Stem cells reside and interact in their native microenvironment in complex three-dimensional (3D) networks with divergent cellular responses. Natural-origin hydrogels represent a valuable mean to investigate such cellular events, including viability, spreading, migration, proliferation, and differentiation regulated by matrix-stiffness. Gellan gum (GG), a natural polymer-based biomaterial is well explored for 3D cell encapsulation. Microscopy-based, high-content assays allow parallel monitoring multiple cell phenotypes within 3D hydrogel microenvironments in an automated fashion. Here, we propose to optimize a confocal microscopy-based automated high-content imaging platform for assessing phenotypic markers representing viability, spreading, proliferation and further osteogenic commitment of human adipose-derived stem cells (hASCs) encapsulated in GG-based hydrogels. The platform is based on a standard 8-well ibidi µ-slide to maximize the compatibility with automated screener (Matrix M4, Leica TCS SP8). Specifically, hASCs (1x 106/mL) were mixed individually with 8 different formulations of pre-polymer solutions of [1) GG 1%, 2) GG 1.5%, 3) GG 1%: Col 1mg/mL (75:25), 4) GG 1%: Col 2mg/mL (75:25), 5) GG 1.5%: Col 1mg/mL (75:25), 6) GG 1.5%: Col 2mg/mL (75:25), 7) Col 1mg/mL and 8) Col 2mg/mL] and 100ul drops of each cell-containing solutions were ionically cross-linked with α-MEM. Control collagen gels were thermally cross-linked at 37 ºC for 30 min. Finally, the hydrogel macrospheres were transferred to their respective wells on the screening µ-slide platform and maintained in α-MEM. The µ-slide platforms were autoscanned with optimized autofocus and assigned jobs for phenotypic markers representing viability (Calcein/Ethidium-homodimer-1) on live cells and spreading (Phalloidin/DAPI/Vinculin), proliferation (EdU/Hoescht 33342) and matrix-stiffness responsive marker expression (YAP, yes-associated protein) in fixed cells within the hydrogels. Finally, the imaging database generated out of MatrixScreener platform was analyzed using CellProfiler to quantify the impact of the hydrogels upon encapsulated hASCs. It was observed that GG 1.5%: Col 1mg/mL hydrogel supported better viability, spreading, proliferation and cytoplasmic-YAP localization compared to other conditions. However, more detailed analysis of stiffness-responsive phenotypic changes in encapsulated hASCs needs to be performed, before studying their osteogenic-lineage commitment. Acknowledgments: This work was supported by the European Research Council grant agreement ERC-2012-ADG 20120216-321266 for project ComplexiTE, Portuguese Foundation for Science and Technology (FCT) R&D grant POCI-01-0145-FEDER-016715 (PTDC/BBB-ECT/4317/2014) for the project MicroLiver (DKB) and IF/00347/2015 grant to RPP.