Periodic Reporting for period 2 - VOLUME-BIO (Volumetric light-driven bioprinting capturing complex physiological shape, size and function in artificial tissues and organoids)
Período documentado: 2022-07-01 hasta 2023-12-31
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 propose to develop 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 comprises: 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 comprises: 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 a novel layer-less, ultra-fast volumetric biofabrication approach to rapidly pattern fragile cells and organoids into centimeter-scale engineered tissue constructs, in a matter of seconds, and with superior viability compared to conventional extrusion techniques. To prevent impairment 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 perfusion culture, tuning organoid maturation and functionality. Next, we designed new approaches to introduce multi-material and multi-cellular complexity within these light-based bioprinting techniques. First, we enabled the possibility to “paint” 3D paths of morphogens and growth factors within bioprinted hydrogels. Bioactive molecules can be precisely patterned into any desired spatial distributions, following a computer aided design, even across centimeter-scale objects, and with a resolution of 50 micrometers. As first demonstration, vascular endothelial growth factor was locally photografted into a bioprinted gel and demonstrated region-dependent enhanced adhesion and network formation of endothelial cells. This technology paves the way toward the precise spatiotemporal biofunctionalization and modification of the chemical composition of bioprinted constructs to better guide cell behavior. Further, we then introduced the possibility to sequentially print multiple materials within the same construct. Importantly, as hydrogels for cell culture usually have limited structural stability and poor mechanical properties (low stiffness), we addressed this challenge by enabling 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 produce a bone marrow-inspired model to enable the culture and expansion of hematopoietic stem cells, the cell type needed for transplant therapies to treat many types of 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 derived cells, and can be grown within the hydrogel bioresins we developed for volumetric printing. Hematopoietic stem cells can home within these bioengineered organoids, and kept in culture in these systems. We will leverage the ability of our new volumetric bioprinting to tune the biochemical and architectural composition of the microenvironment within which these organoids carrying hematopoietic stem cells are grown. The knowledge obtained from this new culture system will help 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 have developed a new strategy to build reinforced, cell-laden hydrogels, converging volumetric bioprinting and melt electrowriting
5) 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
7) We developed stromal vascular organoids to support the homing and culture of human hematopoietic stem cells
We expect that we will deliver the following results before the finalization of the project:
1) Generating a complex bone marrow-niche system for the homing and sustained culture of human hematopoietic stem cells
2) Gaining fundamental insights into what drives HSC maintenance of multipotency, survival and differentiation
3) Creation of centimeter-sized and vascularized organoids sized organs that will remain viable in vitro for >4 weeks while sustaining long-term 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 have developed a new strategy to build reinforced, cell-laden hydrogels, converging volumetric bioprinting and melt electrowriting
5) 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
7) We developed stromal vascular organoids to support the homing and culture of human hematopoietic stem cells
We expect that we will deliver the following results before the finalization of the project:
1) Generating a complex bone marrow-niche system for the homing and sustained culture of human hematopoietic stem cells
2) Gaining fundamental insights into what drives HSC maintenance of multipotency, survival and differentiation
3) Creation of centimeter-sized and vascularized organoids sized organs that will remain viable in vitro for >4 weeks while sustaining long-term hematopoietic stem cells, the cell type most needed for reconstituting all blood lineages during stem cell transplantation therapies