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Engineering Organoid Vascularization


Engineering Organoid Vascularization

Sergei Grebenyuk et al. Front Bioeng Biotechnol.


The development of increasingly biomimetic human tissue analogs has been a long-standing goal in two important biomedical applications: drug discovery and regenerative medicine. In seeking to understand the safety and effectiveness of newly developed pharmacological therapies and replacement tissues for severely injured non-regenerating tissues and organs, there remains a tremendous unmet need in generating tissues with both functional complexity and scale. Over the last decade, the advent of organoids has demonstrated that cells have the ability to reorganize into complex tissue-specific structures given minimal inductive factors. However, a major limitation in achieving truly in vivo-like functionality has been the lack of structured organization and reasonable tissue size. In vivo, developing tissues are interpenetrated by and interact with a complex network of vasculature which allows not only oxygen, nutrient and waste exchange, but also provide for inductive biochemical exchange and a structural template for growth. Conversely, in vitro, this aspect of organoid development has remained largely missing, suggesting that these may be the critical cues required for large-scale and more reproducible tissue organization. Here, we review recent technical progress in generating in vitro vasculature, and seek to provide a framework for understanding how such technologies, together with theoretical and developmentally inspired insights, can be harnessed to enhance next generation organoid development.

Keywords: bioengineering; biofabrication; biomaterials; organoid; vascularization.


Figure 1
Figure 1
An overview of vascularization techniques. (A) Vascular structures are bioprinted by extrusion or droplet deposition of cells suspended in biocompatible gel in a patterned manner. (B) Photo-induced gelation of cell-containing liquid precursors is performed in a layer-by-layer fashion with DMD patterning. (C) 2-photon photopolymerization is used to directly fabricate perfusable tubular networks of arbitrary geometry which can be embedded in a cell-containing hydrogel. (D) A cell-laden extracellular matrix is cast over a sacrificial filament lattice created by bioprinting or stereo-lithography. In an aqueous environment, the sacrificial filaments are dissolved and the resulting hollow perfusable network is perfused with endothelial cells which form a conformal layer around the inner diameter of the vessels. (E) Microchannels fabricated in a cell-laden hydrogel by laser ablation can be directly seeded with endothelial cells to generate functional blood vessels. (F) Organoid angiogenesis can be achieved by grafting in a highly vascularized animal tissue, with the host vasculature infiltrating the organoid. Alternatively, organoid vascularization is also achieved in vitro by endothelial cell co-culture in compartmentalized microfluidic chip. VEGF and hypoxia gradients established on-chip provide spatial guidance cues to direct angiogenic sprouting.
Figure 2
Figure 2
Examples of vascularized constructs and tissues. (A) Schematic vascular network formed by bio-ink extrusion and confocal image of the bio-printed tubular constructs containing green fluorescent beads, perfused with red fluorescent micro-beads inside the lumens. (B) DMD-based bio-printing of cell-laden tissue constructs with HUVECs (red) encapsulated in the intended channels and HepG2 (green) cells encapsulated in the surrounding area (top). Scale bars, 250 μm. HUVECs (Green, CD31) and supportive 10T1/2 mesenchymal cells (Purple, α-SMA) aligned within the patterned channel regions after 1-week in vitro culture (bottom). Insert indicates cross-section of the channel, demonstrating a lumen formed by biodegradation of the gel. Scale bars: 100 μm. (C) Schematic of the vascular lumen, endothelial cells lining the vascular wall, and the interstitial zone containing matrix and encapsulated cells (top). A single sacrificial carbohydrate-glass fiber is encapsulated in a fibrin gel. After immersion in aqueous solution the fiber is dissolved (middle) yielding an open perfusable channel in the fibrin gel (bottom, scale bar, 500 μm). Representative vessel after 9 days in culture demonstrating endothelial monolayer (red) lining the vascular lumen, surrounded by 10T1/2 cells (green). Endothelial cells forming single and multicellular sprouts (arrowheads) from the patterned vasculature. Scale bars, 200 μm. (D) Multi-photon micromachining device generates hydrogel micro-channels allowing for fibroblast adhesion and growth within a Y-shaped feature 9 days after seeding. Scale bar, 100 μm.

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