3D micro tissue-microfluidic platform for development of drug screening for treatment or prevention of chronic kidney disease

Abstract

Purpose: Three-dimensional (3D) micro tissue-on-a chip models that epitomize kidney organ are used for drug screening or disease modeling for treatment or prevention of chronic kidney disease. Renal tissue is composed of various including mesangial cells, renal tubular cells and fibroblast. It is necessary to screen drugs for treatment of renal fibrosis that can affect these various cells. To overcome these problems, we developed a threedimensional (3D) kidney organoid with mesangial cell, renal tubular cell and renal fibroblast, and designed the microfluidic device for kidney organoids culturing and drug screening. Methods: The 96-well format-based microfluidic platform was fabricated by using a conventional soft lithography process with Polydimethylsiloxane (PDMS) material. The 96-well format based microfluidic platform contains 10 individual channels, each with 6 micro tissue compartments chamber in the side wall. The medium perfusion in the 96- well format based microfluidic platform is driven by gravity force. The platform was placed on an automated tilting plate, the two reservoirs are located at different height so medium flows. To construction of 3D kidney organoids, rat kidney fibroblast cell (NRK-49F), rat renal tubular epithelial cell (NRK-52E), and rat mesangial cell (RMC) were cultured together for 2 days or longer at Ubottom 96-well plate. The organoids were cultured in Dulbecco’s modified Eagle medium/nutrient mixture F-12 medium containing 8.2% fetal bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin. The morphology and viability (LIVE/DEAD staining) of cultured from 2 to 7 days 3D kidney organoids were detected using confocal microscope. The cultured 3D kidney organoids for 3 days were pretreated 1 mM of FDA Approved & Pharmacopeial Drug Library for 1 h, and then stimulated using 5 ng/mL TGF-β for 24 h. These supernatants were analyzed using Rat PAI-1 simple step ESLIA Kit according to manufacturer’s instruction. Results: PDMS-based microfluidic device works with gravity-driven flow through automated chip-tilting without any need for additional tubing and external pumps. Kidney organoids were prepared using mesangial cell, renal tubular cell and renal fibroblast. Culturing conditions of kidney organoids were experimentally optimized. Morphology and cell viability of kidney organoids were well maintained in U-bottom plate and microfluidic device for 7 days. To mimic renal fibrosis model, kidney organoids were treated with TGF-β. The secretion of PAI-1, a renal fibrotic factor, was increased by TGF-β treatment in these organoids. A renal fibrosis treatment agent without hepatoxicity can be effectively screened using co-cultured with kidney and liver organoids in microfluidic device. These results showed that kidney organoids on-a-chip could be used as a novel drug screening tool and provide hope for a potential treatment of renal fibrosis.