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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here we present a protocol for training a cell population using electrical and mechanical stimuli emulating cardiac physiology. This electromechanical stimulation enhances the cardiomyogenic potential of the treated cells and is a promising strategy for further cell therapy, disease modeling, and drug screening.

Abstract

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated researchers in the stem cell and bioengineering fields to develop in vitro high-fidelity human myocardium for both basic research and clinical applications. However, the immature phenotype of cardiac cells is a limitation on obtaining tissues that functionally mimic the adult myocardium, which is mainly characterized by mechanical and electrical signals. Thus, the purpose of this protocol is to prepare and mature the target cell population through electromechanical stimulation, recapitulating physiological parameters. Cardiac tissue engineering is evolving toward more biological approaches, and strategies based on biophysical stimuli, thus, are gaining momentum. The device developed for this purpose is unique and allows individual or simultaneous electrical and mechanical stimulation, carefully characterized and validated. In addition, although the methodology has been optimized for this stimulator and a specific cell population, it can easily be adapted to other devices and cell lines. The results here offer evidence of the increased cardiac commitment of the cell population after electromechanical stimulation. Electromechanically stimulated cells show an increased expression of main cardiac markers, including early, structural, and calcium-regulating genes. This cell conditioning could be useful for further regenerative cell therapy, disease modeling, and high-throughput drug screening.

Introduction

Heart function is based on the coupling of electrical excitation and mechanical contraction. Briefly, cardiomyocyte intercellular junctions permit electrical signal propagation to produce almost synchronous contractions of the heart that pump blood systemically and through the pulmonary system. Cardiac cells, thus, undergo both electrical and mechanical forces that regulate gene expression and cellular function. Accordingly, many groups have attempted to develop culture platforms that mimic the cardiac physiological environment to understand the role of mechanical and electrical stimulation on cardiac development, function, and maturation. In vitro electrical....

Protocol

This study uses human cardiac ATDPCs from patient samples. Their use has been approved by the local ethics committee, and all patients gave informed consent. The study protocol conforms to the principles outlined in the Declaration of Helsinki.

1. Preparations

  1. Autoclave two tweezers, 12 platinum PTFE electrodes for electrical stimulation, and some paper towels, at 121 °C for 20 min.
  2. Sterilize 12 PDMS custom-made constructs (Figure 1

Representative Results

Figure 2 represents the general schema followed for the cell stimulation. Briefly, cells were seeded on the PDMS construct and subjected to electromechanical stimulation, with a media change performed twice a week. Nonstimulated cells were used as a control for the electromechanical conditioning. Additionally, we added an extra control to the experiment, and subcutaneous ATDPCs were used as a control for cardiac ATDPCs. Subcutaneous ATDPCs are obtained from s.......

Discussion

Electromechanical stimulation appears to be a safe alternative for preparing cells for a hostile cardiac environment and enhancing their cardiac commitment. Here, a protocol described for cardiac progenitor cells increased the expression of main cardiac markers and was reported to be beneficial for their next implantation on infarcted murine myocardium30. In general, electromechanically stimulated cardiac ATDPCs increased the expression of genes related to early, structural, and calcium regulation.......

Acknowledgements

The authors want to thank the members of the ICREC Research Program (IGTP, Badalona) and the Electronic and Biomedical Instrumentation Group (UPC, Barcelona), especially Prof. J. Rosell-Ferrer. In addition, the authors acknowledge STEM CELLS Translational Medicine journal and AlphaMed Press for permitting the adaptation of previously published figures (Llucià-Valldeperas, et al.30). The development of this prototype and the design of the protocol were supported by Ministerio de Educación y Ciencia (SAF 2008-05144), Ministerio de Economía y Competitividad (SAF 2014-59892), the European Commission 7th Framework Programme (....

Materials

NameCompanyCatalog NumberComments
Stimulator
nickel plated neodymium magnetsSupermagneteQ-10-10-05-N
nickel-plated neodymium magnetsSupermagneteQ-06-04-02-HN
polydimethylsiloxane (PDMS) SYLGAR 184 Silicone Elastomer KitDow Corning Corp184
ruled diffraction grating (1250 grooves/mm)Newport05RG150-1250-2
Motor controllerFaulhaberMCLM-3006-S
LabviewNational Instruments
Cell culture
phosphate-buffered saline (PBS)Gibco70013-065
0.05% trypsin-EDTAGibco25300-120
35 mm cell culture dishBD Falcon45353001
fetal bovine serum (FBS)Gibco10270-106
L-Glutamine 200 mM, 100xGibco25030-024
Penicilina/Streptomicine, 10.000 U/mLGibco15140-122
Minimum essential medium eagle (alfa-MEM)SigmaM4526-24x500ML
Protein & RNA analyses
protease inhibitor cocktailSigmaP8340
QIAzol Lysis ReagentQiagen79306
AllPrep RNA/Protein KitQiagen50980404
Rneasy mini kitQiagen74104
iTaq Universal Probes One-Step KitBio-Rad Laboratories172-5140
Random hexamersQiagen79236
TaqMan PreAmp MasterMix 2XApplied Biosystems4391128
TaqMan Universal PCR MasterMixApplied Biosystems4324018
Immunostaining
10% formalinSigmaHT-501128-4L
horse serumSigmaH1138
Triton X-100SigmaX100-500ML
Bovine Serum Albumina (BSA)SigmaA7906-100G
PARAFILMSigmaP6543
4',6-diamidino-2-phenylindole (DAPI)SigmaD9542
Phalloidin Alexa 568InvitrogenA12380
sodium azideSigmaS8032-100g
Hoechst 33342Sigma14533
Connexin-43 rabbit primary antibodySigmaC6219 lot#061M4823
sarcomeric α-actinin mouse primary antibodySigmaA7811 lot#080M4864
GATA-4 goat primary antibodyR&DAF2606 VAZ0515101
MEF2 rabbit primary antibodySanta Cruzsc-313 lot#E0611
SERCA2 goat primary antibodySanta Cruzsc-8095 lot#D2709
Cy3 secondary antibodyJackson ImmunoResearch711-165-152
Cy3 secondary antibodyJackson ImmunoResearch715-165-151
Cy3 secondary antibodyJackson ImmunoResearch712-165-150
Cy2 secondary antibodyJackson ImmunoResearch715-225-150
Cy2 secondary antibodyJackson ImmunoResearch711-225-152
Cy2 secondary antibodyJackson ImmunoResearch705-225-147

References

  1. McDonough, P. M., Glembotski, C. C. Induction of atrial natriuretic factor and myosin light chain-2 gene expression in cultured ventricular myocytes by electrical stimulation of contraction. Journal of Biological Chemistry. 267, 11665-11668 (1992).
  2. Tandon, N., et al.

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