogeneous tumor model that contained spheroids and human umbilical vein endothelial cells (HUVECs) was then constructed using the established integrated cell printing system in a stepwise or simultaneous fashion. HUVEC-loaded droplets were observed to locate around the preformed tumor spheroids as designed. Cells and spheroids in the model maintained high cell viability and sustained growth throughout the culture period. The ELISA results of albumin production also proved that the spheroids maintained increased cellular function during the culture. These results demonstrated the feasibility of this integrated 3D printing system for the engineering of in vitro heterogeneous tissue models for future biological and pathological studies. STATEMENT OF SIGNIFICANCE: Addressing the challenge of multi-scale printing in the construction of heterogeneous tissue models, a new 3D cell printing system was developed to organize cells/biomaterials of a control precision within 100 μm. AVIFJ was integrated with extrusion printing, thereby achieving the construction of cell interactions between single cells and spheroids, the manipulation of single cells in a 3D microenvironment with high accuracy, and the real-time on-demand printing. The printed heterogeneous tumor model maintained cell viability, sustained cell growth, and increased cell function during 7 days of culture. We believed that this work would benefit the production of functional artificial tissues, enabling the construction of more biomimetic cell arrangements and microenvironment to support cell functions.