Protocol for in vitro co-culture assay for rapid expansion of human T cell acute lymphoblastic leukemia

T cell acute lymphoblastic leukemia (T-ALL) is a rare but aggressive hematological cancer that occurs primarily in children and adolescents. Here, we present a protocol for in vitro co-culture assay that enables robust expansion of primary T-ALL cells. We describe steps for seeding T-ALL and stromal cells in 3D organoids and subsequent flow analysis to capture the T-ALL cell growth for long-term culture. This protocol provides a valuable platform for in vitro functional studies and drug screenings using patient-derived cells. For complete details on the use and execution of this protocol, please refer to Rivera et al.¹.