Abstract
Bottom-up biofabrication approaches combining micro-tissue fabrication techniques with extrusion-based 3D printing of thermoplastic polymer scaffolds are emerging strategies in tissue engineering. These biofabrication strategies support native self-assembly mechanisms observed in developmental stages of tissue or organoid growth as well as promoting cell–cell interactions and cell differentiation capacity. Few technologies have been developed to automate the precise assembly of micro-tissues or tissue modules into structural scaffolds. We describe an automated 3D bioassembly platform capable of fabricating simple hybrid constructs via a two-step bottom-up bioassembly strategy, as well as complex hybrid hierarchical constructs via a multistep bottom-up bioassembly strategy. The bioassembly system consisted of a fluidic-based singularisation and injection module incorporated into a commercial 3D bioprinter. The singularisation module delivers individual micro-tissues to an injection module, for insertion into precise locations within a 3D plotted scaffold. To demonstrate applicability for cartilage tissue engineering, human chondrocytes were isolated and micro-tissues of 1 mm diameter were generated utilising a high throughput 96-well plate format. Micro-tissues were singularised with an efficiency of 96.0 ± 5.1%. There was no significant difference in size, shape or viability of micro-tissues before and after automated singularisation and injection. A layer-by-layer approach or aforementioned bottom-up bioassembly strategy was employed to fabricate a bilayered construct by alternatively 3D plotting a thermoplastic (PEGT/PBT) polymer scaffold and inserting pre-differentiated chondrogenic micro-tissues or cell-laden gelatin-based (GelMA) hydrogel micro-spheres, both formed via high-throughput fabrication techniques. No significant difference in viability between the construct assembled utilising the automated bioassembly system and manually assembled construct was observed. Bioassembly of pre-differentiated micro-tissues as well as chondrocyte-laden hydrogel micro-spheres demonstrated the flexibility of the platform while supporting tissue fusion, long-term cell viability, and deposition of cartilage-specific extracellular matrix proteins. This technology provides an automated and scalable pathway for bioassembly of both simple and complex 3D tissue constructs of clinically relevant shape and size, with demonstrated capability to facilitate direct spatial organisation and hierarchical 3D assembly of micro-tissue modules, ranging from biomaterial free cell pellets to cell-laden hydrogel formulations.
Highlights
In tissue engineering and regenerative medicine, conventional ‘top-down’ fabrication strategies revolve around populating cells within a 3D scaffold [1]
It was essential to design the scaffold in this manner so that the micro-tissues could reliably be inserted into the pore by the automated bioassembly system, while at the same time ensuring they remained securely in place and were not dislodged after the insertion process
We described the development of an automated and integrated biofabrication system for 3D bioassembly of micro-tissues within structural 3D plotted scaffolds
Summary
In tissue engineering and regenerative medicine, conventional ‘top-down’ fabrication strategies revolve around populating cells within a 3D scaffold [1]. With regards to cartilage tissue engineering, the typical paradigm involves in growing chondrocytes or chondroprogenitor cells in combination with cell phenotype modulating growth factors within a biodegradable 3D scaffold in vitro, and implanting the engineered construct into the target site [2]. This approach offers advantages by providing the initial mechanical properties, cells and extracellular matrix (ECM) components required to elicit regeneration and tissue repair [3]. A spectra of techniques have been demonstrated to assemble prefabricated tissue modules into higher order tissue structures. These techniques include: stacking of cell sheets [11], random packing of microgels [12], utilising hydrophobic effects for assembling microgels [13], physical manipulation of individual cell-laden microgels [14], magnetically driven assembly of microgels [15, 16], acoustic wave driven assembly of microgels [17], assembly of microgels with micro-robotic systems [18], vacuum moulding [19, 20], stacking rings on a tube [21], assembling micro-tissues by stacking them on skewers [22], or manual assembly of microtissues within 3D plotted scaffolds [23]
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