AccScience Publishing / IJB / Online First / DOI: 10.36922/ijb.1974

Man vs. machine: Automated bioink mixing device improves reliability and reproducibility of bioprinting results compared to human operators

Dongwei Wu1 Shumin Pang2 Viola Röhrs1 Johanna Berg1 Ahmed S. M. Ali1 Yikun Mei1 Mathias Ziersch1 Beatrice Tolksdorf1 Jens Kurreck1*
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1 Chair of Applied Biochemistry, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany
2 Chair of Advanced Ceramic Materials, Institute of Material Science and Technology, Technische Universität Berlin, Berlin, Germany
IJB 2024, 10(2), 1974
Submitted: 5 October 2023 | Accepted: 14 December 2023 | Published: 12 February 2024
© 2024 by the Author(s). This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International License ( )

The bioink mixing process is highly relevant to the bioink quality, which is the basis for reproducible extrusion-based three-dimensional (3D) bioprinting (EBB). Currently, most bioinks mixed by skilled human operators show variations in terms of cell homogeneity and biological properties as well as other properties. For preparation of many types of bioinks, striking the balance between homogeneity and cell viability remains a major challenge. This study investigates the relationship between bioink homogeneity and mixing parameters, particularly mixing speed and number of exchanges, utilizing a customized automated device. We found that up to a certain point, increasing the rate of mixing led to a better distribution of cells within the bioink, but beyond that point, there was a detrimental effect on cell viability. In contrast, the mixing number had less impact on the physiological properties of the cells in the bioink. Furthermore, a comparison between skilled human and machine bioink mixing revealed that the machine consistently provided better outcomes in terms of bioink homogeneity, cell distribution, and cell viability, highlighting the advantages and importance of standardizing the bioink mixing process. The methodology and approaches in this study can improve the reproducibility and reliability of EBB bioink and may thereby advance the field of 3D bioprinting in various applications.

Hydrogel mixing
Mixing device
Cell viability
This research was supported by the Chinese Scholarship Council (CSC; fellowship No. 201906780024 to D.W., and fellowship No. 201906780023 to S.P.). Financial support from the Einstein Foundation Berlin (Einstein Center 3R, EZ-2020-597-2) is gratefully acknowledged.
  1. Assad H, Assad A, Kumar A. Recent developments in 3D bio-printing and its biomedical applications. Pharmaceutics. 2023;15(1):255. doi: 10.3390/pharmaceutics15010255
  2. Hagenbuchner J, Nothdurfter D, Ausserlechner MJ. 3D bioprinting: novel approaches for engineering complex human tissue equivalents and drug testing. Essays Biochem. 2021;65(3):417-427. doi: 10.1042/ebc20200153
  3. Berg J, Kurreck J. Clean bioprinting - fabrication of 3D organ models devoid of animal components. ALTEX. 2021;38(2):269-288. doi: 10.14573/altex.2009151
  4. Bhattacharyya A, Janarthanan G, Tran HN, Ham HJ, Yoon J, Noh I. Bioink homogeneity control during 3D bioprinting of multicomponent micro/nanocomposite hydrogel for even tissue regeneration using novel twin screw extrusion system. Chem Eng J. 2021;415:128971. doi: 10.1016/j.cej.2021.128971
  5. Wang Y, Yuan X, Yao B, Zhu S, Zhu P, Huang S. Tailoring bioinks of extrusion-based bioprinting for cutaneous wound healing. Bioact. Mater. 2022;17:178-194. doi: 10.1016/j.bioactmat.2022.01.024
  6. Ainsworth MJ, Chirico N, de Ruijter M, et al. Convergence of melt electrowriting and extrusion-based bioprinting for vascular patterning of a myocardial construct. Biofabrication. 2023;15(3):035025. doi: 10.1088/1758-5090/ace07f
