Abstract
Purpose :
Interest in ARPE-19s has been revitalized in recent years due to the ability to differentiate them using nicotinamide media formulation to develop multiple primary cell traits and attributes[1]. Whilst the media formulation is established, culture time and growth surfaces differ throughout the literature. The purpose of this study is to propose a standard minimum time required for ARPE-19s to transition.
Methods :
ARPE-19 cells were plated at confluence in uncoated wells or pretreated with type I collagen (xng/cm2). Culture medium was changed to either DMEM/F12+ 1% FBS, DMEM/F12+ 10% FBS, or MEM NIC[2]+ 1% FBS. On days 1, 3, 7, and 10, cells were utilized for CellTiter-Glo® (mitochondrial function), flow cytometry using JC-1 (mitochondrial membrane potential), and MitoSox Green (ROS production). Cell lysates were probed for RPE65 and TOMM20 by western blotting. Fixed cells were stained with ZO-1 and assessed by immunofluorescence and confocal microscopy. Statistics were performed in JASP 0.18.1.
Results :
Pretreatment of wells with type I collagen had no significant effect on any metric at any time point. Circumferential ZO-1 staining was evident from day 7 in MEM NIC media and by day 10 in all groups. A RPE differentiation marker (RPE65) expression was higher on day 10 in the MEM NIC cells (p < 0.05). Assessment of mitochondrial function was decreased by Tomm20 expression (p < 0.05) and the CellTiter- Glo assay (H = 9.248, 3, p<0.05) by day 10 in the MEM NIC treated group.
Conclusions :
Collagen I is not necessary for ARPE-19 differentiation and its inclusion poses an additional challenge when investigating some wound healing models. Cellular differentiation (ZO-1 and RPE65 expression) occurs by day 7 with MEM NIC media, however reduced mitochondrial activity does not occur until day 10. While some aspects are changed by day 7, it is proposed that 10 days in MEM NIC media be the minimum required.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.