Transporters in the hepatocyte basolateral membrane are responsible for carrier-mediated processes involved in the uptake of xenobiotics from the systemic circulation. Hepatic clearance can be thought to begin with movement of xenobiotics from the blood into hepatocytes by either passive diffusion or active transport through the cell membrane. In hepatocytes, active basolateral uptake has been shown to be mediated by various pathways including organic anion transporters (e.g. OATP1B1, OATP1B3, OATPB2B1, OATP1A2, OAT2, OAT5), organic cation transporters (e.g. OCT1), and the Na-taurocholate cotransporting protein NTCP. Multiple transporters are also known to mediate the excretion of compounds from hepatocytes back into the blood via basolateral membrane efflux transporters or excretion into the bile via outwardly directed efflux transporters including the ATP-binding cassette (ABC) protein family. Collectively these and other transport and metabolism processes work in concert to carry out essential hepatic detoxification drug/xenobiotic clearance functions.

Drug-drug interactions involving hepatic membrane transporters are thought to arise via direct (competitive) or allosteric inhibition of hepatic transporters or via perturbations in transporter cytosolic membrane localization or gene expression levels. The resulting changes in transport activity can alter the drug pharmacokinetics that can result in elevated levels of a co-administered compound due to impaired clearance pathways. Therefore, evaluation of the substrate and inhibitor potential of a drug candidate for the uptake hepatic transporters in vitro can be useful in understanding xenobiotic clearance pathways and predicting the potential for drug-drug interactions (e.g. the hepatitis C drugs alphainterferon and S-acyl-2-thioethyl esters or the HMGCoA inhibitors (statins)).

In addition to drug-drug interactions, hepatic transporters also play an important role in detoxification, and have been linked with toxicities including cholestasis and hyperbilirubinemia. Drug-induced hepatotoxicity is a major liability in drug development and there is growing evidence that inhibition of biliary transporters is a contributing mechanism (1).

Primary hepatocytes in suspension, attached to tissue culture matrices, or in sandwich-culture have all been shown to be useful models to assess hepatic transport potential. The contribution of transporter-mediated uptake to hepatic clearance (CLH) was recognized when CLH was consistently under-predicted for many series of chemotypes using just metabolic stability for the calculations. Factoring in transporter-mediated hepatic uptake, along with metabolic clearance using hepatocytes in suspension, improved these predictions (2).

Important notes

  • Review this protocol, as well as the protocol, Thawing and Use of Plateable and Suspension Cryopreserved Hepatocytes, to ensure you have all the necessary reagents and equipment prior to starting the procedure.
  • The incubation and sampling steps in this experiment are time-sensitive; therefore, it is useful to double-check the experimental set-up to make certain samples can be collected quickly. In addition, once thawed, cryopreserved hepatocytes must be used within a few hours and will not maintain viability if refrozen.
  • Use universal safety precautions and an appropriate biosafety cabinet when handing primary hepatocytes.

Protocol overview

Critical materials and reagents

  • Ca2+ - containing buffer
  • Test articles and positive control substrates (taurocholate, digoxin, estradiol 17ß-glucuronide [E2-17G] and 1-methyl-4-phenylpyridinium [MPP+])
  • Ice
  • Reservoirs
  • Scintillation fluid
  • Scintillation vials/cassettes


  • Water bath set at 37°C (and 4˚C for cold procedure)
  • 6-chanel aspirating manifold
  • Vacuum pump and trap system (radioactive)
  • Positive displacement pipets
  • Multichannel pipettors
  • Slide warmer
  • Ice bucket
  • Liquid scintillation counter


Substrate/inhibitor preparation

  1. Prepare test articles as a solution in the Ca2+ - containing buffer using <0.2% organic solvent in the final incubation mixture.
  2. Final concentration of the positive control substrates taurocholate, digoxin, estradiol 17ß-glucuronide (E2-17G) and 1-methyl-4-phenylpyridinium (MPP+) is suggested to be 1 μM.
  3. If using inhibitors prepare as a substrate/inhibitor mix to use as a co-incubation.
  4. Pre-warm all solutions to 37°C before start of the experiment.


  1. Aspirate all the media off wells being tested.
  2. Add 300 μL of pre-warmed Ca2+ - containing buffer to all the wells being tested, shake the plate briefly then aspirate the plate.
  3. Repeat step 6.
  4. After the second wash aspirate media and add 300 μL of warm Ca2+ - containing buffer to each of the wells and incubate at 37°C for 10 minutes.


  1. Remove plate from 37°C and remove buffer from all the wells being tested.
  2. Initiate reactions with the addition of 300 μL of substrates or substrate/inhibitors.
  3. Incubate plate at 37°C for 10 minutes.
  4. After the 10 minute incubation, aspirate the substrates or substrates/inhibitors and wash the plate 3X with 300μL of cold Ca2+ - containing buffer.
  5. After the addition of each 300 μL of cold Ca2+ - containing buffer, shake the plate for about 20 seconds to allow buffer to wash the sides of the wells.
  6. After final wash remove all the Ca2+ - containing buffer and place the plate on ice.
  7. Store plates at -80°C until ready to sample process, store for a minimum of 15 minutes.

* Important note: Repeat this assay using cold (4˚C) buffer, dose solution, cold wash buffer and incubation temperatures in order to account for non-specific uptake. Plates should be kept on ice for the entire cold procedure. 

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  1. Marion TL, Leslie EM and Brouwer, KL (2007) Use of sandwich-cultured hepatocytes to evaluate impaired bile acid transport as a mechanism of drug-induced hepatotoxicity. Mol Pharm 4:911-918.
  2. Soars MG, Grime K, Sproston JL, Webborn PJ and Riley, RJ (2007) Use of hepatocytes to assess the contribution of hepatic uptake to clearance in vivo. Drug Metab Dispos 35:59-865.
  3. Shitara Y, Hirano M, Sato H and Sugiyama Y (2004) Gemfibrozil and its glucuronide inhibit the organic anion transporter polypeptide 2 (OATP2/OATP1B1:SLC21A6)-mediated hepatic uptake and CYP2C8-mediated metabolism of cerivastatin: analysis of the mechanism of the clinically relevant drug-drug interaction between cerivastatin and gemfibrozil. JPET 311:228-236.
  4. Griffin SJ and Houston JB (2005) Prediction of in vitro intrinsic clearance from hepatocytes: comparison of suspensions and monolayer cultures. Drug Metab Dispos 33:115-120.
LT132                              1-Mar-2011