3D cell culture constructs have emerged as indispensable tools in the field of cell biology and tissue engineering. These innovative scaffold-based platforms offer a more physiologically relevant environment for studying cell behavior, tissue development, disease modeling, and drug testing. In this work, we explore the importance of 3D cell culture scaffold-based constructs, the common biomaterials used for their creation, and prospective research routes that promise to further advance this field. In addition to that, we evaluate the use of multidomain peptides (MDPs) as platforms for 3D cell culture, and their applications for stem cell preservation, T cell-based therapies, and growth factor and nucleobase-modified MDP scaffolds. By providing a soft three-dimensional substrate, neutral MDP hydrogels proved to be successful at inducing stem cell quiescence and at maintaining stem cell phenotype and immunomodulatory properties. Additionally, MDP-based hydrogels demonstrated promising properties to be used as a 3D platform for T cell culture, enabling T cell in vitro activation, expansion, and maintenance of antigen specificity. Because of their chemical tunability, ongoing work explores the development of more complex chemistries by introducing growth factor peptide mimics, as well nucleobases for the generation of higher order supramolecular structures within the MDP hydrogels. This thesis describes the use of multidomain peptides as chemically tunable, biocompatible, versatile biomaterials for the generation of 3D culture scaffolds, with potential applications in the fields of cell therapy, drug delivery, stem cell preservation, and translational medicine.