Abstract
Bottom-up biofabrication approaches for fabricating engineered tissue constructs are emerging strategies in tissue engineering. Few technologies have been developed that are capable of assembling tissue units into 3D Plotted scaffolds. We developed an integrated and automated 3D Bioassembly system for bioassembling engineered tissue constructs. The developed automated bioassembly system consisted of a (i) singularisation module and (ii) an injection module integrated into a commercial 3D bioprinter. The fluidic-based singularisation module delivered single Ø1 mm sized tissue unit at a time to the injection module and the injection module together with the 3D positioning system of the 3D bioprinter delivered the tissue unit into a predefined pore in the 3D Plotted scaffold. The developed automated bioassembly system was capable of either fabricating a construct via a two-step top-down bioassembly approach (fabricating a complete scaffold and insertion of tissue units) or a multistep bottom-up bioassembly approach (alternative layer-by-layer scaffold fabrication and tissue unit co-assembly). The automated bioassembly system was validated for application in cartilage and tumour engineering using tissue units (microspheres and micro-tissues). For cartilage engineering, Ø1 mm sized cartilage micro-tissues were fabricated utilising a previously demonstrated high-throughput 96-well plate format and Ø1 mm sized chondrocytes or chondroprogenitor cells-laden GelMA (gelatin-methacryloyl)-HepMA (methacrylated heparin) (9.5%-0.5%) hydrogel microspheres were fabricated utilising an adopted microfluidic system. For tumour engineering, a co-culture of cancer cells with fibroblasts using a liquid overlay technique was required to fabricate compact spherical Ø1 mm micro-tissues that could be handled by the automated bioassembly system and cancer cell-laden 10% GelMA hydrogel microspheres were fabricated utilising the adopted microfluidic system. Reliable handling of the tissue units was demonstrated by the automated bioassembly system. Bottom-up bioassembly of tissue units into 3D Plotted PEGT/PBT polymer scaffolds was demonstrated with the automated bioassembly system. No difference in viability was observed between the constructs assembled manually and with the automated bioassembly system. The flexibility of the automated tissue bioassembly system was shown by assembling constructs with coloured microspheres (denoting microspheres of different types) in various desired arrangements. The automated bioassembly of an anatomically shaped construct was also demonstrated. Neocartilage formation was observed in the chondrocyte-laden individual microspheres and assembled constructs when cultured in vitro for 35 days. Neocartilage formation was also visualised in the assembled graduated constructs fabricated with human articular chondrocytes (HAC) and mesenchymal stromal cells (MSC). In the in vitro micro-tissue tumour model, individual micro-tissues had higher chemoresistance compared to cells in 2D and the co-culture assembled construct had higher chemoresistance compared to individual co-culture micro-tissues. Similarly, in the in vitro microsphere tumour model, the assembled constructs were the most chemoresistant followed by individual microspheres and the cell in 2D had the lowest chemoresistance. The novel and flexible automated bioassembly technology that we have developed provides a pathway for fabricating a larger number of anatomically shaped clinically relevant constructs with precise control of the spatial position of the tissues units for application in cartilage engineering and for fabricating in vitro cancer models for application for drug discovery and high-throughput screening.