Engineering a multicellular vascular niche to model hematopoietic cell trafficking.

BACKGROUND: The marrow microenvironment and vasculature plays a critical role in regulating hematopoietic cell recruitment, residence, and maturation. Extensive in vitro and in vivo studies have aimed to understand the marrow cell types that contribute to hematopoiesis and the stem cell environment. Nonetheless, in vitro models are limited by a lack of complex multicellular interactions, and cellular interactions are not easily manipulated in vivo. Here, we develop an engineered human vascular marrow niche to examine the three-dimensional cell interactions that direct hematopoietic cell trafficking. METHODS: Using soft lithography and injection molding techniques, fully endothelialized vascular networks were fabricated in type I collagen matrix, and co-cultured under flow with embedded marrow fibroblast cells in the matrix. Marrow fibroblast (mesenchymal stem cells (MSCs), HS27a, or HS5) interactions with the endothelium were imaged via confocal microscopy and altered endothelial gene expression was analyzed with RT-PCR. Monocytes, hematopoietic progenitor cells, and leukemic cells were perfused through the network and their adhesion and migration was evaluated. RESULTS: HS27a cells and MSCs interact directly with the vessel wall more than HS5 cells, which are not seen to make contact with the endothelial cells. In both HS27a and HS5 co-cultures, endothelial expression of junctional markers was reduced. HS27a co-cultures promote perfused monocytes to adhere and migrate within the vessel network. Hematopoietic progenitors rely on monocyte-fibroblast crosstalk to facilitate preferential recruitment within HS27a co-cultured vessels. In contrast, leukemic cells sense fibroblast differences and are recruited preferentially to HS5 and HS27a co-cultures, but monocytes are able to block this sensitivity. CONCLUSIONS: We demonstrate the use of a microvascular platform that incorporates a tunable, multicellular composition to examine differences in hematopoietic cell trafficking. Differential recruitment of hematopoietic cell types to distinct fibroblast microenvironments highlights the complexity of cell-cell interactions within the marrow. This system allows for step-wise incorporation of cellular components to reveal the dynamic spatial and temporal interactions between endothelial cells, marrow-derived fibroblasts, and hematopoietic cells that comprise the marrow vascular niche. Furthermore, this platform has potential for use in testing therapeutics and personalized medicine in both normal and disease contexts.

Micropatterning and Assembly of 3D Microvessels.

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 µm. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.