Skip to main content

Journal for Biophysical Chemistry

Biointerphases Cover Image

Optimization of protein patterns for neuronal cell culture applications

Abstract

In the present study, we fabricated two-component extracellular matrix protein patterned substrates with fibronectin (FN) and laminin (LN) because of our interest in the mechanism of axonal regeneration and injury in the central and peripheral nervous systems. The authors investigated how the patterning order and method of attachment affected the spatial distribution and biological activity of the immobilized proteins. Micro-contact printing (μCP) techniques in concert with reactive surface chemistry were used to modify glass substrates with one- and two-component films of FN and LN, including micrometer-scale patterns of FN and LN. The composition and spatial distributions of both proteins on the patterned surfaces were characterized by x ray photoelectron spectroscopy, epi-fluorescence microscopy, atomic force microscopy, and time-of-flight secondaryion mass spectrometry. The authors also characterized the biological activity of the top-most protein layer in a two-layer protein system as well as the ability of the top-most protein layer to mask the biological activity of an underlying protein layer using a fluorescence-based enzyme-linked immunosorbent assay. The order of protein deposition significantly affected the relative biological activity of the upper-most and underlying immobilized proteins. As a result of these optimization studies, maximum biological activity per surface protein was achieved by first immobilizing FN from solution, followed by μCP of LN on the FN. Addition of μCP LN films was able to mask ~84% of the underlying FN activity, whereas μCP FN films were only able to mask ~27% of the underlying LN activity.

References

  1. 1

    J. C. Harper, R. Polsky, D. R. Wheeler, S. M. Dirk, and S. M. Brozik, Langmuir 23, 8285 (2007).

    Article  CAS  Google Scholar 

  2. 2

    T. H. Park and M. L. Shuler, Biotechnol. Prog. 19, 243 (2003).

    Article  CAS  Google Scholar 

  3. 3

    J. V. Veetil and K. M. Ye, Biotechnol. Prog. 23, 517 (2007).

    Article  CAS  Google Scholar 

  4. 4

    J. H. Hyun, H. W. Ma, P. Banerjee, J. Cole, K. Gonsalves, and A. Chilkoti, Langmuir 18, 2975 (2002).

    Article  CAS  Google Scholar 

  5. 5

    S. Mitragotri and J. Lahann, Nat. Mater. 8, 15 (2009).

    Article  CAS  Google Scholar 

  6. 6

    G. MacBeath and S. L. Schreiber, Science 289, 1760 (2000).

    CAS  Google Scholar 

  7. 7

    A. Kaushansky, J. E. Allen, A. Gordus, M. A. Stiffler, E. S. Karp, B. H. Chang, and G. MacBeath, Nat. Protoc. 5, 773 (2010).

    Article  CAS  Google Scholar 

  8. 8

    C. D. Chin, V. Linder, and S. K. Sia, Lab Chip 7, 41 (2007).

    Article  CAS  Google Scholar 

  9. 9

    C. S. Chen, M. Mrksich, S. Huang, G. M. Whitesides, and D. E. Ingber, Science 276, 1425 (1997).

    Article  CAS  Google Scholar 

  10. 10

    C. S. Chen, M. Mrksich, S. Huang, G. M. Whitesides, and D. E. Ingber, Biotechnol. Prog. 14, 356 (1998).

    Article  CAS  Google Scholar 

  11. 11

    M. Mrksich, Chem. Soc. Rev. 29, 267 (2000).

    Article  CAS  Google Scholar 

  12. 12

    D. Lehnert, B. Wehrle-Haller, C. David, U. Weiland, C. Ballestrem, B. A. Imhof, and M. Bastmeyer, J.Cell Sci. 117, 41 (2004).

