Advertisement

Optimization by Response Surface Methodology of Confluent and Aligned Cellular Monolayers for Nerve Guidance

  • 114 Accesses

  • 10 Citations

Abstract

Anisotropic tissue structures provide guidance for navigating neurons in vitro and in vivo. Here we optimized the generation of comparable anisotropic monolayers of astrocytes, endothelial cells, and Schwann cells as a first step toward determining which properties of anisotropic cells are sufficient for nerve guidance. The statistical experimental design method Design of Experiments (DOE) and the experimental analysis method Response Surface Methodology (RSM) were applied to improve efficiency and utility. Factors investigated included dimensions of microcontact printed protein patterns, cell density, and culture duration. Protein patterning spacing had the strongest influence. When cells initially aligned at borders and proliferated to fill in spaces, space between stripes was most effective when it was comparable to cell size. Maximizing the area of adhesive molecule coverage was also important for confluence of these types of cells. When cells adhered and aligned over the width of a stripe and broadened to fill spaces, space width about half the cell width was most effective. These findings suggest that if the mechanism of alignment, alignment at borders or over the width of the stripe, is predetermined and the cell size determined, the optimal size of the micropatterning for aligned monolayers of other cell types can be predicted. This study also demonstrates the effective use of DOE and RSM to probe cellular responses to various and multiple factors toward determination of optimal conditions for a desired cellular response.

This is a preview of subscription content, log in to check access.

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

Subscribe to journal

Immediate online access to all issues from 2019. Subscription will auto renew annually.

US$ 99

This is the net price. Taxes to be calculated in checkout.

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8

References

  1. 1.

    Alexander, J. K., B. Fuss, and R. J. Colello. Electric field-induced astrocyte alignment directs neurite outgrowth. Neuron Glia Biol. 2:93–103, 2006.

  2. 2.

    Anderson, P. N., and M. Turmaine. Axonal regeneration through arterial grafts. J. Anat. 147:73, 1986.

  3. 3.

    Biran, R., M. D. Noble, and P. A. Tresco. Directed nerve outgrowth is enhanced by engineered glial substrates. Exp. Neurol. 184:141–152, 2003.

  4. 4.

    Borgens, R. B., R. Shi, T. J. Mohr, and C. B. Jaeger. Mammalian cortical astrocytes align themselves in a physiological voltage gradient. Exp. Neurol. 128:41–49, 1994.

  5. 5.

    Brook, G. A., J. M. Lawrence, B. Shah, and G. Raisman. Extrusion transplantation of Schwann cells into the adult rat thalamus induces directional host axon growth. Exp. Neurol. 126:31–43, 1994.

  6. 6.

    Bruder, J. M., A. P. Lee, and D. Hoffman-Kim. Biomimetic materials replicating Schwann cell topography enhance neuronal adhesion and neurite alignment in vitro. J. Biomater. Sci. Polym. Ed. 18:967–982, 2007.

  7. 7.

    Bunge, R. P. and C. Fernandez-Valle. The biology of Schwann cells. In: Neuroglia, edited by H. Kettenmann and B. R. Ransom. Oxford: Oxford University Press, 1995, pp. 44–57.

  8. 8.

    Chen, C. S., M. Mrksich, S. Huang, G. M. Whitesides, and D. E. Ingber. Geometric control of cell life and death. Science 276:1425–1428, 1997.

  9. 9.

    Chiu, D. T., I. Janecka, T. J. Krizek, M. Wolff, and R. E. Lovelace. Autogenous vein graft as a conduit for nerve regeneration. Surgery 91:226–233, 1982.

  10. 10.

    Clark, P., P. Connolly, A. S. Curtis, J. A. Dow, and C. D. Wilkinson. Topographical control of cell behaviour. I. Simple step cues. Development 99:439–448, 1987.

  11. 11.

    Croarkin, C., P. Tobias, and C. Zey, Engineering Statistics Handbook. National Institute of and Technology and International SEMATECH, 2001.

  12. 12.

