Periodic Reporting for period 4 - VOLUME-BIO (Volumetric light-driven bioprinting capturing complex physiological shape, size and function in artificial tissues and organoids)
Periodo di rendicontazione: 2025-07-01 al 2025-12-31
The generation of engineered tissues in laboratory settings has been a long-sought “holy grail” to solve organ donor shortage, and more recently, also to develop three-dimensional (3D) humanized in vitro models of healthy and diseased tissues. These could replace animal experimentation and 2D cell culture in the context of both fundamental biological research and drug development. I developed a novel volumetric 3D bio-printing technology to manufacture living tissues, which recreate in vitro the assembly of multiple cell types within large, complex geometries typical of human anatomy, and, at the same time, help designing the cellular microenvironment in terms of the 3D patterning of morphogenetic biomolecules and matrix stiffness.
Achieving this primary goal, the pioneering work of VOLUME-BIO comprised: 1) Establishing a new versatile light-based bioprinting technology, to overcome the limits of current bioengineering and printing methods, which preclude the generation of fully functional human tissues; 2) Developing a portfolio of materials compatible with the VOLUME-BIO approach, with tunable mechano-biochemical properties, capable to safely embed cells and drive their maturatio; 3) Creating a platform to probe the fundamental question of how the degree of complexity of the engineered organoids (multicellularity, architecture, ECM stiffness and 3D morphogen patterns) influences the emergence of tissue-level functions, in contrast to simplified, less organized models. 4) Introducing perfusable vascular networks to permit long-term survival of biofabricated tissues. 5) Presenting a proof-of concept of functionality of bioprinted organoids, laden with cells displaying diverse characteristics and engineering requirements, specifically, as bio-factories expressing the hematopoietic function of bone.
Achieving this primary goal, the pioneering work of VOLUME-BIO comprised: 1) Establishing a new versatile light-based bioprinting technology, to overcome the limits of current bioengineering and printing methods, which preclude the generation of fully functional human tissues; 2) Developing a portfolio of materials compatible with the VOLUME-BIO approach, with tunable mechano-biochemical properties, capable to safely embed cells and drive their maturatio; 3) Creating a platform to probe the fundamental question of how the degree of complexity of the engineered organoids (multicellularity, architecture, ECM stiffness and 3D morphogen patterns) influences the emergence of tissue-level functions, in contrast to simplified, less organized models. 4) Introducing perfusable vascular networks to permit long-term survival of biofabricated tissues. 5) Presenting a proof-of concept of functionality of bioprinted organoids, laden with cells displaying diverse characteristics and engineering requirements, specifically, as bio-factories expressing the hematopoietic function of bone.
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.
We have delivered on the following breakthroughs that we consider to be beyond state-of-the art:
1) A novel technique for bio-friendly refractive index matching, to enable high resolution light-based bioprinting, also in presence of high cell concentrations.
2) The creation of centimeter-scale with micrometer-scale resolution environments to grow and steer the functionality of bioprinted organoids.
3) We built a new set of fully chemically defined, and bioprintable hydrogels in which multiple types of organoids can be expanded
4) We demonstrated for the first time the possibility to use a single-photon technology to position bioactive molecules and morphogens with micrometer-scale resolution, in any desired 3D pattern, across centimeter-large hydrogels, therefore steering cell behavior in a spatially-controlled fashion
5) We demonstrated the integration of optogenetically controlled cells in (volumetrically) bioprinted tissues.
6) We introduced for the first time the concept of generative, adaptive and context-aware 3D printing (GRACE), and demonstrated its implementation in the field of volumetric bioprinting, using computer vision and AI to automatically build blood vessel-mimetic channels that target every organoid in a bioprinted tissues
7) We pioneered algorithms to enable light-based volumetric 3D printing in presence of occluding, opaque elements that shadow light with complex geometries, taking advantage of the potential of GRACE printing
7) We have developed a new strategy to build reinforced, cell-laden hydrogels, converging volumetric bioprinting and melt electrowriting
8) We developed a new modular, granular hydrogel bioresin for volumetric and light-based printing, which allows multiple cell types to thrive, and combines the strengths of extrusion and volumetric bioprinting in a single process
9) We developed stromal vascular organoids to support the homing and culture of human hematopoietic stem cells
10) We developed fetal liver organoids to support the homing and sustained culture of human hematopoietic stem cells, the cell type most needed for reconstituting all blood lineages during stem cell transplantation therapies
1) A novel technique for bio-friendly refractive index matching, to enable high resolution light-based bioprinting, also in presence of high cell concentrations.
2) The creation of centimeter-scale with micrometer-scale resolution environments to grow and steer the functionality of bioprinted organoids.
3) We built a new set of fully chemically defined, and bioprintable hydrogels in which multiple types of organoids can be expanded
4) We demonstrated for the first time the possibility to use a single-photon technology to position bioactive molecules and morphogens with micrometer-scale resolution, in any desired 3D pattern, across centimeter-large hydrogels, therefore steering cell behavior in a spatially-controlled fashion
5) We demonstrated the integration of optogenetically controlled cells in (volumetrically) bioprinted tissues.
6) We introduced for the first time the concept of generative, adaptive and context-aware 3D printing (GRACE), and demonstrated its implementation in the field of volumetric bioprinting, using computer vision and AI to automatically build blood vessel-mimetic channels that target every organoid in a bioprinted tissues
7) We pioneered algorithms to enable light-based volumetric 3D printing in presence of occluding, opaque elements that shadow light with complex geometries, taking advantage of the potential of GRACE printing
7) We have developed a new strategy to build reinforced, cell-laden hydrogels, converging volumetric bioprinting and melt electrowriting
8) We developed a new modular, granular hydrogel bioresin for volumetric and light-based printing, which allows multiple cell types to thrive, and combines the strengths of extrusion and volumetric bioprinting in a single process
9) We developed stromal vascular organoids to support the homing and culture of human hematopoietic stem cells
10) We developed fetal liver organoids to support the homing and sustained culture of human hematopoietic stem cells, the cell type most needed for reconstituting all blood lineages during stem cell transplantation therapies