Three-dimensional engineered heart tissue from neonatal rat cardiac myocytes

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Abstract
A technique is presented that allows neonatal rat cardiac myocytes to form spontaneously and coherently beating 3-dimensional engineered heart tissue (EHT) in vitro, either as a plane biconcaval matrix anchored at both sides on Velcro-coated silicone tubes or as a ring. Contractile activity was monitored in standard organ baths or continuously in a CO2 incubator for up to 18 days (=26 days after casting). Long-term measurements showed an increase in force between days 8 and 18 after casting and stable forces thereafter. At day 10, the twitch amplitude (TA) of electrically paced EHTs (average length × width × thickness, 11 × 6 × 0.4 mm) was 0.51 mN at length of maximal force development (Lmax) and a maximally effective calcium concentration. EHTs showed typical features of neonatal rat heart: a positive force–length and a negative force–frequency relation, high sensitivity to calcium (EC50 0.24 mM), modest positive inotropic (increase in TA by 46%) and pronounced positive lusitropic effect of isoprenaline (decrease in twitch duration by 21%). Both effects of isoprenaline were sensitive to the muscarinic receptor agonist carbachol in a pertussis toxin-sensitive manner. Adenovirus-mediated gene transfer of β-galactosidase into EHTs reached 100% efficiency. In summary, EHTs retain many of the physiological characteristics of rat cardiac tissue and allow efficient gene transfer with subsequent force measurement. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 68: 106–114, 2000.