Advertisement

Sacrificial layer technique for axial force post assay of immature cardiomyocytes

Abstract

Immature primary and stem cell-derived cardiomyocytes provide useful models for fundamental studies of heart development and cardiac disease, and offer potential for patient specific drug testing and differentiation protocols aimed at cardiac grafts. To assess their potential for augmenting heart function, and to gain insight into cardiac growth and disease, tissue engineers must quantify the contractile forces of these single cells. Currently, axial contractile forces of isolated adult heart cells can only be measured by two-point methods such as carbon fiber techniques, which cannot be applied to neonatal and stem cell-derived heart cells because they are more difficult to handle and lack a persistent shape. Here we present a novel axial technique for measuring the contractile forces of isolated immature cardiomyocytes. We overcome cell manipulation and patterning challenges by using a thermoresponsive sacrificial support layer in conjunction with arrays of widely separated elastomeric microposts. Our approach has the potential to be high-throughput, is functionally analogous to current gold-standard axial force assays for adult heart cells, and prescribes elongated cell shapes without protein patterning. Finally, we calibrate these force posts with piezoresistive cantilevers to dramatically reduce measurement error typical for soft polymer-based force assays. We report quantitative measurements of peak contractile forces up to 146 nN with post stiffness standard error (26 nN) far better than that based on geometry and stiffness estimates alone. The addition of sacrificial layers to future 2D and 3D cell culture platforms will enable improved cell placement and the complex suspension of cells across 3D constructs.

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.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

References

  1. O.J. Abilez, J. Wong, R. Prakash, K. Deisseroth, C.K. Zarins, E. Kuhl, Biophys. J. 101, 6 (2011)

  2. D. Armani, C. Liu, N. Aluru, Re-configurable fluid circuits by PDMS elastomer micromachining. Proc. IEEE MEMS. (1999)

  3. C. Bollensdorff, O. Lookin, P. Kohl, Pflüg. Arch. Eur. J. Phy. 462, 1 (2011)

  4. A.J. Brady, S.T. Tan, N.V. Ricchiuti, Nature 282, 5740 (1979)

  5. M.-A. Bray, S.P. Sheehy, K.K. Parker, Cell Motil. Cytoskeleton 65, 8 (2008)

  6. J.P. Butler, I.M. Tolić-Nørrelykke, B. Fabry, J.J. Fredberg, Am. J. Physiol. Cell Physiol. 282, 3 (2002)

  7. K. Campbell, Pflüg. Arch. Eur. J. Phy. 462, 1 (2011)

  8. O. Cazorla, A. Lacampagne, Pflüg. Arch. Eur. J. Phy. 462, 1 (2011)

  9. Q. Cheng, Z. Sun, G.A. Meininger, M. Almasri, Rev. Sci. Instrum. 81, 10 (2010)

  10. G. Constantinides, Z.I. Kalcioglu, M. McFarland, J.F. Smith, K.J. Van Vliet, J. Biomech. 41, 15 (2008)

  11. M. Curtis, B. Russell, Pflüg. Arch. Eur. J. Phy. 462, 1 (2011)

  12. M. Dembo, T. Oliver, A. Ishihara, K. Jacobson, Biophys. J. 70, 4 (1996)

  13. A.J. Engler, C. Carag-Krieger, C.P. Johnson, M. Raab, H.-Y. Tang, D.W. Speicher, J.W. Sanger, J.M. Sanger, D.E. Discher, J. Cell Sci. 121, 22 (2008)

  14. A. Fabiato, J. Gen. Physiol. 78, 5 (1981)

  15. A.W. Feinberg, A. Feigel, S.S. Shevkoplyas, S. Sheehy, G.M. Whitesides, K.K. Parker, Science 317, 5843 (2007)

  16. D. Fuard, T. Tzvetkova-Chevolleau, S. Decossas, P. Tracqui, P. Schiavone, Microelectron. Eng. 85(5), Äì6 (2008)

  17. M. Ghibaudo, J.-M. Di Meglio, P. Hersen, B. Ladoux, Lab Chip 11, 5 (2011)

  18. A. Grosberg, P.W. Alford, M.L. McCain, K.K. Parker, Lab Chip 11, 24 (2011)

  19. G. Iribe, M. Helmes, P. Kohl, Am. J. Physiol. Heart Circ. Physiol. 292, 3 (2007)

  20. J.P. Holman (McGraw-Hill College, 1988)

  21. J.G. Jacot, A.D. McCulloch, J.H. Omens, Biophys. J. 95, 7 (2008)

  22. J.G. Jacot, H. Kita-Matsuo, K.A. Wei, H.S. Vincent Chen, J.H. Omens, M. Mercola, A.D. McCulloch, Ann. N. Y. Acad. Sci. 1188, 1 (2010)

