Optimization of Renal Organoid and Organotypic Culture for Vascularization, Extended Development, and Improved Microscopy Imaging

Summary

This work describes two methods for studying organ development, an improved xenotransplantation setup on chorioallantoic membrane (CAM) from avian embryos that allows for vascularization of cultured embryonic organs and organoids and a novel fixed z-direction organ culture method with modified experimental conditions that allows for high-resolution time-lapse confocal imaging.

Abstract

Embryonic kidney organotypic cultures, and especially pluripotent stem cell-derived kidney organoids, are excellent tools for following developmental processes and modelling kidney disease. However, the models are limited by a lack of vascularization and functionality. To address this, an improved protocol for the method of xenografting cells and tissues to the chorioallantoic membrane (CAM) of an avian embryo to gain vascularization and restoration of blood flow was developed. The grafts are overlaid with custom-made minireservoirs that fix the samples to the CAM and supply them with culture medium that protects the grafts from drying. The improved culture method allows xenografts to grow for up to 9 days. The manuscript also describes how to provide optimal conditions for long-term confocal imaging of renal organoids and organotypic cultures using the previously published Fixed Z-Direction (FiZD) method. This method gently compresses an embryonic organ or organoid between a glass coverslip and membrane in a large amount of medium and provides excellent conditions for imaging for up to 12 days. Together, these methods allow vascularization and blood flow to renal organoids and organotypic kidney cultures with improved confocal imaging. The methods described here are highly beneficial for studying fundamental and applied functions of kidneys ex vivo. Both methods are applicable to various types of tissues and organoids.

Introduction

Organotypic culture of embryonic kidneys became an important model to study nephrogenesis decades ago^1,2,3. Renal organoids represent an advanced model system for studying development of healthy and diseased kidneys⁴. The main drawback for both methods, however, is that neither method recapitulates the main function of the kidney: blood filtration. Nephrons and renal vasculature develop in renal organoids and organotypic cultures similarly to early stage in vivo development; however, the glomeruli formed in vitro remain avascular⁵

Protocol

Animal care and procedures were in accordance with Finnish national legislation for the use of laboratory animals, the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes (ETS 123), and the EU Directive 86/609/EEC.

1. Fabrication of minireservoirs for cultivation of mouse embryonic kidneys and renal organoids on chicken CAM and setting up xenotransplantation experiments

1. Use transwell cell culture inserts designed for 6 well or 12 well plates.
   NOTE: Depending on the particular experiment, large or small minireservoirs can be used. It is best to use small minirese....

Results

The CAM culture protocol presented here enabled highly efficient vascularization of renal organoids and embryonic kidneys as a result of xenotransplantation on chicken CAM (Figure 1, Movie 1). Minireservoirs containing culture medium supplied nutrients to donor tissue and protected it from drying during the time period preceding proper vascularization. This method provided permissive conditions for donor-derived endothelial cells to grow. Therefore, renal vasculature in the .......

Discussion

Two detailed protocols are presented that refine the classical renal organotypic culture method, and enable vascularization, extended development, and optimal 4D (i.e., 3D image and time) imaging of ex vivo embryonic kidneys and organoids. This section highlights the critical steps in the methods and discusses troubleshooting.

The significant difference between other CAM culture methods and this improved chicken CAM culture method is the use of custom-made minireservoirs in the xenografting of.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Adjustable Spade Drill Bit	Bosch	2609255277	For drilling 20 mm diameter holes
Automatic Egg Turner	OLBA B.V., Netherlands	AT-42	For incubation of eggs before ex ovo setup.
Cell Incubator	Panasonic	13090543	Temperature set at 37° C, 90% humidity, 5% CO2
Cell Incubator	SANYO	10070347	Chicken CAM-culture incubator. Temperature set at 37° C, 90% humidity, 5% CO2
Cellstar 6-well Plate	Greiner	M9062	16 mm depth of wells to match with the inserts
Circular Saw Blade for Dremel Rotary Tool	Dremel	SC690
Confocal Microscope	Zeiss	LSM780
Corning Transwell Multiple Well Plate with Permeable Polycarbonate Membrane Inserts	Corning	10301031	For 6-well plates. 40 µm pore size
Countersink Drill Bit	Craftomat, Bauhaus	22377902	For polishing (20.5 mm, 1/4", HSS)
Disposable Glass Capillary Tube	Blaubrand	7087 33
Disposable Scalpel	Swann-Morton	0501
Dissecting Microscope	Olympus	SZ61
Drilling Machine	Bosch	GSR 18 V-EC Professional
Dulbecco's Modified Eagle's Medium	Sigma	D777	High glucose
Egg Incubator Compact S84	Grumbach, Germany	8012	For incubation of eggs before ex ovo setup. Temperature set at 38° C with relative humidity set above 60%
Ethanol (70%)	VWR
Fertilized Eggs	Haaviston Siitoskanala, Panelia, Finland		Hy-Line White and Nick Chick
Fetal Bovine Serum	HyClone	SH3007003HI Thermo Scientific
Forceps DUMONT #5	Dumont	#5SF
Glass Coverslips	Menzel-Gläser	Menzel BBAD02200220#A1TBCMNZ#0##	22x22 mm
Histoacryl Glue	Braun	1050052
Matrigel	Corning	356230
On-stage Incubator	Okolab		Boldline, custom made
PBS -/-	Corning	20-031-CV
PBS +/+	Biowest	X0520-500	Washing the mini reservoirs.
Penicillin and Streptomycin	Sigma	P4333
Polystyrene Beads	Corpuscular	1000263-10	70 µm in diameter
Rotary Multi Tool System	Dremel	4000
Soldering Iron	Weller	TCP S	Heated glass capillary can also be used
Thincert 12-well Cell Culture Inserts With 0.4 µm-pore Polystyrene Membrane	Greiner Bio-One	665641
Thincert 6-well Cell Culture Inserts With 0.4 µm-pore Polystyrene Membrane	Greiner Bio-One	657610