Abstract
Alginate hydrogel beads are a common platform for generating 3D cell cultures in biomedical research. Simple methods for bead generation using a manual pipettor or syringe are low-throughput and produce beads showing high variability in size and shape. To address these challenges, we designed a 3D printed bead generator that uses an airflow to cleave beads from a stream of hydrogel solution. The performance of the proposed alginate bead generator was evaluated by changing the volume flow rates of alginate (QAlg) and air (QA), the diameter of device nozzle (d) and the concentration of alginate gel solution (C). We identified that the diameter of beads (D = 0.9 -2.8 mm) can be precisely controlled by changing QA and d. Also the bead generation frequency (f) can be tuned by changing QAlg. Finally, we demonstrated that viability and biological function (pericellular matrix deposition) of chondrocytes were not adversely affected by high f using this bead generator. Because 3D printing is becoming a more accessible technique, our unique design will allow greater access to average biomedical research laboratories, STEM education and industries in cost- and time-effective manner.
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K. Alessandri, M. Feyeux, B. Gurchenkov, C. Delgado, A. Trushko, K.-H. Krause, D. Vignjević, P. Nassoy, A. Roux, A 3D printed microfluidic device for production of functionalized hydrogel microcapsules for culture and differentiation of human Neuronal Stem Cells (hNSC). Lab. Chip. 16, 1593–1604 (2016)
K. Alessandri, B.R. Sarangi, V.V. Gurchenkov, B. Sinha, T.R. Kießling, L. Fetler, F. Rico, S. Scheuring, C. Lamaze, A. Simon, S. Geraldo, D. Vignjević, H. Doméjean, L. Rolland, A. Funfak, J. Bibette, N. Bremond, P. Nassoy, Cellular capsules as a tool for multicellular spheroid production and for investigating the mechanics of tumor progression in vitro. Proc. Natl. Acad. Sci. U.S.A. 110, 14843–14848 (2013)
X. Bai, M. Gao, S. Syed, J. Zhuang, X. Xu, X.-Q. Zhang, Bioactive hydrogels for bone regeneration. Bioact. Mater. 3, 401–417 (2018)
B.M. Baker, C.S. Chen, Deconstructing the third dimension – how 3D culture microenvironments alter cellular cues. J. Cell. Sci. 125, 3015–3024 (2012)
S.J. Bidarra, C.C. Barrias, P.L. Granja, Injectable alginate hydrogels for cell delivery in tissue engineering. Acta. Biomater. 10, 1646–1662 (2014)
E.-S. Chan, T.-K. Lim, W.-P. Voo, R. Pogaku, B.T. Tey, Z. Zhang, Effect of formulation of alginate beads on their mechanical behavior and stiffness. Particuology 9, 228–234 (2011)
S.H. Ching, N. Bansal, B. Bhandari, Alginate gel particles–A review of production techniques and physical properties. Crit. Rev. Food Sci. Nutr. 57, 1133–1152 (2017)
C.-H. Choi, J.-H. Jung, Y.W. Rhee, D.-P. Kim, S.-E. Shim, C.-S. Lee, Generation of monodisperse alginate microbeads and in situ encapsulation of cell in microfluidic device. Biomed. Microdevices 9, 855–862 (2007)
C. Cramer, P. Fischer, E.J. Windhab, Drop formation in a co-flowing ambient fluid. Chem. Eng. Sci. 59, 3045–3058 (2004)
F. Davarcı, D. Turan, B. Ozcelik, D. Poncelet, The influence of solution viscosities and surface tension on calcium-alginate microbead formation using dripping technique. Food Hydrocoll. 62, 119–127 (2017)
P. Del Gaudio, P. Colombo, G. Colombo, P. Russo, F. Sonvico, Mechanisms of formation and disintegration of alginate beads obtained by prilling. Int. J. Pharm. 302, 1–9 (2005)
A.G. Erickson, T.D. Laughlin, S.M. Romereim, C.N. Sargus-Patino, A.K. Pannier, A.T. Dudley, A Tunable, Three-Dimensional in Vitro Culture Model of Growth Plate Cartilage Using Alginate Hydrogel Scaffolds (Tissue Eng, Part A, 2017).
