In Vitro Modeling for Three-Dimensional Imaging of the Bone Marrow

Abstract

The bone marrow microenvironment is responsible for the maintenance of hematopoietic stem cell (HSC) activity, and this HSC niche is characterized by complex cellular, chemical, and structural components necessary to maintain hematopoiesis. While the inaccessibility of living bone marrow hampers the study of its pathophysiology, physical and digital models could be powerful tools to study hematopoiesis and test new therapeutics. Current in vitro bone marrow models utilize either simple microfluidic systems or opaque scaffolds with tortuous pores that do not mimic physiologically relevant bone geometry or do not have the ability for longitudinal imaging. Therefore, there is an unmet need for 3D models that are both perfusable and amenable to fluorescence microscopy to visualize the spatiotemporal dynamics of cells in the bone marrow microenvironment in real time. The primary goal of this work is to create a 3D bone marrow model that is able to capture physiologically relevant features of trabecular bone while still being amenable to longitudinal imaging, easily manufactured, and amenable to human cell culture to study disease and potentially test therapeutics. We developed a hybrid injection molding/stereolithography (SLA) fabrication method for rapid prototyping of polystyrene (PS) perfusion cell culture devices, along with a computational fluid dynamics (CFD) model to predict the collection efficiency of perfused human mesenchymal stem cells (hMSCs) on the collectors. hMSC deposition on the collectors and proliferation of cells over 7 days was visualized by fluorescence microscopy. CFD simulations of collection efficiency agreed with experimental measurements within a factor of two. The effect of collector diameter on simulated and experimental cell collection efficiencies followed a trend similar to that predicted by interception theory corrected for intermolecular and hydrodynamic forces. In addition, engraftment of HSCs and acute myeloid leukemia (AML) cells into a mineralized osteoblastic matrix was visualized in a 2D coculture system. This work highlights the utility of CFD simulations and hybrid injection molding for rapid prototyping and optimization of 3D bone marrow models to study cell dynamics in physiologically relevant bone marrow microenvironments and disease states in real time.