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Cell and gene therapies (CGT) have reached new therapeutic targets but have noticeably high prices. Solutions to reduce production costs might be found in CGT storage and transportation since they typically involve cryopreservation, which is a heavily burdened process. Encapsulation at hypothermic temperatures (e.g., 2–8 °C) can be a feasible alternative. In this study, we determined the ability of alginate encapsulation to maintain cell viability, identity, and function in the context of mesenchymal stromal cell (MSC)-based therapy manufacturing. MSC encapsulation was proven possible for a remarkable 12 day period, showing its potential to act as a serious competitor to cryopreservation for short to medium time periods. This study developed at iBB was led by Dr. Ana Fernandes-Platzgummer and Prof. Cláudia Lobato da Silva and first-authored by PhD student André Branco.

Cell-based therapies have been showing unprecedented therapeutic potential, already changing the landscape of medical care. Extracellular vesicles are nanoparticles naturally secreted by cells, which are important mediators of intercellular communication in our organism and are able to mediate therapeutic effects from their cells of origin. These vesicles can also be used as drug delivery vehicles to target multiple diseases. In a paper recently published in Frontiers in Cell and Developmental Biology, iBB researchers in collaboration with iMM Lisboa, CQE-IST and the companies PBS Biotech and AventaCell Biomedical, developed a new strategy for the robust and scalable production of extracellular vesicles from mesenchymal stromal cells, using bioreactors and an animal serum-free cell culture supplement. This strategy, developed within the frame of the PhD Program in Bioengineering of Miguel de Almeida Fuzeta, is a relevant step towards the large scale production of extracellular vesicles form different human tissue sources, which are promising tools for the development of new therapies against a variety of diseases, from cardiovascular diseases to cancer.

Cell-based therapies can enhance the specificity of anti-cancer therapeutic agents. In this context, human mesenchymal stromal cells (MSC) hold a promising future as cell delivery systems for anti-cancer proteins due to their innate tropism for tumors. iBB researchers Marília Silva, Gabriel Monteiro, Arsénio Fialho, Nuno Bernardes and Cláudia Lobato da Silva, engineered human MSC through non-viral methods to secrete a human codon-optimized version of azurin (hazu), a bacterial protein with demonstrated anti-cancer activity towards different cancer models in vitro and in vivo. Upon treatment with conditioned media (CM) from these engineered cells, a decrease in cancer cell proliferation, migration and invasion was seen, and an increase in cell death was observed for breast and lung cancer cell models. The results achieved by SCERG- and BSRG-iBB researchers were published in Frontiers in Cell and Developmental Biology, Stem Cell Research section.

Cell-derived extracellular matrix (ECM) has been employed as scaffolds for tissue engineering. SCERG-iBB researchers working with colleagues from the Rensselaer Polytechnic developed bioactive cell-derived ECM electrospun polycaprolactone (PCL) scaffolds produced from ECM derived from human mesenchymal stem/stromal cells (MSC), human umbilical vein endothelial cells (HUVEC) and their combination based on the hypothesis that the cell-derived ECM incorporated into the PCL fibers would enhance the biofunctionality of the scaffold. The findings show that all cell-derived ECM electrospun scaffolds promoted significant cell proliferation compared to PCL alone, while presenting similar physical/mechanical properties. Additionally, MSC:HUVEC-ECM electrospun scaffolds significantly enhanced osteogenic differentiation of MSCs. The study was published in Materials Science and Engineering: C.

The project "UROPRINT: Urinary bladder bioprinting for fully autologous transplantation" has been recommended for funding by the EU. The goal of UROPRINT is to laser print fully functional immunocompatible urothelial tissue ex vivo and in vivo for bladder augmentation and replacement. The project is led by the Institute of Biomedical Research of the Academy of Athens and involves iBB-SCERG, Asphalion SL, Institute of Comunication and Computer Systems, Optics11 BV and METATISSUE/JFCC-Biosolutions, LDA.

Photo details: National Institute of Standards and Technology, Public domain, via Wikimedia Commons.