    Dalby, M. J., M. O. Riehle, S. J. Yarwood, C. D. Wilkinson, and A. S. Curtis. Nucleus alignment and cell signaling in fibroblasts: response to a micro-grooved topography. Exp. Cell Res. 284:274–282, 2003.

  13. 13.

    David, S., and A. J. Aguayo. Axonal elongation into peripheral nervous system “Bridges” after central nervous system injury in adult rats. Science 214:931–933, 1981.

  14. 14.

    den Braber, E. T., J. E. de Ruijter, L. A. Ginsel, A. F. von Recum, and J. A. Jansen. Quantitative analysis of fibroblast morphology on microgrooved surfaces with various groove and ridge dimensions. Biomaterials 17:2037–2044, 1996.

  15. 15.

    Deumens, R., G. C. Koopmans, C. G. Den Bakker, V. Maquet, S. Blacher, W. M. Honig, R. Jerome, J. P. Pirard, H. W. Steinbusch, and E. A. Joosten. Alignment of glial cells stimulates directional neurite growth of cns neurons in vitro. Neuroscience 125:591–604, 2004.

  16. 16.

    Dubey, N., P. C. Letourneau, and R. T. Tranquillo. Guided neurite elongation and Schwann cell invasion into magnetically aligned collagen in simulated peripheral nerve regeneration. Exp. Neurol. 158:338–350, 1999.

  17. 17.

    El-Malah, Y., S. Nazzal, and N. M. Khanfar. D-optimal mixture design: optimization of ternary matrix blends for controlled zero-order drug release from oral dosage forms. Drug Dev. Ind. Pharm. 32:1207–1218, 2006.

  18. 18.

    Falconnet, D., G. Csucs, H. Michelle Grandin, and M. Textor. Surface engineering approaches to micropattern surfaces for cell-based assays. Biomaterials 27:3044–3063, 2006.

  19. 19.

    Feinberg, A. W., W. R. Wilkerson, C. A. Seegert, A. L. Gibson, L. Hoipkemeier-Wilson, and A. B. Brennan. Systematic variation of microtopography, surface chemistry and elastic modulus and the state dependent effect on endothelial cell alignment. J. Biomed. Mater. Res. A 86A:522–534, 2007.

  20. 20.

    Foidart-Dessalle, M., A. Dubuisson, A. Lejeune, A. Severyns, Y. Manassis, P. Delree, J. M. Crielaard, R. Bassleer, and G. Lejeune. Sciatic nerve regeneration through venous or nervous grafts in the rat. Exp. Neurol. 148:236–246, 1997.

  21. 21.

    Glasby, M. A., S. G. Gschmeissner, R. J. I. Hitchcock, and C. L. H. Huang. The dependence of nerve regeneration through muscle grafts in the rat on the availability and orientation of basement membrane. J. Neurocytol. 15:497–510, 1986.

  22. 22.

    Gomes, F. C., T. C. Spohr, R. Martinez, and V. Moura Neto. Cross-talk between neurons and glia: highlights on soluble factors. Braz. J. Med. Biol. Res. 34:611–620, 2001.

  23. 23.

    Hall, S. Axonal regeneration through acellular muscle grafts. J. Anat. 190:57–71, 1997.

  24. 24.

    Hofstetter, C. P., E. J. Schwarz, D. Hess, J. Widenfalk, A. El Manira, D. J. Prockop, and L. Olson. Marrow stromal cells form guiding strands in the injured spinal cord and promote recovery. Proc. Natl Acad. Sci. USA 99:2199–2204, 2002.

  25. 25.

    Kalil, S. J., F. Maugeri, and M. I. Rodrigues. Response surface analysis and simulation as a tool for bioprocess design and optimization. Process Biochem. 35:539–550, 2000.

  26. 26.

    Keynes, R. J., W. G. Hopkins, and L. H. Huang. Regeneration of mouse peripheral nerves in degenerating skeletal muscle: guidance by residual muscle fibre basement membrane. Brain Res. 295:275–281, 1984.

  27. 27.

    Li, G. N. Y., and D. Hoffman-Kim. Evaluation of neurite outgrowth anisotropy using a novel application of circular analysis. J. Neurosci. Methods 174:202–217, 2008.

