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Development of an In Vitro 3D Brain Tissue Model Mimicking In Vivo-Like Pro-inflammatory and Pro-oxidative Responses

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Abstract

To analyze complex inflammatory responses in an in vitro system, we constructed a new 3D in vitro brain tissue model that exhibits in vivo-like tissue responses (e.g. immune cell phenotypes, and molecular response) to inflammatory stimuli. Finite element modeling of oxygen diffusion and cellular oxygen consumption predicted the oxygen profile within 3D structures, consisting of Type I collagen hydrogel embedded with murine microglia. Viability and cytotoxicity analyses supported the mathematical analysis, determining optimal cell growth conditions for 3D construct development. Real-time RT-PCR and ELISA demonstrated significant up-regulation of pro-inflammatory mediators, such as TNF-α, MCP-1, IL-6 and IL-1β, in lipopolysaccharide (LPS)-stimulated in vitro cell culture (2D and 3D) and in vivo mouse model systems. Interestingly, levels of inflammatory responses from the in vitro 3D model system were more similar to in vivo than in vitro 2D. Additionally, in situ dihydroethidium (DHE) assay and immunofluorescence staining revealed that levels of LPS-stimulated reactive oxygen species (ROS) generation and microglial activation from in vitro 3D model system were closer to in vivo than in vitro 2D. These results demonstrated that an in vitro 3D model provides more physiologically relevant pro-oxidative and pro-inflammatory environments in brain than an in vitro 2D model.

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References

  1. Almendros, I., J. M. Montserrat, M. Torres, C. González, D. Navajas, and R. Farré. Changes in oxygen partial pressure of brain tissue in an animal model of obstructive apnea. Respir. Res. 11:3, 2010.

    Article  PubMed  PubMed Central  Google Scholar 

  2. Ardakani, A. G., U. Cheema, R. A. Brown, and R. J. Shipley. Quantifying the correlation between spatially defined oxygen gradients and cell fate in an engineered three-dimensional culture model. J. R. Soc. Interface 11:20140501, 2014.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Block, M. L., L. Zecca, and J. S. Hong. Microglia-mediated neurotoxicity: uncovering the molecular mechanisms. Nat. Rev. Neurosci. 8:57–69, 2007.

    Article  CAS  PubMed  Google Scholar 

  4. Carreau, A., B. El Hafny-Rahbi, A. Matejuk, C. Grillon, and C. Kieda. Why is the partial oxygen pressure of human tissues a crucial parameter? Small molecules and hypoxia. J. Cell. Mol. Med. 15:1239–1253, 2011.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Cho, H. J., V. S. Sajja, P. J. Vandevord, and Y. W. Lee. Blast induces oxidative stress, inflammation, neuronal loss and subsequent short-term memory impairment in rats. Neuroscience 253:9–20, 2013.

    Article  CAS  PubMed  Google Scholar 

  6. Cox, M. C., L. M. Reese, L. R. Bickford, and S. S. Verbridge. Toward the broad adoption of 3D tumor models in the cnacer drug pipeline. ACS Biomater. Sci. Eng. 1:877–894, 2015.

    Article  CAS  Google Scholar 

  7. Cukierman, E., R. Pankov, D. R. Stevens, and K. M. Yamada. Taking cell-matrix adhesions to the third dimension. Science 294:1708–1712, 2001.

    Article  CAS  PubMed  Google Scholar 

  8. Cullen, D. K., J. A. Wolf, V. N. Vernekar, J. Vukasinovic, and M. C. LaPlaca. Neural tissue engineering and biohybridized microsystems for neurobiological investigation in vitro (Part 1). Crit. Rev. Biomed. Eng. 39:201–240, 2011.

    Article  PubMed  Google Scholar 

  9. DelNero, P., M. Lane, S. S. Verbridge, B. Kwee, P. Kermani, B. Hempstead, A. Stroock, and C. Fischbach. 3D culture broadly regulates tumor cell hypoxia response and angiogenesis via pro-inflammatory pathways. Biomaterials 55:110–118, 2015.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Fang, M., E. L. Goldstein, E. K. Matich, B. G. Orr, and M. M. Holl. Type I collagen self-assembly: the roles of substrate and concentration. Langmuir 29:2330–2338, 2013.

    Article  CAS  PubMed  Google Scholar 

  11. Geckil, H., F. Xu, X. Zhang, S. Moon, and U. Demirci. Engineering hydrogels as extracellular matrix mimics. Nanomedicine (Lond) 5:469–484, 2010.

    Article  CAS  Google Scholar 

  12. Griffith, L. G., and M. A. Swartz. Capturing complex 3D tissue physiology in vitro. Nat. Rev. Mol. Cell. Biol. 7:211–224, 2006.

    Article  CAS  PubMed  Google Scholar 

  13. Grinnell, F. Fibroblast biology in three-dimensional collagen matrices. Trends Cell. Biol. 13:264–269, 2003.

    Article  CAS  PubMed  Google Scholar 

  14. Haw, R. T., C. K. Tong, A. Yew, H. C. Lee, J. B. Phillips, and S. Vidyadaran. A three-dimensional collagen construct to model lipopolysaccharide-induced activation of BV2 microglia. J. Neuroinflamm. 11:134, 2014.

    Article  Google Scholar 

  15. Hu, X., A. K. Liou, R. K. Leak, M. Xu, C. An, J. Suenaga, Y. Shi, Y. Gao, P. Zheng, and J. Chen. Neurobiology of microglial action in CNS injuries: receptor-mediated signaling mechanisms and functional roles. Prog. Neurobiol. 119–120:60–84, 2014.

