Example Metabolic Activity (nmoles/hr/106 cells)
| Substrate | Enzyme | Concentration (µM) | Day 1 | Day 4 | Day 8 |
|---|---|---|---|---|---|
| 7-Ethoxycoumarin | Phase I | 25 | 0.07 | 0.13 | 0.22 |
| 7-Hydroxycoumarin | Phase II | 100 | 9.18 | 19.58 | 9.24 |
| 7-Hydroxycoumarin | Phase III | 100 | 2.5 | 6.9 | 4.11 |
Culture Condition and Morphology
Cryopreserved primary dog hepatocytes were thawed and plated with HUREL PlatinumHeps™ Media supplemented with 10% serum and subsequently changed 24 hours post-seeding to HUREL PlatinumHeps™ Basal 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 96-well at a 10x magnification
Day 7 – Morphology
Phase contrast image in a 96-well at a 10x magnification
Day 7 – Bile Canaliculi
Bile canaliculi assayed via 5-(and-6)-carboxy-2’, 7’-dichlorofluorescein diacetate (CDFDA) stain at a concentration of 5 µM and imaged in the GFP channel in a 96-well at 10x magnification