Real-time drug efflux in human kidney organoid (Version 1.0)

Version

1.0

Notice

This page is the corresponding protocol tomestone page generated as part of the ATLAS-D2K shutdown in July 2025. Many links on this page may be broken.

Authors

Ryuji Morizane; Astia Safitri

Keywords

[‘3D imaging’, ‘3D culture’, ‘hiPSC’, ‘immunofluorescent’, ‘organoid’, ‘nephron’, ‘proximal tubule epithelial cells’]

Subjects

[‘Imaging’]

Release Date

2022-04-20

Abstract

This protocol utilizes Rhodamine123 to observe the MDR1 activities in human iPSCs derived kidney organoid

Procedure

Sample preparation on the plate/chamber slides. A coated 24-well plate (Corning) and coated chamber slides (Ibidi) are used for imaging. For non-coated plate, 1% and 5% Geltrex (Gibco) coating was done on 24-well plate and chamber slides respectively with 1-hour incubation at 37oC. After removal of the Geltrex coating solution, organoids were placed on the bottom of the well/chamber slides and incubated at 37oC for overnight. The attached organoids were observed to have stromal-like cells sprouting and spreading on the surface of the well/chamber slides.

_Live imaging set up. _Plate/chamber slides containing live stained kidney organoids was placed into STX-IXZWX series stage incubator system (Tokai Hit) following the manufacturer manual. Samples were incubated at 37oC with 5% CO2 in the live-imaging chamber that was installed on the confocal microscope stage (Leica Stelaris-8) for real-time imaging.

Drug efflux assay on proximal tubules. The attached organoids were treated with 5µg/ml of wheat germ agglutinin/WGA conjugated-647 (biotium) and incubated at 37oC overnight in orbital shaker at 80 rpm. For inhibitor treatment, 20 µg/ml of PSC-833 (Sigma) was incubated with organoids for 1h. Rhodamine 123 (Rh123, Sigma) 10 µM was added right before live imaging. RPMI-free phenol red (Gibco) is consecutively used as culture medium for live imaging.

Timing

Organoid attachment : 12h Structural live staining (WGA) : 1-12h Incubation with Inhibitor : 1h Rh123 transport : 20min

Consortium

(Re)Building a Kidney (RBK) Consortium