    Article  PubMed  Google Scholar 

  16. Jokinen, J., E. Dadu, P. Nykvist, J. Käpylä, D. J. White, J. Ivaska, P. Vehviläinen, H. Reunanen, H. Larjava, L. Häkkinen, and J. Heino. Integrin-mediated cell adhesion to type I collagen fibrils. J. Biol. Chem. 279:31956–31963, 2004.

    Article  CAS  PubMed  Google Scholar 

  17. Kettenmann, H., U. K. Hanisch, M. Noda, and A. Verkhratsky. Physiology of microglia. Physiol. Rev. 91:461–553, 2011.

    Article  CAS  PubMed  Google Scholar 

  18. Lee, W. H., W. E. Sonntag, M. Mitschelen, H. Yan, and Y. W. Lee. Irradiation induces regionally specific alterations in pro-inflammatory environments in rat brain. Int. J. Radiat. Biol. 86:132–144, 2010.

    Article  PubMed  PubMed Central  Google Scholar 

  19. Luo, X. G., J. Q. Ding, and S. D. Chen. Microglia in the aging brain: relevance to neurodegeneration. Mol. Neurodegener. 5:12, 2010.

    Article  PubMed  PubMed Central  Google Scholar 

  20. O’Connor, S. M., D. A. Stenger, K. M. Shaffer, D. Maric, J. L. Barker, and W. Ma. Primary neural precursor cell expansion, differentiation and cytosolic Ca2+ response in three-dimensional collagen gel. J. Neurosci. Methods 102:187–195, 2000.

    Article  PubMed  Google Scholar 

  21. Okazaki, K., and E. Maltepe. Oxygen, epigenetics and stem cell fate. Regen. Med. 1:71–83, 2006.

    Article  CAS  PubMed  Google Scholar 

  22. Pöttler, M., S. Zierler, and H. H. Kerschbaum. An artificial three-dimensional matrix promotes ramification in the microglial cell-line, BV-2. Neurosci. Lett. 410:137–140, 2006.

    Article  PubMed  Google Scholar 

  23. Riedl, A., M. Schlederer, K. Pudelko, M. Stadler, S. Walter, D. Unterleuthner, C. Unger, N. Kramer, M. Hengstschlager, L. Kenner, D. Pfeiffer, G. Krupitza, and H. Dolznig. Comparison of cancer cells in 2D vs 3D culture reveals differences in AKT-mTOR-S6 K signaling and drug responses. J. Cell Sci. 130:203–218, 2017.

    Article  CAS  PubMed  Google Scholar 

  24. Skardal, A., M. Devarasetty, H. W. Kang, I. Mead, C. Bishop, T. Shupe, S. J. Lee, J. Jackson, J. Yoo, S. Soker, and A. Atala. A hydrogel bioink toolkit for mimicking native tissue biochemical and mechanical properties in bioprinted tissue constructs. Acta Biomater. 25:24–34, 2015.

    Article  CAS  PubMed  Google Scholar 

  25. Song, Q., Z. Jiang, N. Li, P. Liu, L. Liu, M. Tang, and G. Cheng. Anti-inflammatory effects of three-dimensional graphene foams cultured with microglial cells. Biomaterials 35:6930–6940, 2014.

    Article  CAS  PubMed  Google Scholar 

  26. Stamati, K., V. Mudera, and U. Cheema. Evolution of oxygen utilization in multicellular organisms and implications for cell signalling in tissue engineering. J. Tissue Eng. 2:2041731411432365, 2011.

    Article  PubMed  PubMed Central  Google Scholar 

  27. Streeter, I., and U. Cheema. Oxygen consumption rate of cells in 3D culture: the use of experiment and simulation to measure kinetic parameters and optimise culture conditions. Analyst 136:4013–4019, 2011.

    Article  CAS  PubMed  Google Scholar 

  28. Tibbitt, M. W., and K. S. Anseth. Hydrogels as extracellular matrix mimics for 3D cell culture. Biotechnol. Bioeng. 103:655–663, 2009.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Toborek, M., Y. W. Lee, S. Kaiser, and B. Hennig. Measurement of inflammatory properties of fatty acids in human endothelial cells. Methods Enzymol. 352:198–219, 2002.

    Article  CAS  PubMed  Google Scholar 

  30. Verbridge, S. S., N. W. Choi, Y. Zheng, D. J. Brooks, A. D. Stroock, and C. Fischbach. Oxygen-controlled three-dimensional cultures to analyze tumor angiogenesis. Tissue Eng. Part A 16:2133–2141, 2010.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

The authors are thankful to Nabil Boutagy, Ph.D., Department of Medicine, Yale University School of Medicine, for his vital assistance with the Seahorse XF24 analyzer.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, FL, 33136, USA

    Hyung Joon Cho

  2. Department of Biomedical Engineering and Mechanics, Virginia Tech, 337 Kelly Hall (MC-0298), 325 Stanger Street, Blacksburg, VA, 24061, USA

    Scott S. Verbridge, Rafael V. Davalos & Yong W. Lee

Authors
  1. Hyung Joon Cho
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  2. Scott S. Verbridge
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  3. Rafael V. Davalos
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  4. Yong W. Lee
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Correspondence to Yong W. Lee.

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Associate Editor Jennifer West oversaw the review of this article.

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Cho, H.J., Verbridge, S.S., Davalos, R.V. et al. Development of an In Vitro 3D Brain Tissue Model Mimicking In Vivo-Like Pro-inflammatory and Pro-oxidative Responses. Ann Biomed Eng 46, 877–887 (2018). https://doi.org/10.1007/s10439-018-2004-z

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  • DOI: https://doi.org/10.1007/s10439-018-2004-z

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