Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Micropatterned cell cultures on elastic membranes as an in vitro model of myocardium

Abstract

We describe here a new in vitro protocol for structuring cardiac cell cultures to mimic important aspects of the in vivo ventricular myocardial phenotype by controlling the location and mechanical environment of cultured cells. Microlithography is used to engineer microstructured silicon metal wafers. Those are used to fabricate either microgrooved silicone membranes or silicone molds for microfluidic application of extracellular matrix proteins onto elastic membranes (involving flow control at micrometer resolution). The physically or microfluidically structured membranes serve as a cell culture growth substrate that supports cell alignment and allows the application of stretch. The latter is achieved with a stretching device that can deliver isotropic or anisotropic stretch. Neonatal ventricular cardiomyocytes, grown on these micropatterned membranes, develop an in vivo–like morphology with regular sarcomeric patterns. The entire process from fabrication of the micropatterned silicon metal wafers to casting of silicone molds, microfluidic patterning and cell isolation and seeding takes approximately 7 days.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Photolithography, PDMS replication, micropatterning of collagen tracks and cell deposition on micropattern.
Figure 2: Circular and elliptical stretch devices for the culture of micropatterned cells.
Figure 3: Elliptical 'non-equi-biaxial' stretch device for cell cultures.
Figure 4: Mean membrane stretch percentage (relative to control conditions) as function of rotation of the screw-top (angle of rotation) for ten circular stretch devices.
Figure 5: Neonatal cardiac cells cultured on collagen-micropatterned or grooved silicone membranes.
Figure 6: Connexin-43 expression in microfluidic structured myocyte-fibroblast cocultures in control condition and after circular stretch.
Figure 7: Connexin-43 expression in microgrooved neonatal myocytes after elliptical stretch.

References

  1. Gopalan, S.M. et al. Anisotropic stretch-induced hypertrophy in neonatal ventricular myocytes micropatterned on deformable elastomers. Biotechnol. Bioeng. 81, 578–587 (2003).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  3. Davies, M.J. & Pomerance, A. Quantitative study of ageing changes in the human sinoatrial node and internodal tracts. Br. Heart J. 34, 150–152 (1972).

    CAS  Article  Google Scholar 

  4. Shiraishi, I., Takamatsu, T., Minamikawa, T., Onouchi, Z. & Fujita, S. Quantitative histological analysis of the human sinoatrial node during growth and aging. Circulation 85, 2176–2184 (1992).

    CAS  Article  Google Scholar 

  5. Camelliti, P., Borg, T.K. & Kohl, P. Structural and functional characterisation of cardiac fibroblasts. Cardiovasc. Res. 65, 40–51 (2005).

    CAS  Article  Google Scholar 

  6. MacKenna, D., Summerour, S.R. & Villarreal, F.J. Role of mechanical factors in modulating cardiac fibroblast function and extracellular matrix synthesis. Cardiovasc. Res. 46, 257–263 (2000).

    CAS  Article  Google Scholar 

  7. Sun, Y., Kiani, M.F., Postlethwaite, A.E. & Weber, K.T. Infarct scar as living tissue. Basic Res. Cardiol. 97, 343–347 (2002).

    Article  Google Scholar 

  8. Kohl, P. & Noble, D. Mechanosensitive connective tissue: potential influence on heart rhythm. Cardiovasc. Res. 32, 62–68 (1996).

    CAS  Article  Google Scholar 

  9. Kohl, P. Heterogeneous cell coupling in the heart: an electrophysiological role for fibroblasts. Circ. Res. 93, 381–383 (2003).

    CAS  Article  Google Scholar 

  10. Camelliti, P., Green, C.R., LeGrice, I. & Kohl, P. Fibroblast network in rabbit sinoatrial node: structural and functional identification of homogeneous and heterogeneous cell coupling. Circ. Res. 94, 828–835 (2004).

    CAS  Article  Google Scholar 

  11. Camelliti, P., Devlin, G.P., Matthews, K.G., Kohl, P. & Green, C.R. Spatially and temporally distinct expression of fibroblast connexins after sheep ventricular infarction. Cardiovasc. Res. 62, 415–425 (2004).

    CAS  Article  Google Scholar 

  12. Komuro, I. & Yazaki, Y. Control of cardiac gene expression by mechanical stress. Annu. Rev. Physiol. 55, 55–75 (1993).

    CAS  Article  Google Scholar 

  13. Yamazaki, T., Komuro, I. & Yazaki, Y. Molecular mechanism of cardiac cellular hypertrophy by mechanical stress. J. Mol. Cell. Cardiol. 27, 133–140 (1995).

    CAS  Article  Google Scholar 

  14. Zhuang, J., Yamada, K.A., Saffitz, J.E. & Kleber, A.G. Pulsatile stretch remodels cell-to-cell communication in cultured myocytes. Circ. Res. 87, 316–322 (2000).

    CAS  Article  Google Scholar 

  15. Ruwhof, C. & van der Laarse, A. Mechanical stress-induced cardiac hypertrophy: mechanisms and signal transduction pathways. Cardiovasc. Res. 47, 23–37 (2000).

