Electric field stimulation integrated into perfusion bioreactor for cardiac tissue engineering.

We describe herein the features of a novel cultivation system, combining electrical stimulation with medium perfusion for producing thick, functional cardiac patches. A custom-made electrical stimulator was integrated via inserting two carbon rod electrodes into a perfusion bioreactor, housing multiple neonatal Sprague-Dawley rat cardiac cell constructs between two 96% open-pore-area fixing nets. The stimulator produced adjustable stimulation waveform (i.e., duty cycle, number of stimulating channels, maximum stimulation amplitude, etc.), specially designed for cardiac cell stimulation. The cell constructs were subjected to a homogenous fluid flow regime and electrical stimulation under conditions optimal for cell excitation. The stimulation threshold in the bioreactor was set by first determining its value in a Petri dish under a microscope, and then matching the current density in the two cultivation systems by constructing electric field models. The models were built by Comsol Multiphysics software using the exact three-dimensional geometry of the two cultivation systems. These models illustrate, for the first time, the local electric conditions required for cardiomyocyte field excitation and they confirmed the uniformity of the electrical field around the cell constructs. Bioreactor cultivation for only 4 days under perfusion and continuous electrical stimulus (74.4 mA/cm², 2 ms, bipolar, 1 Hz) promoted cell elongation and striation in the cell constructs and enhanced the expression level of Connexin-43, the gap junction protein responsible for cell-cell coupling. These results thus confirm the validity of the electrical field model in predicting the optimal electrical stimulation in a rather complex cultivation system, a perfusion bioreactor.

[1]  Milica Radisic,et al.  Electrical stimulation systems for cardiac tissue engineering , 2009, Nature Protocols.

[2]  Xin Zhang,et al.  Real-time monitoring primary cardiomyocyte adhesion based on electrochemical impedance spectroscopy and electrical cell-substrate impedance sensing. , 2008, Analytical chemistry.

[3]  Milica Radisic,et al.  Functional assembly of engineered myocardium by electrical stimulation of cardiac myocytes cultured on scaffolds , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[4]  M. Yano,et al.  Rapid electrical stimulation of contraction modulates gap junction protein in neonatal rat cultured cardiomyocytes: involvement of mitogen-activated protein kinases and effects of angiotensin II-receptor antagonist. , 2004, Journal of the American College of Cardiology.

[5]  L. Shapiro,et al.  Novel alginate sponges for cell culture and transplantation. , 1997, Biomaterials.

[6]  C. Glembotski,et al.  Induction of atrial natriuretic factor and myosin light chain-2 gene expression in cultured ventricular myocytes by electrical stimulation of contraction. , 1992, The Journal of biological chemistry.

[7]  Milica Radisic,et al.  Cardiac tissue engineering using perfusion bioreactor systems , 2008, Nature Protocols.

[8]  J. Leor,et al.  Autospecies and post-myocardial infarction sera enhance the viability, proliferation, and maturation of 3D cardiac cell culture. , 2006, Tissue engineering.

[9]  Smadar Cohen,et al.  Optimization of cardiac cell seeding and distribution in 3D porous alginate scaffolds. , 2002, Biotechnology and bioengineering.

[10]  Electrical stimulation of cardiac myocytes , 1995, Annals of Biomedical Engineering.

[11]  Tal Dvir,et al.  Activation of the ERK1/2 cascade via pulsatile interstitial fluid flow promotes cardiac tissue assembly. , 2007, Tissue engineering.

[12]  J. Leor,et al.  Bioengineered Cardiac Grafts: A New Approach to Repair the Infarcted Myocardium? , 2000, Circulation.

[13]  Tal Dvir,et al.  Prevascularization of cardiac patch on the omentum improves its therapeutic outcome , 2009, Proceedings of the National Academy of Sciences.

[14]  Tal Dvir,et al.  A novel perfusion bioreactor providing a homogenous milieu for tissue regeneration. , 2006, Tissue engineering.

[15]  Milica Radisic,et al.  Medium perfusion enables engineering of compact and contractile cardiac tissue. , 2004, American journal of physiology. Heart and circulatory physiology.

[16]  L Tung,et al.  Analysis of electric field stimulation of single cardiac muscle cells. , 1992, Biophysical journal.

[17]  C. Cannizzaro,et al.  Characterization of Electrical Stimulation Electrodes for Cardiac Tissue Engineering , 2006, 2006 International Conference of the IEEE Engineering in Medicine and Biology Society.

[18]  Andreas Hess,et al.  Engineered heart tissue grafts improve systolic and diastolic function in infarcted rat hearts , 2006, Nature Medicine.

[19]  Hermann A. Haus,et al.  Electromagnetic Fields And Energy , 1989 .

[20]  D. Atsma,et al.  Forced Alignment of Mesenchymal Stem Cells Undergoing Cardiomyogenic Differentiation Affects Functional Integration With Cardiomyocyte Cultures , 2008, Circulation research.

[21]  Smadar Cohen,et al.  Perfusion cell seeding and cultivation induce the assembly of thick and functional hepatocellular tissue-like construct. , 2009, Tissue engineering. Part A.