Example Metabolic Activity (nmoles/hr/106 cells)
Substrate | Enzyme | Concentration (µM) | Day 1 | Day 4 | Day 8 |
|---|---|---|---|---|---|
| 7-Ethoxycoumarin | Phase I | 100 | 0.074 | 0.183 | 0.181 |
| 7-Hydroxycoumarin | Phase II | 100 | 14.380 | 6.640 | 8.460 |
| 7-Hydroxycoumarin | Phase II | 100 | 3.848 | 2.484 | 1.572 |
Culture Conditions and Morphology
Cryopreserved primary rat hepatocytes were thawed and plated with HUREL PlatinumHeps™ Media supplemented with 10% serum and subsequently changed 24 hours post-seeding to HUREL PlatinumHeps™ maintenance Media. CYP substrate concentrations are given in the table above along with metabolite formation recorded as nmoles/hr/106 cells. All incubations were carried out in triplicate on days D1, D4, and D8 upon cell delivery and incubated for 60 minutes. Reactions took place in a humidified incubator at 37°C, in 5% CO2. Collected supernatants were stored at -20°C until further LC/MS/MS analysis.
Day 1 – Morphology
Phase contrast image in a 24- well at a 10x magnification
Day 7 – Morphology
Phase contrast image in a 24- well at a 10x magnification.
Day 7 – Bile Canaliculi
Bile canaliculi assayed via 5- (and-6)-carboxy-2’, 7’- dichlorofluorescein diacetate (C- DCFDA) stain at a concentration of 5 μM and imaged in the GFP channel in a 96-well at 10x magnification with filters EX/EM 492-495/512-527 nm.