  7. Ozbolat IT, Hospodiuk M. Current advances and future perspectives in extrusion-based bioprinting. Biomaterials. 2016;76:321-343. doi: 10.1016/j.biomaterials.2015.10.076
  8. Kessel B, Lee M, Bonato A, Tinguely Y, Tosoratti E, Zenobi- Wong M. 3D bioprinting of macroporous materials based on entangled hydrogel microstrands. Adv Sci. 2016;7(18):2001419. doi: 10.1002/advs.202001419
  9. Karvinen J, Kellomäki M. Design aspects and characterization of hydrogel-based bioinks for extrusion-based bioprinting. Bioprinting. 2023;32:e00274. doi: 10.1016/j.bprint.2023.e00274
  10. Vu M, Pramanik A, Basak AK, Prakash C, Shankar S. Progress and challenges on extrusion based three dimensional (3D) printing of biomaterials. Bioprinting. 2022; 27:e00223. doi: 10.1016/j.bprint.2022.e00223
  11. Jia R, Zhang M, Yang T, Ma M, Sun Q, Li M. Evolution of the morphological, structural, and molecular properties of gluten protein in dough with different hydration levels during mixing. Food Chem: X. 2022;15:100448. doi: 10.1016/j.fochx.2022.100448
  12. Thayer PS, Orrhult LS, Martínez H. Bioprinting of cartilage and skin tissue analogs utilizing a novel passive mixing unit technique for bioink precellularization. JoVE. 2018;131(131):e56372. doi: 10.3791/56372
  13. Li G, Huang K, Deng J, et al. Highly conducting and stretchable double-network hydrogel for soft bioelectronics. Adv Mater. 2022;34(15):2200261. doi: 10.1002/adma.202200261
  14. Raja N, Park H, Gal CW, Sung A, Choi Y-J, Yun H-s. Support-less ceramic 3D printing of bioceramic structures using a hydrogel bath. Biofabrication. 2023; 15(3):035006. doi: 10.1088/1758-5090/acc903
  15. Zheng Z, Wu J, Liu M, et al. 3D bioprinting of self-standing silk-based bioink. Adv Healthc Mater. 2018;7(6):1701026. doi: 10.1002/adhm.201701026
  16. Guagliano G, Volpini C, Sardelli L, et al. Hep3Gel: a shape-shifting extracellular matrix- based, three-dimensional liver model adaptable to different culture systems. ACS Biomater Sci Eng. 2023;9(1):211-229. doi: 10.1021/acsbiomaterials.2c01226
  17. Dou Z, Tang H, Chen K, et al. Highly elastic and self-healing nanostructured gelatin/clay colloidal gels with osteogenic capacity for minimally invasive and customized bone regeneration. Biofabrication. 2023;15(2):025001. doi: 10.1088/1758-5090/acab36
  18. Berg J, Hiller T, Kissner MS, et al. Optimization of cell-laden bioinks for 3D bioprinting and efficient infection with influenza A virus. Sci Rep. 2018;8(1):13877. doi: 10.1038/s41598-018-31880-x
  19. Wu D, Berg J, Arlt B, et al. Bioprinted cancer model of neuroblastoma in a renal microenvironment as an efficiently applicable drug testing platform. Int J Mol Sci. 2022;23(1):122. doi: 10.3390/ijms23010122
  20. Dani S, Ahlfeld T, Albrecht F, et al. Homogeneous and reproducible mixing of highly viscous biomaterial inks and cell suspensions to create bioinks. Gels. 72021;(4):227. doi: 10.3390/gels7040227
  21. Cavallo A, Al Kayal T, Mero A, et al. Marine collagen-based bioink for 3D bioprinting of a bilayered skin model. Pharmaceutics. 2023;15(5):1331. doi: 10.3390/pharmaceutics15051331
  22. Gillispie G, Prim P, Copus J, et al. Assessment methodologies for extrusion-based bioink printability. Biofabrication. 2020;12(2):022003. doi: 10.1088/1758-5090/ab6f0d
  23. Kang D, Liu Z, Qian C, et al. 3D bioprinting of a gelatin-alginate hydrogel for tissue-engineered hair follicle regeneration. Acta Biomater. 2023;165:19-30. doi: 10.1016/j.actbio.2022.03.011
  24. Isaacson A, Swioklo S, Connon CJ. 3D bioprinting of a corneal stroma equivalent. Exp Eye Res. 2018;173:188-193. doi: 10.1016/j.exer.2018.05.010
