RESUMEN
BACKGROUND: There is great interest to engineer in vitro models that allow the study of complex biological processes of the microvasculature with high spatiotemporal resolution. Microfluidic systems are currently used to engineer microvasculature in vitro, which consists of perfusable microvascular networks (MVNs). These are formed through spontaneous vasculogenesis and exhibit the closest resemblance to physiological microvasculature. Unfortunately, under standard culture conditions and in the absence of co-culture with auxiliary cells as well as protease inhibitors, pure MVNs suffer from a short-lived stability. METHODS: Herein, we introduce a strategy for stabilization of MVNs through macromolecular crowding (MMC) based on a previously established mixture of Ficoll macromolecules. The biophysical principle of MMC is based on macromolecules occupying space, thus increasing the effective concentration of other components and thereby accelerating various biological processes, such as extracellular matrix deposition. We thus hypothesized that MMC will promote the accumulation of vascular ECM (basement membrane) components and lead to a stabilization of MVN with improved functionality. RESULTS: MMC promoted the enrichment of cellular junctions and basement membrane components, while reducing cellular contractility. The resulting advantageous balance of adhesive forces over cellular tension resulted in a significant stabilization of MVNs over time, as well as improved vascular barrier function, closely resembling that of in vivo microvasculature. CONCLUSION: Application of MMC to MVNs in microfluidic devices provides a reliable, flexible and versatile approach to stabilize engineered microvessels under simulated physiological conditions.
RESUMEN
A faithful reconstruction of the native cellular microenvironment is instrumental for tissue engineering. Macromolecular crowding (MMC) empowers cells to deposit their own extracellular matrix (ECM) in greater amounts, and thus contributes to building tissue-specific complex microenvironments in vitro. Dextran sulfate (DxS, 500â¯kDa), a semi-synthetic sulfated polyglucose, was shown previously at a fractional volume occupancy (FVO) of 5.2% (v/v; 100⯵g/ml) to act as a potent molecular crowding agent in vitro. When added to human mesenchymal stromal cell (MSC) cultures, DxS enhanced fibronectin and collagen I deposition several-fold also at concentrations with negligible FVO (<1% v/v). In a cell-free system, incubation of culture media supplemented with fetal bovine serum (FBS), purified fibronectin or collagen I with DxS led to a co-deposition of respective components, exhibiting a similar granular pattern as observed in cell culture. Aggregation of FBS components, fibronectin or collagen I with DxS was confirmed by dynamic light scattering, where an increase in hydrodynamic radius in the respective mixtures was observed. FBS- and fibronectin aggregates could be dissociated with increasing salt concentrations, indicating electrostatic forces to be responsible for the aggregation. Conversely, collagen I-DxS aggregates increased in size with increasing ion concentration, likely caused by charge screening of collagen I, which is net negatively charged at neutral pH, thus permitting weaker intermolecular interactions to occur. The incorporation of DxS into the ECM resulted in altered ECM topography and stiffness. DxS-supplemented cultures exhibited potentiated bioactivity, such as enhanced adipogenic and especially osteogenic differentiation under inductive conditions. We propose an alternative mechanism by which DxS drives ECM deposition via aggregation, and in an independent manner from MMC. A deeper understanding of the underlying mechanism will enable optimized engineering approaches for ECM-rich tissue constructs.