Periodic Reporting for period 1 - Laser-Tissue-Perfuse (Laser biofabrication of 3D multicellular tissue with perfusible vascular network)
Periodo di rendicontazione: 2022-10-01 al 2025-03-31
Building 3D vascularised tissue or organs remains the major unsolved challenge to be overcome in biofabrication and tissue engineering. Establishing blood vessels capable of efficient transport of gas, nutrients, and metabolites to and from cells is a prerequisite for the survival of tissue constructs, both in vitro and when transplanted in vivo. High resolution multi-scale constructs are necessary to replicate the complexity of vascular tree from large scale arteries and veins to micron scale arterioles, venules and capillaries. This functional vascular tree is essential not only for tissue perfusion, but also for stimulation and controlled maturation of engineered tissue constructs. Computer-controlled generation of a 3D vascular capillary tree and its perfusion in centimetre scale cardiac and skin tissue constructs, using the developed biofabrication methodologies, will represent a seminal breakthrough in organ regeneration with widespread long-term impacts across the field of regenerative medicine.
This project aims to develop a set of technologies, including a unique combination of advanced laser bioprinting (LBP) with two-photon polymerisation (2PP) technique, for the generation of a full vascular system with a dense microcapillary network. Different scaffold-based, scaffold-free, sacrificial, and hybrid approaches will be explored. The major challenge is the generation of a complex vasculature with functional cell layers and its connection to a pulsatile perfusion flow system at physiological conditions.
This project aims to develop a set of technologies, including a unique combination of advanced laser bioprinting (LBP) with two-photon polymerisation (2PP) technique, for the generation of a full vascular system with a dense microcapillary network. Different scaffold-based, scaffold-free, sacrificial, and hybrid approaches will be explored. The major challenge is the generation of a complex vasculature with functional cell layers and its connection to a pulsatile perfusion flow system at physiological conditions.
Laser bioprinting of cell spheroids has been investigated, since this approach is generally considered promising for the fabrication of vasculature structures. For laser printing experiments, an automated detection of spheroids and spatial pulse shaping (to reach flat-top or donut-like intensity distributions) was developed. It has been observed that the printability of spheroids strongly depends on the type of applied cells and their bounding. If the cell bounding is too loose, spheroids disintegrate during the printing procedure. Others remain intact and all cells in the spheroid remain viable. However, a considerable amount of bio-ink is also transferred during printing. If two spheroids are printed directly next to each other, the spheroid printed first is sometimes washed away when the second one is printed. The positioning accuracy is also reduced compared to printing of dissociated cells. It has been observed that spheroids consisting only from endothelial cells (ECs) can fuse into continuous strands with the formation of a lumen inside.
As a basis for a tissue perfusion system, an Arduino-controlled microfluidic actuation platform with peristaltic pump and sensors was developed, together with different designs for microfluidic chips for applications with and without scaffolds. For the fabrication of large-scale vascular scaffolds with high structural resolution and at high throughput, a new 2PP machine prototype was designed; currently, customised machine parts are in production, commissioning, and testing. Furthermore, material selection and construction of experimental scaffold-like vascular structures suitable for perfusion has been performed with the existing 2PP system. Scaffolds were protein-coated, pre-colonized with ECs and smooth muscle cells (SMCs), and perfused, partially embedded in tissue. Moreover, vascular networks embedded in tissue, extra-cellular matrix (ECM) with fibroblasts (partially with overlying keratinocyte layers), were printed, cultivated for a few days, and a potential vessel formation was studied; printed tissue with integrated vascular structure was perfused for up to 7 days.
As a basis for a tissue perfusion system, an Arduino-controlled microfluidic actuation platform with peristaltic pump and sensors was developed, together with different designs for microfluidic chips for applications with and without scaffolds. For the fabrication of large-scale vascular scaffolds with high structural resolution and at high throughput, a new 2PP machine prototype was designed; currently, customised machine parts are in production, commissioning, and testing. Furthermore, material selection and construction of experimental scaffold-like vascular structures suitable for perfusion has been performed with the existing 2PP system. Scaffolds were protein-coated, pre-colonized with ECs and smooth muscle cells (SMCs), and perfused, partially embedded in tissue. Moreover, vascular networks embedded in tissue, extra-cellular matrix (ECM) with fibroblasts (partially with overlying keratinocyte layers), were printed, cultivated for a few days, and a potential vessel formation was studied; printed tissue with integrated vascular structure was perfused for up to 7 days.
Laser printing of cell spheroids has been established. It has been demonstrated that spheroids consisting only from endothelial cells (ECs) can fuse into continuous strands with the formation of a lumen inside. Differentiation of all the required blood vessel cells from hiPSCs and their stable transduction has been achieved. It has been observed that inside tubular scaffolds embedded in a hydrogel, ECs self-align as a monolayer on SMCs after prolonged perfusion. Such an arrangement does not occur without perfusion. This can be considered as an important preliminary step towards the realization of vascularized tissue.