Skip to main content
SpringerLink
Log in

A Method to Replicate the Microstructure of Heart Tissue In Vitro Using DTMRI-Based Cell Micropatterning

  • Published:
Annals of Biomedical Engineering Aims and scope Submit manuscript

Abstract

A novel cell culture methodology is described in which diffusion tensor magnetic resonance imaging (DTMRI) and cell micropatterning are combined to fabricate cell monolayers that replicate realistic cross-sectional tissue structure. As a proof-of-principle, neonatal rat ventricular myocyte (NRVM) monolayers were cultured to replicate the tissue microstructure of murine ventricular cross-sections. Specifically, DTMRI-measured in-plane cardiac fiber directions were converted into soft-lithography photomasks. Silicone stamps fabricated from the photomasks deposit fibronectin patterns to guide local cellular alignment. Fibronectin patterns consisted of a matrix of 190 μm2 subregions, each comprised of parallel lines 11–20 μm-wide, spaced 2–8.5 μm apart, and angled to match local DTMRI-measured fiber directions. Within 6 days of culture, NRVMs established confluent, electrically coupled monolayers, and for 18 μm-wide, 5 μm-spaced lines, directions of cell alignment in subregions microscopically replicated DTMRI-measurements with a local error of 7.2 ± 4.1°. By adjusting fibronectin line widths and spacings, cell elongation, gap junctional membrane distribution, and local cellular disarray were altered without changing the dominant directions of cell alignment in individual subregions. Changes in the anisotropy of electrical propagation were assessed by optically mapping membrane potentials. This novel methodology is expected to enable systematic studies of intramural structure–function relationships in both healthy and structurally remodeled hearts.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

References

  1. Badie, N., and N. Bursac. Micropatterned heart slice cultures for studies of intramural cardiac electrophysiology. Paper presented at American Heart Association: Scientific Sessions, Chicago, IL, Nov 15, 2006.

  2. Badie, N., and N. Bursac. Micropatterned ventricular slice: role of realistic tissue microstructure in impulse conduction. Paper presented at American Heart Association: Scientific Sessions, New Orleans, LA, 2008.

  3. Badie, N., and N. Bursac. Novel micropatterned cardiac cell cultures with realistic ventricular microstructure. Biophys. J. 96(9):3873–3885, 2009.

    Article  CAS  PubMed  Google Scholar 

  4. Bub, G., K. Tateno, A. Shrier, and L. Glass. Spontaneous initiation and termination of complex rhythms in cardiac cell culture. J. Cardiovasc. Electrophysiol. 14(10 Suppl):S229–S236, 2003.

    Article  PubMed  Google Scholar 

  5. Bursac, N., F. Aguel, and L. Tung. Multiarm spirals in a two-dimensional cardiac substrate. Proc. Natl. Acad. Sci. USA 101(43):15530–15534, 2004.

    Article  CAS  PubMed  Google Scholar 

  6. Bursac, N., K. K. Parker, S. Iravanian, and L. Tung. Cardiomyocyte cultures with controlled macroscopic anisotropy. A model for functional electrophysiological studies of cardiac muscle. Circ. Res. 91:e45–e54, 2002.

    Article  CAS  PubMed  Google Scholar 

  7. Bursac, N., and L. Tung. Acceleration of functional reentry by rapid pacing in anisotropic cardiac monolayers: formation of multi-wave functional reentries. Cardiovasc. Res. 69(2):381–390, 2006.

    Article  CAS  PubMed  Google Scholar 

  8. Chen, J., S. K. Song, W. Liu, M. McLean, J. S. Allen, J. Tan, S. A. Wickline, and X. Yu. Remodeling of cardiac fiber structure after infarction in rats quantified with diffusion tensor MRI. Am. J. Physiol. Heart Circ. Physiol. 285(3):H946–H954, 2003.

