Recovery of Therapeutically Ablated Engineered Blood-Vessel Networks on a Plug-and-Play Platform.

Limiting the availability of key angiogenesis-promoting factors is a successful strategy to ablate tumor-supplying blood vessels or to reduce excessive vasculature in diabetic retinopathy. However, the efficacy of such anti-angiogenic therapies (AATs) varies with tumor type, and regrowth of vessels is observed upon termination of treatment. The ability to understand and develop AATs remains limited by a lack of robust in vitro systems for modeling the recovery of vascular networks. Here, complex 3D micro-capillary networks are engineered by sequentially seeding human bone marrow-derived mesenchymal stromal cells and human umbilical vein endothelial cells (ECs) on a previously established, synthetic plug-and-play hydrogel platform. In the tightly interconnected vascular networks that form this way, the two cell types share a basement membrane-like layer and can be maintained for several days of co-culture. Pre-formed networks degrade in the presence of bevacizumab. Upon treatment termination, vessel structures grow back to their original positions after replenishment with new ECs, which also integrate into unperturbed established networks. The data suggest that this plug-and-play platform enables the screening of drugs with blood-vessel inhibiting functions. It is believed that this platform could be of particular interest in studying resistance or recovery mechanisms to AAT treatment.

Driving electrochemical reactions at the microscale using CMOS microelectrode arrays.

Precise control of pH values at electrode interfaces enables the systematic investigation of pH-dependent processes by electrochemical means. In this work, we employed high-density complementary metal-oxide-semiconductor (CMOS) microelectrode arrays (MEAs) as miniaturized systems to induce and confine electrochemical reactions in areas corresponding to the pitch of single electrodes (17.5 µm). First, we present a strategy for generating localized pH patterns on the surface of the CMOS MEA with unprecedented spatial resolution. Leveraging the versatile routing capabilities of the switch matrix beneath the CMOS MEA, we created arbitrary combinations of anodic and cathodic electrodes and hence pH patterns. Moreover, we utilized the system to produce polymeric surface patterns by additive and subtractive methods. For additive patterning, we controlled the in situ formation of polydopamine at the microelectrode surface through oxidation of free dopamine above a threshold pH > 8.5. For subtractive patterning, we removed cell-adhesive poly-L-lysine from the electrode surface and backfilled the voids with antifouling polymers. Such polymers were chosen to provide a proof-of-concept application of controlling neuronal growth via electrochemically-induced patterns on the CMOS MEA surface. Importantly, our platform is compatible with commercially available high-density MEAs and requires no custom equipment, rendering the findings generalizable and accessible.

Simple Establishment of a Vascularized Osteogenic Bone Marrow Niche Using Pre-Cast Poly(ethylene Glycol) (PEG) Hydrogels in an Imaging Microplate.

The bone and bone marrow are highly vascularized and structurally complex organs, and are sites for cancer and metastasis formation. In vitro models recapitulating bone- and bone marrow-specific functions, including vascularization, that are compatible with drug screening are highly desirable. Such models can bridge the gap between simplistic, structurally irrelevant two-dimensional (2D) in vitro models and the more expensive, ethically challenging in vivo models. This article describes a controllable three-dimensional (3D) co-culture assay based on engineered poly(ethylene glycol) (PEG) matrices for the generation of vascularized, osteogenic bone-marrow niches. The PEG matrix design allows the development of 3D cell cultures through a simple cell seeding step requiring no encapsulation, thus enabling the development of complex co-culture systems. Furthermore, the matrices are transparent and pre-cast onto glass-bottom 96-well imaging plates, rendering the system suitable for microscopy. For the assay described here, human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) are cultured first until a sufficiently developed 3D cell network is formed. Subsequently, GFP-expressing human umbilical vein endothelial cells (HUVECs) are added. The culture development is followed by bright-field and fluorescence microscopy. The presence of the hBM-MSC network supports the formation of vascular-like structures that otherwise would not form and that remain stable for at least 7 days. The extent of vascular-like network formation can easily be quantified. This model can be tuned toward an osteogenic bone-marrow niche by supplementing the culture medium with bone morphogenetic protein 2 (BMP-2), which promotes the osteogenic differentiation of the hBM-MSCs, as assessed by increased alkaline phosphatase (ALP) activity at day 4 and day 7 of co-culture. This cellular model can be used as a platform for culturing various cancer cells and studying how they interact with bone- and bone marrow-specific vascular niches. Moreover, it is suitable for automation and high-content analyses, meaning it would enable cancer drug screening under highly reproducible culture conditions.

