First, we developed an ultra-fast volumetric biofabrication approach to rapidly pattern fragile cells and organoids into centimeter-scale engineered tissues, in a matter of seconds, and with superior viability compared to conventional extrusion techniques. To prevent loss of printing resolution caused light scattering, we developed optically tuned bioresins, enabling to print with high resolution also in presence of cell densities comparable to those found in soft tissues. Bioprinted architectures modulate fluid flow during culture, tuning organoid maturation and functionality. Next, we designed new approaches to introduce multi-material and multi-cellular elements. First, we enabled the possibility to “paint” 3D paths of morphogens and growth factors within hydrogels, showing how patterned vasculogenic factors guide blood vessel cells growth. Bioactive molecules can be precisely patterned into any desired spatial distributions, even across centimeter-scale objects, and with a resolution of 0.05mm. This technology paves the way toward the precise spatiotemporal modification of the chemical composition of bioprinted tissues to better guide cell behavior. Importantly, as hydrogels for cell culture usually have limited structural stability and poor mechanical properties (low stiffness), we enabled volumetric bioprinting of multimaterial structures also across pre-formed, mechanically strong scaffolds, in the form of thermoplastic meshes printed with melt electrowriting, which endowed the volumetrically printed hydrogels with high mechanical properties. To facilitate cell migration and re-organization within the bioprinted resin, we developed a modular, microgel-based light-printable material. These microgel bioresins can be sculpted within seconds with tomographic light projections into centimetre-scale, granular hydrogel-based, convoluted constructs. Interstitial microvoids enhanced differentiation of multiple stem/progenitor cells, permitting the formation vascular capillaries, as well as neuronal networks in 3D, structure that could not be obtained with conventional bulk hydrogels. With the goal to improve the viability and function of bioprinted organoids, we developed a novel computer vision and AI-driven printing method, which permits to build blood vessels that feed each organoid in a printed tissue, improving their biological performance. With the goal to produce a bone marrow model to enable the culture and expansion of hematopoietic stem cells, the cell type needed for transplant therapies to treat many blood related malignancies and leukemia, we developed stromal vascular organoids as biological building blocks to be patterned with these techniques. These organoids comprise a dense vascular capillary network within a stroma composed by bone marrow cells, and can be grown within the hydrogels we developed for volumetric printing. Additionally, we also developed fetal liver organoids, to study the early stages of development of blood stem cells. Hematopoietic stem cells home within these bioengineered organoids, and thrive in these systems. The technologies developed in VOLUME-BIO are now broadly adopted to build better engineered tissues as drug testing models and for regenerative medicine. The knowledge from the project helps to understand hematopoietic stem cell fate regulation, and to design systems to expand these cells as a replenishable supply for stem cell transplant and therapy.
