Engineering injectable vascularized tissues from the bottom-up: Dynamics of in-gel extra-spheroid dermal tissue assembly.

Modular tissue engineering approaches open up exciting perspectives for the biofabrication of vascularized tissues from the bottom-up, using micro-sized units such as spheroids as building blocks. While several techniques for 3D spheroid formation from multiple cell types have been reported, strategies to elicit the extra-spheroid assembly of complex vascularized tissues are still scarce. Here we describe an injectable approach to generate vascularized dermal tissue, as an example application, from spheroids combining fibroblasts and endothelial progenitors (OEC) in a xeno-free (XF) setting. Short-term cultured spheroids (1 day) were selected over mature spheroids (7 days), as they showed significantly higher angiogenic sprouting potential. Embedding spheroids in fibrin was crucial for triggering cell migration into the external milieu, while providing a 3D framework for in-gel extra-spheroid morphogenesis. Migrating fibroblasts proliferated and produced endogenous ECM forming a dense tissue, while OEC self-assembled into stable capillaries with lumen and basal lamina. Massive in vitro interconnection between sprouts from neighbouring spheroids rapidly settled an intricate vascular plexus. Upon injection into the chorioallantoic membrane of chick embryos, fibrin-entrapped pre-vascularized XF spheroids developed into a macrotissue with evident host vessel infiltration. After only 4 days, perfused chimeric capillaries with human cells were present in proximal areas, showing fast and functional inosculation between host and donor vessels. This method for generating dense vascularized tissue from injectable building blocks is clinically relevant and potentially useful for a range of applications.

Directed self-assembly of spheroids into modular vascular beds for engineering large tissue constructs.

Spheroids can be used as building-blocks for bottom-up generation of artificial vascular beds, but current biofabrication strategies are often time-consuming and complex. Also, pre-optimization of single spheroid properties is often neglected. Here, we report a simple setup for rapid biomanufacturing of spheroid-based patch-like vascular beds. Prior to patch assembly, spheroids combining mesenchymal stem/stromal cells (MSCs) and outgrowth endothelial cells (OECs) at different ratios (10:1; 5:1; 1:1; 1:5) were formed in non-adhesive microwells and monitored along 7 d. Optimal OEC retention and organization was observed at 1:1 MSC/OEC ratio. Dynamic remodelling of spheroids led to changes in both cellular and extracellular matrix components (ECMs) over time. Some OEC formed internal clusters, while others organized into a peripheral monolayer, stabilized by ECM and pericyte-like cells, with concomitant increase in surface stiffness. Along spheroid culture, OEC switched from an active to a quiescent state, and their endothelial sprouting potential was significantly abrogated, suggesting that immature spheroids may be more therapeutically relevant. Non-adhesive moulds were subsequently used for triggering rapid, one-step, spheroid formation/fusion into square-shaped patches, with spheroids uniformly interspaced via a thin cell layer. The high surface area, endothelial sprouting potential, and scalability of the developed spheroid-based patches make them stand out as artificial vascular beds for modular engineering of large tissue constructs.

Growth of Endothelial Cells in Space and in Simulated Microgravity - a Comparison on the Secretory Level.

BACKGROUND/AIMS: Endothelial cells exposed to the Random Positioning Machine (RPM) reveal three different phenotypes. They grow as a two-dimensional monolayer and form three-dimensional (3D) structures such as spheroids and tubular constructs. As part of the ESA-SPHEROIDS project we want to understand how endothelial cells (ECs) react and adapt to long-term microgravity. METHODS: During a spaceflight to the International Space Station (ISS) and a subsequent stay onboard, human ECs (EA.hy926 cell line) were cultured for 12 days in real microgravity inside an automatic flight hardware, specially designed for use in space. ECs were cultivated in the absence or presence of vascular endothelial growth factor, which had demonstrated a cell-protective effect on ECs exposed to an RPM simulating microgravity. After cell fixation in space and return of the samples, we examined cell morphology and analyzed supernatants by Multianalyte Profiling technology. RESULTS: The fixed samples comprised 3D multicellular spheroids and tube-like structures in addition to monolayer cells, which are exclusively observed during growth under Earth gravity (1g). Within the 3D aggregates we detected enhanced collagen and laminin. The supernatant analysis unveiled alterations in secretion of several growth factors, cytokines, and extracellular matrix components as compared to cells cultivated at 1g or on the RPM. This confirmed an influence of gravity on interacting key proteins and genes and demonstrated a flight hardware impact on the endothelial secretome. CONCLUSION: Since formation of tube-like aggregates was observed only on the RPM and during spaceflight, we conclude that microgravity may be the major cause for ECs' 3D aggregation.

Continuous Exposure to Simulated Hypergravity-Induced Changes in Proliferation, Morphology, and Gene Expression of Human Tendon Cells.

Gravity influences physical and biological processes, especially during development and homeostasis of several tissues in the human body. Studies under altered gravity have been receiving great attention toward a better understanding of microgravity-, hypogravity (<1 g)-, or hypergravity (>1 g)-induced alterations. In this work, the influence of simulated hypergravity over human tendon-derived cells (hTDCs) was studied at 5, 10, 15, and 20 g for 4 or 16 h, using a large diameter centrifuge. Main results showed that 16 h of simulated hypergravity limited cell proliferation. Cell area was higher in hTDCs cultured at 5, 10, and 15 g for 16 h, in comparison to 1 g control. Actin filaments were more pronounced in hTDCs cultured at 5 and 10 g for 16 h. Focal adhesion kinase (FAK) was mainly expressed in focal adhesion sites upon hypergravity stimulation, in comparison to perinuclear localization in control cells after 16 h; and FAK number/cell increased with increasing g-levels. A tendency toward an upregulation of tenogenic markers was observed; scleraxis (SCX), tenascin C (TNC), collagen type III (COL3A1), and decorin (DCN) were significantly upregulated in hTDCs cultured at 15 g and COL3A1 and DCN were significantly upregulated in hTDCs cultured at 20 g. Overall, simulated hypergravity affected the behavior of hTDCs, with more pronounced effects in the long-term period (16 h) of stimulation.

Effects of hypergravity on the angiogenic potential of endothelial cells.

Angiogenesis, the formation of blood vessels from pre-existing ones, is a key event in pathology, including cancer progression, but also in homeostasis and regeneration. As the phenotype of endothelial cells (ECs) is continuously regulated by local biomechanical forces, studying endothelial behaviour in altered gravity might contribute to new insights towards angiogenesis modulation. This study aimed at characterizing EC behaviour after hypergravity exposure (more than 1g), with special focus on cytoskeleton architecture and capillary-like structure formation. Herein, human umbilical vein ECs (HUVECs) were cultured under two-dimensional and three-dimensional conditions at 3g and 10g for 4 and 16 h inside the large diameter centrifuge at the European Space Research and Technology Centre (ESTEC) of the European Space Agency. Although no significant tendency regarding cytoskeleton organization was observed for cells exposed to high g's, a slight loss of the perinuclear localization of ß-tubulin was observed for cells exposed to 3g with less pronounced peripheral bodies of actin when compared with 1g control cells. Additionally, hypergravity exposure decreased the assembly of HUVECs into capillary-like structures, with a 10g level significantly reducing their organization capacity. In conclusion, short-term hypergravity seems to affect EC phenotype and their angiogenic potential in a time and g-level-dependent manner.