Study of cell viability during laser-direct writing of cell-alginate suspension.

摘要

Laser-assisted cell printing, developed based on Matrix-assisted pulsed-laser evaporation direct-write (MAPLE DW), a typical LIFT (laser-induced forward transfer) practice, has been emerging as one of the most promising biofabrication techniques. Alginate, particularly sodium alginate, is extensively used as the constituent of bioink in laser-assisted cell printing. However, thus far, studies investigating the effect of alginate gelation on cell viability in laser-assisted cell printing are lacking. The objective of this study is to investigate the effects of gelation, gelation time, sodium alginate concentration, and the effect of operating conditions such as the laser fluence on post-transfer cell viability during laser-assisted cell printing.;Two experimental setups have been designed in this study. Experimental setup A was characterized by laser fluences 800, 1200, and 1600 mJ/cm 2 and a constant alginate concentration of 1% w/v with 5 × 106 NIH3T3 cells/ml in bioink. Experimental setup B was characterized by alginate concentrations of 1, 2, and 3 % w/v with 5 × 106 NIH3T3 cells/ml in bioink and a constant laser fluence of 800 mJ/cm2. Experimental setup A was designed to study the effects of gelation, gelation time, and laser fluence on post-transfer cell viability. Experimental setup B was designed to study effects of gelation and sodium alginate concentration on post-transfer cell viability. Furthermore, cell-laden alginate droplets were subjected to no gelation, two-minute gelation, or ten-minute gelation. Cell viability was evaluated immediately after printing and after 24 hours of incubation.;Process-induced cell injury during alginate gelation in laser-assisted cell printing is systematically elucidated through investigating the effects of operating conditions and material properties on the post-transfer cell viability and cell injury reversibility. Two-minute gelation is observed to increase cell viability over 24 hours because of cushion effect. That is, forming gel membrane has minimized the impact of mechanical stresses generated during droplet landing. Despite ten minutes gelation having a cushion effect during droplet landing, it is observed to decrease cell viability over 24 hours because of the thick gel membrane which reduces nutrient diffusion from culture medium. Also, the longer exposure of encapsulated cells to calcium chloride has resulted in greater cell injury due to Ca2+ ions. Increase in laser fluence as well as alginate concentration is observed to decrease cell viability by introducing greater mechanical stresses during droplet formation. The process-induced cell death is modeled using power-law and Gompertz models. Gompertz model is observed to better predict cell viability than power-law model. However, the two models ignore molecular signaling pathways that govern the cell responses. Hence, future studies have to model process-induced cell death based on molecular signaling pathways.