    Article  CAS  Google Scholar 

  13. 13

    J. A. Hammarback, J. B. McCarthy, S. L. Palm, L. T. Furcht, and P. C. Letourneau, Dev. Biol. 126, 29 (1988).

    Article  CAS  Google Scholar 

  14. 14

    Z. P. Zhang, R. Yoo, M. Wells, T. P. Beebe, R. Biran, and P. Tresco, Biomaterials 26, 47 (2005).

    Article  Google Scholar 

  15. 15

    M. L. Condic and P. C. Letourneau, Nature 389, 852 (1997).

    Article  CAS  Google Scholar 

  16. 16

    G. M. Whitesides, E. Ostuni, S. Takayama, X. Y. Jiang, and D. E. Ingber, Annu. Rev. Biomed. Eng. 3, 335 (2001).

    Article  CAS  Google Scholar 

  17. 17

    G. M. Whitesides, Nature 442, 368 (2006).

    Article  CAS  Google Scholar 

  18. 18

    B. A. Langowski and K. E. Uhrich, Langmuir 21, 10509 (2005).

    Article  CAS  Google Scholar 

  19. 19

    A. Ohl and K. Schroder, Surf. Coat. Technol. 119, 820 (1999).

    Article  Google Scholar 

  20. 20

    B. D. Gates, Q. B. Xu, M. Stewart, D. Ryan, C. G. Willson, and G. M. Whitesides, Chem. Rev. 105, 1171 (2005).

    Article  CAS  Google Scholar 

  21. 21

    Y. N. Xia and G. M. Whitesides, Annu. Rev. Mater. Scie. 28, 153 (1998).

    Article  CAS  Google Scholar 

  22. 22

    A. P. Quist, E. Pavlovic, and S. Oscarsson, Anal. Bioanal. Chem. 381, 591 (2005).

    Article  CAS  Google Scholar 

  23. 23

    A. Bernard, E. Delamarche, H. Schmid, B. Michel, H. R. Bosshard, and H. Biebuyck, Langmuir 14, 2225 (1998).

    Article  CAS  Google Scholar 

  24. 24

    H. W. Ma, J. Hyun, Z. P. Zhang, T. P. Beebe, and A. Chilkoti, Adv. Funct. Mater. 15, 529 (2005).

    Article  CAS  Google Scholar 

  25. 25

    J. Hyun, Y. J.; Zhu, A. Liebmann-Vinson, T. P. Beebe, and A. Chilkoti, Langmuir 17, 6358 (2001).

    Article  CAS  Google Scholar 

  26. 26

    J. H. Hyun, H. W. Ma, Z. P. Zhang, T. P. Beebe, and A. Chilkoti, Adv. Mater. 15, 576 (2003).

    Article  CAS  Google Scholar 

  27. 27

    A. Dolatshahi-Pirouz, S. Skeldal, M. B. Hovgaard, T. Jensen, M. Foss, J. Chevallier, and F. Besenbacher, J. Phys. Chem. C 113, 4406 (2009).

    Article  CAS  Google Scholar 

  28. 28

    G. B. Sigal, M. Mrksich, and G. M. Whitesides, J. Am. Chem. Soc. 120, 3464 (1998).

    Article  CAS  Google Scholar 

  29. 29

    M. H. Lee, P. Ducheyne, L. Lynch, D. Boettiger, and R. J. Composto, Biomaterials 27, 1907 (2006).

    Article  CAS  Google Scholar 

  30. 30

    C. F. Wertz and M. M. Santore, Langmuir 17, 3006 (2001).

    Article  CAS  Google Scholar 

  31. 31

    C. F. Wertz and M. M. Santore, Langmuir 18, 706 (2002).

    Article  CAS  Google Scholar 

  32. 32

    L. Baugh and V. Vogel, J. Biomed. Mater. Res. 69A, 525 (2004).

    Article  CAS  Google Scholar 

  33. 33

    K. E. Michael, V. N. Vernekar, B. G. Keselowsky, J. C. Meredith, R. A. Latour, and A. J. Garcia, Langmuir 19, 8033 (2003).

