Example Metabolic Activity (nmoles/hr/106 cells)
Substrate | Enzyme | Concentration (µM) | Day 1 | Day 4 | Day 8 |
|---|---|---|---|---|---|
| 7-Ethoxycoumarin | Phase I(ECOD) | C100 | 0.05 | 0.05 | 0.05 |
| Phase II(UGT) | 0.05 | 0.05 | 0.05 | ||
| Phase II(SULT) | 0.05 | 0.05 | 0.05 | ||
| 7-Hydroxycoumarin | Phase II(UGT) | 100 | 0.05 | 0.05 | 0.05 |
| Phase II(SULT) | 0.05 | 0.05 | 0.05 |
Culture Conditions and Morphology
Cryopreserved 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/million cells. All incubations were carried out in triplicate on days 1, 4, and 8 (customer day) 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 microns and imaged in the GFP channel in a 96-well at 10X magnifcation with filters EX/EM 492-495/512-527 nm.