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GIBCO® Human Cryopreserved Hepatocytes, Transporter Qualified
How we qualify suspension and plateable human hepatocytes for transporter uptake
- Contain functional membrane receptors and transporters
- Facilitate effective transporter uptake (suspension cells) and basolateral efflux (plated cells)
- Have stringent release specs: ≥80% viability and ≥90% confluency (plated cells)
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).|
GIBCO® Human Cryopreserved Hepatocytes, Induction Qualified
- Prequalified for CYP1A2, CYP2B6, and CYP3A induction
- ≥80% viability and ≥90% confluency
- Minimum specific activities:
- ≥10-fold induction of CYP1A2
- ≥5-fold induction of CYP2B6
- ≥3-fold induction of CYP3A4
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.
Table 1. Substrate probes for the assessment of CYP450 activity in GIBCO® Human Cryopreserved Hepatocytes, Induction Qualified.
| Enzyme || Inducer || [Inducer] || Substrate || [Substrate] || Incubation || Marker metabolite |
|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.|
GIBCO® Human Cryopreserved Hepatocytes, Plated Metabolism Qualified
How we qualify plateable human hepatocytes for metabolism (intrinsic clearance)
- Useful for assessing intrinsic clearance (CLint) in low-turnover compounds
- Prequalified according to CLint of midazolam, tolbutamide, dextromethorphan
- ≥80% viability and ≥75% attachment efficiency
Our plated metabolism-qualified hepatocytes are tested for the enzymatic functions of CYP3A4, CYP2C9, and CYP2D6 using the prototypical CYP450 substrates midazolam, tolbutamide, and dextromethorphan respectively (Figure 3).
Hepatocytes attach to collagen-coated plates prior to substrate incubations in serum-free Williams Medium E. Reactions are stopped with ice-cold acetonitrile at time points indicated in Table 2, and well contents are stored at -70°C prior to analysis. The disappearance of parent compound is monitored by LC-MS/MS and intrinsic clearance values are determined by linear regression.
Table 2. Incubation conditions for CLint in plated cryopreserved human hepatocytes.
| Substrate || Concentration || Incubation time |
|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.|
GIBCO® Human Cryopreserved Hepatocytes, Suspension Metabolism Qualified
How we qualify suspension human hepatocytes for metabolism
- HEP10™ Pooled Lots are characterized for CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A4/5, FMO activity, and NTCP, OATP1B3, and OATP transporter pathways
- Single Donor Lots are characterized for for CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A4/5, FMO activity
- Large inventory of pooled (HEP10™) and single donor cryopreserved human hepatocytes available
- Specialty lots available, including low CYP2D6 metabolizers
GIBCO® metabolism-qualified hepatocytes have passed our test for activity of major CYP450 enzymes following the test conditions outlined in Table 3.
Table 3. Test conditions for determining the metabolic capacity of GIBCO® Human Cryopreserved Suspension Hepatocytes
| Enzyme || Substrate || [Substrate] || Incubation || Marker metabolite |
|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
Table 4. Incubation conditions for the transporter uptake assay in hepatocytes suspensions.
| Substrate || Concentration (µM) || Incubation time (min) |
|Figure 4.Human hepatocytes qualified for suspension metabolism applications.|