    Article  CAS  Google Scholar 

  34. 34

    J. J. Gray, Curr. Opin. Struct. Biol. 14, 110 (2004).

    Article  CAS  Google Scholar 

  35. 35

    B. Wang, J. Feng, and C. Y. Gao, Macromol. Biosci. 5,767 (2005).

    Article  CAS  Google Scholar 

  36. 36

    G. N. Hodgkinson, P.A. Tresco, and V. Hlady, Biomaterials 28, 2590 (2007).

    Article  CAS  Google Scholar 

  37. 37

    M. Song and K. E. Uhrich, Ann. Biomed. Eng. 35, 1812 (2007).

    Article  Google Scholar 

  38. 38

    J. Silver and J. H. Miller, Nat. Rev. Neurosci. 5, 146 (2004).

    Article  CAS  Google Scholar 

  39. 39

    S. S. G. Johansson, K. Wennerberg, A. Armulik, and L. Lohikangas, Front. Biosci. 1, 126 (1997).

    Google Scholar 

  40. 40

    S. K. Powell and H. K. Kleinman, Int. J. Biochem. Cell Biol. 29, 401 (1997).

    Article  CAS  Google Scholar 

  41. 41

    J. Graf, Y. Iwamoto, M. Sasaki, G. R. Martin, H. K. Kleinman, F. A. Robey, and Y. Yamada, Cell 48, 989 (1987).

    Article  CAS  Google Scholar 

  42. 42

    Y. Iwamoto, F. A. Robey, J. Graf, M. Sasaki, H. K. Kleinman, Y. Yamada, and G. R. Martin, Science 238, 1132 (1987).

    Article  CAS  Google Scholar 

  43. 43

    K. Tashiro, G. C. Sephel, B. Weeks, M. Sasaki, G. R. Martin, H. K. Kleinman, and Y. Yamada, J. Biol. Chem. 264, 16174 (1989).

    CAS  Google Scholar 

  44. 44

    M. Q. Zhang and M. Ferrari, Biotechnol. Bioeng. 56, 618 (1997).

    Article  CAS  Google Scholar 

  45. 47

    P. S. Hale, P. Kappen, W. Prissanaroon, N. Brack, P. J. Pigram, and J. Liesegang, Appl. Surf. Sci. 253, 3746 (2007).

    Article  CAS  Google Scholar 

  46. 48

    Y. S. Lo, N. D. Huefner, W. S. Chan, P. Dryden, B. Hagenhoff, and T. P. Beebe, Langmuir 15, 6522 (1999).

    Article  CAS  Google Scholar 

  47. 49

    P. A. Underwood, J. G. Steele, and B. A. Dalton, J. Cell Sci. 104, 793 (1993).

    CAS  Google Scholar 

  48. 50

    S. Scheele, T. Sasaki, A. Arnal-Estape, M. Durbeej, and P. Ekblom, Matrix Biol. 25, 301 (2006).

    Article  CAS  Google Scholar 

  49. 51

    U. M. Wewer, G. Taraboletti, M. E. Sobel, R. Albrechtsen, and L. A. Liotta, Cancer Res. 47, 5691 (1987).

    CAS  Google Scholar 

  50. 52

    A. J. García, P. Ducheyne, and D. Boettiger, Biomaterials 18, 1091 (1997).

    Article  Google Scholar 

  51. 53

    P. A. Underwood, J. G. Steele, and B. A. Dalton, J. Cell Sci. 104, 793 (1993).

    CAS  Google Scholar 

  52. 54

    G. K. Toworfe, R. J. Composto, C. S. Adams, I. M. Shapiro, and P. Ducheyne, J. Biomed. Mater. Res. A 71A, 449 (2004).

    Article  CAS  Google Scholar 

  53. 55

    L. Vroman, Nature 196, 476 (1962).

    Article  CAS  Google Scholar 

  54. 56

    C. Thibault, C. Severac, A. F. Mingotaud, C. Vieu, and M. Mauzac, Langmuir 23, 10706 (2007).

    Article  CAS  Google Scholar 

  55. 57

    B. A. Langowski and K. E. Uhrich, Langmuir 21, 6366 (2005).

    Article  CAS  Google Scholar 

  56. 58

    K. Glasmäster, J. Gold, A.-S. Andersson, D. S. Sutherland, and B. Kasemo, Langmuir 19, 5475 (2003).

    Article  Google Scholar 

  57. 59

    E. Delamarche, M. Geissler, A. Bernard, H. Wolf, B. Michel, J. Hilborn, and C. Donzel, Adv. Mater. 13, 1164 (2001).

    Article  Google Scholar 

Download references

Author information

Affiliations

Authors

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Theilacker, W.M., Bui, H. & Beebe, T.P. Optimization of protein patterns for neuronal cell culture applications. Biointerphases 6, 105–116 (2011). https://doi.org/10.1116/1.3624584

Download citation