Renal Organotypic Culture on Chicken CAM: A Technique for Transplantation of Murine Kidney Explants on Chicken CAM to Induce Formation of Renal Vascular Architecture
Transcrição
Depending on your experiment, take transwell cell culture inserts designed for 6-well or 12-well plates. Cut down the size of the insert using a Dremel tool to create a 2-millimeter high plastic ring with a permeable membrane attached to it.
Polish the edges of the ring from swarf with a scalpel or a sharp knife. Sterilize the minireservoirs in 70% ethanol for at least 1 hour. Then, wash the minireservoirs in autoclave by distilled water and dry them in a laminar hood. Rinse the minireservoirs in PBS and culture medium.
Place the reservoir on a Petri dish on top of a drop of culture medium with the membrane facing upwards. Arrange dissected ex vivo embryonic kidneys or renal organoids on the membrane. Avoid leaving much liquid around the samples and let them attach for 2 to 24 hours in a cell culture incubator.
Xenotransplantation is done to the chorioallantoic membrane of 8-day-old ex vivo embryonic chicken. Transfer the minireservoir so that the membrane is covering the graft. Place them in the periphery of the CAM so that they are not covering the embryo.
Then, add culture medium to the minireservoirs. Cultivate the samples on chicken CAM for 9 days as maximum. Replace the culture medium in the minireservoirs daily. Vascularization of the samples can be observed already 24 to 48 hours after transplantation.
