Optimization of Renal Organoid and Organotypic Culture for Vascularization, Extended Development, and Improved Microscopy Imaging

Kidney organ culture and organelles that are derived from programmed stem cells provide excellent ways to study mechanisms behind kidney development and disease. The current organ culture system do not provide blood flow to the kidney, however. Due to this, reason the current models are incomplete and fail to recapitulate the main function of the kidney, which is blood filtration.

In the this publication, we present an improved protocol for cultivation and visualization of embryo kidneys and organoids on the chorioallantoic membrane of chicken embryo. In our novel experimental set up, we overlay the kidney rudiments transplanted on CAM with permeable meaning reservoirs that are filled with culture medium. This protocol increases significantly the survival of kidney rudiments and also the intrinsic endothelial cells.

The method enables blood flow to the developing glomerulus. Recently we published also a novel fixed set direction organ culture method that offers high resolution confocal 3D time-lapse imaging. The method provided optimal conditions for long term confocal imaging of the organoids and organ rudiments.

In the approach, we gently compress the tissue between a glass cover slip and a permeable membrane. Here we show how the custom designed plates are made and how the culture experiments are set up. Thus, in this publication we present an improved renal organelle tubing culture technique.

This method offers a better way to model organelle genesis and conduct high resolution imaging to study embryonic kidneys and renal organoids ex vivo. Depending on your experiment take transfer cell contra-inserts designed for six-well or 12-well plates. Cut down the sides of the insert using a Dremel tool to create a two 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 mini reservoirs in 70%ethanol for at least one hour. Then wash the mini reservoirs in autoclave by distilled water and dry them in a laminar hood.

Rinse the mini reservoirs in PBS and culture medium. Place the reservoir on a petri dish on top of our 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 leave them attached from two to 24 hours in a cell culture incubator. Exenal transplantation is done to the chorioallantoic membrane of eight-day-old ex ovo embryonic chicken. Transfer the mini reservoirs 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 mini reservoirs. Cultivate the samples on chicken CAM for nine days as maximum.

Replace the culture medium in the mini reservoirs daily. Vascularization of the samples can be observed already 24 to 48 hours after transplantation. To make the custom designed six-well plates for the fixed culture.

You need to start by drilling 20 millimeter holes in the bottom of the plate using a drilling machine with a suitable drill bit. Next, polish the rims of the holes on the upper side of the plate. Finally, sterilize the plates in 70%ethanol for at least one hour.

Then wash the plates in autoclave by distilled water and dry them in lemiar hood. Wash the 22x22 millimeter cover slips in 70%ethanol and let them dry completely. Glue the cover slip on top of a hole in the bottom of a well using tissue glue.

After drying, inspect the plates under sterile microscope and remove the excess glue. First, mix an aliquot of polystyrene beads with an equal volume of hydrogen. Place the transfill insert under a dissecting microscope with membrane facing up and arrange the kidneys on the membrane.

Add a drop of hydro-gel bead mixture next the kidneys carefully. Flip the transfill insert so that the membrane and the kidneys are facing down. Then carefully place the insert into our well of custom designed six-well plate.

Push the insert very gently into the well. Follow the degree of the flattening from the microscope until the kidney is at the level with the beads. Keep the insert slightly pressed to the well with one hand and fix it to the plate by melting plastic at three points at the periphery of the insert with a soldering iron.

Add two milliliter culture medium into the well through an opening on the side. When starting a time-lapse imaging, transfer the plate into the on-stage incubator of an inverted microscope. Our modified CAM capture protocol enables highly efficient vascularization of renal organoids and embryonic kidneys.

Many reservoirs containing culture medium supplies donor tissue and protects it from drying during the initial period of time proceeding the vascularizaton. This movie shows the flow of chicken blood cells in an embryonic mouse kidney center grafted to chicken CAM and cultured for seven days. This figure panel shows that our improved CAM culture method provides permissive conditions for the renal derived endothelial cells.

Figures A and B show embryonic kidneys grown for five days on days interval culture were center grafted to chicken CAM for five days. Staining with mouse specifics CD-31 antibody shows that the endothelial cell network of the kidney center grafted to CAM is more extensive than in the left side control. In the figure C we see that mouse blood vessels stained with mouse specific CD-31 antibody and that's the most with chicken blood vessels.

Figures D and E show cryosections of mouse embryonic kidneys grown on travel culture or chicken CAM for five days and stained with CD-31 and Hoechst. The glomeruli vasculature is more mature in kidneys cultured on chicken CAM than in control cultures. Figure F shows a mouse embryonic kidney cultured on CAM of a transgenic GFP chicken for seven days.

The mouse endothelial cells are stained with CD-31 antibody and we can observe that the vasculature in xenotransplanted mouse kidneys is mostly but not exclusively mouse derived. The novel fixed set direction culture enables culturing of embryonic kidneys and organoids in a restricted set distance. In a traditional travel culture, the cultured organs are placed on a filter that is kept in an air medium interface by a metal grid.

This method allows good conditions for the development and growth but is not optimal for imaging. In the fixed set direction culture, the cultured organ is pressed between a glass bottom of the dish and a membrane and the thickness of it is controlled by the polystyrene beads working as spacers here. The developing organs can be imaged right through the cover glass giving the improved view of the organ.

This figure panel shows that the embryonic kidneys and kidney organoids can be cultured in the fixed set direction culture method. Bright field images of embryonic kidneys and kidney organoids on day seven show normal development. In the figure C, GFB expression has been activated in the developing nephrons of a kidney organoid by the Figure D shows a kidney with trauma one staining on the euretric bud and six two staining marking the nephron precursor cells.

The figures E and F present the mouse kidney cultured for 12 days. The trauma one and nephron staining show the eurethric bud and podocites. The movie too, presents an embryonic kidney of MTMG, Taiwan cree origin, where the Taiwan cree induces GFB expression in endothelial cells.

We can see that the renal endothelial cells are present in the developing embryonic kidney and the endothelial network can also develop in the culture method. In the panel on the right, you can see how endothelial cells migrate into the vascular cleft of a S-shaped stage nephron. Here we have presented the improved protocol for xenotransplantation of renal organoids and embryonic kidneys to chicken CAM.

This protocol increases significantly the survival of kidney rudiments and also their intrinsic endothelial cells. The method enables blood flow to the developing glomerulus. The other protocol presented here is the fixed set direction organ culture method which improves the imaging quality and helps to study organelle genesis at the single cell level.

The high resolution images are suitable for automated cell segmentation and computer based study of cellular behavior in renal organoids and kidney cultures.