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. 2022 Nov 30;12(1):20699.
doi: 10.1038/s41598-022-24945-5.

Creating a kidney organoid-vasculature interaction model using a novel organ-on-chip system

Amanda Bas-Cristóbal Menéndez  1   2 , Z Du  3 , T P P van den Bosch  4 , A Othman  5 , N Gaio  5 , C Silvestri  5 , W Quirós  5 , H Lin  6 , S Korevaar  3 , A Merino  3 , J Mulder  7   8 , M J Hoogduijn  3
Affiliations

Creating a kidney organoid-vasculature interaction model using a novel organ-on-chip system

Amanda Bas-Cristóbal Menéndez et al. Sci Rep. .

Abstract

Kidney organoids derived from human induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study kidney development and disease. However, the lack of vascularization of these organoids often leads to insufficient oxygen and nutrient supply. Vascularization has previously been achieved by implantation into animal models, however, the vasculature arises largely from animal host tissue. Our aim is to transition from an in vivo implantation model towards an in vitro model that fulfils the advantages of vascularization whilst being fully human-cell derived. Our chip system supported culturing of kidney organoids, which presented nephron structures. We also showed that organoids cultured on chip showed increased maturation of endothelial populations based on a colocalization analysis of endothelial markers. Moreover, we observed migration and proliferation of human umbilical vein endothelial cells (HUVECs) cultured in the channels of the chip inside the organoid tissue, where these HUVECs interconnected with endogenous endothelial cells and formed structures presenting an open lumen resembling vessels. Our results establish for the first-time vascularization of kidney organoids in HUVEC co-culture conditions using a microfluidic organ-on-chip. Our model therefore provides a useful insight into kidney organoid vascularization in vitro and presents a tool for further studies of kidney development and drug testing, both for research purposes and pre-clinical applications.

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Conflict of interest statement

C. Silvestri and N. Gaio and W. Quiros Solano are the founders of BIOND Solutions B.V. (BIOND). A. Othman is an employee of BIOND solutions B.V. All other authors declare no competing interests.

Figures

Figure 1
Figure 1
Organoids cultured on chip present all major nephron structures. (A) Schematic representation of the organoid generation protocol timeline, indicating days, addition of factors and the different phases of culture. (B) Schematic representation of the BIOND chip indicating main parts of the device. (C) Kidney organoid on chip (indicated with arrow) at day 20 of the protocol before collection, scale bar = 3 mm. (D) Immunohistochemical staining of sections of kidney organoids cultured both on transwell and on chip. Stainings show both organoids present glomeruli (WT1+), podocytes (PODXL+), proximal tubuli (Villin+) and distal tubuli (E-cadherin+), scale bar = 200 µm.
Figure 2
Figure 2
Kidney organoids cultured on chip show expanded EC populations and directed endothelial maturation patterns. (A) Immunofluorescent images of DAPI-MCAM-PECAM co-staining showing kidney organoids cultured on transwell and on chip, scale bars = 200 µm. (B) Statistical analysis of MCAM and PECAM expression in kidney organoids based on percentage of total area. Organoids cultured on chip (n = 8) showed higher areas positive for both MCAM and PECAM in comparison to organoids cultured on transwell (n = 6). Each data point = one organoid. * = P < 0.05. Error bar = SD. (C) Colocalization analysis showing percentages of colocalized and non-colocalized markers in kidney organoids cultured on transwell and on chip.
Figure 3
Figure 3
GFP+ HUVECs seeded on microfluidic chip channels form synthetic 3D vessels. (A) fluorescence image of the chip’s three microfluidic channels after 48 h of static culture, showing GFP+ HUVECs forming a monolayer inside these channels. (B) Fluorescence image of a chip channel after 48 h of static culture + 24 h of flow, showing HUVECs remained in the channels after exercising fluidic stress and adopted a directionality concurrent with the flow direction. (C) Detail images of the chip channels after 48 h of static culture + 24 h of flow, in which the directionality acquired by the HUVECs can be observed (indicated with arrow). (D) 3D render of confocal stack of a chip channel lined with GFP+ HUVECs showing establishment of a 3D synthetic vessel presenting cells in all planes. Scale bars = 500 µm (A, B, D) and 250 µm (C).
Figure 4
Figure 4
GFP+ HUVECs migrate from the chip channels into the organoids and establish integrated vascular structures presenting an open lumen. (A) Schematic representation of the co-culture timeline of HUVECs and kidney organoids, indicating static culture, exercise of flow and addition and collection points of kidney organoids. (B) Immunofluorescent images of DAPI-PECAM-GFP of organoids co-cultured with GFP+ HUVECs on the microfluidic chip system for 9 days. Shown in the images are vascular structures derived from the GFP+ HUVECs integrated in the organoid tissue. (B1) Whole organoid presenting vascular structures derived from GFP+ HUVECs. (B2) Detail image of large vascular structure on longitudinal section. Open lumen can be appreciated throughout the structure. (B3) Detail image showing continuity of vascular structures formed by HUVECs (PECAM+, GFP+) with native ECs (PECAM+, GFP-). (B4) Detail image of a transversal cut of several vascular structures with clearly visible open lumens. Scale bars = 200 µm.
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