Use of Cryopreserved Transiently Transfected Cells in High-Throughput Pregnane X Receptor Transactivation Assay

Abstract

Cryopreserved, transiently transfected HepG2 cells were compared to freshly transfected HepG2 cells for use in a pregnane X receptor (PXR) transactivation assay. Assay performance was similar for both cell preparations; however, cryopreserved cells demonstrated less interassay variation. Validation with drugs of different PXR activation potencies and efficacies demonstrated an excellent correlation (r(2) > 0.95) between cryopreserved and fresh cells. Cryopreservation did not change the effect of known CYP3A4 inducers that have poor cell permeability, indicating that cryopreservation had little effect on membrane permeability. In addition, cryopreserved HepG2 cells did not exhibit enhanced susceptibility to cytotoxic compounds compared to transiently transfected control cells. The use of cryopreserved cells enables this assay to run with enhanced efficiency.

A High-Content Glucocorticoid Receptor Translocation Assay for Compound Mechanism-of-Action Evaluation

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Induction of Xenobiotic Receptors, Transporters, and Drug Metabolizing Enzymes by Oxycodone

“…After 30 minutes incubation with luciferase, the plate was read on a Packard TopCount plate reader (GMI Inc., Ramsey, MN) for luminescence intensity. Cytotoxicity was assessed using Alamar blue as previously described (Zhu et al, 2007).…”

Tools and Strategies for the Assessment of Enzyme Induction in Drug Discovery and Development

“…The promoter element can be derived from a gene of interest, CYP3A4 , CYP1A2 , CYP2B6 , and so on, or from a general promoter, commonly thymidine kinase ( TK ) or cytomegalovirus ( CMV ). Opinions vary on the advantages and disadvantages of each, but for drug metabolism induction studies, the core promoter from a gene of interest is the most commonly used (Raucy et al, 2002 ;Zhu et al, 2007 ;Cui et al, 2008 ). The important element is the enhancer element, which is the element to which the receptor of interest will bind.…”