    CAS  Article  Google Scholar 

  16. Kohl, P., Hunter, P. & Noble, D. Stretch-induced changes in heart rate and rhythm: clinical observations, experiments and mathematical models. Prog. Biophys. Mol. Biol. 71, 91–138 (1999).

    CAS  Article  Google Scholar 

  17. Clark, P., Connolly, P., Curtis, A.S., Dow, J.A. & Wilkinson, C.D. Cell guidance by ultrafine topography in vitro. J. Cell Sci. 99, 73–77 (1991).

    PubMed  Google Scholar 

  18. Motlagh, D., Hartman, T.J., Desai, T.A. & Russell, B. Microfabricated grooves recapitulate neonatal myocyte connexin 43 and N-cadherin expression and localization. J. Biomed. Mater. Res. A 67, 148–157 (2003).

    Article  Google Scholar 

  19. Motlagh, D., Senyo, S.E., Desai, T.A. & Russell, B. Microtextured substrata alter gene expression, protein localization and the shape of cardiac myocytes. Biomaterials 24, 2463–2476 (2003).

    CAS  Article  Google Scholar 

  20. Yamazaki, T. et al. Role of ion channels and exchangers in mechanical stretch-induced cardiomyocyte hypertrophy. Circ. Res. 82, 430–437 (1998).

    CAS  Article  Google Scholar 

  21. Park, J.S. et al. Differential effects of equiaxial and uniaxial strain on mesenchymal stem cells. Biotechnol. Bioeng. 88, 359–368 (2004).

    CAS  Article  Google Scholar 

  22. Jo, S. & Park, K. Surface modification using silanated poly(ethylene glycol)s. Biomaterials 21, 605–616 (2000).

    CAS  Article  Google Scholar 

  23. Belus, A. & White, E. Streptomycin and intracellular calcium modulate the response of single guinea-pig ventricular myocytes to axial stretch. J. Physiol. (Lond.) 546, 501–509 (2003).

    CAS  Article  Google Scholar 

  24. Lee, A.A. et al. An equibiaxial strain system for cultured cells. Am. J. Physiol. 271, C1400–C1408 (1996).

    CAS  Article  Google Scholar 

  25. Folch, A., Ayon, A., Hurtado, O., Schmidt, M.A. & Toner, M. Molding of deep polydimethylsiloxane microstructures for microfluidics and biological applications. J. Biomech. Eng. 121, 28–34 (1999).

    CAS  Article  Google Scholar 

  26. Bhatia, S.N. in Methods of Tissue Engineering (eds. A. Atala & R.P. Lanza) 121–129 (Acadamic Press, San Diego, 2002).

    Google Scholar 

  27. Lee, A.A., Delhaas, T., McCulloch, A.D. & Villarreal, F.J. Differential responses of adult cardiac fibroblasts to in vitro biaxial strain patterns. J. Mol. Cell. Cardiol. 31, 1833–1843 (1999).

    CAS  Article  Google Scholar 

  28. Gudi, S.R., Lee, A.A., Clark, C.B. & Frangos, J.A. Equibiaxial strain and strain rate stimulate early activation of G proteins in cardiac fibroblasts. Am. J. Physiol. 274, C1424–C1428 (1998).

    CAS  Article  Google Scholar 

  29. Sotoudeh, M. et al. Induction of apoptosis in vascular smooth muscle cells by mechanical stretch. Am. J. Physiol. Heart Circ. Physiol. 282, H1709–H1716 (2002).

    CAS  Article  Google Scholar 

  30. Kaunas, R., Nguyen, P., Usami, S. & Chien, S. Cooperative effects of Rho and mechanical stretch on stress fiber organization. Proc. Natl. Acad. Sci. USA 102, 15895–15900 (2005).

    CAS  Article  Google Scholar 

  31. Sotoudeh, M., Jalali, S., Usami, S., Shyy, J.Y. & Chien, S. A strain device imposing dynamic and uniform equi-biaxial strain to cultured cells. Ann. Biomed. Eng. 26, 181–189 (1998).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank the following collaborators who contributed to the development of these protocols: S. Bhatia (Massachusetts Institute of Technology, Boston, Massachusetts), C. Flaim (University of California, San Diego, La Jolla, California), S. Gopalan and T. Borg (University of South Carolina, Columbia, South Carolina). Supported by the National Heart Lung and Blood Institute (R21 HL072160 and HL46345 to A.D.M.) and the UK Biotechnology and Biological Sciences Research Council (18561 to P.K.); P.C. is a Junior Research Fellow at Christ Church, Oxford; and P.K. is a British Heart Foundation Research Fellow.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Peter Kohl.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Camelliti, P., Gallagher, J., Kohl, P. et al. Micropatterned cell cultures on elastic membranes as an in vitro model of myocardium. Nat Protoc 1, 1379–1391 (2006). https://doi.org/10.1038/nprot.2006.203

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2006.203

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing