Objective: To compare the effects of enzyme digestion with differential attachment and enzyme digestion with differential attachment plus 5'-bromodeoxyuridine (5-BrdU) (conventional) method for isolating sino-atrial node cells from newborn rats for patch clamp studies.
Methods: The sino-atrial node tissue were removed from neonatal Wistar rats, digested and separated into single sino-atrial node cells with the above two methods. The obtained cells were observed for morphology, structure, beat frequency, degree of difficulty for patch clamp experiment and the success rate. The atrial cells isolated with enzyme digestion with differential attachment served as the control cells.
Results: The spindle-shaped cells constituted the greatest proportion of the cultured sino-atrial cells, and the cultured atrial cells showed mostly a triangular morphology. Both of the sino-atrial cells separated with the two methods contained mostly spindle-shaped cells, in which spontaneous action potential was recorded. The spindle-shaped cells isolated with enzyme digestion and differential attachment showed a greater viability with a higher success rate in patch clamp studies.
Conclusion: The sino-atrial node cells can be successfully isolated with enzyme digestion and differential attachment, which requires simple procedures and well preserves the cell viability for better performance in patch clamp experiment.