Droplet｜一种利用热喷墨生物打印技术制备尺寸可调的载细胞水凝胶微粒的简便方法

(¹HP-NTU Digital Manufacturing Corporate Lab, Nanyang Technological University (NTU), Singapore; ²Singapore Centre for 3D Printing (SC3DP), School of Mechanical and Aerospace Engineering, Nanyang Technological University (NTU), Singapore; ³HP Inc., Palo Alto, California, USA)

This study investigates the application of a drop-on-demand (DOD) thermal inkjet (TIJ)-based bioprinting system for the fabrication of cell-laden hydrogel microparticles (HMPs) with tunable sizes. The TIJ bioprinting technique involves the formation of vapor bubbles within the print chamber through thermal energy, expelling small droplets of bio-ink onto a substrate. The study employs a heat-treated saponified gelatin-based bio-ink, HSP-GelMA. This bio-ink is modified through methacrylic anhydride functionalization and undergoes subsequent saponification and heat treatment processes. Various concentrations of SPAN 80 surfactant in mineral oil were evaluated to assess their influence on HMP size and stability. The results indicate a direct correlation, with higher SPAN 80 concentrations resulting in smaller and more stable HMPs. The study further investigates the influence of jetting volume on HMP size distribution, revealing that larger jetting volumes lead to increased HMP sizes, attributed to droplet coalescence. This is supported by our further study via a Monte Carlo simulation, which shows that the mean droplet diameter grows approximately linear with the number of dispensed droplets. In addition, the study demonstrates the capability of the TIJ bioprinting system to achieve multimaterial encapsulation within HMPs, exemplified by staining living cells with distinct cytoplasmic membrane dyes. The presented approach provides insights into the controlled fabrication of cell-laden HMPs, highlighting the versatility of the TIJ bioprinting system for potential applications in tissue engineering and drug delivery.

Fig.1 Fabrication of cell-laden HMPs of tunable sizes by controlling the jetting volume of the hydrogel-based droplets inside the mineral oil containing SPAN 80 surfactant within the well plates.

Fig.2 Influence of SPAN 80 concentration in mineral oil on the size of HMPs. (a) Representative images depict HMP sizes in mineral oil containing varying SPAN 80 concentrations (0, 2, 3, and 5%, v/v) at an arbitrary jetting volume of 450 nL; scale bar = 200 µm. (b) Measured HMP sizes at varying SPAN 80 surfactant concentrations (n = 5). Data are represented as mean ± standard deviation, ***p < 0.001, *p < 0.05. (c) A simplified schematic drawing illustrating the influence of SPAN 80 surfactant concentration (depicted by orange surfactant molecules with hydrophobic heads and hydrophilic tails) on bio-ink droplet behavior (represented by blue spherical shapes). At higher SPAN 80 concentrations, a complete monolayer forms at the water–oil interface, mitigating potential coalescence when smaller HMPs come into proximity.

Fig.3 Simulation studies to evaluate mean normalized droplet radius and normalized standard deviation of droplet radius. (a) Mean droplet radius scaled by the dispensed (initial) droplet radius    as a function of the number of droplets dispensed, and as a function of the scaled width of the dispensing distribution. The scaled distribution width is the standard deviation of the spatial distribution of the droplets as dispensed from the nozzle, scaled by the dispensed (initial) droplet radius   . The mean droplet radius increases sublinearly with the number of droplets dispensed. For a large number of droplets dispensed, the mean normalized droplet radius scales inversely with the width of the dispensing distribution. (b) The standard deviation of droplet radius scaled by dispensed (initial) droplet radius    as a function of the number of droplets dispensed, and as a function of the scaled width of the dispensing distribution. The standard deviation of droplet radius increases approximately linearly with the number of droplets dispensed. (c) Mean normalized droplet radius (  ) grows inversely as a function of the normalized standard deviation of the dispense distribution   . (d) For a large number of droplets dispensed, the normalized standard deviation of droplet radius scales approximately inversely with the width of the dispensing distribution.

Fig.4 Influence of jetting volume on the size and stability of fabricated HMPs in mineral oil containing 2% (v/v) SPAN 80 surfactant. (a) Representative images showcase the fabricated HMPs at varying jetting volumes, with a scale bar of 400 µm. (b) The size distribution of fabricated HMPs is presented across varying jetting volumes.

Fig.5 A proof-of-concept study demonstrating the fabrication of multimaterial HMPs of tunable sizes and ratios. (a) The fabrication process involved the deposition of the first cell-laden bio-ink (green) into a 96-well plate containing mineral oil with 2% (v/v) SPAN 80 surfactant with a 30-s pause, followed by the subsequent jetting of the second cell-laden bio-ink (red) at the same spot. UV crosslinking was performed to maintain the structural integrity of the multimaterial HMPs. (b) Representative images of fabricated multimaterial HMPs of tunable sizes and ratios by controlling the volume of deposited bio-inks—(top) optical bright-field images, (bottom) fluorescence images; scale bars = 200 µm.