Background:

Hematopoietic stem cell transplantation (HSCT) is a well-established therapy for various hematological malignancies. Umbilical cord blood (UCB) HSCs offer an alternative source but are limited by a small number of CD34 + cells, delaying hematopoietic and immunologic recovery. Recent evidence underscores the importance of closely recapitulating the bone marrow niche for ex vivo stem cell expansion.. Methods We describe a novel 3D microfluidic chip for dynamic co-culture of MSCs and HSCs within a demineralized bone matrix (DBM) scaffold. Co-culture was conducted in both dynamic and static 3D conditions without exogenous cytokines for seven days. Results Scanning electron microscopy images revealed that CD34 + CD38- cells primarily lodged beneath the MSCs layer rather than on its surface. MSCs repopulated the DBM scaffold and sustained HSC expansion for seven days. The proportion of CD34 + cells increased significantly (1.35-fold), with CD34 + CD38- cells nearly doubling in the microfluidic 3D co-culture compared to the control group. Microfluidic conditions promoted CXCR4 transcription (4.7-fold), colony-forming potency (3.6-fold), and osteogenic properties of DBM (9.4-fold) compared to the control group. Conclusion Indirect and direct crosstalk among HSCs, MSCs, and extracellular matrix (ECM) proteins can enhance UCB HSC expansion potency in an engineered bone marrow chip. Our microfluidic-based chip represents a significant step toward overcoming current limitations in UCB HSC numbers.