Speaker
Description
The combination of 3D bioprinting and tailored bioreactor designs allows the fabrication of advanced in 3D in vitro models. However, there is a lack of biomimic vessel models focusing on chemotaxis, which enable the investigation of the effect of simultaneous external stimuli. In this study, we propose a novel bioreactor system combined with bioprinted microvessels embedded in fibrin-based extracellular matrix substitutes (ECM) for that purpose. The long-term stability of fibrin-collagen and fibrin-gelatin blends was investigated using an in vitro degradation test. Hydrogel samples were incubated in PBS and the weight was measured for up to 28 days. The permeability of fibrin-collagen and fibrin-gelatin blends were investigated using a tailored transwell assay. The transwell membranes were covered with hydrogel blends and the diffusion of fluorescein isothiocyanate labelled albumin from bovine serum (FITC-BSA) from the upper well into the PBS-filled lower well was measured using a microplate reader. The release of growth factors typical for inflammation (tumor necrosis factor α (TNF-α), stromal derived factor 1 (SDF-1)) from the hydrogel blends was investigated using an ELISA-kit. Microvessels mimicking arteries were fabricated using coaxial bioprinting technique by combining an endothelial cell-laden (HUVEC) sacrificial gelatin core with a smooth muscle cell-laden (SMC) fibrin-based shell. The channels were embedded into a fibrin-based ECM cultivated using a perfusion pump and a tailored, 3D printed bioreactor system for up to 28 days. The middle part of the bioreactor incorporated the final hydrogel construct of 3 mm thickness. Two exterior reservoirs were separated from the hydrogel construct by permeable membranes. Medium containing cytokines and chemical stimuli were injected into the exterior reservoirs and diffuse into the hydrogel construct and the perfusable microvessel substitute. The cellular organization of HUVECs and SMCs was investigated using immunostaining and confocal and two-photon microscopy. All hydrogel blends showed hydrolytic stability for at least 21 days. Swelling and shrinking of fibrin-gelatin blends was tuned by heat pretreatment of the gelatin component. Fibrin-collagen blends initially shrunk while the shrinking was reduced by increasing thrombin concentration and control of pH and temperature. Fibrin-gelatin blends provided twice the permeability of fibrin-collagen blends within the first 10 h. However, both blends levelled at a similar maximum permeability after 48 h. Gels with higher polymer concentration and hence denser microstructure showed lower permeability within the first hours compared to lower concentrated gels. The release of cytokines was distinctly higher from fibrin-gelatin blends (10-20 ng/ml for TNF-α; ~10 ng/ml for SDF-1) compared to fibrin-collagen blends (~5 ng/ml for TNF-α; ~3 ng/ml for SDF-1) after 24 h. A functional HUVEC monolayer lined the inner lumen of perfusable channels of approx. 500 µm in diameter. SMC showed high viability (<80%) and characteristic stretching inside the gels. In conclusion, we present a novel tailored bioreactor system which can be used to investigate the effect of external chemoattractants on angiogenesis, chemotaxis, intravasation, or extravasation. In combination with bioprinted microvessel substitutes, it represents a versatile and easy-to-use approach and can be used for a broad variety of tissue engineering applications.
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