  23. A. Kajzar, C.M. Cesa, N. Kirchgeflner, B. Hoffmann, R. Merkel, Biophys. J. 94, 5 (2008)

  24. K. Kim, R.E. Taylor, J.Y. Sim, S.-J. Park, J. Norman, G. Fajardo, D. Bernstein, B.L. Pruitt, Micro Nano Lett. 6, 5 (2011)

  25. N.M.P. King, M. Methawasin, J. Nedrud, N. Harrell, C.S. Chung, M. Helmes, H. Granzier, J. Gen. Physiol. 137, 1 (2011)

  26. E. Kolossov, Z. Lu, I. Drobinskaya, N. Gassanov, Y. Duan, H. Sauer, O. Manzke, W. Bloch, H. Bohlen, J.r. Hescheler, B.K. Fleischmann. Faseb J. (2005)

  27. M.L. Lam, M. Bartoli, W.C. Claycomb, Mol. Cell. Biochem. 229, 1 (2002)

  28. J.Y. Le Guennec, N. Peineau, J.A. Argibay, K.G. Mongo, D. Garnier, J. Mol. Cell. Cardiol. 22, 10 (1990)

  29. W.R. Legant, A. Pathak, M.T. Yang, V.S. Deshpande, R.M. McMeeking, C.S. Chen, Proc. Natl. Acad. Sci. U. S. A. 106, 25 (2009)

  30. G. Lin, K.S.J. Pister, K.P. Roos, J. Electrochem. Soc. 142, 3 (1995)

  31. G. Lin, K.S.J. Pister, K.P. Roos, J. Microelectromech. Syst. 9, 1 (2000)

  32. Z. Liu, J.L. Tan, D.M. Cohen, M.T. Yang, N.J. Sniadecki, S.A. Ruiz, C.M. Nelson, C.S. Chen, Proc. Natl. Acad. Sci. U. S. A. 107, 22 (2010)

  33. J.M. Mann, R.H.W. Lam, S. Weng, Y. Sun, J. Fu, Lab Chip 12, 4 (2012)

  34. A. Mata, A. Fleischman, S. Roy, Biomed. Microdevices 7, 4 (2005)

  35. M. McCain, K. Parker, Pflüg. Arch. Eur. J. Phy. 462, 1 (2011)

  36. L. Miao, S. Jianren, S. Ying, B. Christopher, C. Quanfang, J. Micromech. Microeng. 19, 3 (2009)

  37. S. Nishimura, S.-i. Yasuda, M. Katoh, K.P. Yamada, H. Yamashita, Y. Saeki, K. Sunagawa, R. Nagai, T. Hisada, S. Sugiura, Am. J. Physiol. Heart Circ. Physiol. 287(1) (2004)

  38. T. Okano, N. Yamada, H. Sakai, Y. Sakurai, J. Biomed. Mater. Res. 27, 10 (1993)

  39. S.J. Park, M.B. Goodman, B.L. Pruitt, Proc. Natl. Acad. Sci. USA. (2007)

  40. J.R. Pratt, D.T. Smith, D.B. Newell, J.A. Kramar, E. Whetenton, J. Mater. Res. 19, (2004)

  41. A.G. Rodriguez, S.J. Han, M. Regnier, N.J. Sniadecki, Biophys. J. 101, 10 (2011)

  42. V.L. Roger, A.S. Go, D.M. Lloyd-Jones, R.J. Adams, J.D. Berry, T.M. Brown, M.R. Carnethon, S. Dai, G. de Simone, E.S. Ford, C.S. Fox, H.J. Fullerton, C. Gillespie, K.J. Greenlund, S.M. Hailpern, J.A. Heit, P.M. Ho, V.J. Howard, B.M. Kissela, S.J. Kittner, D.T. Lackland, J.H. Lichtman, L.D. Lisabeth, D.M. Makuc, G.M. Marcus, A. Marelli, D.B. Matchar, M.M. McDermott, J.B. Meigs, C.S. Moy, D. Mozaffarian, M.E. Mussolino, G. Nichol, N.P. Paynter, W.D. Rosamond, P.D. Sorlie, R.S. Stafford, T.N. Turan, M.B. Turner, N.D. Wong, J. Wylie-Rosett, Circulation 123, 4 (2011)

