Hepatic uptake studies typically measure the rate of appearance of substrate into cells after a short incubation (in most cases from 15 sec to 3 min).
We functionally test for NTCP, OATP1B3, and OATP transporter pathway activity using the substrates taurocholate, digoxin, and estradiol 17β glucuronide (E2-17G). We also test phase I and phase II metabolic activities. Visualization of the bile canalicular networks is attained by fluorescence microscopy using 5-(6)-carboxy-2', 7'-dichlorofluorescein diacetate (CDFDA), as shown in Figure 1.
Figure 1. Visualization of functional bile canaliculi networks showing the accumulation of 5-(6)-carboxy-2', 7'-dichlorofluorescein (CDF).
How we qualify plateable human hepatocytes for enzyme induction
GIBCO® induction-qualified hepatocytes have passed our test for specific activity and mRNA levels in response to prototypical inducers. Cryopreserved human hepatocytes are cultured in collagen-coated plates and dosed in triplicate with vehicle (0.1% DMSO), omeprazole (OMP), phenobarbital (PB), and rifampicin (RIF) for 72 hr.
Once monolayers are washed, they are incubated with substrates phenacetin, bupropion, and testosterone to determine CYP1A2, CYP2B6, and CYP3A activities, respectively (Table 1). Fold induction of specific activity is expressed as the ratio of induced activity to vehicle activity (Figure 2). mRNA content is also determined by TaqMan® qRT-PCR analysis after 48 hr treatment.
|CYP1A2||Omeprazole||50 µM||Phenacetin||100 µM||15 min||Acetaminophen|
|CYP2B6||Phenobarbital||1,000 µM||Bupropion||500 µM||20 min||Hydroxybupropion|
|CYP3A||Rifampicin||10 µM||Testosterone||200 µM||14 min||6β-Hydroxytestosterone|
Figure 2. Example of CYP3A induction test results. The fold induction value (inducer/control) is the number shown above the red bars.
|Midazolam||0.50 µM||0, 1, 2, 4, 6, 8 hr|
|Tolbutamide||1.00 µM||0, 4, 6, 8, 18, 24 hr|
|Dextromethorphan||1.00 µM||0, 1, 2, 4, 6, 8 hr|
Figure 3. Plated metabolism qualified human hepatocytes. CLint results for this lot were midazolam, 14.6; tolbutamide, 1.3; dextromethorphan, 7.2 µL/1 x 106 cells/min.
|CYP1A2||Phenacetin||100 µM||15 min||Acetaminophen|
|CYP2B6||Bupropion||500 µM||20 min||Hydroxybupropion|
|CYP2C8||Paclitaxel||20 µM||45 min||6α-Hydroxypaclitaxel|
|CYP2C9||Diclofenac||25 µM||15 min||4'-Hydroxydiclofenac|
|CYP2C19||(S)-mephenytoin||250 µM||30 min||4'-Hydroxymephenytoin|
|CYP2D6||Dextromethorphan||15 µM||15 min||Dextrorphan|
|CYP2E1||Chlorzoxazone||250 μM||15 min||6-Hydroxychlorzoxazone|
|CYP3A||Testosterone||200 µM||14 min||6β-Hydroxytestosterone|
|CYP3A||Midazolam||10 µM||10 min||1-hydroxymidazolam|
|Phase I and II||7-Ethoxycoumarin||100 µM||30 min||7-HCG, 7-HCS, 7-HC*|
|FMO||Benzydamine||250 μM||30 min||Benzydamine-N-Oxide|
*7-hydroxycoumarin glucuronide, 7-hydroxycoumarin sulfate, and 7-hydroxycourmarin
|Substrate||Concentration (µM)||Incubation time (min)|
Figure 4. Human hepatocytes qualified for suspension metabolism applications.
For Research Use Only. Not for use in diagnostic procedures.