The SOLVO hepatocyte uptake assays are performed with single or multiple donor cryopreserved human hepatocytes. Accumulation of the TA in cryopreserved human hepatocytes can be determined by incubating the TA at two concentrations at two or four incubation times at 37°C and on ice.
The incubation at 37°C represents active uptake, while incubation on ice represents passive diffusion of TA. Oil spin method (recommended for radiolabeled TAs) Hepatocytes will be separated from substrate buffer by using the oil spin method. Briefly hepatocytes will be layered on top of a layer of oil in a thin centrifugation tube. The tube will be spinned in a tabletop centrifuge at high speed, causing the hepatocytes to migrate through the oil layer and end up in a 2N NaOH solution in the lower part of the tube.
The lower part will be cut off and subjected to liquid scintillation counting. Filterplate method (recommended for cold TAs) Hepatocytes will be filtered on a filterplate on a vacuum manifold, followed by several wash steps to remove excess probe substrate. To all wells of the filterplate a solvent will be added to lyse the cells. Subsequently the solvent will be collected in a 96-wells plate by applying vaccuum. The TA will be detected by liquid scintillation counting or LC/MS.