  43. S. Sivaramakrishnan, E. Ashley, L. Leinwand, J. Spudich, J. Cardiovasc. Transl. Res. 2, 4 (2009)

  44. N.J. Sniadecki, C.S. Chen, in Microfabricated Silicone Elastomeric Post Arrays for Measuring Traction Forces of Adherent Cells. Methods in Cell Biology, ed. by W. Yu-li and E.D. Dennis (Academic Press, 2007), pp. 313–328

  45. J.L. Tan, J. Tien, D.M. Pirone, D.S. Gray, K. Bhadriraju, C.S. Chen, Proc. Natl. Acad. Sci. U. S. A. 100, 4 (2003)

  46. X. Tang, P. Bajaj, R. Bashir, T.A. Saif, Soft Matter 7, (2011)

  47. R.E. Taylor, K. Kim, B.L. Pruitt, Proc Solid State Sens, Actuators, Microsystems Workshop. (Hilton Head, SC., 2010)

  48. R.E. Taylor, A. Ribeiro, G. Fajardo, H. Razavi, D. Bernstein, B.L. Pruitt, Proc MicroTAS. (Seattle, WA., 2011)

  49. M. Théry, J. Cell Sci. 123, 24 (2010)

  50. M. Théry, A. Pépin, E. Dressaire, Y. Chen, M. Bornens, Cell Motil. Cytoskeleton 63, 6 (2006)

  51. E. White, Pflüg. Arch. Eur. J. Phy. 462, 1 (2011)

  52. J. Xi, J.J. Schmidt, C.D. Montemagno, Nat. Mater. 4, 2 (2005)

  53. W. Xu, N. Chahine, T. Sulchek, Langmuir 27, 13 (2011)

  54. L. Yang, M.H. Soonpaa, E.D. Adler, T.K. Roepke, S.J. Kattman, M. Kennedy, E. Henckaerts, K. Bonham, G.W. Abbott, R.M. Linden, L.J. Field, G.M. Keller, Nature 453, 7194 (2008)

  55. S.-I. Yasuda, S. Sugiura, N. Kobayakawa, H. Fujita, H. Yamashita, K. Katoh, Y. Saeki, H. Kaneko, Y. Suda, R. Nagai, H. Sugi, Am. J. Physiol. Heart. Circ. 281, 3 (2001)

  56. M. Yazawa, B. Hsueh, X. Jia, A.M. Pasca, J.A. Bernstein, J. Hallmayer, R.E. Dolmetsch, Nature 471, 7337 (2011)

  57. Y. Zhao, X. Zhang, Appl. Phys. Lett. 87, 14 (2005)

  58. Y. Zhao, X. Zhang, Sensor. Actuator. A 125, 2 (2006)

  59. Y. Zhao, C.C. Lim, D.B. Sawyer, R. Liao, X. Zhang, Cell Motil. Cytoskeleton 64, 9 (2007)

Download references

Acknowledgments

The authors acknowledge support from the National Science Foundation (EFRI-CBE 073555, EFRI-MIKS 1136790, ECS-0449400), the National Institutes of Health (R33 HL089027, RC1 HL099117, RC1AG036142, R01 EB006745, R01 HL061535, and DP2OD004437), the California Institute for Regenerative Medicine (CIRM RC1-00151-1, CIRM RB3-05129, and CIRM TR3-05556), Stanford Center for Integrated Systems, Stanford University (Bio-X Interdisciplinary Initiatives Award, Bio-X Graduate Fellowships, Ilju foundation scholarship, Stanford Graduate Fellowship and a Stanford DARE Doctoral Fellowship), and Natural Sciences and Engineering Research Council of Canada Postdoctoral Fellowship.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Author information

Correspondence to Beth L. Pruitt.