  25. Cadena M, Ning L, King A, et al. 3D bioprinting of neural tissues. Adv Healthc Mater. 2021;10(15):2001600. doi: 10.1002/adhm.202001600
  26. Graham AD, Olof SN, Burke MJ, et al. High-resolution patterned cellular constructs by droplet-based 3D printing. Sci Rep. 2017;7(1):7004. doi: 10.1038/s41598-017-06358-x
  27. Pretorius V, Smuts TW. Turbulent flow chromatography. A new approach to faster analysis. Anal Chem. 1966;38(2):274-281. doi: 10.1021/ac60234a030
  28. Dimotakis PE. Turbulent mixing. Annu Rev Fluid Mech. 2005;37(1):329-356. doi: 10.1146/annurev.fluid.36.050802.122015
  29. Hiller T, Berg J, Elomaa L, et al. Generation of a 3D liver model comprising human extracellular matrix in an alginate/ gelatin-based bioink by extrusion bioprinting for infection and transduction studies. Int J Mol Sci. 2018;19(10):3129. doi: 10.3390/ijms19103129
  30. Berg J, Weber Z, Fechler-Bitteti M, et al. Bioprinted multi-cell type lung model for the study of viral inhibitors. Viruses. 2021;13(8):1590. doi: 10.3390/v13081590
  31. Al-Zeer MA, Prehn F, Fiedler S, et al. Evaluating the suitability of 3D bioprinted samples for experimental radiotherapy: a pilot study. Int J Mol Sci. 2022;23(17):9951. doi: 10.3390/ijms23179951
  32. Senturk E, Bilici C, Afghah F, et al. 3D bioprinting of tyramine modified hydrogels under visible light for osteochondral interface. Biofabrication. 2023;15(3):034102. doi: 10.1088/1758-5090/acd6bf
  33. Xin S, Deo KA, Dai J, et al. Generalizing hydrogel microparticles into a new class of bioinks for extrusion bioprinting. Sci Adv. 2021;7(42):eabk3087. doi: 10.1126/sciadv.abk3087
  34. Tibbitt MW, Anseth KS. Hydrogels as extracellular matrix mimics for 3D cell culture. Biotechnol Bioeng. 2009;103(4):655-663. doi: 10.1002/bit.22361
  35. Nam S, Stowers R, Lou J, Xia Y, Chaudhuri O. Varying PEG density to control stress relaxation in alginate-PEG hydrogels for 3D cell culture studies. Biomaterials. 2019;200:15-24. doi: 10.1016/j.biomaterials.2019.02.004
  36. Pasturel A, Strale P-O, Studer V. Tailoring common hydrogels into 3D cell culture templates. Adv Healthc Mater. 2020;9(18):2000519. doi: 10.1002/adhm.202000519
  37. Cui X, Li J, Hartanto Y, et al. Advances in extrusion 3D bioprinting: a focus on multicomponent hydrogel-based bioinks. Adv Healthc Mater. 2020;9(15):1901648. doi: 10.1002/adhm.201901648
  38. Unagolla JM, Jayasuriya AC. Hydrogel-based 3D bioprinting: a comprehensive review on cell-laden hydrogels, bioink formulations, and future perspectives. Appl Mater Today. 2020;18:100479. doi: 10.1016/j.apmt.2019.100479
  39. Ho T-C, Chang C-C, Chan H-P, et al. Hydrogels: properties and applications in biomedicine. Molecules. 2022;27(9):2902. doi: 10.3390/molecules27092902
  40. Puertas-Bartolomé M, Włodarczyk-Biegun MK, del Campo A, Vázquez-Lasa B, San Román J. 3D printing of a reactive hydrogel bio-ink using a static mixing tool. Polymers. 2020;12(9):1986. doi: 10.3390/polym12091986
  41. Jeon O, Lee YB, Lee SJ, Guliyeva N, Lee J, Alsberg E. Stem cell-laden hydrogel bioink for generation of high resolution and fidelity engineered tissues with complex geometries. Bioact Mater. 2022;15:185-193. doi: 10.1016/j.bioactmat.2021.11.025
  42. Semba JA, Mieloch AA, Tomaszewska E, Cywoniuk P, Rybka JD. Formulation and evaluation of a bioink composed of alginate, gelatin, and nanocellulose for meniscal tissue engineering. Int J Bioprint. 2023;9(1):621. doi: 10.18063/ijb.v9i1.621
  43. Pagan E, Stefanek E, Seyfoori A, et al. A handheld bioprinter for multi-material printing of complex constructs. Biofabrication. 2023;15(3):035012. doi: 10.1088/1758-5090/acc42c
