The chicken chorioallantoic membrane (CAM) is a highly vascularized and immunodeficient layer that serves as an excellent model for xenografting. This study outlines the methods for renal organotypic culture using CAM, detailing the process of transplanting murine kidney explants onto the CAM.
Chicken chorioallantoic membrane, or CAM, is a highly vascularized and naturally immunodeficient layer present beneath the eggshell. CAM serves as an ideal model for xenografting - a heterologous transfer of tissue from a donor organism to the recipient of another species.
To perform renal organotypic culture on chicken CAM, begin by taking a mini-reservoir with a permeable membrane present at the base. Invert the mini-reservoir and position it over a drop of media present in a Petri dish. This ensures that the membrane is in direct contact with the media. Transfer the murine kidney explants to the pre-wet membrane. Aspirate the excess media around the kidneys.
Incubate to facilitate the attachment of the kidney explants to the membrane. Next, take an ex-ovo culture from an embryonic chicken exhibiting a fully developed CAM. Locate the prominent blood vessels peripheral to the embryo. Take the reservoir with attached kidney explants and invert it over the CAM such that the membrane presses the kidney explants against the CAM tissue.
Supplement the reservoir with media to support the growth of kidney explants and prevent their shrinkage. Cultivate for a prolonged duration. The CAM blood vessels infiltrate the transplanted kidney and induce vascularization within the kidney explants, stimulating the functional kidney's recapitulation on CAM tissue.
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.
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