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Three-dimensional neuronal cell culture: in pursuit of novel treatments for neurodegenerative disease

  • Sarah-Sophia D. Carter (a1) (a2), Xiao Liu (a1), Zhilian Yue (a1) and Gordon G. Wallace (a1)


To gain a better understanding of the underlying mechanisms of neurological disease, relevant tissue models are imperative. Over the years, this realization has fuelled the development of novel tools and platforms, which aim at capturing in vivo complexity. One example is the field of biofabrication, which focuses on fabrication of three-dimensional (3D) biologically functional products in a controlled and automated manner. Herein, we provide a general overview of classical 3D cell culture platforms, particularly in the context of neurodegenerative disease. Subsequently, the focus is put on bioprinting-based biofabrication, its potential to advance 3D neuronal cell culture and, to conclude, the relevant translational bottlenecks, which will need to be considered as the field evolves.

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Corresponding author

Address all correspondence to Gordon G. Wallace at


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1.Lee, J., Cuddihy, M.J., and Kotov, N.A.: Three-dimensional cell culture matrices: state of the art. Tissue Eng. B: Rev. 14, 61 (2008).
2.Edmondson, R., Broglie, J.J., Adcock, A.F., and Yang, L.: Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors. Assay Drug Dev. Technol. 12, 207 (2014).
3.Pampaloni, F., Reynaud, E.G., and Stelzer, E.H.: The third dimension bridges the gap between cell culture and live tissue. Nat. Rev. Mol. Cell Biol. 8, 839 (2007).
4.Santos, E., Hernández, R.M., Pedraz, J.L., and Orive, G.: Novel advances in the design of three-dimensional bio-scaffolds to control cell fate: translation from 2D to 3D. Trends Biotechnol. 30, 331 (2012).
5.Antoni, D., Burckel, H., Josset, E., and Noel, G.: Three-dimensional cell culture: a breakthrough in vivo. Int. J. Mol. Sci. 16, 5517 (2015).
6.McMurtrey, R.J.: Novel advancements in three-dimensional neural tissue engineering and regenerative medicine. Neural Regener. Res. 10, 352 (2015).
7.Caliari, S.R. and Burdick, J.A.: A practical guide to hydrogels for cell culture. Nat. Methods 13, 405 (2016).
8.Banerjee, S. and Bhat, M.A.: Neuron-glial interactions in blood-brain barrier formation. Annu. Rev. Neurosci. 30, 235 (2007).
9.Struzyna, L.A., Katiyar, K., and Cullen, D.K.: Living scaffolds for neuroregeneration. Curr. Opin. Solid State Mater. Sci. 18, 308 (2014).
10.Miller, J.P. and Jacobs, G.A.: Relationships between neuronal structure and function. J. Exp. Biol. 112, 129 (1984).
11.Araque, A. and Navarrete, M.: Glial cells in neuronal network function. Phil. Trans. R. Soc. B 365, 2375 (2010).
12.Barros, C.S., Franco, S.J., and Müller, U.: Extracellular matrix: functions in the nervous system. Cold Spring Harbor Perspect. Biol. 3, a005108 (2011).
13.Bradke, F. and Dotti, C.G.: Establishment of neuronal polarity: lessons from cultured hippocampal neurons. Curr. Opin. Neurobiol. 10, 574 (2000).
14.Kleinman, H.K., Philp, D., and Hoffman, M.P.: Role of the extracellular matrix in morphogenesis. Curr. Opin. Biotechnol. 14, 526 (2003).
15.Cullen, D.K., Wolf, J.A., Vernekar, V.N., Vukasinovic, J., and LaPlaca, M.C.: Neural tissue engineering and biohybridized microsystems for neurobiological investigation in vitro (Part 1). Crit. Rev. Biomed. Eng. 39, 201 (2011).
16.Liedmann, A., Frech, S., Morgan, P.J., Rolfs, A., and Frech, M.J.: Differentiation of human neural progenitor cells in functionalized hydrogel matrices. BioRes. Open Access 1, 16 (2012).
17.Tang-Schomer, M.D., White, J.D., Tien, L.W., Schmitt, L.I., Valentin, T.M., Graziano, D.J., Hopkins, A.M., Omenetto, F.G., Haydon, P.G., and Kaplan, D.L.: Bioengineered functional brain-like cortical tissue. Proc. Natl Acad. Sci. USA 111, 13811 (2014).
