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In vitro bioengineered model of cortical brain tissue

Abstract

A bioengineered model of 3D brain-like tissue was developed using silk-collagen protein scaffolds seeded with primary cortical neurons. The scaffold design provides compartmentalized control for spatial separation of neuronal cell bodies and neural projections, which resembles the layered structure of the brain (cerebral cortex). Neurons seeded in a donut-shaped porous silk sponge grow robust neuronal projections within a collagen-filled central region, generating 3D neural networks with structural and functional connectivity. The silk scaffold preserves the mechanical stability of the engineered tissues, allowing for ease of handling, long-term culture in vitro and anchoring of the central collagen gel to avoid shrinkage, and enabling neural network maturation. This protocol describes the preparation and manipulation of silk-collagen constructs, including the isolation and seeding of primary rat cortical neurons. This 3D technique is useful for mechanical injury studies and as a drug screening tool, and it could serve as a foundation for brain-related disease models. The protocol of construct assembly takes 2 d, and the resulting tissues can be maintained in culture for several weeks.

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Figure 1: The preparation process of 3D bioengineered brain tissue.
Figure 2: Neuronal compartmentalized outgrowth in the 3D brain-like tissue model.
Figure 3: Functional evaluation of the constructs.
Figure 4: Neurite density in relation to the number of seeded cells.

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References

  1. Choi, S.H. et al. A three-dimensional human neural cell culture model of Alzheimer's disease. Nature 515, 274–278 (2014).

    Article  CAS  Google Scholar 

  2. Lancaster, M.A. et al. Cerebral organoids model human brain development and microcephaly. Nature 501, 373–379 (2013).

    Article  CAS  Google Scholar 

  3. Hopkins, A.M., DeSimone, E., Chwalek, K. & Kaplan, D.L. 3D in vitro modeling of the central nervous system. Prog. Neurobiol. 125C, 1–25 (2015).

    Article  Google Scholar 

  4. Tang-Schomer, M.D. et al. Bioengineered functional brain-like cortical tissue. Proc. Natl. Acad. Sci. USA 111, 13811–13816 (2014).

    Article  CAS  Google Scholar 

  5. Rockwood, D.N. et al. Materials fabrication from Bombyx mori silk fibroin. Nat. Protoc. 6, 1612–1631 (2011).

    Article  CAS  Google Scholar 

  6. Vepari, C. & Kaplan, D.L. Silk as a biomaterial. Prog. Polym. Sci. 32, 991–1007 (2007).

    Article  CAS  Google Scholar 

  7. Devine, M.J. et al. Parkinson's disease induced pluripotent stem cells with triplication of the α-synuclein locus. Nat. Commun. 2, 440 (2011).

    Article  Google Scholar 

  8. Shi, Y., Kirwan, P. & Livesey, F.J. Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks. Nat. Protoc. 7, 1836–1846 (2012).

    Article  CAS  Google Scholar 

  9. Li, X., Liu, X., Zhang, N. & Wen, X. Engineering in situ cross-linkable and neurocompatible hydrogels. J. Neurotrauma 31, 1431–1438 (2014).

    Article  Google Scholar 

  10. Lutolf, M.P. et al. Synthetic matrix metalloproteinase-sensitive hydrogels for the conduction of tissue regeneration: engineering cell-invasion characteristics. Proc. Natl. Acad. Sci. USA 100, 5413–5418 (2003).

    Article  CAS  Google Scholar 

  11. Tsurkan, M.V. et al. Defined polymer-peptide conjugates to form cell-instructive starPEG-heparin matrices in situ. Adv. Mater. 25, 2606–2610 (2013).

    Article  CAS  Google Scholar 

  12. Dubois-Dauphin, M.L. et al. The long-term survival of in vitro engineered nervous tissue derived from the specific neural differentiation of mouse embryonic stem cells. Biomaterials 31, 7032–7042 (2010).

    Article  CAS  Google Scholar 

  13. Hogberg, H.T. et al. Toward a 3D model of human brain development for studying gene/environment interactions. Stem Cell Res. Ther. 4 (suppl. 1) S4 (2013).

    Article  Google Scholar 

  14. Muguruma, K., Nishiyama, A., Kawakami, H., Hashimoto, K. & Sasai, Y. Self-organization of polarized cerebellar tissue in 3D culture of human pluripotent stem cells. Cell. Rep. 10, 537–550 pii: S2211-1247(14)01104-8 (2015).

    Article  CAS  Google Scholar 

  15. Kato-Negishi, M., Morimoto, Y., Onoe, H. & Takeuchi, S. Millimeter-sized neural building blocks for 3D heterogeneous neural network assembly. Adv. Healthc. Mater. 2, 1564–1570 (2013).

    Article  CAS  Google Scholar 

  16. van Vliet, E. et al. Electrophysiological recording of re-aggregating brain cell cultures on multi-electrode arrays to detect acute neurotoxic effects. Neurotoxicology 28, 1136–1146 (2007).

    Article  CAS  Google Scholar 

  17. Zurich, M.G., Honegger, P., Schilter, B., Costa, L.G. & Monnet-Tschudi, F. Involvement of glial cells in the neurotoxicity of parathion and chlorpyrifos. Toxicol. Appl. Pharmacol. 201, 97–104 (2004).

    Article  CAS  Google Scholar 

  18. Aurand, E.R., Wagner, J.L., Shandas, R. & Bjugstad, K.B. Hydrogel formulation determines cell fate of fetal and adult neural progenitor cells. Stem Cell Res. 12, 11–23 (2014).

    Article  CAS  Google Scholar 

  19. Watanabe, K., Nakamura, M., Okano, H. & Toyama, Y. Establishment of three-dimensional culture of neural stem/progenitor cells in collagen type-1 gel. Restor. Neurol. Neurosci. 25, 109–117 (2007).

    CAS  PubMed  Google Scholar 

  20. Pacifici, M. & Peruzzi, F. Isolation and culture of rat embryonic neural cells: a quick protocol. J. Vis. Exp. 63, e3965 (2012).

    Google Scholar 

  21. Leal-Egana, A. & Scheibel, T. Interactions of cells with silk surfaces. J. Mater. Chem. 22, 14330–14336 (2012).

    Article  CAS  Google Scholar 

  22. An, B. et al. Physical and biological regulation of neuron regenerative growth and network formation on recombinant dragline silks. Biomaterials 48, 137–146 (2015).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank S. Moss from Tufts University for providing embryonic rat brain tissues. This work was funded by a US National Institutes of Health (NIH) P41 Tissue Engineering Resource Center Grant (EB002520) and by the German Research Foundation (DFG; CH 1400/2-1, Postdoctoral Fellowship for K.C.).

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Contributions

K.C. performed experimental work, data analysis and wrote the paper; M.D.T.-S., D.L.K. and F.G.O. conceived the idea; M.D.T.-S. developed the design and protocol; and D.L.K and F.G.O. supervised the project. All authors commented on the results and the manuscript.

Corresponding author

Correspondence to David L Kaplan.

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The authors declare no competing financial interests.

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Chwalek, K., Tang-Schomer, M., Omenetto, F. et al. In vitro bioengineered model of cortical brain tissue. Nat Protoc 10, 1362–1373 (2015). https://doi.org/10.1038/nprot.2015.091

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