  28. 28.

    Li, Y., Y. Sauve, D. Li, R. D. Lund, and G. Raisman. Transplanted olfactory ensheathing cells promote regeneration of cut adult rat optic nerve axons. J. Neurosci. 23:7783–7788, 2003.

  29. 29.

    Li, X., T. Xu, X. Ma, K. Guo, L. Kai, Y. Zhao, X. Jia, and Y. Ma. Optimization of culture conditions for production of cis-epoxysuccinic acid hydrolase using response surface methodology. Bioresour. Technol. 99:5391–5396, 2008.

  30. 30.

    Lumor, S. E., and C. C. Akoh. Enzymatic incorporation of stearic acid into a blend of palm olein and palm kernel oil: optimization by response surface methodology. J. Am. Oil Chem. Soc. 82:421–426, 2005.

  31. 31.

    Makita, T., H. M. Sucov, C. E. Gariepy, M. Yanagisawa, and D. D. Ginty. Endothelins are vascular-derived axonal guidance cues for developing sympathetic neurons. Nature 452:759–763, 2008.

  32. 32.

    Malek, A. M., and S. Izumo. Mechanism of endothelial cell shape change and cytoskeletal remodeling in response to fluid shear stress. J. Cell Sci. 109(Pt 4):713–726, 1996.

  33. 33.

    Maniotis, A. J., C. S. Chen, and D. E. Ingber. Demonstration of mechanical connections between integrins, cytoskeletal filaments, and nucleoplasm that stabilize nuclear structure. Proc. Natl Acad. Sci. USA 94:849–854, 1997.

  34. 34.

    Miller, C., H. Shanks, A. Witt, G. Rutkowski, and S. Mallapragada. Oriented Schwann cell growth on micropatterned biodegradable polymer substrates. Biomaterials 22:1263–1269, 2001.

  35. 35.

    Nerem, R. M., M. J. Levesque, and J. F. Cornhill. Vascular endothelial morphology as an indicator of the pattern of blood flow. J. Biomech. Eng. 103:172–176, 1981.

  36. 36.

    Pettigrew, D. B., and K. A. Crutcher. White matter of the cns supports or inhibits neurite outgrowth in vitro depending on geometry. J. Neurosci. 19:8358–8366, 1999.

  37. 37.

    Recknor, J. B., J. C. Recknor, D. S. Sakaguchi, and S. K. Mallapragada. Oriented astroglial cell growth on micropatterned polystyrene substrates. Biomaterials 25:2753–2767, 2004.

  38. 38.

    Recknor, J. B., D. S. Sakaguchi, and S. K. Mallapragada. Growth and differentiation of astrocytes and neural progenitor cells on micropatterned polymer films. Ann. N. Y. Acad. Sci. 1049:24–27, 2005.

  39. 39.

    Richardson, P. M., U. M. McGuinness, and A. J. Aguayo. Axons from cns neurons regenerate into pns grafts. Nature 284:264–265, 1980.

  40. 40.

    Sabourin, L. A., and M. A. Rudnicki. The molecular regulation of myogenesis. Clin. Genet. 57:16, 2000.

  41. 41.

    Schmalenberg, K. E., and K. E. Uhrich. Micropatterned polymer substrates control alignment of proliferating Schwann cells to direct neuronal regeneration. Biomaterials 26:1423–1430, 2005.

  42. 42.

    Schmidt, A., K. Brixius, and W. Bloch. Endothelial precursor cell migration during vasculogenesis. Circ. Res. 101:125–136, 2007.

  43. 43.

    Silver, J., M. A. Edwards, and P. Levitt. Immunocytochemical demonstration of early appearing astroglial structures that form boundaries and pathways along axon tracts in the fetal brain. J. Comp. Neurol. 328:415–436, 1993.

  44. 44.

    Singer, M., R. H. Nordlander, and M. Egar. Axonal guidance during embryogenesis and regeneration in the spinal cord of the newt: the blueprint hypothesis of neuronal pathway patterning. J. Comp. Neurol. 185:1–21, 1979.

  45. 45.