18.Haycock, J.W.: 3D cell culture: a review of current approaches and techniques. 3D Cell Cult.: Methods Protoc. 695, 1 (2011).
19.Knight, E. and Przyborski, S.: Advances in 3D cell culture technologies enabling tissue-like structures to be created in vitro. J. Anat. 227, 746 (2015).
20.Pereira, J.F., Awatade, N.T., Loureiro, C.A., Matos, P., Amaral, M.D., and Jordan, P.: The third dimension: new developments in cell culture models for colorectal research. Cell. Mol. Life Sci. 73, 3971 (2016).
21.Liu, Y., Gill, E., and Huang, Y.Y.S.: Microfluidic on-chip biomimicry for 3D cell culture: a fit-for-purpose investigation from the end user standpoint. Future Sci. OA 3, FSO173 (2017).
22.Melchels, F.P., Domingos, M.A., Klein, T.J., Malda, J., Bartolo, P.F., and Hutmacher, D.W.: Additive manufacturing of tissues and organs. Prog. Polym. Sci. 37, 1079 (2012).
23.Malda, J., Visser, J., Melchels, F.P., Jüngst, T., Hennink, W.E., Dhert, W.J., Groll, J., and Hutmacher, D.W.: 25th anniversary article: engineering hydrogels for biofabrication. Adv. Mater. 25, 5011 (2013).
24.Groll, J., Boland, T., Blunk, T., Burdick, J.A., Cho, D.-W., Dalton, P.D., Derby, B., Forgacs, G., Li, Q., Mironov, V.A., Moroni, L., Nakamura, M., Shu, W., Takeuchi, S., Vozzi, G., Woodfield, T.B.F., Xu, T., Yoo, J.J., and Malda, J.: Biofabrication: reappraising the definition of an evolving field. Biofabrication 8, 013001 (2016).
25.Derby, B.: Bioprinting: inkjet printing proteins and hybrid cell-containing materials and structures. J. Mater. Chem. 18, 5717 (2008).
26.Przedborski, S., Vila, M., and Jackson-Lewis, V.: Series introduction: neurodegeneration: what is it and where are we? J. Clin. Invest. 111, 3 (2003).
27.Yiannopoulou, K.G. and Papageorgiou, S.G.: Current and future treatments for Alzheimer's disease. Therap. Adv. Neurol. Disord. 6, 19 (2013).
28.Korolev, I.O.: Alzheimer's disease: a clinical and basic science review. Med. Stud. Res. J. 4, 24 (2014).
29.Dharmarajan, T.S. and Gunturu, S.G.: Alzheimer's disease: a healthcare burden of epidemic proportion. Am. Health Drug Benefits 2, 3 (2009).
30.Cuny, G.D.: Neurodegenerative diseases: challenges and opportunities. Future Med. Chem. 4, 1647 (2012).
31.Wimo, A., Jönsson, L., Bond, J., Prince, M., and Winblad, B.: The worldwide economic impact of dementia 2010. Alzheimers Dement. 9, 1 (2013).
32.Donev, R., Kolev, M., Millet, B., and Thome, J.: Neuronal death in Alzheimer's disease and therapeutic opportunities. J. Cell. Mol. Med. 13, 4329 (2009).
33.Deleglise, B., Magnifico, S., Duplus, E., Vaur, P., Soubeyre, V., Belle, M., Vignes, M., Viovy, J.L., Jacotot, E., Peyrin, J.M., and Brugg, B.: β-amyloid induces a dying-back process and remote trans-synaptic alterations in a microfluidic-based reconstructed neuronal network. Acta Neuropathol. Commun. 2, 145 (2014).
34.Heneka, M.T., Carson, M.J., El Khoury, J., Landreth, G.E., Brosseron, F., Feinstein, D.L., Jacobs, A.H., Wyss-Coray, T., Vitorica, J., Ransohoff, R.M., Herrup, K., Frautschy, S.A., Finsen, B., Brown, G.C., Verkhratsky, A., Yamanaka, K., Koistinaho, J., Latz, E., Halle, A., Petzold, G.C., Town, T., Morgan, D., Shinohara, M.L., Perry, V.H., Holmes, C., Bazan, N.G., Brooks, D.J., Hunot, S., Joseph, B., Deigendesch, N., Garaschuk, O., Boddeke, E., Dinarello, C.A., Breitner, J.C., Cole, G.M., Golenbock, D.T., and Kummer, M.P.: Neuroinflammation in Alzheimer's disease. Lancet Neurol. 14, 388 (2015).
