A microfluidic bone marrow model combining CFD and organ-on-a-chip technologies to study leukemia niche dynamics.

B-cell acute lymphoblastic leukemia (B-ALL) disrupts the architecture and function of the bone marrow niche. However, current in vitro and in vivo models fail to fully capture the spatial, biochemical, and mechanical complexity of the native microenvironment. Here, we present a biomimetic bone marrow on-a-chip that integrates organ-on-a-chip technology, 3D hydrogel culture, and computational modeling to recreate the perivascular, central, and endosteal niches of human bone marrow. The Computational Fluid Dynamics (CFD) was used to guide the design and operation of the microdevice by predicting physiological interstitial flow within the culture system, enabling consistent mechanical stimuli as in vivo under conditions compatible with bone marrow physiology. The microdevice was fabricated using high-resolution 3D printing and soft lithography, and incorporates phaseguide structures for hydrogel confinement, the establishment of three distinct niches and continuous perfusion. Co-cultures of endothelial, stromal, osteoblast, and leukemic cells were maintained in a type I collagen matrix under dynamic conditions. The platform supported high cell viability and enabled compartmentalized spatial organization of multicellular co-cultures. The presence of leukemic cells was associated with changes in soluble signaling molecules within the microenvironment, including increased levels of cytokines, chemokines, and growth factors such as IL-10, IL-13, TNF-α, CCL2, CCL3, CCL5, FGF, G-CSF, and GM-CSF. These patterns are consistent with signaling processes linked to immunoregulation, leukemic supportive signaling, and therapeutic resistance in B-ALL. Together, these findings indicate that the bone-marrow-on-a-chip captures relevant aspects of niche-associated signaling and provides a versatile platform for investigating leukemia microenvironment interactions, with potential in drug screening and preclinical model development.