**2. Methods**

72 Toxicity and Drug Testing

give an integrated readout of effects of drugs on the electrophysiology of the intact cardiac myocyte or the intact heart as a multicellular organ consisting of a functional syncytium of cardiac myocytes and all other cardiac cell types that make up normal heart tissue (e.g.

Over the past decade, techniques have been developed to generate cardiac tissue-like 3 dimensional constructs *in vitro* (Eschenhagen & Zimmermann, 2005). The field of cardiac tissue engineering opened the possibility for many applications. Artificial hart constructs may serve as means for cell-based cardiac repair and as improved *in vitro* models for predictive toxicology and target validation, taking advantage of a more physiological cellular environment. Previous studies used different approaches to construct engineered tissues: Cell seeding onto solid, preformed scaffolds (Carrier et al., 1999; Engelmayr et al., 2008; Leor et al., 2000; Li et al., 2000; Ott et al., 2008; Radisic et al., 2004), matrix-free generation of tissues from stackable cell sheets (Shimizu et al., 2002) or the generation of constructs in preformed casting moulds using hydrogels such as collagen I, matrigel, fibronectin or fibrin (Bian et al., 2009; Eschenhagen et al., 1997; Huang et al., 2007; Naito et al., 2006; Zimmermann et al., 2002). The hydrogel technique has been shown to be suitable for both, cardiac repair *in vivo* (Zimmermann et al., 2006) and target validation *in vitro* (El-Armouche et al., 2007). Circular engineered heart tissues (EHTs) were made by casting neonatal rat heart cells, collagen I and matrigel into circular casting moulds and develop a high degree of cellular differentiation, longitudinal orientation, intercellular coupling and force generation (Zimmerman et al., 2002). It turned out that several factors improve tissue quality and force generation of EHT such as phasic (Fink et al., 2000) or auxotonic stretch, increased ambient oxygen concentration during culture and supplementation with insulin (Zimmermann et al., 2006). Others demonstrated beneficial effects of electrical stimulation (Radisic et al., 2004). The possibility to generate cardiac myocytes from human embryonic stem cells (Kehat et al., 2001) or induced pluripotent stem cells (Zhang et al., 2009) have opened the realistic and exciting perspective to use these techniques for the validation of hypotheses and testing drugs in healthy and diseased human heart muscles (Zimmermann

The current techniques to generate engineered cardiac tissues are either not suitable for this purpose (stacked cell sheet technique) or exhibit drawbacks that limit their usefulness. Extensive handling steps preclude routine execution of large series in an at least medium through put scale and are always a source of variability. Furthermore, the EHT technique in the ring format requires relatively high numbers of cells and turned out to be difficult to

In this chapter we describe a new EHT technique that was driven by the intention to miniaturize the EHT-format for multi-well-testing and automated evaluation and to determine the suitability of EHTs for drug screening and predictive toxicology. The main results have been published in a recent original paper (Hansen et al. 2010). An essentiel change was to use fibrin(ogen) instead of collagen I as a matrix. Fibrinogen is part of the blood clotting cascade. It is a glycoprotein with a size of 340 kDa. Physiologically it achieves plasma concentrations of 1.5 to 4 g/l and can be relative easily purified from different species. An important mechanical property is its nonlinear elasticity. Due to this, fibrin polymers have a high elastic modulus under shear stress combined with a beneficial

fibroblasts, endothelial cells and smooth muscle cells).

**1.1 Cardiac tissue engineering** 

& Eschenhagen 2007).

miniaturize.