L.P. Ferreira, V.M. Gaspar, J.F. Mano, Design of spherically structured 3D in vitro tumor models -Advances and prospects. Acta. Biomater. 75, 11–34 (2018)
P. Garstecki, M.J. Fuerstman, H.A. Stone, G.M. Whitesides, Formation of droplets and bubbles in a microfluidic T-junction—scaling and mechanism of break-up. Lab. Chip. 6, 437–446 (2006)
D. J. Hadley, K. T. Campbell, M. H. Gabriel and E. A. Silva. Open-source 3D printed air-jet for generating monodispersed alginate microhydrogels. bioRxiv 804849, (2019)
A.G. Håti, D.C. Bassett, J.M. Ribe, P. Sikorski, D.A. Weitz, B.T. Stokke, Versatile, cell and chip friendly method to gel alginate in microfluidic devices. Lab. Chip. 16, 3718–3727 (2016)
S. Iwamoto, K. Nakagawa, S. Sugiura, M. Nakajima, Preparation of gelatin microbeads with a narrow size distribution using microchannel emulsification. AAPS PharmSciTech 3, 72–76 (2002)
D. Jain, D. Bar-Shalom, Alginate drug delivery systems: application in context of pharmaceutical and biomedical research. Drug Dev. Ind. Pharm. 40, 1576–1584 (2014)
B.-B. Lee, P. Ravindra, E.-S. Chan, A CRITICAL REVIEW: SURFACE AND INTERFACIAL TENSION MEASUREMENT BY THE DROP WEIGHT METHOD. Chem. Eng. Commun. 195, 889–924 (2008)
B.-B. Lee, P. Ravindra, E.-S. Chan, Size and shape of calcium alginate beads produced by extrusion dripping. Chem. Eng. Technol. 36, 1627–1642 (2013)
W. Lee, N. Kalashnikov, S. Mok, R. Halaoui, E. Kuzmin, A.J. Putnam, S. Takayama, M. Park, L. McCaffrey, R. Zhao, R.L. Leask, C. Moraes, Dispersible hydrogel force sensors reveal patterns of solid mechanical stress in multicellular spheroid cultures. Nat. Commun. 10, 144 (2019)
R. Martinez-Duarte and M. Madou. SU-8 photolithography and its impact on microfluidics. Microfluid. Nanofluid. Handb. 231–268, (2011).
Y.T. Matsunaga, Y. Morimoto, S. Takeuchi, Molding Cell Beads for Rapid Construction of Macroscopic 3D Tissue Architecture. Adv. Mater. 23, H90–H94 (2011)
D.J. McClements, Recent progress in hydrogel delivery systems for improving nutraceutical bioavailability. Food Hydrocoll. 68, 238–245 (2017)
E. Mohagheghian, J. Luo, J. Chen, G. Chaudhary, J. Chen, J. Sun, R.H. Ewoldt, N. Wang, Quantifying compressive forces between living cell layers and within tissues using elastic round microgels. Nat. Commun. 9, 1878 (2018)
Y. Morimoto, W.-H. Tan, S. Takeuchi, Three-dimensional axisymmetric flow-focusing device using stereolithography. Biomed. Microdevices 11, 369–377 (2009)
U. Prüsse, L. Bilancetti, M. Bučko, B. Bugarski, J. Bukowski, P. Gemeiner, D. Lewińska, V. Manojlovic, B. Massart, C. Nastruzzi, V. Nedovic, D. Poncelet, S. Siebenhaar, L. Tobler, A. Tosi, A. Vikartovská, K.-D. Vorlop, Comparison of different technologies for alginate beads production. Chem. Pap. 62, 364 (2008)
Y. Senuma, C. Lowe, Y. Zweifel, J.G. Hilborn, I. Marison, Alginate hydrogel microspheres and microcapsules prepared by spinning disk atomization. Biotechnol. Bioeng. 67, 616–622 (2000)
R.F. Shepherd, J.C. Conrad, S.K. Rhodes, D.R. Link, M. Marquez, D.A. Weitz, J.A. Lewis, Microfluidic Assembly of Homogeneous and Janus Colloid-Filled Hydrogel Granules. Langmuir 22, 8618–8622 (2006)