    Stepien, E., J. Stanisz, and W. Korohoda. Contact guidance of chick embryo neurons on single scratches in glass and on underlying aligned human skin fibroblasts. Cell Biol. Int. 23:105–116, 1999.

  46. 46.

    Thompson, D. M., and H. M. Buettner. Schwann cell response to micropatterned laminin surfaces. Tissue Eng. 7:247–265, 2001.

  47. 47.

    Thompson, D. M., and H. M. Buettner. Oriented Schwann cell monolayers for directed neurite outgrowth. Ann. Biomed. Eng. 32:1120–1130, 2004.

  48. 48.

    Thompson, D. M., and H. M. Buettner. Neurite outgrowth is directed by Schwann cell alignment in the absence of other guidance cues. Ann. Biomed. Eng. 34:161–168, 2006.

  49. 49.

    Tomanek, R. J. Formation of the coronary vasculature during development. Angiogenesis 8:273–284, 2005.

  50. 50.

    Vartanian, K. B., S. J. Kirkpatrick, S. R. Hanson, and M. T. Hinds. Endothelial cell cytoskeletal alignment independent of fluid shear stress on micropatterned surfaces. Biochem. Biophys. Res. Commun. 371:787–792, 2008.

  51. 51.

    Walboomers, X. F., H. J. Croes, L. A. Ginsel, and J. A. Jansen. Contact guidance of rat fibroblasts on various implant materials. J. Biomed. Mater. Res. 47:204–212, 1999.

  52. 52.

    Walsh, J. F., M. E. Manwaring, and P. A. Tresco. Directional neurite outgrowth is enhanced by engineered meningeal cell-coated substrates. Tissue Eng. 11:1085–1094, 2005.

  53. 53.

    Webb, A., P. Clark, J. Skepper, A. Compston, and A. Wood. Guidance of oligodendrocytes and their progenitors by substratum topography. J. Cell Sci. 108(Pt 8):2747–2760, 1995.

  54. 54.

    Wood, A., and P. Thorogood. An ultrastructural and morphometric analysis of an in vivo contact guidance system. Development 101:363–381, 1987.

  55. 55.

    Wu, C. C., Y. S. Li, J. H. Haga, R. Kaunas, J. J. Chiu, F. C. Su, S. Usami, and S. Chien. Directional shear flow and rho activation prevent the endothelial cell apoptosis induced by micropatterned anisotropic geometry. Proc. Natl Acad. Sci. USA 104:1254–1259, 2007.

  56. 56.

    Yim, E. K., R. M. Reano, S. W. Pang, A. F. Yee, C. S. Chen, and K. W. Leong. Nanopattern-induced changes in morphology and motility of smooth muscle cells. Biomaterials 26:5405–5413, 2005.

Download references

Acknowledgments

The authors would like to thank Christina Johnson and Grace Li for helpful discussion of the initial experimental set-up. They would also like to thank Carmichael Ong, Liane Livi, Cameron Rementer, and Jesse Thon for assistance with imaging and analysis. Si wafers were fabricated at the BioMEMS Resource Center with the generous assistance of Octavio Hurtado. This work was funded by an NSF Career Award and NIH R01 EB005722-01A2 to DHK and an AAUW Selected Professions dissertation fellowship to CK.

Author information

Correspondence to Diane Hoffman-Kim.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (MOV 25785 kb)

Supplementary material 2 (MOV 23687 kb)

Supplementary material 3 (MOV 35560 kb)

Supplementary material 1 (MOV 25785 kb)

Supplementary material 2 (MOV 23687 kb)

Supplementary material 3 (MOV 35560 kb)

Supplementary material 4 (DOCX 100 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kofron, C.M., Hoffman-Kim, D. Optimization by Response Surface Methodology of Confluent and Aligned Cellular Monolayers for Nerve Guidance. Cel. Mol. Bioeng. 2, 554 (2009) doi:10.1007/s12195-009-0087-1

Download citation

Keywords

  • Design of Experiments (DOE)
  • D-optimal
  • Schwann cell
  • Astrocyte
  • Endothelial cell