  44. Mörö A, Samanta S, Honkamäki L, et al. Hyaluronic acid based next generation bioink for 3D bioprinting of human stem cell derived corneal stromal model with innervation. Biofabrication. 2023;15(1):015020. doi: 10.1088/1758-5090/acab34
  45. Hunsberger J, Simon C, Zylberberg C, et al. Improving patient outcomes with regenerative medicine: how the Regenerative Medicine Manufacturing Society plans to move the needle forward in cell manufacturing, standards, 3D bioprinting, artificial intelligence-enabled automation, education, and training. Stem Cells Transl Med. 2020;9(7):728-733. doi: 10.1002/sctm.19-0389
  46. Brown A. 3D printing in instructional settings: identifying a curricular hierarchy of activities. TechTrends. 2015; 59(5):16-24. doi: 10.1007/s11528-015-0887-1
  47. Boogert NJ, Madden JR, Morand-Ferron J, Thornton A. Measuring and understanding individual differences in cognition. Philos Trans R Soc Lond B Biol Sci. 2018;373(1756):20170280. doi: 10.1098/rstb.2017.0280
  48. Spurk JH, Aksel N, eds. Fluid Mechanics. Cham: Springer International Publishing; 2020: 223-249. doi: 10.1007/978-3-030-30259-7_7
  49. Li J, Shelby T, Alizadeh HV, Shelby H, Yang YP. Development and characterization of an automated active mixing platform for hydrogel bioink preparation. Int J Bioprint. 2023;9(4):4. doi: 10.18063/ijb.705
  50. Samokhin AS. Syringe pump created using 3D printing technology and arduino platform. J Anal Chem. 2020;75(3):416-421. doi: 10.1134/S1061934820030156
  51. Campbell T, Jones JFX. Design and implementation of a low cost, modular, adaptable and open-source XYZ positioning system for neurophysiology. HardwareX. 2020; 7:e00098. doi: 10.1016/j.ohx.2020.e00098
  52. Hadisujoto B, Wijaya R. Development and accuracy test of a fused deposition modeling (FDM) 3D printing using H-Bot mechanism. IJoCED. 2021;3(1):46-53. doi: 10.35806/ijoced.v3i1.148
  53. Manolescu VD, Secco EL. Design of an assistive low-cost 6 d.o.f. robotic arm with gripper. In: Yang XS, Sherratt S, Dey N, Joshi A, eds. Proceedings of Seventh International Congress on Information and Communication Technology. Singapore: Springer Nature Singapore; 2023: 39-55.
  54. Othman SA, Soon CF, Ma NL, et al. Alginate-gelatin bioink for bioprinting of hela spheroids in alginate-gelatin hexagon shaped scaffolds. Polym Bull. 2021;78(11):6115-6135. doi: 10.1007/s00289-020-03421-y
  55. Freeman FE, Pitacco P, van Dommelen LHA, et al. 3D bioprinting spatiotemporally defined patterns of growth factors to tightly control tissue regeneration. Sci Adv. 2020;6(33):eabb5093. doi: 10.1126/sciadv.abb5093
  56. Mirek A, Belaid H, Bartkowiak A, et al. Gelatin methacrylate hydrogel with drug-loaded polymer microspheres as a new bioink for 3D bioprinting. Biomater Adv. 2023;150:213436. doi: 10.1016/j.bioadv.2023.213436
  57. Seo JW, Kim GM, Choi Y, Cha JM, Bae H. Improving printability of digital-light-processing 3D bioprinting via photoabsorber pigment adjustment. Int J Mol Sci. 2022;23(10):5428. doi: 10.3390/ijms23105428
  58. Ding Y-W, Zhang X-W, Mi C-H, Qi X-Y, Zhou J, Wei D-X. Recent advances in hyaluronic acid-based hydrogels for 3D bioprinting in tissue engineering applications. Smart Mater Med. 2023;4:59-68. doi: 10.1016/j.smaim.2022.07.003
  59. Serafin A, Culebras M, Oliveira JM, Koffler J, Collins MN. 3D printable electroconductive gelatin-hyaluronic acid materials containing polypyrrole nanoparticles for electroactive tissue engineering. Adv Compos Hybrid Mater. 2023;6(3):109. doi: 10.1007/s42114-023-00665-w
Conflict of interest
The authors declare no conflicts of interest.
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International Journal of Bioprinting, Electronic ISSN: 2424-8002 Print ISSN: 2424-7723, Published by AccScience Publishing