    CAS  PubMed  Google Scholar 

  9. de Diego, C., R. K. Pai, A. S. Dave, A. Lynch, M. Thu, F. Chen, L. H. Xie, J. N. Weiss, and M. Valderrabano. Spatially discordant alternans in cardiomyocyte monolayers. Am. J. Physiol. Heart Circ. Physiol. 294(3):H1417–H1425, 2008.

    Article  PubMed  Google Scholar 

  10. Entcheva, E., S. N. Lu, R. H. Troppman, V. Sharma, and L. Tung. Contact fluorescence imaging of reentry in monolayers of cultured neonatal rat ventricular myocytes. J. Cardiovasc. Electrophysiol. 11(6):665–676, 2000.

    Article  CAS  PubMed  Google Scholar 

  11. Fast, V. G., and R. E. Ideker. Simultaneous optical mapping of transmembrane potential and intracellular calcium in myocyte cultures. J. Cardiovasc. Electrophysiol. 11(5):547–556, 2000.

    Article  CAS  PubMed  Google Scholar 

  12. Fast, V. G., and A. G. Kleber. Cardiac tissue geometry as a determinant of unidirectional conduction block: assessment of microscopic excitation spread by optical mapping in patterned cell cultures and in a computer model. Cardiovasc. Res. 29(5):697–707, 1995.

    CAS  PubMed  Google Scholar 

  13. Gao, X., X. He, B. Luo, L. Peng, J. Lin, and Z. Zuo. Angiotensin II increases collagen I expression via transforming growth factor-beta1 and extracellular signal-regulated kinase in cardiac fibroblasts. Eur. J. Pharmacol. 606(1–3):115–120, 2009.

    Article  CAS  PubMed  Google Scholar 

  14. Geerts, L., P. Bovendeerd, K. Nicolay, and T. Arts. Characterization of the normal cardiac myofiber field in goat measured with MR-diffusion tensor imaging. Am. J. Physiol. Heart Circ. Physiol. 283(1):H139–H145, 2002.

    CAS  PubMed  Google Scholar 

  15. Grinnell, F., H. Fukamizu, P. Pawelek, and S. Nakagawa. Collagen processing, crosslinking, and fibril bundle assembly in matrix produced by fibroblasts in long-term cultures supplemented with ascorbic acid. Exp. Cell Res. 181(2):483–491, 1989.

    Article  CAS  PubMed  Google Scholar 

  16. Helm, P., M. F. Beg, M. I. Miller, and R. L. Winslow. Measuring and mapping cardiac fiber and laminar architecture using diffusion tensor MR imaging. Ann. NY Acad. Sci. 1047:296–307, 2005.

    Article  PubMed  Google Scholar 

  17. Holmes, A. A., D. F. Scollan, and R. L. Winslow. Direct histological validation of diffusion tensor MRI in formaldehyde-fixed myocardium. Magn. Reson. Med. 44(1):157–161, 2000.

    Article  CAS  PubMed  Google Scholar 

  18. Hooks, D. A., M. L. Trew, B. J. Caldwell, G. B. Sands, I. J. Legrice, and B. H. Smaill. Laminar arrangement of ventricular myocytes influences electrical behavior of the heart. Circ. Res., 2007.

  19. Hsu, E. W., A. L. Muzikant, S. A. Matulevicius, R. C. Penland, and C. S. Henriquez. Magnetic resonance myocardial fiber-orientation mapping with direct histological correlation. Am. J. Physiol. 274(5 Pt 2):H1627–H1634, 1998.

    CAS  PubMed  Google Scholar 

  20. Iravanian, S., Y. Nabutovsky, C. R. Kong, S. Saha, N. Bursac, and L. Tung. Functional reentry in cultured monolayers of neonatal rat cardiac cells. Am. J. Physiol. Heart Circ. Physiol. 285(1):H449–H456, 2003.

    CAS  PubMed  Google Scholar 

  21. Jiang, Y., K. Pandya, O. Smithies, and E. W. Hsu. Three-dimensional diffusion tensor microscopy of fixed mouse hearts. Magn. Reson. Med. 52(3):453–460, 2004.

