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A Novel Technology for Simultaneous Tensile Loading and High-Resolution Imaging of Cells

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Abstract

A variety of methods have been used to study the tensile properties of cells or the influence of tensile loading on cellular function. Such methods are frequently limited either by cellular detachment or by an inability to image cells at high temporal or spatial resolution. Previously, we preserved cellular adhesion during loading and imaging by using a flexible silicone membrane inverted over a glass coverslip. This enabled high magnification real-time imaging of subcellular structures but chemical and physical access to the cells was limited due to geometric constraints. In this study, we present a method to integrate thin films made from poly(dimethylsiloxane) (PDMS) into a novel device. The optically clear PDMS thin films allow simultaneous tensile loading and high magnification microscopy without the need to invert the cells, maintaining physical access during experiments. To characterize the utility of this technology, we evaluated fabrication conditions for optimizing the geometry, durability, and uniformity of these films. Additionally, we demonstrate the suitability of this device for use in high-magnification, live-cell fluorescence microscopy by examining the response of the cytoskeletal protein actin, expressed in cultured primary sensory neurons, to a tensile load. This technology offers considerable potential for extending our understanding of mechanical influences on cellular function at a variety of spatial and temporal scales.

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References

  1. Armbruster, C., M. Schneider, S. Schumann, K. Gamerdinger, M. Cuevas, S. Rausch, G. Baaken, and J. Guttmann. Characteristics of highly flexible pdms membranes for long-term mechanostimulation of biological tissue. J. Biomed. Mater. Res. B 91:700–705, 2009.

    Google Scholar 

  2. Baker, B. M., R. P. Shah, A. H. Huang, and R. L. Mauck. Dynamic tensile loading improves the functional properties of mesenchymal stem cell-laden nanofiber-based fibrocartilage. Tissue Eng. A 17:1445–1455, 2011.

    Article  Google Scholar 

  3. Barbee, K. A., E. J. Macarak, and L. E. Thibault. Strain measurements in cultured vascular smooth muscle cells subjected to mechanical deformation. Ann. Biomed. Eng. 22:14–22, 1994.

    Article  Google Scholar 

  4. Bieler, F. H., C. E. Ott, M. S. Thompson, R. Seidel, S. Ahrens, D. R. Epari, U. Wilkening, K. D. Schaser, S. Mundlos, and G. N. Duda. Biaxial cell stimulation: a mechanical validation. J. Biomech. 42:1692–1696, 2009.

    Article  Google Scholar 

  5. Bischofs, I. B., and U. S. Schwarz. Cell organization in soft media due to active mechanosensing. Proc. Natl. Acad. Sci. USA 100:9274–9279, 2003.

    Article  Google Scholar 

  6. Bueno, F. R., and S. B. Shah. Implications of tensile loading for the tissue engineering of nerves. Tissue Eng. Part B Rev. 14:219–233, 2008.

    Article  Google Scholar 

  7. Caille, N., Y. Tardy, and J. J. Meister. Assessment of strain field in endothelial cells subjected to uniaxial deformation of their substrate. Ann. Biomed. Eng. 26:409–416, 1998.

    Article  Google Scholar 

  8. Camelliti, P., A. D. McCulloch, and P. Kohl. Microstructured cocultures of cardiac myocytes and fibroblasts: a two-dimensional in vitro model of cardiac tissue. Microsc. Microanal. 11:249–259, 2005.

    Article  Google Scholar 

  9. Cesa, C. M., N. Kirchgessner, D. Mayer, U. S. Schwarz, B. Hoffmann, and R. Merkel. Micropatterned silicone elastomer substrates for high resolution analysis of cellular force patterns. Rev. Sci. Instrum. 78:034301, 2007.

    Article  Google Scholar 

  10. Chetta, J., C. Kye, and S. B. Shah. Cytoskeletal dynamics in response to tensile loading of mammalian axons. Cytoskeleton (Hoboken) 67:650–665, 2010.

    Article  Google Scholar 

  11. Chetta, J., and S. B. Shah. A novel algorithm to generate kymographs from dynamic axons for the quantitative analysis of axonal transport. J. Neurosci. Methods 199:230–240, 2011.

    Article  Google Scholar 

  12. Chun, H., D. S. Lee, and H. C. Kim. Bio-cell chip fabrication and applications. Methods Mol. Biol. 509:145–158, 2009.

    Article  Google Scholar 

  13. Davis, M. J., J. A. Donovitz, and J. D. Hood. Stretch-activated single-channel and whole cell currents in vascular smooth muscle cells. Am. J. Physiol. 262:C1083–C1088, 1992.

    Google Scholar 

  14. Folch, A., and M. Toner. Cellular micropatterns on biocompatible materials. Biotechnol. Prog. 14:388–392, 1998.

    Article  Google Scholar 

  15. Formigli, L., E. Meacci, C. Sassoli, R. Squecco, D. Nosi, F. Chellini, F. Naro, F. Francini, and S. Zecchi-Orlandini. Cytoskeleton/stretch-activated ion channel interaction regulates myogenic differentiation of skeletal myoblasts. J. Cell. Physiol. 211:296–306, 2007.

    Article  Google Scholar 

  16. Garvin, J., J. Qi, M. Maloney, and A. J. Banes. Novel system for engineering bioartificial tendons and application of mechanical load. Tissue Eng. 9:967–979, 2003.

    Article  Google Scholar 

  17. Gerstmair, A., G. Fois, S. Innerbichler, P. Dietl, and E. Felder. A device for simultaneous live cell imaging during uni-axial mechanical strain or compression. J. Appl. Physiol. 107:613–620, 2009.

    Article  Google Scholar 

  18. Gilchrist, C. L., S. W. Witvoet-Braam, F. Guilak, and L. A. Setton. Measurement of intracellular strain on deformable substrates with texture correlation. J. Biomech. 40:786–794, 2007.

    Article  Google Scholar 

  19. Hanein, Y., O. Tadmor, S. Anava, and A. Ayali. Neuronal soma migration is determined by neurite tension. Neuroscience 172:572–579, 2011.

    Article  Google Scholar 

  20. Harris, A. K., P. Wild, and D. Stopak. Silicone rubber substrata: a new wrinkle in the study of cell locomotion. Science 208:177–179, 1980.

    Article  Google Scholar 

  21. Haston, W. S., J. M. Shields, and P. C. Wilkinson. The orientation of fibroblasts and neutrophils on elastic substrata. Exp. Cell Res. 146:117–126, 1983.

    Article  Google Scholar 

  22. Hochmuth, R. M. Micropipette aspiration of living cells. J. Biomech. 33:15–22, 2000.

    Article  Google Scholar 

  23. Houtchens, G. R., M. D. Foster, T. A. Desai, E. F. Morgan, and J. Y. Wong. Combined effects of microtopography and cyclic strain on vascular smooth muscle cell orientation. J. Biomech. 41:762–769, 2008.

    Article  Google Scholar 

  24. Huang, L., P. S. Mathieu, and B. P. Helmke. A stretching device for high-resolution live-cell imaging. Ann. Biomed. Eng. 38:1728–1740, 2010.

    Article  Google Scholar 

  25. Jean, R. P., D. S. Gray, A. A. Spector, and C. S. Chen. Characterization of the nuclear deformation caused by changes in endothelial cell shape. J. Biomech. Eng. 126:552–558, 2004.

    Article  Google Scholar 

  26. Kartalov, E. P., W. F. Anderson, and A. Scherer. The analytical approach to polydimethylsiloxane microfluidic technology and its biological applications. J. Nanosci. Nanotechnol. 6:2265–2277, 2006.

    Article  Google Scholar 

  27. Kaunas, R., S. Usami, and S. Chien. Regulation of stretch-induced JNK activation by stress fiber orientation. Cell. Signal. 18:1924–1931, 2006.

    Article  Google Scholar 

  28. Kim, B. S., and D. J. Mooney. Scaffolds for engineering smooth muscle under cyclic mechanical strain conditions. J. Biomech. Eng. 122:210–215, 2000.

    Article  Google Scholar 

  29. Knight, M. M., Z. Bomzon, E. Kimmel, A. M. Sharma, D. A. Lee, and D. L. Bader. Chondrocyte deformation induces mitochondrial distortion and heterogeneous intracellular strain fields. Biomech. Model. Mechanobiol. 5:180–191, 2006.

    Article  Google Scholar 

  30. Koschwanez, J. H., R. H. Carlson, and D. R. Meldrum. Thin PDMS films using long spin times or tert-butyl alcohol as a solvent. PLoS ONE 4:e4572, 2009.

    Article  Google Scholar 

  31. Lawrence, C. J. The mechanics of spin coating of polymer-films. Phys. Fluids 31:2786–2795, 1988.

    Article  Google Scholar 

  32. Lee, C. F., C. Haase, S. Deguchi, and R. Kaunas. Cyclic stretch-induced stress fiber dynamics—dependence on strain rate, rho-kinase and MLCK. Biochem. Biophys. Res. Commun. 401:344–349, 2010.

    Article  Google Scholar 

  33. Lindqvist, N., Q. Liu, J. Zajadacz, K. Franze, and A. Reichenbach. Retinal glial (muller) cells: Sensing and responding to tissue stretch. Invest. Ophthalmol. Vis. Sci. 51:1683–1690, 2010.

    Article  Google Scholar 

  34. Liu, M., J. R. Sun, Y. Sun, C. Bock, and Q. F. Chen. Thickness-dependent mechanical properties of polydimethylsiloxane membranes. J. Micromech. Microeng. 19:035028, 2009.

    Article  Google Scholar 

  35. Loverde, J. R., V. C. Ozoka, R. Aquino, L. Lin, and B. J. Pfister. Live imaging of axon stretch growth in embryonic and adult neurons. J. Neurotrauma 28:2389–2403, 2011.

    Article  Google Scholar 

  36. 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.

    Article  Google Scholar 

  37. McDonald, J. C., and G. M. Whitesides. Poly(dimethylsiloxane) as a material for fabricating microfluidic devices. Acc. Chem. Res. 35:491–499, 2002.

    Article  Google Scholar 

  38. Merkel, R., N. Kirchgessner, C. M. Cesa, and B. Hoffmann. Cell force microscopy on elastic layers of finite thickness. Biophys. J . 93:3314–3323, 2007.

    Article  Google Scholar 

  39. Mizutani, T., H. Haga, and K. Kawabata. Development of a device to stretch tissue-like materials and to measure their mechanical properties by scanning probe microscopy. Acta Biomater. 3:485–493, 2007.

    Article  Google Scholar 

  40. Norrman, K., A. Ghanbari-Siahkali, and N. B. Larsen. 6 studies of spin-coated polymer films. Annu. Rep. Sect. C Phys. Chem. 101:174–201, 2005.

    Article  Google Scholar 

  41. Paten, J. A., R. Zareian, N. Saeidi, S. A. Melotti, and J. W. Ruberti. Design and performance of an optically accessible, low-volume, mechanobioreactor for long-term study of living constructs. Tissue Eng. Part C Methods 17:775–788, 2011.

    Article  Google Scholar 

  42. Pfister, B. J., A. Iwata, D. F. Meaney, and D. H. Smith. Extreme stretch growth of integrated axons. J. Neurosci. 24:7978–7983, 2004.

    Article  Google Scholar 

  43. Ryu, K. S., X. Wang, K. Shaikh, and C. Liu. A method for precision patterning of silicone elastomer and its applications. J. Microelectromech. Syst. 13:568–575, 2004.

    Article  Google Scholar 

  44. Shah, S. B., and R. L. Lieber. Simultaneous imaging and functional assessment of cytoskeletal protein connections in passively loaded single muscle cells. J. Histochem. Cytochem. 51:19–29, 2003.

    Article  Google Scholar 

  45. Shi, Y., H. Li, X. Zhang, Y. Fu, Y. Huang, P. P. Lui, T. Tang, and K. Dai. Continuous cyclic mechanical tension inhibited runx2 expression in mesenchymal stem cells through rhoa-erk1/2 pathway. J. Cell. Physiol. 226:2159–2169, 2011.

    Article  Google Scholar 

  46. Sia, S. K., and G. M. Whitesides. Microfluidic devices fabricated in poly(dimethylsiloxane) for biological studies. Electrophoresis 24:3563–3576, 2003.

    Article  Google Scholar 

  47. Sleep, J., D. Wilson, R. Simmons, and W. Gratzer. Elasticity of the red cell membrane and its relation to hemolytic disorders: An optical tweezers study. Biophys. J. 77:3085–3095, 1999.

    Article  Google Scholar 

  48. Smith, D. H., J. A. Wolf, T. A. Lusardi, V. M. Lee, and D. F. Meaney. High tolerance and delayed elastic response of cultured axons to dynamic stretch injury. J. Neurosci. 19:4263–4269, 1999.

    Google Scholar 

  49. Smith, D. H., J. A. Wolf, and D. F. Meaney. A new strategy to produce sustained growth of central nervous system axons: continuous mechanical tension. Tissue Eng. 7:131–139, 2001.