35.Lunn, J.S., Sakowski, S.A., Hur, J., and Feldman, E.L.: Stem cell technology for neurodegenerative diseases. Ann. Neurol. 70, 353 (2011).
36.Achilli, T.M., Meyer, J., and Morgan, J.R.: Advances in the formation, use and understanding of multi-cellular spheroids. Expert Opin Biol. Ther. 12, 1347 (2012).
37.Simian, M. and Bissell, M.J.: Organoids: a historical perspective of thinking in three dimensions. J. Cell Biol. 216, 31 (2017).
38.Fang, Y. and Eglen, R.M.: Three-dimensional cell cultures in drug discovery and development. SLAS DISCOV.: Adv. Life Sci. R&D. doi: 10.1177/2472555217696795, Published online 17 March 2017.
39.Mehta, G., Hsiao, A.Y., Ingram, M., Luker, G.D., and Takayama, S.: Opportunities and challenges for use of tumor spheroids as models to test drug delivery and efficacy. J. Control. Release 164, 192 (2012).
40.Fennema, E., Rivron, N., Rouwkema, J., van Blitterswijk, C., and de Boer, J.: Spheroid culture as a tool for creating 3D complex tissues. Trends Biotechnol. 31, 108 (2013).
41.Dingle, Y-T. L., Boutin, M.E., Chirila, A.M., Livi, L.L., Labriola, N.R., Jakubek, L.M., Morgan, J.R., Darling, E.M., Kauer, J.A., and Hoffman-Kim Diane, D.: Three-dimensional neural spheroid culture: an in vitro model for cortical studies. Tissue Eng. C: Methods 21, 1274 (2015).
42.Boutin, M.E., Kramer, L.L., Livi, L.L., Brown, T., Moore, C., and Hoffman-Kim, D.: A three-dimensional neural spheroid model for capillary-like network formation. J. Neurosci. Methods. doi 10.1016/j.jneumeth.2017.01.014, Published online 29 January 2017.
43.Ranga, A., Gjorevski, N., and Lutolf, M.P.: Drug discovery through stem cell-based organoid models. Adv. Drug Delivery. Rev. 69, 19 (2014).
44.Fatehullah, A., Tan, S.H., and Barker, N.: Organoids as an in vitro model of human development and disease. Nat. Cell Biol. 18, 246 (2016).
45.Schneeberger, K., Spee, B., Costa, P., Sachs, N., Clevers, H., and Malda, J.: Converging biofabrication and organoid technologies: the next frontier in hepatic and intestinal tissue engineering? Biofabrication 9, 013001 (2017).
46.Raja, W.K., Mungenast, A.E., Lin, Y.T., Ko, T., Abdurrob, F., Seo, J., and Tsai, L.H.: Self-Organizing 3D human neural tissue derived from induced pluripotent stem cells recapitulate Alzheimer's disease phenotypes. PLoS ONE 11, e0161969 (2016).
47.O'brien, F.J.: Biomaterials & scaffolds for tissue engineering. Mater. Today 14, 88 (2011).
48.Breslin, S. and O'Driscoll, L.: Three-dimensional cell culture: the missing link in drug discovery. Drug Discov. Today 18, 240 (2013).
49.Chan, B.P. and Leong, K.W.: Scaffolding in tissue engineering: general approaches and tissue-specific considerations. Eur. Spine J. 17, 467 (2008).
50.Murphy, A.R., Laslett, A., O'Brien, C.M., and Cameron, N.R.: Scaffolds for 3D in vitro culture of neural lineage cells. Acta Biomater. 54, 1 (2017).
51.Frampton, J.P., Hynd, M.R., Shuler, M.L., and Shain, W.: Fabrication and optimization of alginate hydrogel constructs for use in 3D neural cell culture. Biomed. Mater. 6, 015002 (2011).
52.Lai, Y., Cheng, K., and Kisaalita, W.: Three dimensional neuronal cell cultures more accurately model voltage gated calcium channel functionality in freshly dissected nerve tissue. PLoS ONE 7, e45074 (2012).
53.Choi, S.H., Kim, Y.H., Hebisch, M., Sliwinski, C., Lee, S., D'Avanzo, C., Chen, H., Hooli, B., Asselin, C., Muffat, J., and Klee, J.B.: A three-dimensional human neural cell culture model of Alzheimer's disease. Nature 515, 274 (2014).