    Article  PubMed  Google Scholar 

  22. Karlon, W. J., J. W. Covell, A. D. McCulloch, J. J. Hunter, and J. H. Omens. Automated measurement of myofiber disarray in transgenic mice with ventricular expression of ras. Anat. Rec. 252(4):612–625, 1998.

    Article  CAS  PubMed  Google Scholar 

  23. Kawara, T., R. Derksen, J. de Groot, R. Coronel, S. Tasseron, A. Linnenbank, R. Hauer, H. Kirkels, M. Janse, and J. de Bakker. Activation delay after premature stimulation in chronically diseased human myocardium relates to the architecture of interstitial fibrosis. Circulation 104:3069–3075, 2001.

    Article  CAS  PubMed  Google Scholar 

  24. Klinger, R., and N. Bursac. Cardiac cell therapy in vitro: reproducible assays for comparing the efficacy of different donor cells. IEEE Eng. Med. Biol. Mag. 27(1):72–80, 2008.

    Article  PubMed  Google Scholar 

  25. Koura, T., M. Hara, S. Takeuchi, K. Ota, Y. Okada, S. Miyoshi, A. Watanabe, K. Shiraiwa, H. Mitamura, I. Kodama, and S. Ogawa. Anisotropic conduction properties in canine atria analyzed by high-resolution optical mapping: preferential direction of conduction block changes from longitudinal to transverse with increasing age. Circulation 105(17):2092–2098, 2002.

    Article  PubMed  Google Scholar 

  26. Kucera, J. P., A. G. Kleber, and S. Rohr. Slow conduction in cardiac tissue, II: effects of branching tissue geometry. Circ. Res. 83(8):795–805, 1998.

    CAS  PubMed  Google Scholar 

  27. LeGrice, I. J., B. H. Smaill, L. Z. Chai, S. G. Edgar, J. B. Gavin, and P. J. Hunter. Laminar structure of the heart: ventricular myocyte arrangement and connective tissue architecture in the dog. Am. J. Physiol. 269(2 Pt 2):H571–H582, 1995.

    CAS  PubMed  Google Scholar 

  28. Lijnen, P. J., V. V. Petrov, and R. H. Fagard. Induction of cardiac fibrosis by transforming growth factor-beta(1). Mol. Genet. Metab. 71(1–2):418–435, 2000.

    Article  CAS  PubMed  Google Scholar 

  29. Lim, Z. Y., B. Maskara, F. Aguel, R. Emokpae, Jr., and L. Tung. Spiral wave attachment to millimeter-sized obstacles. Circulation 114(20):2113–2121, 2006.

    Article  PubMed  Google Scholar 

  30. McSpadden, L. C., R. D. Kirkton, and N. Bursac. Electrotonic loading of anisotropic cardiac monolayers by unexcitable cells depends on connexin type and expression level. Am. J. Physiol. 2009.

  31. Munoz, V., K. R. Grzeda, T. Desplantez, S. V. Pandit, S. Mironov, S. M. Taffet, S. Rohr, A. G. Kleber, and J. Jalife. Adenoviral expression of IKs contributes to wavebreak and fibrillatory conduction in neonatal rat ventricular cardiomyocyte monolayers. Circ. Res. 101(5):475–483, 2007.

    Article  CAS  PubMed  Google Scholar 

  32. Pedrotty, D. M., R. Y. Klinger, N. Badie, S. Hinds, A. Kardashian, and N. Bursac. Structural coupling of cardiomyocytes and noncardiomyocytes: quantitative comparisons using a novel micropatterned cell pair assay. Am. J. Physiol. Heart Circ. Physiol. 295(1):H390–H400, 2008.

    Article  CAS  PubMed  Google Scholar 

  33. Rohmer, D., A. Sitek, and G. T. Gullberg. Reconstruction and visualization of fiber and laminar structure in the normal human heart from ex vivo diffusion tensor magnetic resonance imaging (DTMRI) data. Invest. Radiol. 42(11):777–789, 2007.

