An immunocompetent bone marrow-on-a-chip model for studying human hematological malignancies and preclinical therapeutic screening.

The complex pathophysiology of leukemia and other bone marrow-related hematological malignancies underscores the critical need for immunocompetent bone marrow models that can accurately recapitulate key disease microenvironment and patient therapeutic responses. Existing bone marrow models often lack the fidelity required to recapitulate patient-specific pathophysiology or to emulate the intricate dynamics of in vivo immune responses. This limitation ultimately constrains their utility in critical areas such as leukemia immunotherapy development. Here, to overcome this challenge, we present a detailed protocol for building a 3D microfluidic human bone marrow-on-a-chip microphysiological system that recapitulates the anatomical organization and function of the native tissue. Its concentric three-compartment design replicates the marrow's spatial architecture, co-culturing stromal and hematopoietic cells within a vascularized niche to establish a physiologically relevant, immunocompetent microenvironment. To model patient-specific biology, the platform incorporates patient-derived samples, enabling the personalized evaluation of therapeutic interventions such as chimeric antigen receptor T cell therapy and chemotherapy. The platform supports multiplexed readouts through live imaging, immunofluorescence, cytokine profiling, flow cytometry and single-cell sequencing, enabling a comprehensive analysis of treatment response. The protocol requires ~7 d to establish the functional bone marrow chip, comprising microfluidic device fabrication (1 d), cell seeding (1 d) and progressive development of the bone marrow model (5 d). Therapeutic testing is conducted over a variable timeframe, typically ranging from 2 d to 14 d. The protocol is designed for researchers with basic experience in cell culture and fluorescence imaging,who have basic microfluidics fabrication and tissue culture experience.