    Article  Google Scholar 

  50. Sniadecki, N. J., A. Anguelouch, M. T. Yang, C. M. Lamb, Z. Liu, S. B. Kirschner, Y. Liu, D. H. Reich, and C. S. Chen. Magnetic microposts as an approach to apply forces to living cells. Proc. Natl. Acad. Sci. USA 104:14553–14558, 2007.

    Article  Google Scholar 

  51. Sonobe, T., T. Inagaki, D. C. Poole, and Y. Kano. Intracellular calcium accumulation following eccentric contractions in rat skeletal muscle in vivo: role of stretch-activated channels. Am. J. Physiol. Regul. Integr. Comp. Physiol. 294:R1329–R1337, 2008.

    Article  Google Scholar 

  52. Suresh, S. Biomechanics and biophysics of cancer cells. Acta Biomater. 3:413–438, 2007.

    Article  MathSciNet  Google Scholar 

  53. Tondon, A., H. J. Hsu, and R. Kaunas. Dependence of cyclic stretch-induced stress fiber reorientation on stretch waveform. J. Biomech. 45:728–735, 2011.

    Article  Google Scholar 

  54. Venkataraman, S. K., L. Coyne, F. Chambon, M. Gottlieb, and H. H. Winter. Critical extent of reaction of a polydimethylsiloxane polymer network. Polymer 30:2222–2226, 1989.

    Article  Google Scholar 

  55. Wall, M. E., P. S. Weinhold, T. Siu, T. D. Brown, and A. J. Banes. Comparison of cellular strain with applied substrate strain in vitro. J. Biomech. 40:173–181, 2007.

    Article  Google Scholar 

  56. Wang, D., Y. Xie, B. Yuan, J. Xu, P. Gong, and X. Jiang. A stretching device for imaging real-time molecular dynamics of live cells adhering to elastic membranes on inverted microscopes during the entire process of the stretch. Integr. Biol. (Camb). 2:288–293, 2010.

    Article  Google Scholar 

  57. Wang, J. H., and B. Li. Mechanics rules cell biology. Sports Med. Arthrosc. Rehabil. Ther. Technol. 2:16, 2010.

    Article  Google Scholar 

  58. Wang, Y. L., and R. J. Pelham, Jr. Preparation of a flexible, porous polyacrylamide substrate for mechanical studies of cultured cells. Methods Enzymol. 298:489–496, 1998.

    Article  Google Scholar 

  59. Xia, Y. N., and G. M. Whitesides. Soft lithography. Annu. Rev. Mater. Sci. 28:153–184, 1998.

    Article  Google Scholar 

  60. Zhang, H., F. Landmann, H. Zahreddine, D. Rodriguez, M. Koch, and M. Labouesse. A tension-induced mechanotransduction pathway promotes epithelial morphogenesis. Nature 471:99–103, 2011.

    Article  Google Scholar 

  61. Zheng, J., P. Lamoureux, V. Santiago, T. Dennerll, R. E. Buxbaum, and S. R. Heidemann. Tensile regulation of axonal elongation and initiation. J. Neurosci. 11:1117–1125, 1991.

    Google Scholar 

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Acknowledgments

We gratefully acknowledge technical assistance by staff of the Maryland Nanocenter Micro and Nano Fabrication Laboratory and helpful discussions with the Neuromuscular Bioengineering Laboratory. This research was supported by funding from the National Science Foundation (CBET0932590 and CMMI1130997) and the State of Maryland Stem Cell Research Commission.

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Authors and Affiliations

  1. Fischell Department of Bioengineering, University of Maryland, College Park, MD, USA

    Bao-Ngoc B. Nguyen, Joshua Chetta & Sameer B. Shah

  2. Departments of Orthopaedic Surgery and Bioengineering, University of California, San Diego, 9500 Gilman Drive, Mail Code 0863, La Jolla, CA, 92093, USA

    Sameer B. Shah

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  1. Bao-Ngoc B. Nguyen
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  2. Joshua Chetta
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  3. Sameer B. Shah
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Correspondence to Sameer B. Shah.

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Associate Editor Joyce Wong oversaw the review of this article.

B.-N. B. Nguyen and J. Chetta contributed equally to this work.

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Nguyen, BN.B., Chetta, J. & Shah, S.B. A Novel Technology for Simultaneous Tensile Loading and High-Resolution Imaging of Cells. Cel. Mol. Bioeng. 5, 504–513 (2012). https://doi.org/10.1007/s12195-012-0245-8

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