Transporters in the hepatocyte basolateral membrane are responsible for carrier-mediated processes involved in the uptake of xenobiotics from the systemic circulation. Hepatic clearance starts with transport into hepatocytes by passive diffusion though the cell membrane and by active transport. In hepatocytes, uptake is mediated by basolateral membrane organic anion transporters including the organic anion transporters OATP1B1, OATP1B3, OATPB2B1, OATP1A2, OAT2, OAT5, the organic cation transporter OCT1 and the Na-taurocholate cotransporting NTCP. Multiple transporters mediate the excretion of compounds from hepatocytes back into systemic circulation via basolateral membrane efflux transporters or out of circulation into the bile via outwardly directed efflux transporters of the ATP-binding cassette (ABC) protein family. Thus, multiple membrane transporters in hepatocytes work in concert with the drug metabolizing enzymes to mediate hepatic drug clearance and these may contribute to drug-drug interactions.
Drug-drug interaction involving hepatic membrane transporters can occur by competition for the same substrate-binding site of the transporter, or tight or allosteric binding leading to inhibition of transporter activity, or by change in expression level of transporters. This has the potential to alter the blood concentration time profiles of drugs, leading to elevated levels of a co-administered compound. Evaluating the substrate potential of a drug candidate for the uptake hepatic transporters in vitro is particularly beneficial when the liver is the drug target. For example, the hepatitis C drugs alpha-interferon and S-acyl-2-thioethyl esters or the HMGCoA inhibitors (statins) must achieve adequate concentrations in the liver for pharmacological activity.
In addition to drug-drug interactions, hepatic transporters also play a role in toxicities including cholestasis and hyperbilirubinemia. Drug-induced hepatotoxicity is a major problem in drug development and there is growing evidence that inhibition of bile acid transporters is a contributing mechanism (1).
Hepatocytes in suspension, attached to tissue culture dishes, or in primary sandwich-culture are all good models of hepatic transport. The contribution of transportermediated 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).
- 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 immediately and will not maintain viability if refrozen.
- Use universal safety precautions and appropriate biosafety cabinet when handing primary hepatocytes.
Hepatic uptake studies typically measure the rate of appearance of substrate into cells after a relatively short incubation period, in most cases from 10 seconds to 3 minutes. After incubation, hepatocytes are centrifuged in microfuge tubes through an oil layer (which prevents passage of the buffer and also traps lipophilic compounds that may be on the outer cell membrane) into a layer of 2N potassium hydroxide or cesium chloride for digestion (3). This bottom layer, along with the digested trapped hepatocytes, is counted in a liquid scintillation counter or analyzed by mass spectrometry. This methodology has provided robust, mechanistic data that has been used to successfully predict the in vivo clearance of drugs (4).
Critical materials and reagents
- Silicone and mineral oils
- 2N potassium hydroxide (KOH)
- Krebs-Henseleit buffer
- 1M HEPES
- Suspension hepatocytes prequalified for transporter uptake (Life Technologies Cat. No. HMCSTS)
- CHRM® medium (Life Technologies Cat. No. CM7000)
- Test articles and positive control substrates (taurocholate, digoxin, estradiol 17β-glucuronide [E2-17G] and 1-methyl-4-phenylpyridinium [MPP+])
- Ice / dry ice-isopropanol
- 2N HCL
- Scintillation fluid
- 24-well plates
- 500 μL microcentrifuge tubes
- Scintillation vials
- Water bath set at 37°C, with shaker
- Microcentrifuge, temperature regulated
- Scintillation counter
- Microcentrifuge tube cutter
- Reciprocal shaker
- Prepare oil mixture
- Mix 74.5 : 25.5 silicon oil : mineral oil to achieve a final density of 1.015 g/mL
- Prepare microcentrifuge tubes with oil and digestive solutions
- Pipette 20 μL of 2N KOH* (or alternatively, 3% cesium chloride or 1% Triton X100) into 500 μL microcentrifuge tubes. We recommend triplicate incubations for each timepoint and condition.
