Bio-inks for drop-on-demand cell printing (2013)

A rapidly growing synergy between biological science and engineering technology is currently re-shaping the way we view the challenge of treating injury and disease. In particular, emerging biofabrication techniques that allow the precise construction of complex biological structures have reinvigorated the effort to engineer replacement tissues and organs. In addition to the potential for direct therapeutic approaches, advanced engineered tissues promise to significantly improve in vitro studies of fundamental cell biology and disease processes, and expedite drug development.\nDrop-on-demand cell printing technologies are at the forefront of these advances in biofabrication. These approaches offer the ability to place living cells, biomaterials and other factors in defined arrangements in two or three dimensions in order to reproduce the complex spatial interplay that regulates tissue function. Significant progress towards this goal has been made over the last decade, but the design of bioinks remains a key challenge due to the need to simultaneously satisfy disparate engineering and biological requirements. The aim of this thesis was to develop bioinks for drop-on-demand cell printing that enable the robust deposition of living cells. Specifically, a suitable bio-ink should be non-cytotoxic, prevent cell settling and aggregation, possess optimal fluid properties (i.e. viscosity and surface tension) for drop-on-demand printing and contain minimal dry mass.\nBio-inks were developed by forming gellan gum (GG) microgel suspensions in cell culture media by applying shear during gelation. At a low polymer concentration (0.05% w/v) the bio-ink showed a yield stress (~ 43 mPa), while exhibiting a low viscosity (~ 1.7 mPa.s) at high shear rates (103 s-1). These properties were shown to prevent cell settling and aggregation without affecting printability. Surfactants were added to the formulation to achieve surface tension reduction for inkjet printing. Addition of the fluorosurfactant, Novec FC-4430, allowed a suitable surface tension (~ 30 mN/m at 0.05% v/v) to be achieved, while Poloxamer 188 (P188) was included (0.1% v/v) for its reported cell-protecting qualities. Neither surfactant significantly affected the bio-ink structure or rheology, and C2C12 (skeletal muscle) and PC12 (pheocromocytoma) cells exposed to the surfactant-containing bio-ink for 2 hr exhibited normal viability, proliferation and differentiation.\nThe bio-ink formulations, with and without surfactants, proved suitable for cell deposition by microvalve and inkjet printing, respectively. The bio-ink enabled reproducible cell output over 1 hr printing periods from both a microvalve printer (Deerac Equator GX1) and multiple-nozzle piezoelectric inkjet print heads (Xaar-126). Printed cells exhibited phenotypic responses that were comparable to controls. It was also demonstrated that P188 had a protective effect on cells during inkjet printing.\nInkjet cell printing using the bio-ink was applied to the fabrication of two dimensional cell constructs. Cell microarrays were printed on glass slides and subsequently analysed by a novel surface-sampling mass spectrometry technique. The combination of the two techniques allowed the detection of characteristic lipid profile ‘fingerprints’ from single printed C2C12, PC12 and L929 (fibroblast) cells. The bio-ink also enabled simultaneous printing of two cells types (C2C12 and PC12) from two inkjet print heads to create patterned co-cultures of viable cells on collagen substrates.\nBoth cell printing techniques were applied in approaches to fabricate cell-laden hydrogel constructs. In a novel method, a microvalve printer tip dispensed cell-laden solutions while immersed in GG solutions. In another approach, cell-laden bio-ink and GG solutions were printed simultaneously from separate inkjet print heads. In both cases, cell-encapsulating hydrogels were formed through ionic crosslinking of GG by the cell solution. Encapsulated cells did not interact with the hydrogel matrix, however, and this observation was repeated in standard culture. Modification strategies to impart biofunctionality to GG hydrogels were therefore explored. Covalent conjugation of the peptide G4RGDSY to purified GG was optimised. The peptide conferred significant improvement in the attachment, proliferation and differentiation of surface-seeded and encapsulated C2C12 cells, which was attributed to specific binding with the RGD domain.\nThe work in this thesis shows that smarter design of bio-ink materials can yield important advances in drop-on-demand cell printing approaches. The bio-inks address some of the key challenges in the continuing evolution of these techniques towards becoming clinically relevant biofabrication tools.