Efficacy of a low volume recirculating superfusion chamber for long term administration of expensive drugs and dyes

Abstract

We have characterized the efficacy of a low volume (2 ml) two-compartment experimental chamber in which a gas inflow equilibrates and recirculates the bathing fluid. This type of chamber is suitable for experiments employing en bloc preparations that require the administration of expensive molecular probes. The fluid in the chamber is pumped from a compartment holding the preparation to an elevated reservoir compartment using gas bubbles. The fluid returns via gravity along a different path. The flow rate of superfusate in the chamber was 30 ml min −1. To determine the effectiveness of the chamber in dissolving gas, we filled the chamber with bicarbonate-buffered physiological saline and measured pH and P O 2 with ion-selective and Clark-style microelectrodes. Steady state values of pH and P O 2 in the chamber were almost identical to those in an external tonometer bubbled vigorously with the same gas mixture. When CO 2 was increased from 2 to 4.4%, the chamber pH fell with a time constant of 56 s (about twice that of the tonometer). To determine the effectiveness of gas exchange between a brain preparation and the fluid in the chamber we measured pH and P O 2 depth profiles of the in vitro tadpole brainstem. We found virtually no unstirred layer owing to excellent mixing and the high flow created by the recirculating mechanism. We demonstrate that despite the high flow rates, preparations are mechanically stable allowing intracellular electrophysiological recordings.