Objective: To locate sinoatrial node (SAN) in suckling pigs, to develop a reliable method for isolation, purification and cultivation of SAN cells and to observe the compatibility of SAN cells and Col I fiber scaffold.
Methods: Five newborn purebred ChangBaiShan suckling pigs (male and female), aged less than 1-day-old and weighing 0.45-0.55 kg, were used. Multi-channels electrophysiological recorder was applied to detect the original site of atrial waves. Primary SAN cells harvested from that area were cultured by the conventional culture method and the purification culture method including differential velocity adherent technique and 5-BrdU treatment, respectively. Atrial myocytes isolated from the left atrium underwent purified culture. Cell morphology, time of cell attachment, time of unicellular pulsation, and pulsation frequency were observed using inverted microscope. The purified cultured SAN cells (5 x 10(5) cells/mL) were co-cultured with pre-wetted Col I fiber scaffold for 5 days, and then the cells were observed by HE staining and scanning electron microscope (SEM).
Results: The atrial waves occurred firstly at the area of SAN. The purified cultured SAN cells were spindle, triangular, and irregular in morphology, and the spindle cells comprised the greatest proportion. Atrial myocytes were not spindle-shaped, but primarily triangular and irregular. The proportion of spindle cells in the conventional cultured SAN cells was decreased from 73.0% +/- 2.9% in the purified cultured SAN cells, to 44.7% +/- 2.3% (P < 0.01), and the proportion of irregular cells increased from 7.0% +/- 1.7% in the purified cultured SAN cells to 36.1% +/- 2.6% (P < 0.01) . The proportion of the triangular cells in the purified and the conventional cultured SAN cells was 20.0% +/- 2.1% and 19.2% +/- 2.5%, respectively (P > 0.05). At 5 days after co-culture, HE staining displayed lots of SAN cells in Col I fiber scaffold, and SEM demonstrated conglobata adherence of the cells to the surface and lateral pore wall of scaffold, mutual connections of the cell processes, or attachment of cells to lateral pore wall of scaffold through pseudopodia.
Conclusion: With accurate SAN location, the purification culture method containing differential velocity adherent technique and 5-BrdU treatment can increase the proportion of spindle cells and is a reliable method for the purification and cultivation of SAN cells. The SAN cells and Col I fiber scaffold have a good cellular compatibility.