The porous bottom culture dishes (PBCDs) and related devices developed in this laboratory for the sterile measurement of electrophysiological parameters of cell layers are used in over 50 laboratories in the U.S.A. Two companies have devoted considerable effort toward producing commercial versions of the PBCDs using cellulose ester membranes (Millipore Corp.) and plasma treated polycarbonate membranes (Becton-Dickinson Corp.). Many types of epithelial cell layers are being grown on the PBCDs using a variety of membrane materials. Recently, endothelial cell layers have been grown here at NIH on our PBCDs with transparent collagen membranes. A defined medium has been developed for use with the PBCDs to optimize a differentiated function (transepithelial Na transport) rather than to maximize growth. Since the PBCDs facilitate the study of this differentiated function, this medium has obvious advantages. The major role played by Ca in the regulation of many cellular processes has prompted us to improve methods of measuring free Ca activity inside cells. Microelectrodes need to be as small as 0.1 microns in diameter to enter many cells without causing damage which makes reliable readings extremely difficult. Electrodes of this size have walls so thin that they may be totally hydrated near the tip and become Na and K electrodes due to the monovalent metals contained in the glass. This conflict between Ca, Na, and K sensitivities is believed to cause the poor performance often seen for electrodes smaller than 1 micron in diameter at Ca activities below micromolal. We have therefore designed, constructed, and used unique anular burners for making micropipettes to fused quartz which is free of metals other than the silicon of the silicone dioxide. An improved silanization method is being developed for these pipettes so that the advantages of the quartz can be fully realized.
