Development of highly-differentiated human primary proximal tubule MPS model (aProximate MPS™ Flow)

Introduction

The proximal tubule (PT) is the key nephron segment mediating renal drug elimination and is the primary site of drug induced nephrotoxicity. A kidney MPS model has been developed to model various segments of the kidney nephron, including the proximal tube. However, current animal studies have proved poorly predictive of outcome in a human kidney. To address this, there has been a recent upsurge in physiological relevant microphysiological systems (MPS) of PT to recapitulate differentiation and function in vitro. Here we present results from our recently developed Proximal Tubule aProximate MPS^TM human PT platform (Patent No:G001336.GB), in which primary PT cells are subject to fluidic media flow and a shear stress between 0.1-2 dynes/cm².

Methods

To develop the highly-differentiated human primary proximal tubule MPS model (aProximate MPSTM Flow), Computational Fluid Dynamics (CFD) was used to optimise uniformity of the shear stress within the flow chambers. Using CAD software and 3D printing, we were able to rapidly prototype different plates with different format to determine optimal performance for primary cells within the flowplate system. Primary PT cells were seeded on the underside of Corning ThinCert™ and incubated overnight. After flipping, the flowplate was placed on a rocking platform at angle θ allowing passive liquid levelling creating lateral flow without the use of a pumping system.

Results

A characterisation and barrier function assessment was carried out during the development of the PT MPS model. qPCR was used to determined gene expression levels of key transporters involved in primary PT cells function. IHC was used to confirm expression levels and to determine phenotypic differences between static and FSS-exposed cells. Barrier function and integrity were assessed using TEER and Lucifer Yellow assays.

There were also Functional Assays on aProximate MPS™ Flow carried out. There was a Creatinine uptake at 90 minutes then a FITC-Albumin Uptake at one hour with large dose range; a nephrotoxicity assessment was carried out using cisplatin treatment on stimulated cells vs control and there was a ATP release measured at 72 hours following treatment. TEER and Renal Injury Biomarker Release was measured at 72 hours following treatment, with the latter treatment using MSD.

Review the full results here.

Conclusion

This dataset suggests that growing human PT cells on ThinCert™ with media flow across the apical membrane, significantly improves phenotype and function and has significant benefit to the utility and near-physiology of the model.