T. Suksamran, P. Opanasopit, T. Rojanarata, T. Ngawhirunpat, U. Ruktanonchai, P. Supaphol, Biodegradable alginate microparticles developed by electrohydrodynamic spraying techniques for oral delivery of protein. J. Microencapsulation 26, 563–570 (2009)
W.-H. Tan, S. Takeuchi, Monodisperse Alginate Hydrogel Microbeads for Cell Encapsulation. Adv. Mater. 19, 2696–2701 (2007)
S.K. Tang, G.M. Whitesides, Basic microfluidic and soft lithographic techniques, in Optofluidics: Fundamentals, Devices and Applications. ed. by Y. Fainman, L.P. Lee, D. Psaltis, C. Yang (McGraw-Hill, New York, 2010), pp. 7–31
S. Tendulkar, S.-H. Mirmalek-Sani, C. Childers, J. Saul, E.C. Opara, M.K. Ramasubramanian, A three-dimensional microfluidic approach to scaling up microencapsulation of cells. Biomed. Microdevices 14, 461–469 (2012)
N. Träber, K. Uhlmann, S. Girardo, G. Kesavan, K. Wagner, J. Friedrichs, R. Goswami, K. Bai, M. Brand, C. Werner, D. Balzani, J. Guck, Polyacrylamide Bead Sensors for in vivo Quantification of Cell-Scale Stress in Zebrafish Development. Sci. Rep. 9, 17031 (2019)
A.S. Utada, A. Fernandez-Nieves, H.A. Stone, D.A. Weitz, Dripping to Jetting Transitions in Coflowing Liquid Streams. Phys. Rev. Lett. 99, 094502 (2007)
S. Utech, R. Prodanovic, A.S. Mao, R. Ostafe, D.J. Mooney, D.A. Weitz, Microfluidic Generation of Monodisperse, Structurally Homogeneous Alginate Microgels for Cell Encapsulation and 3D Cell Culture. Adv. Healthcare Mater. 4, 1628–1633 (2015)
M. Workamp, S. Alaie, J.A. Dijksman, Coaxial air flow device for the production of millimeter-sized spherical hydrogel particles. Rev. Sci. Instrum. 87, 125113 (2016)
Y. Xia, G.M. Whitesides, SOFT LITHOGRAPHY. Annu. Rev. Mater. Sci. 28, 153–184 (1998)
L. Yu, C. Ni, S.M. Grist, C. Bayly, K.C. Cheung, Alginate core-shell beads for simplified three-dimensional tumor spheroid culture and drug screening. Biomed. Microdevices 17, 33 (2015)
H. Zhang, E. Tumarkin, R.M.A. Sullan, G.C. Walker, E. Kumacheva, Exploring Microfluidic Routes to Microgels of Biological Polymers. Macromol. Rapid Commun. 28, 527–538 (2007)
J. Zhang, K.L. Tan, G.D. Hong, L.J. Yang, H.Q. Gong, Polymerization optimization of SU-8 photoresist and its applications in microfluidic systems and MEMS. J. Micromech. Microeng. 11, 20–26 (2000)
Y. Zhang, Y. Yong, D. An, W. Song, Q. Liu, L. Wang, Y. Pardo, V.R. Kern, P.H. Steen, W. Hong, Z. Liu, M. Ma, A drip-crosslinked tough hydrogel. Polymer 135, 327–330 (2018)
Z.-Q. Zhang, Y.H. Mori, Formulation of the Harkins-Brown correction factor for drop-volume description. Ind. Eng. Chem. Res. 32, 2950–2952 (1993)
Acknowledgements
We thank Dr. Bin Duan for providing access to the 3D printer. This study was supported by the University of Nebraska Medical Center (UNMC), and grant AR070242 from the NIH/NIAMS.
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Lee, D., Greer, S.E., Kuss, M.A. et al. 3D printed alginate bead generator for high-throughput cell culture. Biomed Microdevices 23, 22 (2021). https://doi.org/10.1007/s10544-021-00561-4
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DOI: https://doi.org/10.1007/s10544-021-00561-4