We report a simple in vitro model of cardiac tissue that mimics three-dimensional (3-D) environment and mechanical load conditions and, as such, may serve as a convenient method to study stem cell engraftment or address developmental questions such as cytoskeleton or intercalated disk maturation. To create in vitro cardiac fibers we used Matrigel, a commercially available basement membrane preparation. A semisolid pillow from concentrated Matrigel was overlaid with a suspension of rat neonatal cardiomyocytes in a diluted Matrigel solution. This created an environment in which the multicellular fibers continuously contracted against a mechanical load. The described approach allows continuous structural and functional monitoring of 20–300-micron-thick cardiac fibers and provides easy access to epitopes for immunostaining purposes.

Introduction

Standard cell culture techniques are of limited value when it comes to issues of cell engraftment or structural maturation of cardiac muscle. This is because the formation of electromechanical junctions and the maturation of cardiomyocytes are highly dependent on the three-dimensional (3-D) environment, continuous mechanical load, and cellular interaction with the extracellular matrix (1,2,3,4,5). These factors are missing from conventional cardiomyocyte cultures, that is, when cells are plated on a two-dimensional (2-D) flat surface, whether plastic or glass. In contrast, here we describe an in vitro approach that produces a net of 3-D cardiac fibers contracting against a mechanical load. The model uses standard cell culture equipment and is accessible for physiological or epitope monitoring. Notably, in recent years, several novel strategies have been successful in developing tissue-engineered cardiac muscle constructs (6,7,8,9,10,11). However, all of these strategies require specialized equipment including custom-made stretch devices, synthesis of particular scaffolds, continuous external stimulation, bioreactors, and/or special perfusion systems. Therefore, use of these new techniques remains largely limited to researchers working in the field of tissue engineering. In contrast, we attempted to avoid any special devices and developed an in vitro model that is usable in any lab with standard cell culture equipment. As such, it may serve as a helpful new technique to study cell engraftment into a cardiac syncytium as well as to address developmental changes in cytoskeleton, myofibril, or intercalated disk structures.

Materials and Methods

Cell Culture

Cardiomyocytes were obtained from two-day-old Sprague-Dawley rats (Hilltop Lab Animals, Inc., Scottdale, PA, USA) using a standard enzymatic digestion procedure (12). They were plated on top of Matrigel pillows as detailed in the Results and Discussion section (Matrigel is from BD Biosciences, Franklin Lakes, NJ, USA). Samples were kept in standard culture conditions in Dulbecco-modified minimum essential medium supplemented with 5% fetal bovine serum, 10 U/mL penicillin, 10 µg/mL gentamycin, and 1 µg/mL streptomycin (all from Sigma-Aldrich, St. Louis, MO, USA). Matrigel is a basement membrane preparation extracted from the Engelbreth-Holm-Swarm mouse sarcoma, which self-polymerizes upon warming to room temperature. Its major components include laminin, type IV collagen, heparan sulfate proteoglycans, and growth factors. When reconstituted, it retains structural and functional characteristics resembling those of basement membranes in vivo (13). The Young's modulus of polymerized Matrigel was measured using a Tissue Elastometer Model 0502 (ARTANN Labs, West Trenton, NJ, USA).

Image Acquisition

The bright field images and videos of live unstained samples in culture media were taken using a Nikon CoolPix 4300 digital camera (Nikon, Tokyo, Japan) mounted to an upright WPI (World Precise Instruments, Sarasota, FL, USA) cell culture microscope ((Figure 1), (Figure 2)A, and Supplementary Movie S1–S3 available online at www.BioTechniques.com). For physiological and immunohisto-chemical assessment the specimens were examined using a Zeiss LSM510 confocal imaging system ((Figure 2)B and (Figure 3)). To visualize myocyte membranes and/or record cell electrical activity the samples were stained with the potentiometric probe RH237 (10 µmol/L for 5 min). To acquire calcium transients, samples were loaded for 1 h with 5 µmol/L Fluo-4AM (both RH237 and Fluo-4AM were purchased from Molecular Probes, Invitrogen, Carlsbad, CA, USA). Each spontaneous or paced action potential was associated with an increase in Ca^2 +_in recorded as a Ca^2 + transient. Bipolar pacing was accomplished using a pair of platinum electrodes (Grass SD9 stimulator, Harvard Instruments, Holliston, MA, USA). To reveal epitope localization, the samples were fixed by an ice-cold methanol-acetone mixture, permeabilized with 0.1% Triton-X100, and blocked with 1% BSA (Sigma-Aldrich). The samples were then stained with rabbit-anti-pan-Cadherin, rabbit-anti-connexin43, or mouse-anti-actinin (Sigma-Aldrich; 1:500 dilution, overnight at 4°C). Secondary antibodies linked to goat-anti-rabbit or goat-anti-mouse Cy3 or Cy5 (Jackson Immunoresearch Lab, West Grove, PA, USA) were diluted 1:1000. The immunohistological images shown are representatives of at least four separate experiments.

Figure 1.