ABSTRACT
In vitro models that can faithfully replicate critical aspects of kidney tubule function such as directional drug transport are in high demand in pharmacology and toxicology. Accordingly, development and validation of new models is underway. The objective of this study was to characterize physiological and transport functions of various sources of human renal proximal tubule epithelial cells (RPTECs). We tested TERT1-immortalized RPTEC, including OAT1-, OCT2- or OAT3-overexpressing variants, and primary RPTECs. Cells were cultured on transwell membranes in static (24-well transwells) and fluidic (transwells in PhysioMimix{trade mark, serif} T12 organ-on-chip with 2 mL/s flow) conditions. Barrier formation, transport, and gene expression were evaluated. We show that two commercially available primary RPTECs were not suitable for studies of directional transport on transwells because they formed a substandard barrier even though they exhibited higher expression of transporters, especially under flow. TERT1-parent, -OAT1 and -OAT3 cells formed robust barriers, but were unaffected by flow. TERT1-OAT1 cells exhibited inhibitable para-aminohippurate transport, it was enhanced by flow. However, efficient tenofovir secretion and perfluorooctanoic acid reabsorption by TERT1-OAT1 cells were not modulated by flow. Gene expression showed that TERT1 and TERT1-OAT1 cells were most correlated with human kidney than other cell lines, but that flow did not have noticeable effects. Overall, our data show that addition of flow to in vitro studies of the renal proximal tubule may afford benefits in some aspects of modeling kidney function, but that careful consideration of the impact such adaptations would have on the cost and throughput of the experiments is needed. Significance Statement The topic of reproducibility and robustness of the complex microphysiological systems is looming large in the field of biomedical research; therefore, the uptake of these new models by the end-users is slow. This study systematically compared various RPTEC sources and experimental conditions, aiming to identify the level of model complexity needed for testing renal tubule transport. We demonstrate that while tissue chips may afford some benefits, their throughput and complexity need careful consideration in each context of use.
ABSTRACT
Microphysiological systems are an emerging area of in vitro drug development, and their independent evaluation is important for wide adoption and use. The primary goal of this study was to test reproducibility and robustness of a renal proximal tubule microphysiological system, OrganoPlate 3-lane 40, as an in vitro model for drug transport and toxicity studies. This microfluidic model was compared with static multiwell cultures and tested using several human renal proximal tubule epithelial cell (RPTEC) types. The model was characterized in terms of the functional transport for various tubule-specific proteins, epithelial permeability of small molecules (cisplatin, tenofovir, and perfluorooctanoic acid) versus large molecules (fluorescent dextrans, 60-150 kDa), and gene expression response to a nephrotoxic xenobiotic. The advantages offered by OrganoPlate 3-lane 40 as compared with multiwell cultures are the presence of media flow, albeit intermittent, and increased throughput compared with other microfluidic models. However, OrganoPlate 3-lane 40 model appeared to offer only limited (eg, MRP-mediated transport) advantages in terms of either gene expression or functional transport when compared with the multiwell plate culture conditions. Although OrganoPlate 3-lane 40 can be used to study cellular uptake and direct toxic effects of small molecules, it may have limited utility for drug transport studies. Overall, this study offers refined experimental protocols and comprehensive comparative data on the function of RPETCs in traditional multiwell culture and microfluidic OrganoPlate 3-lane 40, information that will be invaluable for the prospective end-users of in vitro models of the human proximal tubule.
Subject(s)
Kidney Tubules, Proximal , Microphysiological Systems , Humans , Reproducibility of Results , Prospective Studies , KidneyABSTRACT
Establishing the functionality, reproducibility, robustness, and reliability of microphysiological systems is a critical need for adoption of these technologies. A high throughput microphysiological system for liver studies was recently proposed in which induced pluripotent stem cell-derived hepatocytes (iHeps) and non-parenchymal cells (endothelial cells and THP-1 cells differentiated with phorbol 12-myristate 13-acetate into macrophage-like cells) were co-cultured in OrganoPlate® 2-lane 96 devices. The goal of this study was to evaluate this platform using additional cell types and conditions and characterize its utility and reproducibility. Primary human hepatocytes or iHeps, with and without non-parenchymal cells, were cultured for up to 17 days. Image-based cell viability, albumin and urea secretion into culture media, CYP3A4 activity and drug metabolism were assessed. The iHeps co-cultured with non-parenchymal cells demonstrated stable cell viability and function up to 17 days; however, variability was appreciable both within and among studies. The iHeps in monoculture did not form clusters and lost viability and function over time. The primary human hepatocytes in monoculture also exhibited low cell viability and hepatic function. Metabolism of various drugs was most efficient when iHeps were co-cultured with non-parenchymal cells. Overall, we found that the OrganoPlate® 2-lane 96 device, when used with iHeps and non-parenchymal cells, is a functional liver microphysiological model; however, the high-throughput nature of this model is somewhat dampened by the need for replicates to compensate for high variability.
