A facile method to fabricate cell-laden hydrogelmicroparticles of tunable sizes using thermal inkjet bioprinting

Abstract

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 SPAN80 concentrations resulting in smaller and more stableHMPs. The study further investigates the influence of jetting volume onHMPsize distribution, revealing that larger jetting volumes lead to increasedHMPsizes, attributed to droplet coalescence. This is supported by our further study via aMonteCarlo 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 cytoplasmicmembrane 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.

References

[1] Highley CB, Song KH, Daly AC, Burdick JA. Jammed microgel inks for 3D printing applications. Adv Sci. 2019;6:1801076.

[2] Griffin DR, Weaver WM, Scumpia PO, Di Carlo D, Segura T. Accelerated wound healing by injectable microporous gel scaffolds assembled from annealed building blocks. Nat Mater. 2015;14:737-744.

[3] Mealy JE, Chung JJ, Jeong HH, et al. Injectable granular hydrogels with multifunctional properties for biomedical applications. Adv Mater. 2018;30:1705912.

[4] Sideris E, Griffin DR, Ding Y, et al. Particle hydrogels based on hyaluronic acid building blocks. ACS Biomater Sci Eng. 2016;2:2034-2041.

[5] Daly AC, Riley L, Segura T, Burdick JA. Hydrogel microparticles for biomedical applications. Nat Rev Mater. 2020;5:20-43.

[6] Franco C, Price J, West J. Development and optimization of a dual-photoinitiator, emulsion-based technique for rapid generation of cell-laden hydrogel microspheres. Acta Biomater. 2011;7:3267-3276.

[7] Leong W, Lau TT, Wang D-A. A temperature-cured dissolvable gelatin microsphere-based cell carrier for chondrocyte delivery in a hydrogel scaffolding system. Acta Biomater. 2013;9:6459-6467.

[8] Xu Q, Hashimoto M, Dang TT, et al. Preparation of monodisperse biodegradable polymer microparticles using a microfluidic flow-focusing device for controlled drug delivery. Small. 2009;5:1575-1581.

[9] Sinclair A, O’Kelly MB, Bai T, Hung HC, Jain P, Jiang S. Self-healing zwitterionic microgels as a versatile platform for malleable cell constructs and injectable therapies. Adv Mater. 2018;30:1803087.

[10] Hinton TJ, Jallerat Q, Palchesko RN, et al. Three-dimensional printing of complex biological structures by freeform reversible embedding of suspended hydrogels. Sci Adv. 2015;1:e1500758.

[11] Thorsen T, Roberts RW, Arnold FH, Quake SR. Dynamic pattern formation in a vesicle-generating microfluidic device. Phys Rev Lett. 2001;86:4163.

[12] Anna SL, Bontoux N, Stone HA. Formation of dispersions using “flow focusing”in microchannels. Appl Phys Lett. 2003;82:364-366.

[13] De Geest BG, Urbanski JP, Thorsen T, Demeester J, De Smedt SC. Synthesis of monodisperse biodegradable microgels in microfluidic devices. Langmuir. 2005;21:10275-10279.

[14] Pittermannová A, Ruberová Z, Zadražil A, Bremond N, Bibette J, Štěpánek F. Microfluidic fabrication of composite hydrogel microparticles in the size range of blood cells. RSC Adv. 2016;6:103532-103540.

[15] Helgeson ME, Chapin SC, Doyle PS. Hydrogel microparticles from lithographic processes: novel materials for fundamental and applied colloid science. Curr Opin Colloid Interface Sci. 2011;16:106-117.

[16] Lee SA, Chung SE, Park W, Lee SH, Kwon S. Three-dimensional fabrication of heterogeneous microstructures using soft membrane deformation and optofluidic maskless lithography. Lab Chip. 2009;9:1670-1675.

[17] Chung SE, Park W, Shin S, Lee SA, Kwon S. Guided and fluidic self-assembly of microstructures using railed microfluidic channels. Nat Mater. 2008;7:581-587.