Multifunctional 3D electrode platform for real-time in situ monitoring and stimulation of cardiac tissues.

Cardiovascular diseases are a major cause of death around the world underlining the importance of efficient treatments including novel medication. In vitro models of cardiac tissues are highly desired for monitoring electrophysiological response to screen pharmaceutical compounds at an early stage. Here, we report a platinum based 3D pillar electrode platform with cell growth guiding channel, which allows integrated, continuous electrical stimulation and recording of the cardiac tissues. This platform was successfully used to culture beating cardiomyocytes over weeks. The Pt-PDMS pillar electrode showed an impedance of 2.5 ±â€¯0.3 kΩ at 10 Hz that is stable for using in cell cultures at 37 °C. Electrically pacing the cells improved maturation of the cardiac tissues. Contraction activities of 3D cardiac tissues were monitored in real-time through the pillar electrodes to evaluate physiological parameters. Adding 100 nM Isoproterenol clearly increased the spontaneous beating rate and decreased the extracellular field potential duration of the microtissues observed with electrical recording through the pillar electrodes. Overall, this platform has a potential to be applied in drug screening for in situ monitoring the biophysical parameters of the heart tissue in real-time.

Soft Hydrogels Featuring In-Depth Surface Density Gradients for the Simple Establishment of 3D Tissue Models for Screening Applications.

Three-dimensional (3D) cell culture models are gaining increasing interest for use in drug development pipelines due to their closer resemblance to human tissues. Hydrogels are the first-choice class of materials to recreate in vitro the 3D extra-cellular matrix (ECM) environment, important in studying cell-ECM interactions and 3D cellular organization and leading to physiologically relevant in vitro tissue models. Here we propose a novel hydrogel platform consisting of a 96-well plate containing pre-cast synthetic PEG-based hydrogels for the simple establishment of 3D (co-)culture systems without the need for the standard encapsulation method. The in-depth density gradient at the surface of the hydrogel promotes the infiltration of cells deposited on top of it. The ability to decouple hydrogel production and cell seeding is intended to simplify the use of hydrogel-based platforms and thus increase their accessibility. Using this platform, we established 3D cultures relevant for studying stem cell differentiation, angiogenesis, and neural and cancer models.

Surface presentation of functional peptides in solution determines cell internalization efficiency of TAT conjugated nanoparticles.

Functionalizing nanoparticles with cell-penetrating peptides is a popular choice for cellular delivery. We investigated the effects of TAT peptide concentration and arrangement in solution on functionalized nanoparticles' efficacy for membrane permeation. We found that cell internalization correlates with the positive charge distribution achieved prior to nanoparticle encountering interactions with membrane. We identified a combination of solution based properties required to maximize the internalization efficacy of TAT-functionalized nanoparticles.

Coagulation at the blood-electrode interface: the role of electrochemical desorption and degradation of fibrinogen.

The influence of electrochemistry on the coagulation of blood on metal surfaces was demonstrated several decades ago. In particular, the application of cathodic currents resulted in reduced surface thrombogenicity, but no molecular mechanism has been so far proposed to explain this observation. In this article we used for the first time the quartz crystal microbalance with dissipation monitoring technique coupled with an electrochemical setup (EQCM-D) to study thrombosis at the blood-electrode interface. We confirmed the reduced thrombus deposition at the cathode, and we subsequently studied the effect of cathodic currents on adsorbed fibrinogen (Fg). Using EQCM and mass spectrometry, we found that upon applying currents Fg desorbed from the electrode and was electrochemically degraded. In particular, we show that the flexible N-terminus of the α-chain, containing an important polymerization site, was cleaved from the protein, thus affecting its clottability. Our work proposes a molecular mechanism that at least partially explains how cathodic currents reduce thrombosis at the blood-electrode interface and is a relevant contribution to the rational development of medical devices with reduced thrombus formation on their surface.

Electrochemical control of the enzymatic polymerization of PEG hydrogels: formation of spatially controlled biological microenvironments.

Control of pH gradient profile at the electrode-electrolyte interfaces allows the control of the enzymatic PEG-hydrogel polymerization. By tuning the solution pH, buffer capacity, and the applied current, the extent of the local inhibition and confinement of the Factor XIII-mediated polymerization of PEG are controlled. This technology opens new perspectives for the production of 3D-structured biological microenvironments.