    Article  PubMed  Google Scholar 

  34. Rohr, S., J. P. Kucera, and A. G. Kleber. Slow conduction in cardiac tissue, I: effects of a reduction of excitability versus a reduction of electrical coupling on microconduction. Circ. Res. 83(8):781–794, 1998.

    CAS  PubMed  Google Scholar 

  35. Rohr, S., and B. Salzberg. Characterization of impulse propagation at the microscopic level across geometrically defined expansions of excitable tissue: multiple site optical recording of transmembrane voltage (MSORTV) in patterned growth heart cell cultures. J. Gen. Physiol. 104:287–309, 1994.

    Article  CAS  PubMed  Google Scholar 

  36. Schmid, P., T. Jaermann, P. Boesiger, P. F. Niederer, P. P. Lunkenheimer, C. W. Cryer, and R. H. Anderson. Ventricular myocardial architecture as visualised in postmortem swine hearts using magnetic resonance diffusion tensor imaging. Eur. J. Cardiothorac. Surg. 27(3):468–472, 2005.

    Article  PubMed  Google Scholar 

  37. Scollan, D. F., A. Holmes, R. Winslow, and J. Forder. Histological validation of myocardial microstructure obtained from diffusion tensor magnetic resonance imaging. Am. J. Physiol. 275(6 Pt 2):H2308–H2318, 1998.

    CAS  PubMed  Google Scholar 

  38. Scollan, D. F., A. Holmes, J. Zhang, and R. L. Winslow. Reconstruction of cardiac ventricular geometry and fiber orientation using magnetic resonance imaging. Ann. Biomed. Eng. 28(8):934–944, 2000.

    Article  CAS  PubMed  Google Scholar 

  39. Spach, M. S., and J. P. Boineau. Microfibrosis produces electrical load variations due to loss of side-to-side cell connections: a major mechanism of structural heart disease arrhythmias. Pacing Clin. Electrophysiol. 20(2 Pt 2):397–413, 1997.

    Article  CAS  PubMed  Google Scholar 

  40. Spach, M. S., and P. C. Dolber. Relating extracellular potentials and their derivatives to anisotropic propagation at a microscopic level in human cardiac muscle. Evidence for electrical uncoupling of side-to-side fiber connections with increasing age. Circ. Res. 58(3):356–371, 1986.

    CAS  PubMed  Google Scholar 

  41. Valderrabano, M. Influence of anisotropic conduction properties in the propagation of the cardiac action potential. Prog. Biophys. Mol. Biol. 94(1–2):144–168, 2007.

    Article  CAS  PubMed  Google Scholar 

  42. Wu, M. T., W. Y. Tseng, M. Y. Su, C. P. Liu, K. R. Chiou, V. J. Wedeen, T. G. Reese, and C. F. Yang. Diffusion tensor magnetic resonance imaging mapping the fiber architecture remodeling in human myocardium after infarction: correlation with viability and wall motion. Circulation 114(10):1036–1045, 2006.

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

The authors would like to thank Dr. Yi Jiang and Dr. Edward Hsu for supplying the DTMRI data, as well as Weining Bian for her assistance with the optical profile acquisition of the template wafer. This work was supported by the American Heart Association Predoctoral Fellowship (No. 0715178U) to Nima Badie.

Author information

Authors and Affiliations

  1. Department of Biomedical Engineering, Duke University, 3000 Science Drive, 136 Hudson Hall, Durham, NC, 27708, USA

    Nima Badie, Lisa Satterwhite & Nenad Bursac

Authors
  1. Nima Badie
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lisa Satterwhite
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nenad Bursac
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Nenad Bursac.

Electronic supplementary material

Below is the link to the electronic supplementary material.

(DOC 463 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Badie, N., Satterwhite, L. & Bursac, N. A Method to Replicate the Microstructure of Heart Tissue In Vitro Using DTMRI-Based Cell Micropatterning. Ann Biomed Eng 37, 2510–2521 (2009). https://doi.org/10.1007/s10439-009-9815-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10439-009-9815-x

Keywords

Search

Navigation