Renal Organotypic Culture on Chicken CAM: A Technique for Transplantation of Murine Kidney Explants on Chicken CAM to Induce Formation of Renal Vascular Architecture

Protocollo

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board

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
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.

Materiali

Name	Company	Catalog Number	Comments
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
Dulbecco's Modified Eagle's Medium	Sigma	D777	High glucose
Ethanol (70%)	VWR
Fetal Bovine Serum	HyClone	SH3007003HI Thermo Scientific
Forceps DUMONT #5	Dumont	#5SF
PBS -/-	Corning	20-031-CV
PBS +/+	Biowest	X0520-500	Washing the mini reservoirs.
Penicillin and Streptomycin	Sigma	P4333
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