Event Abstract Back to Event Generation of scaffold-supported microtissues inside cell-instructive hydrogels Sílvia J. Bidarra1, 2, Aureliana Sousa1, 2, Ana L. Torres1, 2, 3 and Cristina Barrias1, 2 1 i3S - Instituto de Inovação e Investigação em Saúde, Portugal 2 INEB – Instituto de Engenharia Biomédica, Portugal 3 ICBAS – Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Portugal Introduction: Cell-based therapies are important tools for regenerative medicine. Yet, clinical trials have not yet demonstrated consistent therapeutic benefits, which has been attributed to poor survival/engraftment of transplanted cells, usually administered as single-cell suspensions. Some of the experienced failures may be related to the way cells are delivered, and not to a lack of intrinsic therapeutic potential, pointing to the urgent need for improved cell delivery strategies. A promising approach is the use of cell-aggregates, or microtissues, where cell-cell/cell-matrix interactions are preserved, improving survival, retention and pro-regeneration properties[1]. This work describes a bioengineering approach to generate scaffold-supported microtissues inside molecularly designed cell-instructive hydrogels. Materials and Methods: In situ forming alginate hydrogel matrices loaded with mesenchymal stem cells (MSC) were prepared by internal gelation as described previously[2],[3]. To produce hydrogels with tailored composition and stiffness, pre-gel solutions were prepared using binary mixtures of low and high molecular weight sodium alginate, at different polymer concentrations and various densities of alginate-grafted cell-adhesion RGD peptides. Hydrogels viscoelastic properties were characterized by oscillation rheometry. Cell behavior along in vitro culture was characterized at different levels, namely in terms of viability, spatial rearrangement, proliferation, osteogenic differentiation and production of endogenous ECM and angiogenic factors. The angiogenic potential was also evaluated in vivo, using the CAM assay. Results and Discussion: MSC aggregation and microtissue formation occurred only in more compliant matrices (G’≤120 Pa), independently of the presence/amount of cell-adhesion RGD ligands. Fibronectin assembly stabilized cell-cell contacts within aggregates, even in non-adhesive matrices. However, MSC were only able to substantially contract the artificial matrix when RGD was present. Compliant matrices facilitated MSC proliferation and supported osteogenic differentiation, even in the absence of RGD. Cell interactions with the artificial matrix appear to become less important over time, as cells produced their own ECM. Yet, the intrinsic properties of the artificial matrix and the initial cell-matrix interactions were critical determinates of cell fate. After one week of culture, hydrogels with aggregated MSC expressed significantly higher amounts of pro-angiogenic factors (VEGF) in vitro, and showed higher angiogenic capacity in vivo (CAM assay), as compared to freshly-prepared hydrogels with individually dispersed cells. Conclusions: MSC entrapped within rationally designed alginate hydrogels can be induce to aggregate and form microtissue-like structures, which present interesting therapeutic features. This work provided new clues to the design of cell-instructive 3D matrices and established a new biomaterial-driven strategy to generate scaffold-supported microtissues. FCT/MEC through National Funds co-financed by FEDER through the PT2020 Partnership Agreement under the 4293 Unit I&D; FCT project IF/00939/2013/CP1179/CT000; FCT fellowships (SFRH/BPD/80571/2011 to SJB; SFRH/BPD/90047/2012 to AS; SFRH/BD/94306/2013 to ALT); CCB Research position (IF2013, FCT).