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2014
Journal Article
Titel
Organotypic tissue culture extends cold preservation and enables long term investigation of human failing myocardium
Titel Supplements
Abstract
Abstract
Problem: Thin tissue slices prepared from normal or hyper-trophic human myocardium can be maintained in culture and permit studies of contractility and electrophysiology for many days. However, the availability of surgical tissue specimen is low. Diseased myocardium can be procured in higher quantities from the explants of donor heart recipients, but displays high variability and its suitability for slice preparation is unknown. Moreover, its availability is restricted to transplantation clinics. To resolve these limitations, we sought to establish conditions for the transport and slicing of explant specimen, to set up quality and viability controls, and to describe the functional and morphological alterations that slices undergo in tissue culture. Methods: Specimen of ventricular tissue from explanted failing myocardium were obtained at 3 major German trans-plantation clinics, and were immersed in cold BDM-containing modified Tyrode solution during 4-48 h of transport. The tissue was cut into 300 mm thick slices of about 1x1 cm2 surface area, which were either analyzed immediately or cultured at a liquid-air interface for up to 28 d. Characterization included determination of intracellular membrane potential, contractility, and cellular viability and differentiation. Results: MTT was established as a sensitive test of slice viability which was optimal after cold preservation of tis-sue specimen for less than 12 h. Fresh slices performed paced contractions at up to 180 bpm, and displayed contractility similar to hypertrophic myocardium (2.7 mN/mm2) with strong responses to strain and adrenoceptor agonist isoproterenol. Action potentials (APs) displayed normal values of diastolic potential (-80 mV), amplitude, and duration (330-380 ms), and responded to HERG and KAT P channel manipulation. While contractility declined within the first 7 d of culture, electrophysiological parameters were maintained for up to 28 d. Slices obtained after 12-36 h of transport were prone to partial contracture upon BDM washout which negatively correlated with twitch force and survival in culture. Slices with less than 2% fractional contracture recovered upon 1 d cultivation in BDM-containing medium, and showed no impairment of contractility in comparison to slices with shorter cold preservation. Cultured slices from failing myocardium were successfully used to demonstrate AP pro-longation by dofetilide, and to quantify the negative inotropic action of adenosine. Conclusions: Slices of human failing myocardium can be obtained from about 80% of tissue specimen even after 36 h of cold preservation. Tissue culture enables studies of contractility and electrophysiology for about 7 days, and has been employed to refine the conditions of cold tissue preservation and recovery.