Electronic supplementary material

Online Resources 1 and 2 are movies showing a murine neonatal cardiomyocyte and a human embryonic stem cell-derived cardiomyocyte suspended across posts and beating while being paced at 1 Hz.

Beating primary neonatal rat cardiomyocyte on force sensor (MPG 1,062 kb)

Beating hESC-derived cardiomyocyte on force sensor (MPG 460 kb)

ESM 1

Beating primary neonatal rat cardiomyocyte on force sensor (MPG 1,062 kb)

ESM 2

Beating hESC-derived cardiomyocyte on force sensor (MPG 460 kb)

Appendix

Appendix

The natural frequency of the cantilever is related to its stiffness and mass using the classical equation for a translational mechanical harmonic oscillator, where k c is the bending stiffness of the cantilever for a point load applied to its tip and m eff is the mass of the cantilever:

$$ {\omega_n} = \frac{1}{{2\pi }}\sqrt {{\frac{{{k_c}}}{{{m_{{eff}}}}}}} $$
(2)

We substitute cantilever mass with density times volume. In the following equation, ρ is the density of silicon and L, W, and T are the cantilever length, width, and thickness, respectively:

$$ {\omega_n} = \frac{1}{{2\pi }}\sqrt {{\frac{{{k_c}}}{{0.24\rho LWT}}}} $$
(3)

The effective mass is only 0.24 times the overall mass of the cantilever beam due to the mode shape of the first eigenmode. If stiffness is calibrated from a higher-order eigenmode, then the effective mass should be calculated accordingly. Laser Doppler vibrometry was used to determine the natural frequency, ω n , 2.37 kHz, and when we rearrange the previous equation we obtain the following expression for kc:

$$ {k_c} = 0.24\rho LWT{\left( {2\pi {\omega_n}} \right)^2} $$
(4)

The force F applied by the cantilever is calculated from the amplifier output-referred differential voltage, ΔV, and the cantilever force sensitivity, S f :

$$ {F_c} = \frac{{\varDelta V}}{{G{S_f}}} $$
(5)

where G is the overall gain of the signal-conditioning circuit. The force sensitivity scales linearly with the Wheatstone bridge bias voltage and depends on the cantilever design parameters. The force sensitivity is calculated from the product of the cantilever stiffness and displacement sensitivity.

The micropost stiffness, k p , is the quotient of the applied force, F, and the optical displacement of the micropost. Since we measured micropost displacement in pixels, we multiply our resolution [microns/pixel], R, and our pixel deflection, Δx, to obtain the deflection in microns:

$$ {k_p} = \frac{{\varDelta V}}{{G{S_f}R\varDelta x}} $$
(6)

Finally, we must express force sensitivity, S f , in terms of the displacement sensitivity, S d , since we do not measure force sensitivity directly during cantilever calibration. The displacement sensitivity is determined by driving the cantilever on resonance and measuring the ratio of the circuit voltage output to the tip deflection, again using laser Doppler vibrometry (Park et al. 2007). The micropost stiffness can finally be written in terms of experimentally measured parameters as follows:

$$ {k_p} = \frac{{{k_c}\varDelta V}}{{{V_b}G{S_d}R\varDelta x}} $$
(7)
$$ {k_p} = \frac{{0.24\rho LWT{{\left( {2\pi {\omega_n}} \right)}^2}\varDelta V}}{{{V_b}G{S_d}R\varDelta x}} $$
(8)

As we have detailed previously (Kim et al. 2011), uncertainty in the cantilever stiffness contributes to the accuracy or systematic error in our reported forces and is calculated using a root mean sum of squares analysis of the measurement uncertainties (Holman 1988). Lithographic variations and uncertainty in displacement sensitivity contribute to this device uncertainty (Park et al. 2007). We convolve this error with the error in optical measurements, which is limited by pixel resolution.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Taylor, R.E., Kim, K., Sun, N. et al. Sacrificial layer technique for axial force post assay of immature cardiomyocytes. Biomed Microdevices 15, 171–181 (2013) doi:10.1007/s10544-012-9710-3

Download citation

Keywords

  • Force posts
  • Thermoresponsive
  • Sacrificial layer
  • Cardiomyocytes
  • PDMS
  • Stem cells