Abstract
The timely establishment of functional neo-vasculature is pivotal for successful tissue development and regeneration, remaining a central challenge in tissue engineering. In this study, we present a novel (micro)vascularization strategy that explores the use of specialized "vascular units" (VUs) as building blocks to initiate blood vessel formation and create perfusable, stroma-embedded 3D microvascular networks from the bottom-up. We demonstrate that VUs composed of endothelial progenitor cells and organ-specific fibroblasts exhibit high angiogenic potential when embedded in fibrin hydrogels. This leads to the formation of VUs-derived capillaries, which fuse with adjacent capillaries to form stable microvascular beds within a supportive, extracellular matrix-rich fibroblastic microenvironment. Using a custom-designed biomimetic fibrin-based vessel-on-chip (VoC), we show that VUs-derived capillaries can inosculate with endothelialized microfluidic channels in the VoC and become perfused. Moreover, VUs can establish capillary bridges between channels, extending the microvascular network throughout the entire device. When VUs and intestinal organoids (IOs) are combined within the VoC, the VUs-derived capillaries and the intestinal fibroblasts progressively reach and envelop the IOs. This promotes the formation of a supportive vascularized stroma around multiple IOs in a single device. These findings underscore the remarkable potential of VUs as building blocks for engineering microvascular networks, with versatile applications spanning from regenerative medicine to advanced in vitro models.
| Original language | English |
|---|---|
| Pages (from-to) | 499-511 |
| Number of pages | 13 |
| Journal | Bioactive Materials |
| Volume | 38 |
| DOIs | |
| Publication status | Published - Aug 2024 |
Bibliographical note
Publisher Copyright:© 2024 The Authors
Funding
The work was developed under the scope of the EndoSWITCH project (PTDC/BTMORG/5154/2020), supported by the Portuguese Foundation for Science and Technology (FCT), and the REMODEL project (European Union\u2019s Horizon 2020 research and innovation programme, grant agreement 7857491). The authors thanks FCT for Iasmim Orge\u2019s PhD scholarship SFRH/BD/2020.07458 and S\u00EDlvia Bidarra\u2019s research contract DL 57/2016/CP1360/CT0006. The authors also acknowledge the support of i3S Scientific Platforms: \u201CBioimaging\u201D member of the PPBI (Grant No: PPBI-POCI-01-0145-FEDER-022122), \u201CBiointerfaces and Nanotechnology\u201D (Grant No: UID/BIM/04293/2019) and \u201CBioSciences Screening\u201D (member of the PT-OPENSCREEN (NORTE-01-0145-FEDER-085468) and PPBI (PPBI-POCI-01-0145-FEDER-022122)). The presence of a well-developed vascular network in bioengineered tissues and organs is crucial for their functionality and applicability in therapeutic regeneration and disease modelling. The vascular system is among the first systems to develop during embryogenesis, as it is key to supporting and nourishing other tissues and organs [1,2]. It presents an intricate branching pattern, with larger diameter vessels giving rise to intricate networks of progressively smaller ones, ensuring that nearly every cell in the human body is located within a distance of only a few hundreds of micrometers from a capillary [3]. Capillaries, the most prevalent vessels, are lined by a single layer of endothelial cells (EC), supported by basement membrane (BM) and pericytes, and their diameter typically ranges from 5 to 10 \u03BCm [4]. Capillary networks play a central role in tissue homeostasis, enabling the exchange of oxygen, carbon dioxide, nutrients, and metabolites between the bloodstream and the adjacent tissues. Therefore, establishing a functional (micro)vasculature down to the capillary scale in engineered tissues and organs is key to preserving cell viability and function, remaining a central challenge in regenerative medicine strategies.The polylactic acid (PLA) core of the chamber was 3D printed using the Fused Deposition Modeling method and the final design was optimized based on a previously reported set-up [21\u201323] (Fig. S1A, Supporting Information). Briefly, melted PLA layers of 100 \u03BCm height were deposited in precise locations in a layer-by-layer approach to create the object. Then, two fluid dispensing tips of 18 G (Nordson EFD) were inserted into both lateral holes on the sides of the chamber and fixed with epoxy glue. The chamber was then bonded to a glass slide with the same glue and left to dry overnight at room temperature. Pipette tips were used as reservoirs by inserting them into the chamber inlets. For the formation of a perfusable channel in the central reservoir of the microfluidic chamber, an acupuncture needle (300 \u03BCm diameter) was inserted inside the tip inlet and the reservoir was filled with the fibrinogen-thrombin solution for in situ fibrin formation. For studies with VUs, these were added to the hydrogel-precursor solution before loading into the chamber (Fig. S1, Supporting Information). After crosslinking, the needle was carefully removed, and the resulting hollow channel was coated with a fibronectin-collagen solution [24]. In some experiments, we used an alternative VoC setup, incorporating two perfusable hollow channels instead of only one (Fig. S1, Supporting Information). For endothelization, ECFC and hIF (10:1 ratio) suspended in EC growth medium were loaded into the channel at C1 = 1 \u00D7 105, C2 = 5 \u00D7 105 or C3 = 10 \u00D7 105 cells per channel. After 2 h, medium was added to the central well and the tip reservoirs. The chips were cultured under static conditions for the first 24 h after channel endothelization and then transferred to a rocking tilting platform (minimum speed of 4 rpm), which was maintained at 37 \u00B0C under a 5 % v/v CO2 humidified atmosphere for 1\u20132 weeks. Medium (EGM-2MV) inside the channels and central chamber was refreshed daily.The work was developed under the scope of the EndoSWITCH project (PTDC/BTMORG/5154/2020), supported by the Portuguese Foundation for Science and Technology (FCT). The authors thanks FCT for Iasmim Orge's PhD scholarship SFRH/BD/2020.07458, S\u00EDlvia Bidarra's research contract DL 57/2016/CP1360/CT0006 and Silvia Ferreira's research contract CEECINST/00132/2021/CP1774/CT0001. Iasmim Orge thanks the training provided under the scope of the REMODEL project (European Union's Horizon 2020 research and innovation programme, grant agreement 7857491). The authors also acknowledge the support of i3S Scientific Platforms: \u201CBioimaging\u201D member of the PPBI (Grant No: PPBI-POCI-01-0145-FEDER-022122), \u201CBiointerfaces and Nanotechnology\u201D (Grant No: UID/BIM/04293/2019) and \u201CBioSciences Screening\u201D (member of the PT-OPENSCREEN (NORTE-01-0145-FEDER-085468) and PPBI (PPBI-POCI-01-0145-FEDER-022122)).
| Funders | Funder number |
|---|---|
| PPBI | |
| Horizon 2020 | |
| Fundação para a Ciência e a Tecnologia | DL 57/2016/CP1360/CT0006, CEECINST/00132/2021/CP1774/CT0001, SFRH/BD/2020.07458 |
| Fundação para a Ciência e a Tecnologia | |
| Horizon 2020 Framework Programme | DL 57/2016/CP1360/CT0006, 7857491, PPBI-POCI-01-0145-FEDER-022122, UID/BIM/04293/2019, SFRH/BD/2020.07458 |
| Horizon 2020 Framework Programme |
Keywords
- Engineered tissue
- Microtissue
- Organ-on-chip
- Spheroid endothelial colony-forming cells