3D bioprinting is transforming tissue engineering by enabling spatial arrangement of cells and cell aggregates within supportive hydrogels. Among available materials, alginate remains widely used for its biocompatibility, printability, and cost-effectiveness. However, its bioinert nature and lack of adhesive moieties restrict its capacity to support essential processes like cell adhesion, migration, and proliferation. In this study, we propose a comprehensive approach to enhance alginate hydrogels focusing on stem, stromal, and endothelial cell types to support extended growth and vascular network formation. Key innovations include the incorporation of the TYRAY peptide in 3D alginate hydrogels─its first application in this context─to promote cell adhesion and migration, accompanied by Ca(OH)2-modified surfaces for stable hydrogel anchoring and an ultrasonic mist cross-linking to preserve 3D structure fidelity. Functionalization with the TYRAY peptide significantly enhanced cell proliferation, promoted multicellular spheroid fusion, and supported endothelial network development in comparative culture setting. Together, these findings establish this defined, xeno-free alginate system as a versatile bioink material suitable for 3D culture and bioprinting applications.

Department of Biomedical Engineering Technion Israel Institute of Technology Haifa 32000 Israel

Department of Burns and Plastic Surgery University Hospital Brno and Faculty of Medicine Masaryk University Jihlavska 20 Brno 62500 Czech Republic

Department of Histology and Embryology Faculty of Medicine Masaryk University Kamenice 5 Brno 62500 Czech Republic

Department of Molecular Cell Biology Institute for Cancer Research Oslo University Hospital 0379 Oslo Norway

Department of Plastic and Aesthetic Surgery St Anne's University Hospital and Faculty of Medicine Masaryk University Pekarska 53 Brno 656 91 Czech Republic

Institute of Macromolecular Chemistry Czech Academy of Sciences Heyrovskeho nam 2 Prague 16200 Czech Republic

International Clinical Research Center St Anne's University Hospital Brno Pekarska 53 Brno 656 91 Czech Republic

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