Organ Explant Culture of Neonatal Rat Ventricles: A New Model to Study Gene and Cell Therapy

Abstract

Testing cardiac gene and cell therapies in vitro requires a tissue substrate that survives for several days in culture while maintaining its physiological properties. The purpose of this study was to test whether culture of intact cardiac tissue of neonatal rat ventricles (organ explant culture) may be used as a model to study gene and cell therapy. We compared (immuno) histology and electrophysiology of organ explant cultures to both freshly isolated neonatal rat ventricular tissue and monolayers. (Immuno) histologic studies showed that organ explant cultures retained their fiber orientation, and that expression patterns of α-actinin, connexin-43, and α-smooth muscle actin did not change during culture. Intracellular voltage recordings showed that spontaneous beating was rare in organ explant cultures (20%) and freshly isolated tissue (17%), but common (82%) in monolayers. Accordingly, resting membrane potential was -83.9±4.4 mV in organ explant cultures, −80.5±3.5 mV in freshly isolated tissue, and −60.9±4.3 mV in monolayers. Conduction velocity, measured by optical mapping, was 18.2±1.0 cm/s in organ explant cultures, 18.0±1.2 cm/s in freshly isolated tissue, and 24.3±0.7 cm/s in monolayers. We found no differences in action potential duration (APD) between organ explant cultures and freshly isolated tissue, while APD of monolayers was prolonged (APD at 70% repolarization 88.8±7.8, 79.1±2.9, and 134.0±4.5 ms, respectively). Organ explant cultures and freshly isolated tissue could be paced up to frequencies within the normal range for neonatal rat (CL 150 ms), while monolayers could not. Successful lentiviral (LV) transduction was shown via Egfp gene transfer. Co-culture of organ explant cultures with spontaneously beating cardiomyocytes increased the occurrence of spontaneous beating activity of organ explant cultures to 86%. We conclude that organ explant cultures of neonatal rat ventricle are structurally and electrophysiologically similar to freshly isolated tissue and a suitable new model to study the effects of gene and cell therapy.

Controlling the structural and functional anisotropy of engineered cardiac tissues

“…Simultaneously, longitudinal-to-transverse velocity anisotropy ratio was increased to ~1.7, a value similar to those of neonatal rat and dog myocardium (1.7-2.1)[41, 42]. While longer elliptical pores would be expected to further increase uniformity of global cell alignment, longitudinal conduction velocity, and electrical anisotropy, reaching the adult heart properties would require significant increase in cardiomyocyte size, acquisition of rod cell shape, and changes in gap junctional distribution.…”

Use of Myocardial Matrix in a Chitosan-Based Full-Thickness Heart Patch

“…49 Furthermore, Western blot analysis found a significant higher level of Cx-43 in heart matrix/containing hydrogels compared with gelatin containing hydrogels after 7 days in culture. Both APD 50 and APD 80 of NRVM on heart matrix hydrogels were similar to gelatin hydrogels. Our results were comparable with previous reports that measured the APDs of NRVM (APD 70 = 134 -5 ms 50 ; APD 90 = 140 -12 ms 27 ).…”

“…Both APD 50 and APD 80 of NRVM on heart matrix hydrogels were similar to gelatin hydrogels. Our results were comparable with previous reports that measured the APDs of NRVM (APD 70 = 134 -5 ms 50 ; APD 90 = 140 -12 ms 27 ). The average contractile stress of NRVM on heart matrix hydrogels was significantly higher than on gelatin hydrogels.…”

“…The average conduction velocity of heart matrix scaffolds was significantly faster in gelatin scaffolds (*p < 0.05, n = 11). There was no significant difference in both APD 50 and APD 80 ( p > 0.05, n = 4). Color images available online at www.liebertpub.com/tea…”

“…6D) showed that the average conduction velocity of NRVM on heart matrix hydrogels was significantly faster than on gelatin hydrogels (12.6 -4.9 in heart matrix and 7.9 -4.4 in gelatin in cm/s; p < 0.05, n = 11). There was no significant difference in either APD 50 or APD 80 (APD 50 ; 81 -28 in heart matrix and 92 -29 in gelatin in milliseconds; APD 80 ; 154 -35 in heart matrix and 161 -41 in gelatin in ms; p > 0.05, n = 4 for both APD 50 and APD 80 ) (Fig. 6C).…”