Protocol to analyze bioenergetics in single human induced-pluripotent-stem-cell-derived kidney organoids using Seahorse XF96

Abstract

Metabolic derangement is a key culprit in kidney pathophysiology. Organoids have emerged as a promising in vitro tool for kidney research. Here, we present a fine-tuned protocol to analyze bioenergetics in single human induced-pluripotent-stem-cell (iPSC)-derived kidney organoids using Seahorse XF96. We describe the generation of self-organized three-dimensional kidney organoids, followed by preparation of organoids for Seahorse XF96 analysis. We then detail how to carry out stress tests to determine mitochondrial and glycolytic rates in single kidney organoids.

Keywords: Cell Differentiation; Metabolism; Organoids; Stem Cells.

Graphical abstract

Figure 1

Overview of the iPSC maintenance culture, passaging and aggregation for making kidney organoids (A) Representative bright field image of iPSC colonies with recommended size before cell passaging. Scale bar, 1,000 μm. (B) Representative bright field image of iPSC colonies with bigger size than recommended before cell passaging. Scale bar, 1,000 μm. (C) Representative bright field image of iPSC colonies with smaller size than recommended before cell passaging. Scale bar, 1,000 μm. (D) Representative bright field image with required iPSC colony dissociation degree during cell passaging for iPSC maintenance culture. Scale bar, 400 μm. (E) Representative bright field image with iPSC colony dissociation into single cells. Scale bar, 1,000 μm. (F) iPSC pellet in a 1.5 mL tube prior to plating to generate cell aggregates which will derive into kidney organoids. (G) Picking-up the iPSC pellet from (F) using a 200 μL wide-bore tip. (H) Picked-up iPSC pellet from (F) in the bottom of a 200 μL wide-bore tip. (I) Depositing of intact iPSC aggregates on a Transwell filter. (J) Depositing of damaged iPSC aggregates on a Transwell filter.

Figure 2

Schematic of the iPSC-derived kidney organoid culture protocol After iPSC differentiation into either anterior intermediate mesoderm (AIM) (3d) or posterior intermediate mesoderm (PIM) (5d), cells are cultured in a 3D air-liquid interface Transwell system for an additional 18 days using a mixture of growth factors as indicated to stimulate morphogenic cues essential for nephrogenesis. BMP7, bone morphogenic protein 7; CHIR, CHIR 99021; FGF9, fibroblast growth factor 9; hEGF, human epidermal growth factor; SDF1β, Stromal cell-derived factor-1 β.

Figure 3

Seahorse sensor cartridge hydration and injection port loading Seahorse sensor cartridge on top of a utility plate with injection ports A–D, with details of the O₂- and pH-sensitive probe tips of a Seahorse sensor cartridge and substrate/inhibitor stock solutions loading in the injection ports of the Seahorse sensor cartridge.

Figure 4

Overview of kidney organoid re-plating for Seahorse analysis (A) iPSC-derived kidney organoids cultured in a 6-well Transwell plate on an air/liquid interface. (B) Representative bright field image of a kidney organoid. Scale bar, 250 μm. (C) Schematic depicting the strategy individualize every kidney organoid by cutting the porous membrane closely around each one by using a scalpel blade. (D) Individualized kidney organoid by cutting the Transwell porous membrane. (E) Re-plated kidney organoids on a Seahorse XF96 cell culture microplate. (F) Empty well of a Seahorse XF96 cell culture microplate showing its three nodes at the bottom. (G) Correct Geltrex™ immobilized kidney organoid in the middle of a well of a Seahorse microplate. (H) Disrupted Geltrex™ immobilized kidney organoid in the middle of a well of a Seahorse microplate.

Figure 5

Mitochondrial stress test parameters in single kidney organoids (A) Representative curve of OCR dynamics with commonly used reagents used for mitochondrial stress test. (B) OCR associated to a mitochondrial stress test of kidney organoids treated with aristolochic acid (2 ng/mL) for 72 h. (C) Bar chart showing mitochondrial respiration function parameters from (B). Data are normalized by the relative kidney organoid cell number. The difference between two independent groups was analyzed with the nonparametric Mann-Whitney test. Data are reported as mean ± standard error of mean (SEM), n = 3 biological replicates. A P-value of 0.05 or less was considered statistically significant. ∗p < 0.05 compared with the control condition. Data were analyzed using GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA).

Figure 6

Glycolysis stress test parameters in single kidney organoids (A) Representative curve of ECAR dynamics with commonly used reagents used for glycolysis stress test. (B) ECAR associated to a glycolysis stress test of kidney organoids treated with aristolochic acid (2 ng/mL) for 72 h. (C) Bar chart showing glycolysis function parameters from (B). Data are normalized by the relative kidney organoid cell number. The difference between two independent groups was analyzed with the nonparametric Mann-Whitney test. Data are reported as mean ± standard error of mean (SEM), n = 3 biological replicates. A P-value of 0.05 or less was considered statistically significant. ∗p < 0.05 compared with the control condition. Data were analyzed using GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA).