Microvasculature-directed thrombopoiesis in a 3D in vitro marrow microenvironment

Abstract

Vasculature is an interface between the circulation and the hematopoietic tissue providing the means for hundreds of billions of blood cells to enter the circulation every day in a regulated fashion. The precise mechanisms that control the interactions of hematopoietic cells with the vessel wall are largely undefined. Here, we report on the development of an in vitro 3D human marrow vascular microenvironment (VME) to study hematopoietic trafficking and the release of blood cells, specifically platelets. We show that mature megakaryocytes from aspirated marrow as well as megakaryocytes differentiated in culture from CD34+ cells can be embedded in a collagen matrix containing engineered microvessels to create a thrombopoietic VME. These megakaryocytes continue to mature, penetrate the vessel wall, and release platelets into the vessel lumen. This process can be blocked with the addition of antibodies specific for CXCR4, indicating that CXCR4 is required for megakaryocyte migration, though whether it is sufficient is unclear. The 3D marrow VME system shows considerable potential for mechanistic studies defining the role of marrow vasculature in thrombopoiesis. Through a stepwise addition or removal of individual marrow components, this model provides potential to define key pathways responsible for the release of platelets and other blood cells.

Engineered 3D Microvascular Networks for the Study of Ultrasound-Microbubble-Mediated Drug Delivery

“…We successfully developed a perfusable 3D microvascular network (Figure ) that was fabricated with biological materials, including collagen I and human endothelial cells. Collagen I is a key component of the native extracellular matrix and is used extensively for various tissue culture applications; − ,− ,− it provides good mechanical support to the vessels and allows biomolecular diffusion within it. In addition, the collagen I matrix allows HUVECs to exhibit in vivo-like cell-matrix interaction .…”

“…Organ-specific endothelial cells can be used to further improve the physiological relevance of the model. We had previously used organ-specific endothelial cells to simulate the human kidney peritubular capillaries and the marrow vascular microenvironment. , In a future study, we may incorporate organ-specific endothelial cells into the MVN and investigate whether the efficiency of ultrasound-mediated drug delivery efficiency is organ-dependent. As it stands, however, organ-specific endothelial cells are donor-based and therefore limited in supply.…”

“…We had previously used organ-specific endothelial cells to simulate the human kidney peritubular capillaries and the marrow vascular microenvironment. 58,63 In a future study, we may incorporate organ-specific endothelial cells into the MVN and investigate phenotype and much more difficult to culture. These challenges will need to be addressed before a sonoporation study of MVN with the use of organ-specific cells is practically feasible.…”

“…We previously developed an in vitro microvascular network (MVN) model that captured the spatial characteristics (tubular architecture and vessel branching), physical environment (flow and shear conditions), and biological interactions (intercellular and matrix-cell communication) found in vivo. − This collagen-based three-dimensional (3D) microvascular network was made using microfabrication and injection molding techniques and cellularized with human umbilical cord endothelial cells (HUVECs). The diameter of the microvascular channels (150 μm) is comparable to that of a human arteriole or a dilated tumor capillary. , We have demonstrated that HUVECs in MVNs exhibit in vivo-like behaviors and provided evidence that they are suitable for studying vascular biology, pathology, and pharmacology. − ,− Our main hypothesis is that the development of an in vitro model that closely resembles physiology will serve as an important research platform to study sonoporation. Here we have further utilized and exploited the MVN by perfusing it with microbubbles at physiological flow rates and interrogating it with ultrasound to observe in real-time the interactions between microbubbles and the vascular endothelium.…”

Latest culture techniques: cracking the secrets of bone marrow to mass-produce erythrocytes and platelets <i>ex vivo

“…Using a different approach, involving soft gel lithography, Kotha and colleagues were able to use collagen hydrogels as a scaffold to create a microvascular network. 103 Endothelial cells were seeded in the microvascular network to form endothelial vessels with a lumen, while megakaryocytes were encapsulated directly in the type I collagen hydrogel. Megakaryocytes were able to migrate to the microvascular network and to extend proplatelets.…”