Project Portugal 2030

Summary

This proposal aims to provide on-demand solutions for fabricating mature cell-rich constructs using tissue-specific inks that can be printed with the patient’s own cells, providing sustainable and efficient solutions for tissue regeneration and in vitro models with minimal operator intervention. LEGO-specific objectives (SO) comprise: SO1: Provide immunologically compatible solutions: Most of the works performed in 3D bioprinting using microcarriers rely on animal-derived proteins such as gelatin,(Seymour et al., 2021) or synthetic materials such as poly(ethylene glycol) (PEG),(Xin et al., 2021) because of their intrinsic viscosity. Nevertheless, these materials are still controversial for human application. LEGO's final products will be mainly composed of patient-derived cells and their own extracellular matrix. Immune-privileged human/plant-based materials will be employed to produce the microcarriers (which will comprise a minor fraction of the therapy), avoiding adverse inflammatory reactions. SO2: Increased therapeutic efficacy: By ensuring that cells will, by themselves, organize into microtissues in a dynamic culture system, the final product will have unprecedented biomimetic features. This ensures that a much more mature state of therapy is reached, increasing the efficacy of tissue regeneration, compared to injection of “separate” cells encapsulated in a supporting matrix.(Daly et al., 2021) SO3: Use of easy-to-handle advanced technologies: One of the major difficulties in implementing 3D bioprinting techniques in clinical practice is the difficulty in producing bioinks on demand.(Bliley et al., 2022) LEGO will develop an easy-to-handle platform with the possibility of choosing the microcarrier, according to the final intended application. The final products (microtissues) will be used directly as bioinks on a 3D bioprinter, thus minimizing time-consuming expansion and encapsulation steps. SO4: Ensure sustainable production systems: Cell cultures will be performed in dynamic systems that can and will be reusable; cell culture media will be progressively changed during culture time, avoiding the use of costly reagents and disposable plastics during cell culture. Additionally, AI-assisted biofabrication will allow real-time monitoring and improvement during 3D printing, also contributing to efficient use of the produced inks and preventing inaccurate 3D printings with consequent annihilation of the produced products. SO5: Improve precision medicine state-of-the-art, by validating the proposed strategy on the development of advanced perfusable vascularized tissue matrices: attempts to develop in vitro blood vessels usually yield products with insufficient mechanical stability and lack of tissue mimetic structures. In contrast, cell-derived extracellular matrices obtained through the dynamic cultures will hold a collection of diverse biomolecules, providing a favorable microenvironment for vascular cell maturation upon a 1-step co-axial blood vessel 3D printing into a mechanically robust fibroblastic matrix. The interdependencies between the SO’s and the tasks can be found in Figure 1. By joining together knowledge from different lines of investigation and several proof-of-concept studies that demonstrate the feasibility of separate parts of the proposal, it is intended to enable the realistic fabrication of lab-made tissues, essential to keep healthcare standards on a growing and aging world population, without harming the planet