Comprehensive characterization and optimization of Caco-2 cells enabled the development of a miniaturized 96-well permeability assay

Abstract

1. 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.

2. 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.

3. 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.

4. 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.

Keywords:

• Caco-2
• 96-well
• Pgp
• targeted proteomics
• high-throughput
• flow cytometry
• ADME profiling
• permeability
• LC-MS/MS

Acknowledgements

The authors thank Dr. Hong Shen and Yueping Zhang for sharing their insight and experience in proteomics’ application in permeability assay.

Disclosure statement

No potential conflict of interest was reported by the author(s).