To begin, place a 50 milliliter tube on ice and add collagen one solution along with other components sequentially. Shake the tube to mix thoroughly. Using a P10 pipette, add one molar sodium hydroxide solution dropwise while shaking the mix solution to adjust the pH to 7.3.
Add 1.25 milliliters of basement membrane matrix to the five milliliter collagen one mixture and mix the solution gently. Next place a 12-well plate on ice for two minutes. Using wide bore tips, add the collagen one basement membrane matrix mixture slowly to the 12-well plate.
Place the 12-well plate in the 37 degree Celsius incubator for two hours to solidify the first layer of hydrogel. Keep the remaining collagen one basement membrane matrix mixture on ice for later use. Then transfer approximately 60 cell aggregates into a 1.5 milliliter microcentrifuge tube.
And cool the tube on ice for five minutes. Using a P200 pipette tip, carefully remove the medium from the tube. Add 1.5 milliliters of the collagen one basement membrane mixture, dropwise to the cell aggregates, and gently mix them using a wide bore P200 pipette tip to resuspend.
Transfer the plate with the first layer of hydrogel from the incubator to the biosafety cabinet. Add 1.5 milliliters of the aggregate suspension onto the cured hydrogel layer and gently shake the plate to distribute the aggregates evenly. Incubate the plate at 37 degrees Celsius with 5%carbon dioxide for two hours.
Prewarm vascular differentiation medium two in a 37 degrees Celsius water bath for 20 minutes. Add two milliliters of medium to each well and culture the cells at 37 degrees Celsius with 5%carbon dioxide. Change the medium every three days with fresh pre-warmed vascular differentiation medium two.
Organoids expressed CD31, ERG1, and PDGFR beta, indicating the presence of vascular endothelial cells and parasites. Vascular networks were visualized via whole mount immunofluorescence microscopy after tissue clearing. Mature organoids stained on day 17 showed vascular networks encapsulating the spheroids within the gel block.
Organoids retrieved on day 28 had vascular networks wrapping around the spheroids instead of radially expanding.