54.Kraus, D., Boyle, V., Leibig, N., Stark, G.B., and Penna, V.: The neuro-spheroid—a novel 3D in vitro model for peripheral nerve regeneration. J. Neurosci. Methods 246, 97 (2015).
55.van der Meer, A.D. and van den Berg, A.: Organs-on-chips: breaking the in vitro impasse. Integr. Biol. 4, 461 (2012).
56.Bhatia, S.N. and Ingber, D.E.: Microfluidic organs-on-chips. Nat. Biotechnol. 32, 760 (2014).
57.Yi, Y., Park, J., Lim, J., Lee, C.J., and Lee, S.H.: Central nervous system and its disease models on a chip. Trends Biotechnol. 33, 762 (2015).
58.Choi, Y.J., Chae, S., Kim, J.H., Barald, K.F., Park, J.Y., and Lee, S.H.: Neurotoxic amyloid beta oligomeric assemblies recreated in microfluidic platform with interstitial level of slow flow. Sci. Rep. 3, 1921 (2013).
59.Cho, H., Hashimoto, T., Wong, E., Hori, Y., Wood, L.B., Zhao, L., Haigis, K.M., Hyman, B.T., and Irimia, D.: Microfluidic chemotaxis platform for differentiating the roles of soluble and bound amyloid-β on microglial accumulation. Sci. Rep. 3, 1823 (2013).
60.Kunze, A., Meissner, R., Brando, S., and Renaud, P.: Co-pathological connected primary neurons in a microfluidic device for Alzheimer studies. Biotechnol. Bioeng. 108, 2241 (2011).
61.Song, H.L., Shim, S., Kim, D.H., Won, S.H., Joo, S., Kim, S., Jeon, N.L., and Yoon, S.Y.: β-Amyloid is transmitted via neuronal connections along axonal membranes. Ann. Neurol. 75, 88 (2014).
62.Dujardin, S., Lécolle, K., Caillierez, R., Bégard, S., Zommer, N., Lachaud, C., Carrier, S., Dufour, N., Aurégan, G., Winderickx, J., Hantraye, P., Déglon, N., Colin, M., and Buée, L.: Neuron-to-neuron wild-type Tau protein transfer through a trans-synaptic mechanism: relevance to sporadic tauopathies. Acta Neuropathol. Commun. 2, 14 (2014).
63.Calafate, S., Buist, A., Miskiewicz, K., Vijayan, V., Daneels, G., De Strooper, B., De Wit, J., Verstrekenand, P., and Moechars, D.: Synaptic contacts enhance cell-to-cell tau pathology propagation. Cell Rep. 11, 1176 (2015).
64.Murphy, S.V. and Atala, A.: 3D bioprinting of tissues and organs. Nat. Biotechnol. 32, 773 (2014).
65.Hölzl, K., Lin, S., Tytgat, L., Van Vlierberghe, S., Gu, L., and Ovsianikov, A.: Bioink properties before, during and after 3D bioprinting. Biofabrication 8, 032002 (2016).
66.Xu, T., Jin, J., Gregory, C., Hickman, J.J., and Boland, T.: Inkjet printing of viable mammalian cells. Biomaterials 26, 93 (2005).
67.Xu, T., Gregory, C.A., Molnar, P., Cui, X., Jalota, S., Bhaduri, S.B., and Boland, T.: Viability and electrophysiology of neural cell structures generated by the inkjet printing method. Biomaterials 27, 3580 (2006).
68.Lorber, B., Hsiao, W.K., Hutchings, I.M., and Martin, K.R.: Adult rat retinal ganglion cells and glia can be printed by piezoelectric inkjet printing. Biofabrication 6, 015001 (2013).
69.Tse, C., Whiteley, R., Yu, T., Stringer, J., MacNeil, S., Haycock, J.W., and Smith, P.J.: Inkjet printing Schwann cells and neuronal analogue NG108-15 cells. Biofabrication 8, 015017 (2016).
70.Lee, Y.B., Polio, S., Lee, W., Dai, G., Menon, L., Carroll, R.S., and Yoo, S.S.: Bio-printing of collagen and VEGF-releasing fibrin gel scaffolds for neural stem cell culture. Exp. Neurol. 223, 645 (2010).
71.Hsieh, F.Y., Lin, H.H., and Hsu, S.H.: 3D bioprinting of neural stem cell-laden thermoresponsive biodegradable polyurethane hydrogel and potential in central nervous system repair. Biomaterials 71, 48 (2015).