[18] Nichol JW, Koshy ST, Bae H, Hwang CM, Yamanlar S, Khademhosseini A. Cell-laden microengineered gelatin methacrylate hydrogels. Biomaterials. 2010;31:5536-5544.

[19] Panda P, Ali S, Lo E, et al. Stop-flow lithography to generate cell-laden microgel particles. Lab Chip. 2008;8:1056-1061.

[20] Naqvi SM, Vedicherla S, Gansau J, McIntyre T, Doherty M, Buckley CT. Living cell factories-electrosprayed microcapsules and microcarriers for minimally invasive delivery. Adv Mater. 2016;28:5662-5671.

[21] Gansau J, Kelly L, Buckley CT. Influence of key processing parameters and seeding density effects of microencapsulated chondrocytes fabricated using electrohydrodynamic spraying. Biofabrication. 2018;10:035011.

[22] Pancholi K, Ahras N, Stride E, Edirisinghe M. Novel electrohydrodynamic preparation of porous chitosan particles for drug delivery. J Mater Sci Mater Med. 2009;20:917-923.

[23] Qayyum AS, Jain E, Kolar G, Kim Y, Sell SA, Zustiak SP. Design of electrohydrodynamic sprayed polyethylene glycol hydrogel microspheres for cell encapsulation. Biofabrication. 2017;9:025019.

[24] Young CJ, Poole-Warren LA, Martens PJ. Combining submerged electrospray and UV photopolymerization for production of synthetic hydrogel microspheres for cell encapsulation. Biotechnol Bioeng. 2012;109:1561-1570.

[25] Ng WL, Chua CK, Shen Y-F. Print me an organ! Why we are not there yet. Prog Polym Sci. 2019;97:101145.

[26] Lee JM, Ng WL, Yeong WY. Resolution and shape in bioprinting: strategizing towards complex tissue and organ printing. Appl Phys Rev. 2019;6:011307.

[27] Ng WL, Chan A, Ong YS, Chua CK. Deep learning for fabrication and maturation of 3D bioprinted tissues and organs. Virtual Phys Prototyp. 2020;15:340-358.

[28] Ng WL, An J, Chua CK. Process, material, and regulatory considerations for 3D printed medical devices and tissue constructs. Engineering. 2024;36:146-166.

[29] Ng WL, Shkolnikov V. Jetting-based bioprinting: process, dispense physics, and applications. Bio-Des Manuf.2024.

[30] Ng WL, Shkolnikov V. Optimizing cell deposition for inkjet-based bioprinting. Int J Bioprint. 2024;10:2135.

[31] Ng WL, Lee JM, Yeong WY, Win Naing M. Microvalve-based bioprinting—process, bio-inks and applications. Biomater Sci. 2017;5:632-647.

[32] Ng WL, Yeong WY, Naing MW. Polyvinylpyrrolidone-based bio-ink improves cell viability and homogeneity during drop-on-demand printing. Materials. 2017;10:190.

[33] Zgeib R, Wang X, Zaeri A, Zhang F, Cao K, Chang RC. Development of a low-cost quad-extrusion 3D bioprinting system for multi-material tissue constructs. Int J Bioprint. 2023;10:0159.

[34] Oh D, Shirzad M, Kim MC, Chung E-J, Nam SY. Rheology-informed hierarchical machine learning model for the prediction of printing resolution in extrusion-based bioprinting. Int J Bioprint. 2023;9:1280.

[35] Liu S, Zhang H, Ahlfeld T, et al. Evaluation of different crosslinking methods in altering the properties of extrusion-printed chitosan-based multi-material hydrogel composites. Bio-Des Manuf. 2023;6:150-173.

[36] Chand R, Muhire BS, Vijayavenkataraman S. Computational fluid dynamics assessment of the effect of bioprinting parameters in extrusion bioprinting. Int J Bioprint. 2022;8:545.

