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. Vascularization of ex vivo embryonic kidneys and renal organoids was previously demonstrated in transplantation experiments only under in vivo conditions. For example, transplantation of human pluripotent stem cell-derived renal organoids under a mouse kidney capsule allows development of the nephrons in the organoid to a functional stage⁶.

An intermediate approach between purely in vitro cultures and in vivo transplantation methods is xenotransplantation to the CAM of avian embryos. Vascularization of intact mouse kidney primordia has been demonstrated previously using this system^7,8. However, it was also shown that the renal vasculature in the xenotransplanted murine kidney was derived from the host endothelium, not the graft⁹. This observation significantly reduced the potential of chimeric (avian-mammalian) models of embryonic kidney to study development of the renal vasculature, because the experimental conditions were nonpermissive for the survival of donor-derived endothelial cells.

Presented in the first part of this protocol is an improved method for cultivation of mouse embryonic kidneys on CAM of avian eggs, combining microenvironmental conditions of organotypic culture and xenotransplantation. The main improvement to previous methods is that instead of placing the mouse embryonic kidneys and renal organoids directly on the CAM, the implantation area is overlaid with permeable minireservoirs filled with culture medium that supply the transplanted tissue with nutrients and protect it from drying. The success rate of the experiments significantly increases and the conditions for development of donor-derived vasculature improve. Application of this method to xenotransplant cultures results in the development of glomerular vasculature comprised of endogenous endothelial cells from donor kidneys.

Detailed analysis of cellular morphogenesis is another important application of kidney culture models. Previously reported methods of time-lapse image acquisition of kidney cultures are sufficient only for analysis of overall morphology and patterning of embryonic kidney, but not for tracking individual cells¹⁰. Recently, a novel Fixed Z-Direction (FiZD) method aimed for high-resolution confocal 3D time-lapse imaging of renal organoids and organotypic cultures was described¹¹. In this method, the organoids and embryonic organs are gently compressed between a glass coverslip and a permeable membrane of a transwell insert in a custom-designed plate until the thickness of the sample reaches 70 µm, providing optimal optical conditions for imaging. In the second part of the methods, a detailed protocol for the fabrication of a custom-designed plate and the setup of FiZD experiments for long-term organoid imaging is described.

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 minireservoirs for xenografting up to eight embryonic kidneys or when several minireservoirs will be placed on same chicken embryo (e.g., experiment and control samples on same chicken CAM).
2. Cut down the sides of the inserts using a rotary multitool with a circular saw blade or a hand saw to create a 2 mm high plastic ring with a permeable membrane attached to it.
3. Polish the edges of these minireservoirs with a scalpel or a sharp knife.
4. Sterilize the minireservoirs in 70% ethanol for at least 1 h.
5. Wash the minireservoirs in autoclaved double-distilled water and let them dry in a laminar hood.
6. Rinse the minireservoirs in PBS and culture medium (high-glucose DMEM/10% FBS/1% penicillin-streptomycin).
7. Place the reservoir in a Petri dish, on top of a drop (600 µL/400 µL) of culture medium with the membrane facing up.
8. Arrange dissected ex vivo embryonic kidneys or renal organoids evenly on the membrane with the help of a pipette or a glass capillary tube. Avoid leaving a lot of liquid around the kidneys.
9. Let the samples attach to the membrane for 2–24 h in a cell culture incubator at 37 °C and 5% CO₂.
   NOTE: Up to eight E11.5 embryonic mouse kidneys or kidney organoids can be arranged on the small insert. The large insert is recommended if either bigger pieces of embryonic tissue will be used for xenotransplant or a cell-hydrogel mixture on a larger area of the CAM.
10. Take 8-day-old ex ovo cultured embryonic chickens prepared according to previously published methods out of the cell culture incubator^12,13.
11. Transfer the minireservoirs to the CAM so that the grafts are facing the CAM, and the membrane overlays them. Place the minireservoirs in the periphery of the CAM, so that they are not covering the chicken embryo.
    NOTE: When using the smaller minireservoirs fabricated from transwell inserts for 12 well plates, up to three minireservoirs can be placed on one CAM.
12. Add 500 µL (6 well insert) or 300 µL (12 well insert) of culture medium to the minireservoirs.
13. Cultivate the samples on chicken CAM for a maximum of 9 days. Replace the culture medium in the minireservoirs daily.
    NOTE: Vascularization of the samples can be observed as soon as 24–48 h after transplantation.

2. Fabrication of custom-designed plates and setting up FiZD cultures

1. Drill 20 mm diameter holes in the bottom of a 6 well plate.
   NOTE: For FiZD experiments, use 6 well plates with a 16 mm well depth (specified in Table of Materials).
2. Polish the rims of the holes (especially on the upper side) using an electric drill with a countersink drill bit, a scalpel, or a sharp knife.
3. Sterilize the plates in 70% ethanol for at least 1 h.
4. Wash the plates in autoclaved double-distilled water and let them dry in sterile conditions.
5. Thoroughly wash 22 mm x 22 mm glass coverslips with 70% ethanol or clean them according to the published protocol by Saarela et al.¹¹.
6. Glue the coverslips to the upper side of the holes in 6 well plates using a nontoxic tissue glue. Apply glue around the hole and gently place the coverslip to cover it. Let the glue dry.
7. Inspect the plates under a stereomicroscope and remove excess dried glue from the surface of the coverslips if needed.
   NOTE: Check that there is no glue above the coverslip to ensure even compression of the samples.
8. Mix polystyrene beads (70 µm particle size) with an equal volume of hydrogel. A volume of 50–100 µL per well is sufficient.
   NOTE: Keep the mixture of hydrogel and polystyrene beads on ice.
9. Place a transwell insert under a dissecting microscope with the membrane up.
10. Arrange the samples (e.g., ex vivo embryonic kidneys or renal organoids) evenly on the membrane.
11. Add the hydrogel/polystyrene bead mixture next to the samples carefully to avoid damage to fragile tissues.
12. Flip the transwell insert so that the membrane with the samples assembled on it is facing down.
13. Gently place the insert into a well of the modified 6 well plate and gently press it down so the samples are compressed to the level of the beads.
    NOTE: Follow the progress of the compression using a microscope.
14. Keep the insert slightly pressed to the well with one hand and fix it to the plate by melting plastic with a soldering iron at three points at the periphery of the insert.
15. When the insert is fixed to the plate, add 2 mL of culture medium (DMEM/10% FBS/1% penicillin-streptomycin) to the well and continue assembling the remaining wells in the plate in the same way.
16. Transfer the finished plate to an on-stage incubator of an inverted microscope for time-lapse imaging.
17. Perform time-lapse imaging of the samples using appropriate experimental settings.
    NOTE: Changing the culture medium in the wells of the plate during time-lapse experiments is not required but can be easily done through the holes on the sides of the insert.

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