Efficient Vascularization of Kidney Organoids through Intracelomic Transplantation in Chicken Embryos

Kidney organoids cultured in vitro remain immature, limiting their applicability. And this method of intracoelomic transplantation induces vascularization and enhances maturation of the organoids. The procedure is very efficient, allowing for the transplantation and analysis of large number of organoids in one experiment.

After preparing HIPSC-derived kidney organoids and incubating the fertilized leghorn eggs create a window in the eggshell on day three of incubation. Place a small piece of transparent tape on the pointed tip of the egg. Make a small hole in the eggshell in the middle of the transparent tape by tapping it with the sharp end of a pair of dissecting scissors.

Next, insert a 19-gauge needle on a five-milliliter syringe into the hole at a 45-degree angle. Aspirate two to three milliliters of albumin from the egg to lower the embryo inside the egg. Conceal the hole with a second piece of transparent tape, then place a large piece of transparent tape on the pencil marked upward facing side of the egg.

Make a hole in the eggshell in the middle of the transparent tape by tapping it with the sharp end of a pair of dissecting scissors. Cut a small circular window in the eggshell from this hole using curved dissecting scissors. Look through this window to locate the embryo.

Then, enlarge the window to optimize access to the embryo. Remove any large pieces of eggshell that may have fallen on top of the embryo using forceps. Remove smaller pieces by placing a few drops of DPBS with calcium and magnesium on the embryo with a plastic transfer pipette, then aspirating the DPBS with the eggshell into the pipette.

Using a plastic transfer pipette, add three drops of DPBS, supplemented with 0.5%penicillin streptomycin to the egg. Carefully seal the window with a large piece of transparent tape before placing the egg back in the incubator until day four of incubation. Cut the tape from the window with curved dissecting scissors.

Place the egg under a dissecting microscope on a rubber holder or egg carton. The chicken embryo is now in Hamburger-Hamilton Stage 23 to 24 and lying on its left side with its right side facing the viewer. Locate the right wing and leg bud of the embryo as the coelom will be assessed between these two limb buds.

In the area between the right wing and leg bud, create an opening consecutively in the vitelline membrane, the chorion, and the amnion by holding them with two pairs of dissecting forceps and gently pulling them in opposite directions. Check whether there’s unobstructed access to the coelomic cavity. Gently take hold of the edge of the body wall between the wing and limb bud with a pair of dissecting forceps and pull it slightly toward the viewer.

The coelomic cavity must be clearly visible. Carefully insert a blunt but slim instrument into the coelomic cavity. Place half an organoid inside the egg on top of the allantois using a dura dissector.

Using dissecting forceps, carefully take hold of the edge of the body wall and pull it slightly toward the viewer to make the opening to the coelum visible. Gently move the organoid toward and through the opening in the body wall into the coelum with a blunt tungsten wire and a micro scalpel holder. Push the organoid slightly cranially to lodge it inside the coelum.

It is now visible just behind the wing bud. Add three drops of DPBS to the egg using a plastic transfer pipette. Carefully seal the window with a large piece of transparent tape before placing the egg back in the incubator until day 12 of incubation.

Cut the tape from the window with curved dissecting scissors. Place the egg under a dissecting microscope in a rubber holder. Evaluate the vasculature and locate the veins, which can be distinguished from the arteries by their slightly brighter red color.

To improve access to the vasculature, carefully enlarge the window by cutting it with curved dissecting scissors. After selecting the vein, insert the tip of the micro capillary needle into the vein at a zero to 20-degree angle. Ensure the needle is in the vein by gently moving the tip from side to side.

Gently and steadily blow into the injection system to inject the lens culinaris agglutinin. And place the egg back in the incubator for 10 minutes to let it circulate. Place the egg in a rubber holder on the bench.

Then, cut through the membranes surrounding the embryo with curved dissecting scissors. Scoop the embryo up from the egg with a perforated spoon and immediately decapitate the embryo using scissors. Under the dissection microscope, place the body of the embryo in a Petri dish.

Place the embryo on its back in the Petri dish and spread its limbs. Carefully open the abdominal wall of the embryo along the longitudinal axis using forceps. Locate the organoid inside the embryo.

The organoid most frequently becomes attached to the right liver lobe, either at the coddle tip or cranially, just below the rib cage. So, it’s advisable to start by looking at these locations. Once the organoid is located, remove it from the embryo by cutting around it with micro scissors.

Place the organoid in the chicken tissue that is inevitably attached to it in a Petri dish under the dissecting microscope. Remove as much chicken tissue as possible with a double-edged stainless steel razor blade. Immunofluorescent images of an non-transplanted kidney organoid and a transplanted kidney organoid both contain glomerular and tubular structures.

In the untransplanted organoids, some human endothelial cells are present. In the transplanted organoid, a perfused vascular network is visible throughout the organoid. Magnified images of the boxed areas demonstrate the three types of endothelial cells that can be distinguished in transplanted organoids:perfused human endothelial cells, unperfused human endothelial cells, and perfused chicken-derived endothelial cells.

In untransplanted organoids, endothelial cells surround glomerular structures but do not invade them. However, in transplanted organoids, glomerular structures are vascularized by perfused capillaries. Transplanted organoids can be analyzed using different techniques depending on the aim of the experiment.

Direct comparison to untransplanted controls can provide insight into the process of vascularization and maturation.