Introduction: A key limitation preventing the widespread use of cell-based additive manufacturing is the lack of suitable bio-inks that are cell-compatible and have the required properties for printing[1]. Current challenges of many commonly used bio-inks include difficulty maintaining a homogeneous cell suspension within the ink cartridge, preventing long-term exposure to photo-activators, avoiding cell damage during extrusion, customizing the printed matrix properties to facilitate cell-matrix interactions, and printing within a bath to prevent cell dehydration[2]. In response, we designed a new family of tunable biomaterials specifically for cell-based additive manufacturing.
Materials and Methods: These hydrogel-based bio-inks are produced from a blend of recombinantly engineered protein and peptide-modified alginate polysaccharide. This design is advantageous due to the ability of the bio-ink to undergo two-stages of crosslinking: (i) weak, peptide-based, self-assembly to homogeneously encapsulate cells within the ink cartridge and (ii) electrostatic crosslinking of alginate upon printing within a bath to rapidly stabilize the construct. The use of engineered proteins provides control of ligand presentation for cellular attachment and signaling, while the alginate enables cyto-compatible, rapid crosslinking post-printing. Bio-ink mechanical properties were evaluated using oscillatory rheometry; cell-based printing was achieved with an extrusion-based, pressure-controlled (2 PSI) BioBots Printer; and quantitative cell analysis was performed with Live/Dead analysis of membrane rupture, metabolic analysis of cell health, DNA analysis of cell proliferation, and immunocytochemistry followed by confocal microscopy to determine cell homogeneity and morphology within printed 3D constructs. Cell types included a 3T3 mouse embryo fibroblast line and primary human adipose-derived stem cells (hASCs) obtained with informed consent as approved by IRB oversight.
Results and Discussion: While many bio-inks are viscous liquids within the ink cartridge, the bio-inks designed here are weak hydrogels (storage moduli, G' ~10 Pa) prior to printing. Compared to viscous cell/ink mixtures of pure alginate, which experienced significant cell settling within only one hour, our self-assembled hydrogels maintained a homogeneous, 95% viable, cell/ink mixture for up to 3 hours (longest time point tested). During extrusion through fine gauge nozzles (G = 27-32), the self-assembled inks were found to provide significant mechanical shielding to encapsulated 3T3 cells and hASCs. This resulted in an 80% decrease in the number of cells with membrane damage compared to printing with viscous alginate inks. Post-printing, exposure to calcium ions induced secondary crosslinking and a 100-fold increase in scaffold stiffness (G' ~1 kPa). One week post-printing, the encapsulated hASCs remained >95% viable and homogeneously distributed and adopted a well-spread morphology.
Conclusion: As advances in bio-printer design enable the manufacturing of more complex structures, the requirements for bio-inks will become more stringent. Longer print times and faster print speeds lead to cell settling within the ink cartridge, cell damage during printing, and hence printed constructs with inhomogeneous cell distribution and poor cell viability. By designing a bio-ink that combines weak crosslinking within the ink cartridge and ionic crosslinking post-printing, we have introduced a self-assembled bio-ink that overcomes these common challenges.
National Science Foundation DMR-1508006; California Institute for Regenerative Medicine RT3-07948
References:
[1] Murphy, S. V. & Atala A. (2014). 3D bioprinting of tissues and organs. Nature Biotechnology, 32, 773-785.
[2] Malda J. et al. (2013). 25th Anniversary Article: Engineering Hydrogels for Biofabrication. Advanced Materials, 25, 5011–5028.