Umbilical cord blood (UCB) is an accepted and appealing alternative source of hematopoietic stem/progenitor cells (HSPC) for hematopoietic cell transplants. However, low UCB volume recovered from births results in an unsatisfactory cell dose for transplants in adults, having initially limited transplants of a single UCB unit to pediatric patients. Ex vivo expansion of HSPC based on the addition of exogenous cytokines.has been pursued to address this problem. Notably, selection of individual cytokines and their concentrations for an expansion cocktail has differed between existing strategies. To improve the effectiveness of these platforms, namely targeting clinical approval, iBB researchers optimized the cytokine cocktails in two clinically relevant expansion platforms for HSPC, a liquid suspension culture system (CS_HSPC) and a co-culture system with bone marrow derived mesenchymal stromal cells (CS_HSPC/MSC).. The tailored and novel optimized cocktails determined made it possible to individually maximize cytokine contribution in both studied platforms, leading to an increase in the expansion platform performance, while allowing a rational side-by-side comparison between them and enhancing our knowledge on the impact of cytokine supplementation on the HSPC expansion process. The results achieved were published in Frontiers in Bioengineering and Biotechnology, Stem Cell Systems Bioengineering section.

Bioreactors that provide different biophysical stimuli have been used in tissue engineering approaches aimed at enhancing the quality of the cartilage tissue generated. However, such systems are often highly complex, costly and not very versatile. In a recent study published in Biotechnology Journal, researchers from SCERG-iBB working with colleagues from the Polytechnic Institute of Leiria and Rensselaer Polytechnic Institute (USA) developed a novel, cost-effective and customizable perfusion bioreactor fabricated by additive manufacturing (AM) to study of the effect of fluid flow on the chondrogenic differentiation of human bone-marrow mesenchymal stem/stromal cells (hBMSCs) in 3D porous poly (ε-caprolactone) (PCL) scaffolds. Results suggest that the chondrogenic differentiation of hBMSCs was enhanced in cell-scaffold constructs cultured under perfusion and highlights the potential of customizable AM platforms for developing more reliable in vitro models and improved personalized cartilage repair strategies.

Cultured cell-derived extracellular matrix (ECM) has been used as a scaffold for tissue engineering settings to create a biomimetic microenvironment, providing physical, chemical and mechanical cues to cells and support cellular activities, mimicking the composition and organization of native ECM microenvironment. In a recent study published in Journal of Tissue Engineering and Regenerative Medicine, researchers from SCERG-iBB working with colleagues from the Rensselaer Polytechnic Institute developed a new strategy to produce different combinations of decellularized cultured cell-derived ECM obtained from different cultured cell types, namely mesenchymal stem/stromal cells (MSC) and human umbilical vein endothelial cells (HUVEC), as well as the co-culture of MSC:HUVEC and investigate the effects of its various compositions on osteogenic differentiation and angiogenic properties of human bone marrow (BM)-derived MSC, vital features for adult bone tissue regeneration and repair. These results suggest that decellularized ECM derived from a co-culture of MSC:HUVEC impacts the osteogenic and angiogenic capabilities of BM MSC, suggesting the potential use of MSC:HUVEC ECM as a therapeutic product to improve clinical outcomes in bone regeneration. 

There is a high demand for functional bone grafts worldwide partly due to the increased life expectancy. Bone matrix proteins, especially osteopontin (OPN) and osteocalcin (OC), have been reported to regulate some physiological process, such as cell migration and bone mineralization. However, the effects of OPN and OC on cell proliferation, osteogenic differentiation, mineralization and angiogenesis are still undefined.  In a recent study published in Journal of Cellular and Biochemistry, researchers from SCERG-iBB working with colleagues from the Rensselaer Polytechnic Institute assessed the exogenous effect of OPN and OC supplementation on human bone marrow mesenchymal stem/stromal cells proliferation and osteogenic differentiation.  These results suggest that OC and OPN stimulate bone regeneration by inducing stem cell proliferation, osteogenesis and by enhancing angiogenic properties. The synergistic effect of OC and OPN observed in this study can be applied as an attractive strategy for bone regeneration therapeutics by targeting different vital cellular processes.

Peripheral artery disease (PAD) is a debilitating condition characterized by the blockage of arteries, which leads to limb amputation in more severe cases. Researchers from SCERG- and BERG-iBB propose the use of human bone marrow (BM) MSC transiently transfected with minicircles encoding for VEGF as an ex vivo gene therapy strategy to enhance angiogenesis in PAD patients. The data shows that VEGF overexpression improved the angiogenic potential of MSC in vitro, as confirmed by endothelial cell tube formation and cell migration assays.These results suggest that minicircle-mediated VEGF gene delivery, combined with the unique properties of human MSC, could represent a promising ex vivo gene therapy approach to improve angiogenesis in the context of PAD. The work was published in Human Gene Therapy.