| abstract |
In many miniaturized high-throughput assays, effective stirring is difficult to achieve, and its consequences difficult to predict. Most microtitre plate-based in vitro assays of membrane permeability, such as Caco-2 and PAMPA, of sparingly soluble lipophilic test compounds in pharmaceutical research and development report permeability of water, and not of the intended membrane barrier. This is so because the aqueous boundary layer on both sides of the membrane barrier is rate limiting for these highly permeable molecules. The thickness of this water layer can be 1500-4000 μm in unstirred assays. Under in vivo conditions, however, the aqueous boundary layer is believed to be 30-100 μm thick in the intestine, and <1 μm at the blood-brain barrier. Using plate shakers to stir microtitre plates cannot lower the thickness of the water layer to match that found in vivo. We have discovered a high-throughput method and a simple, “robotics-friendly” device where individual-well stirring at speeds of about 600 rpm can lower the aqueous boundary layer thickness to <15 μm, which had not been previously achieved for plate-based permeability assays. |