[37] Ng WL, Yeong WY, Naing MW. Development of polyelectrolyte chitosan-gelatin hydrogels for skin bioprinting. Procedia CIRP. 2016;49:105-112.

[38] Ng WL, Yeong WY, Naing MW. Polyelectrolyte gelatin-chitosan hydrogel optimized for 3D bioprinting in skin tissue engineering. Int J Bioprint. 2016;2:53-62.

[39] Zhuang P, Ng WL, An J, Chua CK, Tan LP. Layer-by-layer ultraviolet assisted extrusion-based (UAE) bioprinting of hydrogel constructs with high aspect ratio for soft tissue engineering applications. PLoS One. 2019;14:e0216776.

[40] Ng WL, Lee JM, Zhou M, et al. Vat polymerization-based bioprinting–process, materials, applications and regulatory challenges. Biofabrication. 2020;12:022001.

[41] Ng WL, Lee JM, Zhou M, Yeong WY. Hydrogels for 3-D bioprinting-based tissue engineering. In: R Narayan, ed. Rapid Prototyping of Biomaterials. Elsevier;2020:183-204.

[42] Huang X, Ng WL, Yeong WY. Predicting the number of printed cells during inkjet-based bioprinting process based on droplet velocity profile using machine learning approaches. J Intell Manuf. 2024;35:2349-2364.

[43] Ng WL, Yeong WY, Naing MW, Microvalve bioprinting of cellular droplets with high resolution and consistency. Proceedings of the International Conference on Progress in Additive Manufacturing, Singapore, 2016:397-402.

[44] Ng WL, Ayi TC, Liu Y-C, Sing SL, Yeong WY, Tan B-H. Fabrication and characterization of 3D bioprinted triple-layered human alveolar lung models. Int J Bioprint. 2021;7:332.

[45] Ng WL, Goh MH, Yeong WY, Naing MW. Applying macromolecular crowding to 3D bioprinting: fabrication of 3D hierarchical porous collagen-based hydrogel constructs. Biomater Sci. 2018;6:562-574.

[46] Ng WL, Tan ZQ, Yeong WY, Naing MW. Proof-of-concept:3D bioprinting of pigmented human skin constructs. Biofabrication. 2018;10:025005.

[47] Saunders RE, Derby B. Inkjet printing biomaterials for tissue engineering: bioprinting. Int Mater Rev. 2014;59:430-448.

[48] Zheng J, Zhu M, Ferracci G, Cho NJ, Lee BH. Hydrolytic stability of methacrylamide and methacrylate in gelatin methacryloyl and decoupling of gelatin methacrylamide from gelatin methacryloyl through hydrolysis. Macromol Chem Phys. 2018;219:1800266.

[49] Suntornnond R, Ng WL, Huang X, Yeow ECH, Yeong WY. Improving printability of hydrogel-based bio-inks for thermal inkjet bioprinting applications via saponification and heat treatment process. J Mater Chem B. 2022;10:5989-6000.

[50] Ng WL, Huang X, Shkolnikov V, Suntornnond R, Yeong WY. Polyvinylpyrrolidone-based bioink: influence of bioink properties on printing performance and cell proliferation during inkjet-based bioprinting. Bio-Des Manuf. 2023;6:676-690.

[51] Ng WL, Huang X, Shkolnikov V, Goh GL, Suntornnond R, Yeong WY. Controlling droplet impact velocity and droplet volume: key factors to achieving high cell viability in sub-nanoliter droplet-based bioprinting. Int J Bioprint. 2021;8:424.

[52] Harris CR, Millman KJ, Van Der Walt SJ, et al. Array programming with NumPy. Nature. 2020;585:357-362.

RIGHTS & PERMISSIONS

2024 The Author(s). Droplet published by Jilin University and John Wiley & Sons Australia, Ltd.