Note: KOH can not be used for mass-spectrometry, cesium chloride is recommended.
- Spin the tubes to ensure that solution is at bottom.
- Carefully add 125 μL of the oil mixture on top of this digestive layer.
- Spin the tubes to ensure material is at the bottom and to remove any bubbles that are introduced from pipetting.
- Prepare uptake buffer
- Mix 9.6g Krebs-Henseleit Buffer with 15 mL 1M HEPES. Bring up to 1L with ddH2O, and adjust to pH 7.4 with NaOH. Store at 4°C.
- Prepare hepatocytes using cryopreserved suspension hepatocytes prequalified for transporter uptake, such as Life Technologies Cat. No. HMCSTS (or alternatively, freshly isolated suspension hepatocytes).
- Preferred hepatocytes have viabilities >80%. Cryopreserved cells must be stored in the vapor phase of liquid nitrogen until immediately prior to use, then thawed for <2 min at 37°C. Follow these hyperlinked protocols for thawing, removing the cryoprotectant with CHRM® medium, and counting the hepatocytes:
- Protocol for Thawing and Use of Plateable and Suspension Cryopreserved Hepatocytes
- Protocol for Counting Primary Hepatocytes using Trypan Blue Exclusion Analysis
- Dilute hepatocytes to a final concentration of 1 x 106 viable cells/mL in the uptake buffer.
- Prepare test articles as a solution in the uptake buffer using < 0.2% organic solvent in the final incubation mixture.
- Radiolabeled substrates should be 4-fold that of the final incubation.
- Final concentration of the positive control substrates taurocholate, digoxin, estradiol 17β-glucuronide (E2-17G) and 1-methyl-4-phenylpyridinium (MPP+) is recommended to be 1 μM.
- Half of the substrate volume should be warmed to 37°C; the other half will be used at the control temperature of 4°C.
- Pre-incubate 150 μL of hepatocyte suspension with 300 μL of uptake buffer in multiple wells of two 24-well plates.
- Incubate one plate at 37°C for 10 min in a water bath with shaking (~150 rpm).
- Incubate the other plate on ice.
- Initiate reactions with the addition of 150 μL warm substrates to the 37°C incubations; continue shaking at 150 rpm.
- In parallel, add 150 μL of 4°C substrates to the incubations on ice.
- Incubation timepoints are usually short—between 15 sec and 5 min.
- 1 min incubations for positive controls taurocholate, digoxin, and E2-17.
- 5 min incubation for positive control MPP+.
- At desired timepoints, remove 100 μL from the wells into the prepared microcentrifuge tubes containing the oil and digestive layers.
- Immediately spin in the microcentrifuge for 30 sec at 14,000 rpm.
- Snap-freeze tubes on a dry ice/isopropanol mixture for a minimum of 30 min.
- Remove samples from the dry ice and use a tube cutter to clip the tips which contain the KOH/hepatocytes layer into liquid scintillation vials.
- Add 3 mL of liquid scintillation cocktail to each vial.
- Neutralize the KOH by adding 20 μL of 2N HCL to each vial.
- Read on a scintillation counter.
- Protein content can also be determined by using the BCA Protein Assay Kit from Pierce (Cat. No. 23227) with left-over suspension from the 24-well plates after the 100 μL is put into the oil tube.
- Transporter-mediated uptake is the uptake measured at 37°C minus the uptake measured at 4°C.
Note for use of inhibitors: If using inhibitors, prepare in uptake buffer at 2-fold that of the final incubation. We do not recommend pre-incubation of the inhibitor. Coincubation of the inhibitor and substrate works best when the inhibitor is not pre-incubated.
For questions related to this protocol, contact us at:
Phone: +1 919 237 4500 (Toll)
Phone: +1 866 952 3559 (U.S. toll-free)
- Marion TL, L 1. eslie 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.
- 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.
- 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.
- 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.