The Construction of Blood Vessel Organoids - A Convenient and Efficient New Technology

Abstract Cardiovascular diseases remain among the primary causes of death in modern society, primarily due to vascular changes. Traditional research methodologies, such as cell culture, tissue samples, and animal models, have provided a foundation for studying vascular disorders. Despite these approaches, they cannot fully replicate the complexity and physiological environment of human blood vessels. Organoid technology enables the creation of organ-like constructs with similar tissue composition and functional characteristics outside the body, significantly accelerating advancements in disease research. Induced from artificial hematopoietic stem cells are three-dimensional human blood vessel organoids (BVOs) derived from extra-organ tissues, opening up new avenues for vascular research. However, the existing protocols for BVOs preparation present operational complexities, expensive inducer reagents, and intricate experimental conditions, thereby limiting their widespread adoption and utilization in routine experiments. This study proposes a novel synthesis scheme through optimizing experimental design. We directly construct human BVOs in 96-well ultra-low adsorption plates, avoiding fusion between organoid spheroids as well as the need for repeated resuspension. An additional improvement lies in the direct encapsulation of cell spheres within ultra-low adsorption 96-well plates at U-bottom, obviating the need for dissection as per the original protocol and enabling the flexible adjustment of the number of desired organoids needed for culture. Furthermore, we employed mTeSR1 culture medium in lieu of the original protocol's concentrated medium, replacing Transfection Reagents ReLeSR for EDTA and Accutase. Our scheme optimized operational procedures, lowered experimental costs, shortened preparation cycles, making the construction of BVOs more economical and efficient. This progression holds promise in promoting the widespread application of BVOs technology, fostering advancements in basic research and clinical translation.