How were CYP enzyme activities measured in Cellartis enhanced hiPS-HEP cells
How were CYP enzyme activities measured in Cellartis enhanced hiPS-HEP cells
Liquid chromatography/mass spectrometry (LC/MS) was used to measure the formation of these specific metabolites: acetaminophen (CYP1A), 4-OH-Diclofenac (CYP2C9), OH-Bufuralol (CYP2D6), and 1-OH-Midazolam (CYP3A). The CYP activities of Cellartis Enhanced hiPS-HEP cells were analyzed at Days 4, 12, and 19 after thawing as part of routine quality control. LC/MS analysis was performed at Pharmacelsus GmbH, Germany.
To measure CYP activity, Cellartis Enhanced hiPS-HEP cells were washed twice with prewarmed William medium E (+0.1% PEST). Then, the activity assay was started by adding 110 µl per cm2 culture area of prewarmed William medium E containing 0.1% PEST, 25 mM HEPES, 2 mM L-Glutamine, and the following probe-substrate cocktail:
-CYP1A, substrate: Phenacetin, concentration: 10 µM
-CYP2B6, substrate: Bupropion, concentration: 10 µM
-CYP2C9, substrate: Diclofenac, concentration: 10 µM
-CYP2D6, substrate: Bufuralol, concentration: 10 µM
-CYP3A, substrate: Midazolam, concentration: 5 µM
After two hours at 37°C, 100 µl of supernatant was collected and kept at –80°C until LC/MS analysis. The metabolite concentrations measured by LC/MS were normalized to the amount of protein per well (determined using the Pierce BCA Protein Assay Kit) and assay duration (120 min).