Engineered heart tissues (EHTs) are three-dimensional, hydrogel-based muscle constructs that can be generated from isolated heart cells of chicken, rat, mouse, human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC; Eschenhagen et al., 1997, Zimmermann et al., 2002, Stöhr et al., 2013, Schaaf et al. 20011, Stöhr et al. 2014). The method for the generation of EHTs was introduced in 1997 and has not been principally changed since then. It requires (i) heart cells, (ii) a liquid hydrogel that solidifies and promotes tissue formation, (iii) a casting mold that determines the 3D shape of the developing tissue and (iv) a support structure to which the cell-containing hydrogel attaches or that otherwise provides mechanical restrain for the developing heart tissue. The continuous mechanical strain orients and aligns cells along force lines and is the most critical factor of the method.


The new generation of fibrin-based miniaturized EHTs (Hansen/Eder et al., 2010) uses fibrin (fibrinogen/ thrombin) as a hydrogel, standard 24-well culture dishes, rectangular casting molds in agarose, and two elastic silicone (PDMS) posts per well to which the growing muscle tissue attaches. Muscle contractions (either spontaneous or electrically stimulated) deflect the posts and can be evaluated by video-optical recording. Casting molds are produced by putting a Teflon spacer in liquid agarose. After solidification of the agarose and removal of the spacer, silicone racks with two posts per well are manually positioned in the casting molds. A reconstitution mix of cardiomyocytes, fibrinogen and thrombin is poured into the casting mold around the silicone posts and incubated for polymerization of the fibrin at 37 °C. After 1.5 h the silicone racks with attached EHTs can be transferred to a 24-well- culture dish where the EHTs will start coherent beating after approximately 5-7 days.


Contraction analysis of engineered heart tissue (EHT) is based on a gas- and temperature-controlled incubator with a glass roof and a video camera mounted onto a motorized xyz-axis system above. The EHTs in a NUNC® 24-well-dish are positioned in the incubator on top of a LED panel. A proprietary software controls positioning of the camera above each well of the culture dish.

The camera records the movement of the EHT for a given time. Real time contraction analysis is based on an automated figure recognition algorithm detecting the edges of the two posts, length and elasticity (young modulus) of the elastic silicone posts and the actual deflection by the EHT contractions. The results of the automatic measurement of up to 24 EHTs in serial analysis are summarized in a pdf data sheet containing: number of beats, rate in beats/min, force of contraction, contraction time T1, relaxation time T2, maximal velocities of contraction and relaxation, and rhythmicity.

Electrical stimulation

Spontaneous contractions of EHTs can be recorded any time, but a thorough analysis of contractile function and its modulation by drugs requires pacing at a constant rate. A pacing system made from stainless steal and carbon electrodes can be easily inserted in the 24-well dish and generates an electrical field around the EHT. Continuous pacing for up to 14 days has been successfully tested (Hirt et al. 2014). Pacing signals are integrated in the software to allow exact determination of contraction kinetics and the generation of average peaks, i.e. the mean of a chosen number of single twitches.