SOLVO uses the gold-standard oil-spin method to evaluated active uptake of a compound in hepatocytes. In addition, it is also possible to use plateable cells, to run the assay.
In the Oil spin method, hepatocytes are incubated with the Test article in suspension and will be separated from substrate buffer by using the oil spin method. This is done by layering the hepatocyte suspension on top of a layer of oil in a thin centrifugation tube. The tube will be spun down in a tabletop centrifuge at high speed, causing the hepatocytes to migrate through the oil layer and end up in the bottom solution in the lower part of the tube, which has a higher density and therefore does not invert during centrifugation..
The lower part will be cut off and subjected to liquid scintillation counting or can be sampled using LC-MS analysis.
The plated hepatocyte assay relies upon having platable hepatocytes from a single donor in tissue culture plates. The cultures are washed to remove any hepatocytes that have not adhered and the Test Article (TA) containing buffer is added to start uptake. After incubation the solution is removed and the plates are washed to remove non-accumulated TA from the wells. Hepatocytes are lysed in a suitable agent and TA will be detected by liquid scintillation counting or LC/MS.
Human cryopreserved hepatocytes used for a study are typically pooled donor, mixed-gender hepatocytes to get a general experiment, however on request a specific lot with characteristics specific for project needs can be requested (e.g. single or multiple donor, gender, etc.). In case of preclinical species it is generally thought to be less critical using several donors as animals are homogeneous and kept under standard laboratory conditions. As a general recommended, cryopreserved hepatocyte lots characterized for transporter mediated uptake are used. Major substrates for which characterization data is available include the following prototypical substrates:
A single or multiple probe substrates can be run along with a test article as positive control.
Accumulation of the TAs in cryopreserved human hepatocytes is generally determined by incubating TA at one concentrations and four incubation time points. The incubation time ranges from several seconds to a few minutes, the concentration is typically around 1-10 μM. Assays are conducted in triplicate at 37°C and on ice, where intracellular accumulation of TAs at 37°C represents active uptake and incubation on ice represents passive diffusion of the TAs.
The assay design is however flexible and can be tailored to your project needs to for example determine kinetics or assess uptake in the presence of inhibitors