72.Lozano, R., Stevens, L., Thompson, B.C., Gilmore, K.J., Gorkin, R., Stewart, E.M., in het Panhuis, M., Romero-Ortega, M., and Wallace, G.G.: 3D printing of layered brain-like structures using peptide modified gellan gum substrates. Biomaterials 67, 264 (2015).
73.Gu, Q., Tomaskovic-Crook, E., Lozano, R., Chen, Y., Kapsa, R.M., Zhou, Q., Wallace, G.G., and Crook, J.M.: Functional 3D neural mini-tissues from printed gel-based bioink and human neural stem cells. Adv. Healthc. Mater. 5, 1429 (2016).
74.Gu, Q., Tomaskovic-Crook, E., Wallace, G.G., and Crook, J.M.: 3D bioprinting human induced pluripotent stem cell constructs for in situ cell proliferation and successive multilineage differentiation. Adv. Healthc. Mater. 6, 1700175 (2017).
75.Mironov, V., Visconti, R.P., Kasyanov, V., Forgacs, G., Drake, C.J., and Markwald, R.R.: Organ printing: tissue spheroids as building blocks. Biomaterials 30, 2164 (2009).
76.Mironov, V., Khesuani, Y.D., Bulanova, E.A., Koudan, E.V., Parfenov, V.A., Knyazeva, A.D., Mitryashkin, A.N., Replyanski, N., Kasyanov, V.A., and DAS, F.P.: Patterning of tissue spheroids biofabricated from human fibroblasts on the surface of electrospun polyurethane matrix using 3D bioprinter. Int. J. Bioprint. 2, 45 (2016).
77.Yi, H.G., Lee, H., and Cho, D.W.: 3D printing of organs-on-chips. Bioengineering 4, 10 (2017).
78.Johnson, B.N., Lancaster, K.Z., Hogue, I.B., Meng, F., Kong, Y.L., Enquist, L.W., and McAlpine, M.C.: 3D printed nervous system on a chip. Lab. Chip 16, 1393 (2016).
79.Rezende, R.A., Selishchev, S.V., Kasyanov, V.A., da Silva, J.V.L., and Mironov, V.A.: An organ biofabrication line: enabling technology for organ printing. Part I: from BIOCAD to biofabricators of spheroids. Biomed. Eng. 47, 116 (2013).
80.Atala, A. and Yoo, J.J.: Essentials of 3D Biofabrication and Translation (Academic Press, Cambridge, Massachusetts, 2015), pp. 1941.
81.Chimene, D., Lennox, K.K., Kaunas, R.R., and Gaharwar, A.K.: Advanced bioinks for 3D printing: a materials science perspective. Ann. Biomed. Eng. 44, 2090 (2016).
82.Gao, B., Yang, Q., Zhao, X., Jin, G., Ma, Y., and Xu, F.: 4D bioprinting for biomedical applications. Trends Biotechnol. 34, 746 (2016).
83.Li, Y.C., Zhang, Y.S., Akpek, A., Shin, S.R., and Khademhosseini, A.: 4D bioprinting: the next-generation technology for biofabrication enabled by stimuli-responsive materials. Biofabrication 9, 012001 (2016).
84.Hourd, P., Medcalf, N., Segal, J., and Williams, D.J.: A 3D bioprinting exemplar of the consequences of the regulatory requirements on customized processes. Regen. Med. 10, 863 (2015).
85.Chhaya, M.P., Poh, P.S., Balmayor, E.R., van Griensven, M., Schantz, J.T., and Hutmacher, D.W.: Additive manufacturing in biomedical sciences and the need for definitions and norms. Expert Rev. Med. Devices 12, 537 (2015).
86.Baker, H.B., McQuilling, J.P., and King, N.M.: Ethical considerations in tissue engineering research: case studies in translation. Methods 99, 135 (2016).
87.Otto, I.A., Breugem, C.C., Malda, J., and Bredenoord, A.L.: Ethical considerations in the translation of regenerative biofabrication technologies into clinic and society. Biofabrication 8, 042001 (2016).
88.Gilbert, F., O'Connell, C.D., Mladenovska, T., and Dodds, S.: Print me an organ? Ethical and regulatory issues emerging from 3D bioprinting in medicine. Sci. Eng. Ethics. doi: 10.1007/s11948-017-9874-6, Published online 09 February 2017.
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