Matrix Stiffness Regulates Diffuse Large B-Cell Lymphoma (DLBCL) Spheroid Formation in a Tunable 3D Chitosan Hydrogel Model

Diffuse large B-cell lymphoma (DLBCL) represents the most frequent subtype of non-Hodgkin lymphoma; however, conventional 2D cultures and murine models fail to fully recapitulate the complexity of the human tumor microenvironment (TME). To address these limitations, we developed and characterized three chitosan-based hydrogels with distinct physicochemical and mechanical properties as tunable three-dimensional (3D) platforms for DLBCL culture. U2932 lymphoma cells were encapsulated either alone or in co-culture with WPMY-1 stromal cells to investigate the effects of matrix stiffness and stromal support on lymphoma growth. Furthermore, co-culture with stromal cells promoted rapid spheroid formation and generated larger cellular aggregates than mono-cultures, with the most pronounced effect observed in the 2% chitosan hydrogel. Live/dead assays demonstrated high cell viability within the selected culture condition. Overall, the 2% chitosan formulation provided the most suitable microenvironment for supporting DLBCL growth and organization, highlighting its potential as a physiologically relevant 3D in vitro model for investigating lymphoma biology and evaluating novel therapeutic strategies.