Multi-niche human bone marrow-on-a-chip for plasma cell survival and differentiation

Introduction:: Antibody-secreting cells (ASCs) are terminally differentiated immune cells that are are responsible for producing and secreting antigen-specific immunoglobulin antibodies to neutralize and help eliminate pathogens. ASCs differentiate from activated B cells in lymph nodes and egress to the bone marrow (BM), eventually maturing to long-lived plasma cells (LLPCs). As LLPCs can produce specific antibodies for years after an initial infection or immunization, generating LLPCs is the main objective of a vaccine¹. However, a major limitation to studying these cells is the inability to culture them in vitro, due to the complexities of the BM niche required to sustain them. To circumvent these limitations, we have bioengineered a BM-chip that incorporates the endosteal, central marrow, and perivascular niches of the human BM where LLPCs differentiate and reside². Herein, we studied how the multi-niche BM-chip may support the survival and maturation of ASCs towards LLPCs.

Materials and Methods:: The human BM-chip device was assembled with a bottomless 96-well plate as previously described. Then, human mesenchymal stromal cells (MSCs) were differentiated into osteoblasts (OBs) in osteogenic medium. After 21 days of differentiation, human umbilical vein endothelial cells (HUVECs) and additional MSCs were loaded in a fibrin-collagen I hydrogel atop the OB layer. Normal human lung fibroblasts (NHLFs) were similarly seeded into the outer channels. Finally, the media channels were hydrated with endothelial growth medium supplemented with additional vascular endothelial growth factor and angiopoietin-1. The NHLFs and media induced the HUVECs to form microvascular networks within the hydrogel, supported by the perivascular MSCs. After 4 days of vasculogenesis, CD19+ human B cells enriched from B cell organoids derived from human tonsil samples were perfused into the BM-chip through the microvascular network. At experimental endpoints, the BM-chip devices were either fixed and stained for confocal microscopy or the hydrogels were degraded and the cells were harvested for flow cytometry.


Results, Conclusions, and Discussions:: Confocal imaging reveals that the BM-chip expresses key components of the BM niche essential for ASC retention and survival including CXC12, APRIL, hyaluronan, and ECM components including collagen-I collagen-IV, and fibronectin. Next, CD19+ human B cells were perfused into the BM-chip in media containing B cell-activating factor and IL21, and allowed to culture for up to 7 days. After 7 days of culture in the BM-chip, the cells took on a CD19loCD27+CD38++ phenotype compared to day 0, indicating potential differentiation towards ASCs. Additionally, CD19+ cells depleted of CD138 expression gain CD138 after culture in the BM-chip, further suggesting ASC maturation. Our results indicate that this BM-chip has the potential to support plasma-like cells ex vivo and provides a novel system for studying the BM microenvironmental cues required for plasma cell maturation and maintenance.

Acknowledgements (Optional): : Funding provided by the Wellcome Leap HOPE program.


References (Optional): : ¹Nguyen DC. Nat Com. 2018; ²Nelson MR. Biomat. 2021.