Recovery of Therapeutically Ablated Engineered Blood-Vessel Networks on a Plug-and-Play Platform.

Limiting the availability of key angiogenesis-promoting factors is a successful strategy to ablate tumor-supplying blood vessels or to reduce excessive vasculature in diabetic retinopathy. However, the efficacy of such anti-angiogenic therapies (AATs) varies with tumor type, and regrowth of vessels is observed upon termination of treatment. The ability to understand and develop AATs remains limited by a lack of robust in vitro systems for modeling the recovery of vascular networks. Here, complex 3D micro-capillary networks are engineered by sequentially seeding human bone marrow-derived mesenchymal stromal cells and human umbilical vein endothelial cells (ECs) on a previously established, synthetic plug-and-play hydrogel platform. In the tightly interconnected vascular networks that form this way, the two cell types share a basement membrane-like layer and can be maintained for several days of co-culture. Pre-formed networks degrade in the presence of bevacizumab. Upon treatment termination, vessel structures grow back to their original positions after replenishment with new ECs, which also integrate into unperturbed established networks. The data suggest that this plug-and-play platform enables the screening of drugs with blood-vessel inhibiting functions. It is believed that this platform could be of particular interest in studying resistance or recovery mechanisms to AAT treatment.

Simple Establishment of a Vascularized Osteogenic Bone Marrow Niche Using Pre-Cast Poly(ethylene Glycol) (PEG) Hydrogels in an Imaging Microplate.

The bone and bone marrow are highly vascularized and structurally complex organs, and are sites for cancer and metastasis formation. In vitro models recapitulating bone- and bone marrow-specific functions, including vascularization, that are compatible with drug screening are highly desirable. Such models can bridge the gap between simplistic, structurally irrelevant two-dimensional (2D) in vitro models and the more expensive, ethically challenging in vivo models. This article describes a controllable three-dimensional (3D) co-culture assay based on engineered poly(ethylene glycol) (PEG) matrices for the generation of vascularized, osteogenic bone-marrow niches. The PEG matrix design allows the development of 3D cell cultures through a simple cell seeding step requiring no encapsulation, thus enabling the development of complex co-culture systems. Furthermore, the matrices are transparent and pre-cast onto glass-bottom 96-well imaging plates, rendering the system suitable for microscopy. For the assay described here, human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) are cultured first until a sufficiently developed 3D cell network is formed. Subsequently, GFP-expressing human umbilical vein endothelial cells (HUVECs) are added. The culture development is followed by bright-field and fluorescence microscopy. The presence of the hBM-MSC network supports the formation of vascular-like structures that otherwise would not form and that remain stable for at least 7 days. The extent of vascular-like network formation can easily be quantified. This model can be tuned toward an osteogenic bone-marrow niche by supplementing the culture medium with bone morphogenetic protein 2 (BMP-2), which promotes the osteogenic differentiation of the hBM-MSCs, as assessed by increased alkaline phosphatase (ALP) activity at day 4 and day 7 of co-culture. This cellular model can be used as a platform for culturing various cancer cells and studying how they interact with bone- and bone marrow-specific vascular niches. Moreover, it is suitable for automation and high-content analyses, meaning it would enable cancer drug screening under highly reproducible culture conditions.