Comprehensive characterization and optimization of Caco-2 cells enabled the development of a miniaturized 96-well permeability assay.
Xenobiotica
; 52(7): 742-750, 2022 Jul.
Article
en En
| MEDLINE
| ID: mdl-36217915
ABSTRACT
Assessment of compound permeability through a Caco-2 cell monolayer is a well-accepted model to evaluate its in-vivo permeability potential and transporter interaction. While this assay has commonly been conducted using a 24-well assay plate format, a miniaturised 96-well assay format is highly desirable to achieve greater capacity and higher efficiency.Previous attempts to convert this assay from 24-well to 96-well format at our lab, however, had met with varied efflux capacities and unacceptable efflux ratios for digoxin, a substrate of P-glycoprotein (Pgp), which indicated inadequate Pgp transporter expression in the 96-well format.These challenges in converting the assays were attributed to the heterogeneous and unstable nature of the Caco-2 cells. To overcome the challenges, single-cell sorting of Caco-2 cells was conducted by flow cytometry to obtain a more homogeneous and stable cell population. The sorted cells were then seeded to 96-well transwell plates and the Pgp expression under various cell culture conditions was monitored by a LC-MS/MS-based targeted proteomics method.Through cell sorting and direct Pgp expression measurement, Caco-2 cells with adequate and sustained Pgp expression in a 96-well format were obtained, which led to the successful development and implementation of a 96-well Caco-2 assay with significant efficiency gain and faster turnaround time than the historical 24-well assay.
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Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Proteómica
/
Espectrometría de Masas en Tándem
Tipo de estudio:
Prognostic_studies
Límite:
Humans
Idioma:
En
Revista:
Xenobiotica
Año:
2022
Tipo del documento:
Article
País de afiliación:
Estados Unidos