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Bioengineering

Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing

Published: March 19th, 2013

DOI:

10.3791/50288

1Cardiovascular Research Center, Massachusetts General Hospital and Harvard Medical School, 2Harvard Stem Cell Institute

The generation of aligned myocardial tissue is a key requirement for adapting the recent advances in stem cell biology to clinically useful purposes. Herein we describe a microcontact printing approach for the precise control of cell shape and function. Using highly purified populations of embryonic stem cell derived cardiac progenitors, we then generate anisotropic functional myocardial tissue.

Advanced heart failure represents a major unmet clinical challenge, arising from the loss of viable and/or fully functional cardiac muscle cells. Despite optimum drug therapy, heart failure represents a leading cause of mortality and morbidity in the developed world. A major challenge in drug development is the identification of cellular assays that accurately recapitulate normal and diseased human myocardial physiology in vitro. Likewise, the major challenges in regenerative cardiac biology revolve around the identification and isolation of patient-specific cardiac progenitors in clinically relevant quantities. These cells have to then be assembled into functional tissue that resembles the native heart tissue architecture. Microcontact printing allows for the creation of precise micropatterned protein shapes that resemble structural organization of the heart, thus providing geometric cues to control cell adhesion spatially. Herein we describe our approach for the isolation of highly purified myocardial cells from pluripotent stem cells differentiating in vitro, the generation of cell growth surfaces micropatterned with extracellular matrix proteins, and the assembly of the stem cell-derived cardiac muscle cells into anisotropic myocardial tissue.

Despite recent advances in medical therapy, advanced heart failure remains a leading cause of mortality and morbidity in the developed world. The clinical syndrome arises from a loss of functional myocardial tissue and subsequently an inability of the failing heart to meet the metabolic demands of affected individuals. Since the heart has a limited regenerative capacity, autologous heart transplantation is the only current clinically accepted therapy directly aimed at replenishing lost functional heart tissue. Significant drawbacks of heart transplantation, including a limited number of donor hearts and the need for long-term immunosuppressive therapy, preclude the wi....

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The protocol to generate aligned functional myocardial tissue can be divided into three major parts. The fabrication of the micropatterned master using soft lithography techniques is not considered part of the following protocol but can be made based on established method6.

1. Microcontact Printing of Fibronectin onto PDMS Substrates

  1. Mix Sylgard 184 (Dow Corning) PDMS elastomer at a 10:1 base to curing agent ratio and degas the assembly using a desiccator to eliminate.......

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FACS-purification of in vitro differentiated ES cells revealed four distinct populations of progenitors (Figure 1). The presence of micropatterned Fibronectin was confirmed by immunofluorescence microscopy that displayed a complete transfer of continuous Fibronectin lines (Figure 2). Plating of FACS-isolated progenitors onto Fibronectin micropatterns resulted in alignment of the R+G+ and part of the R-G+ populations. Although Pl.......

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In this protocol, we presented a method to isolate purified populations of cardiogenic progenitors and to seed them on micropatterned Fibronectin substrates what allows them align and take a cardiac myocyte-like rod shape. Normal cellular organization is critical for normal tissue function8,10 , in particular for myocardial tissue. Cardiac myocytes have been shown to develop improved mechanical11,12 and electrophysiological properties11,13 when forming anisotropic cell arrays. Sinc.......

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This work was performed in part at the Center for Nanoscale Systems (CNS), a member of the National Nanotechnology Infrastructure Network (NNIN), which is supported by the National Science Foundation under NSF award no. ECS-0335765. CNS is part of Harvard University.

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Name Company Catalog Number Comments
Name of Material Company Catalogue Number Comments
L-Ascorbic Acid Sigma-Aldrich A4544 Prepare 0.1M solution in ddH2O
Desiccator, Vacuum 10 inch Nova-Tech International, Inc. 55206
4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI) Life Technologies D1306
Dulbecco's Modified Eagle Medium Thermo Scientific SH30022.01
DPBS Gibco 14190
FACSAria II Flow Cytometer BD Biosciences
Fetal Bovine Serum ES cell-grade Gemini Bio-Products 100-106
Fetal Bovine Serum Differentiation-grade Gemini Bio-Products 100-500
Fibronectin from bovine plasma Sigma-Aldrich F4759 Solubilize to 1 mg/ml in ddH2O
Fisherfinest Premium Cover Glass 22x22mm Fisher Scientific 12-548-B
Gelatin from porcine skin Sigma-Aldrich G1890 Prepare 0.1% solution in ddH2O
Headway Spin Coater Headway Research, Inc. PWM32-PS-CB15
Iscove's Modified Dulbecco's Medium Thermo Scientific SH30228.01
Leukemia Inhibitory Factor Self-prepared from LIF-secreting cell lines Prepare 500x stock solution
MEM Non-Essential Amino Acids Gibco 11140-050
2-Mercaptoethanol Sigma-Aldrich M6250
Mouse Embryonic Fibroblasts Harvested from day 12-15 mouse embryos
Penicillin-Streptomycin Gibco 15140-122
Pluronic F-127 Sigma-Aldrich P2443 Prepare 1% solution in ddH2O
Round-Bottom Tube with 35 μm cell strainer BD Biosciences 352235
SPI Plasma-Prep II Plasma Cleaner SPI Supplies 11005-AB
Sylgard 184 Silicone Elastomer Kit Dow Corning 184 SIL ELAST KIT 0.5KG
0.25% Trypsin-EDTA Gibco 25200-056
Vacuum Gauge SPI Supplies 11019-AB

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