Pre-processing involves preparing the building block tissues that are used to ultimately fabricate the 3D
tissue. Here, cellular spheroids are the building blocks being used as the ‘bio-ink.’ Spheroids are attractive for tissue fabrication techniques due to having precise control over cell and extracellular matrix (ECM) composition, the ability for upscaled production and repeatability, their three-dimensional nature and the fact that spheroids will produce their own ECM over time. Spheroids with tissue-specific functions have been fabricated for cardiac, vascular, cartilage, bone, hepatic and pancreatic applications, demonstrating their potentially broad impact for tissue fabrication. In addition to tissue engineering applications, spheroids are used in studying cancer biology and also in pharmaceuticals with high throughput toxicology and drug testing.
There are many pre-processing methods used to prepare cellular spheroids, which include the pellet or re-aggregation culture, spinner culture, rotating wall vessels, cell sheet techniques, liquid overlay, microfluidics, external forces, the hanging drop technique, and micro-molded hydrogels [5
]. The most popular spheroid pre-processing methods are re-aggregation, hanging drop and micro-molds. In the re-aggregation method, cells from a confluent flask are collected and centrifuged down to form a cell pellet in a tube [5
]. This pellet can be removed from the tube, cut into fragments and then incubated until they form rounded spheroids. In the hanging drop technique, small volumes (15-30 µL) of cell suspensions are pipetted onto the lids of petri dishes [5
]. The lid is then inverted, with the drops staying attached due to surface tension. Gravity
causes the cell suspension to concentrate at the bottom of the droplet, forming a spheroid. Micro-mold pre-processing utilizes computer software for rapid prototyping of ‘templates’ for creating micro-molds with an array of cylindrical pegs with rounded tops. These micro-molds are then used to cast non-adhesive hydrogels into which cells can be seeded into. Due to the non-adhesive surface, the cells will aggregate and form a spheroid.
Spheroids can be fabricated with a either single or multiple cell types, which demonstrates the potential for fabricating many types of tissues
. Even more, endothelial cells can be co-cultured with other cell types, like smooth muscle cells, to create uniluminal spheroids [6
]. This is important for tissue engineering because it highlights the potential for using uniluminal vascular spheroids as modules for fabricating blood vessels. ECM proteins, like collagen or fibronectin, can be incorporated into cellular spheroids to promote cell-matrix interactions and provide an early structural network for the developing spheroids
. Magnetic nanoparticles (MNPs) have also been incorporated into cellular spheroids to allow for the ability to pattern and align the spheroids for tissue assembly. This pre-processing development has allowed for tissues to be manipulated with physical magnetic forces at a distance in the processing step and has potential for being used for maturation techniques.
Millions of tissue spheroids will be required for organ fabrication [7
]. High throughput methods for spheroid fabrication have been developed for biological research, therapeutic testing and 3D tissue construction [8
]. Micro-mold arrays are leading this thrust, with hanging drop and round-bottom well plate designs for fabricating up to 384 spheroids per standard 96 well plate. The use of these approaches, partnered with a robotic dispenser increases the productivity of spheroid biofabrication [7
]. After formation, spheroids can be stored in bio-cartridges (micropipettes) for dispensing in the processing step, or stored in cell culture medium for a desired incubation period. Preventing unwanted fusion of stored tissue spheroids will be challenge that develops with upscaling spheroid fabrication.
Ultimately, the pre-processing step allows for customization of the spheroid for specific applications because of the control over cell type, size, and ECM content. Given this precise control over composition and their versatility, spheroids are a desirable candidate for the pre-processing step in rapid fabrication of tissues.