Establishment of ion channel and ABC transporter assays in 3D-cultured ReNcell VM on a 384-pillar plate for neurotoxicity potential

Abstract

Neurotoxicity potential of compounds by inhibition of ion channels and efflux transporters has been studied traditionally using two-dimensionally (2D) cultured cell lines such as CHO and HEK-293 overexpressing the protein of interest. However, these approaches are time consuming and do not recapitulate the activity of ion channels and efflux transporters indigenously expressed in neural stem cells (NSCs) in vivo. To overcome these issues, we established ion channel and transporter assays on a 384-pillar plate with three-dimensionally (3D) cultured ReNcell VM and demonstrated high-throughput measurement of ion channel and transporter activity. RNA sequencing analysis identified major ion channels and efflux transporters expressed in ReNcell VM, followed by validating 3D ReNcell-based ion channel and transporter assays with model compounds. Major ion channel activities were measured by specifically inhibiting potassium channels Kv 7.2 with XE-991 and Kv 4.3 with fluoxetine, and a calcium channel with 2-APB. Activities of major efflux transporters, MDR1, MRP1, and BCRP, were assessed using their respective blockers, verapamil, probenecid, and novobiocin. From this study, we demonstrated that 3D-cultured ReNcell VM on the 384-pillar plate could be a good alternative to rapidly identify environmental chemicals and therapeutic compounds for their role in modulating the activity of ion channels and efflux transporters, potentially leading to neurotoxicity.