Comprehensive characterization and optimization of Caco-2 cells enabled the development of a miniaturized 96-well permeability assay. (3rd July 2022)

• Record Type: Journal Article
• Authors: Cai, Xianmei; Patel, Shivani; Huang, Chunli; Paiva, Anthony; Sun, Yongnian; Barker, Gregory; Weller, Harold; Shou, Wilson
• Is Part Of: Xenobiotica. Volume 52:Number 7(2022)
• Page Start: 742
• Page End: 750
• Publication Date: 2022-07-03
• Subjects: Caco-2 -- 96-well -- Pgp -- targeted proteomics -- high-throughput -- flow cytometry -- ADME profiling -- permeability -- LC-MS/MS; Metabolism -- Periodicals; Drugs -- Physiological effect -- Periodicals; Food additives -- Periodicals; Chemicals -- Physiological effect -- Periodicals; Biochemistry -- Periodicals; Pharmaceutical Preparations -- metabolism -- Periodicals; Metabolism -- Periodicals; 574.133
• DOI: 10.1080/00498254.2022.2133648
• Languages: English
• ISSNs: 0049-8254

Abstract

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.