GIBCO® Human Cryopreserved Hepatocytes, Transporter Qualified

  • 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)

How we qualify suspension and plateable human hepatocytes for transporter uptake

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
CYP1A2Omeprazole50 µMPhenacetin100 µM15 minAcetaminophen
CYP2B6Phenobarbital1,000 µMBupropion500 µM20 minHydroxybupropion
CYP3ARifampicin10 µMTestosterone200 µM14 min6β-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

  • Useful for assessing intrinsic clearance (CLint) in low-turnover compounds
  • Prequalified according to CLint of midazolam, tolbutamide, dextromethorphan
  • ≥80% viability and ≥75% attachment efficiency

How we qualify plateable human hepatocytes for metabolism (intrinsic clearance)
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
Midazolam0.50 µM 0, 1, 2, 4, 6, 8 hr
Tolbutamide1.00 µM 0, 4, 6, 8, 18, 24 hr
Dextromethorphan1.00 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

  • 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

How we qualify suspension human hepatocytes for metabolism
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
CYP1A2Phenacetin100 µM15 minAcetaminophen
CYP2B6Bupropion500 µM20 minHydroxybupropion
CYP2C8Paclitaxel20 µM45 min6α-Hydroxypaclitaxel
CYP2C9Diclofenac25 µM15 min4'-Hydroxydiclofenac
CYP2C19(S)-mephenytoin250 µM30 min4'-Hydroxymephenytoin
CYP2D6Dextromethorphan15 µM15 minDextrorphan
CYP2E1
Chlorzoxazone
250 μM
15 min
6-Hydroxychlorzoxazone
CYP3ATestosterone200 µM14 min6β-Hydroxytestosterone
CYP3A
Midazolam
10 µM10 min
1-hydroxymidazolam
Phase I and II7-Ethoxycoumarin100 µM30 min7-HCG, 7-HCS, 7-HC*
FMO
Benzydamine
250 μM
30 minBenzydamine-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)
Taurocholoate11
Digoxin11
E2-17G11


Figure 4. Human hepatocytes qualified for suspension metabolism applications.