A SUPERFUSION SYSTEM TO STUDY BORDER ZONES IN CONFLUENT CULTURES OF NEONATAL RAT-HEART CELLS

We present a new experimental method to study intracellular ion regula tion in cultured cardiomyocytes at a border zone separating two differ ent and distinct environments. Our system uses a dual-flow superfusion chamber to produce two different but adjacent environments over a mon olayer of cardiomyocytes. Fluorescent microscopy of fluorescein showed that the transition between the two environments was nearly linear an d was 220-320 mu m wide depending on fluid viscosity and velocity. We superfused cultured monolayers on one side with a solution at pH 6.5 a nd on the other side with a solution at pH 7.4. We observed a sharply demarcated difference in intracellular pH (pH(i)) between the two halv es of the cell monolayer as measured with the fluorescent pH(i) indica tor carboxy-seminaphthorhodafluor-1. The demarcation of pH(i) correspo nded well with the demarcation of the border measured with fluorescein . We conclude that our superfusion system will facilitate the study of intercellular communication and interactions across boundaries of car diac tissue where different ionic or metabolic conditions are present, for example, between ischemic and nonischemic myocardium.