3D-Cultured Vascular-Like Networks Enable Validation of Vascular Disruption Properties of Drugs In Vitro.

Vascular-disrupting agents are an interesting class of anticancer compounds because of their combined mode of action in preventing new blood vessel formation and disruption of already existing vasculature in the immediate microenvironment of solid tumors. The validation of vascular disruption properties of these drugs in vitro is rarely addressed due to the lack of proper in vitro angiogenesis models comprising mature and long-lived vascular-like networks. We herein report an indirect coculture model of human umbilical vein endothelial cells (HUVECs) and human dermal fibroblasts (HDFs) to form three-dimensional profuse vascular-like networks. HUVECs embedded and sandwiched in the collagen scaffold were cocultured with HDFs located outside the scaffold. The indirect coculture approach with the vascular endothelial growth factor (VEGF) producing HDFs triggered the formation of progressively maturing lumenized vascular-like networks of endothelial cells within less than 7 days, which have proven to be viably maintained in culture beyond day 21. Molecular weight-dependent Texas red-dextran permeability studies indicated high vascular barrier function of the generated networks. Their longevity allowed us to study the dose-dependent response upon treatment with the three known antiangiogenic and/or vascular disrupting agents brivanib, combretastatin A4 phosphate (CA4P), and 6´-sialylgalactose (SG) via semi-quantitative brightfield and qualitative confocal laser scanning microscopic (CLSM) image analysis. Compared to the reported data on in vivo efficacy of these drugs in terms of antiangiogenic and vascular disrupting effects, we observed similar trends with our 3D model, which are not reflected in conventional in vitro angiogenesis assays. High-vascular disruption under continuous treatment of the matured vascular-like network was observed at concentrations ≥3.5 ng·ml-1 for CA4P and ≥300 nM for brivanib. In contrast, SG failed to induce any significant vascular disruption in vitro. This advanced model of a 3D vascular-like network allows for testing single and combinational antiangiogenic and vascular disrupting effects with optimized dosing and may thus bridge the gap between the in vitro and in vivo experiments in validating hits from high-throughput screening. Moreover, the physiological 3D environment mimicking in vitro assay is not only highly relevant to in vivo studies linked to cancer but also to the field of tissue regeneration.

Endothelial, smooth muscle and fibroblast cell sheet fabrication from self-assembled thermoresponsive poly(glycidyl ether) brushes.

In this study, we introduce a platform to fabricate human dermal fibroblast (HDF), human aortic smooth muscle cell (HAoSMC) and human umbilical vein endothelial cell (HUVEC) sheets using thermoresponsive poly(glycidyl ether) coatings. Copolymer brushes based on glycidyl methyl ether (GME) and ethyl glycidyl ether (EGE) were self-assembled onto polystyrene (PS) culture substrates via the physical adsorption of a hydrophobic, photoreactive benzophenone anchor block based on the monomer 4-[2-(2,3-epoxypropoxy)ethoxy]benzophenone (EEBP). The directed self-assembly of well-defined, end-tethered poly(GME-ran-EGE)-block-poly(EEBP) (PGE) brushes was achieved via the selective, EEBP-driven adsorption of the asymmetric block copolymer from dilute aqueous solution below its cloud point temperature (CPT). Subsequently, the PGE brush layers were covalently immobilized onto the PS surfaces by irradiation with UV light and characterized by ellipsometry, static water contact angle (CA) measurements and atomic force microscopy (AFM). We found that, by decreasing the temperature from 37 to 20 °C, the coatings undergo a pancake-to-brush transition, which triggers cell sheet detachment. In addition, cell culture parameters were optimized to allow proper adhesion and controlled detachment of confluent HDF, HAoSMC and HUVEC sheets, which can be applied in vascular tissue engineering.

A versatile modular bioreactor platform for Tissue Engineering.

Tissue Engineering (TE) bears potential to overcome the persistent shortage of donor organs in transplantation medicine. Additionally, TE products are applied as human test systems in pharmaceutical research to close the gap between animal testing and the administration of drugs to human subjects in clinical trials. However, generating a tissue requires complex culture conditions provided by bioreactors. Currently, the translation of TE technologies into clinical and industrial applications is limited due to a wide range of different tissue-specific, non-disposable bioreactor systems. To ensure a high level of standardization, a suitable cost-effectiveness, and a safe graft production, a generic modular bioreactor platform was developed. Functional modules provide robust control of culture processes, e.g. medium transport, gas exchange, heating, or trapping of floating air bubbles. Characterization revealed improved performance of the modules in comparison to traditional cell culture equipment such as incubators, or peristaltic pumps. By combining the modules, a broad range of culture conditions can be achieved. The novel bioreactor platform allows using disposable components and facilitates tissue culture in closed fluidic systems. By sustaining native carotid arteries, engineering a blood vessel, and generating intestinal tissue models according to a previously published protocol the feasibility and performance of the bioreactor platform was demonstrated.

Ultrathin Poly(glycidyl ether) Coatings on Polystyrene for Temperature-Triggered Human Dermal Fibroblast Sheet Fabrication.

The fabrication of cell sheets is a major requirement for bottom-up tissue engineering purposes (e.g., cell sheet engineering) and regenerative medicine. Employing thermoresponsive polymer coatings as tissue culture substrates allows for the mild, temperature-triggered detachment of intact cell sheets along with their extracellular matrix (ECM). It has been shown before that biocompatible, thermoresponsive poly(glycidyl ether) monolayers on gold substrates can be utilized to harvest confluent cell sheets by simply reducing the temperature to 20 °C. Herein, we report on the first poly(glycidyl ether)-based coating on an application-relevant tissue culture plastic substrate. We devised a simple, substrate-geometry-independent method to functionalize polystyrene (PS) surfaces from dilute ethanolic solution via the physical adsorption process of a thermoresponsive poly(glycidyl ether) block copolymer (PGE) bearing a short, hydrophobic, and photoreactive benzophenone (BP) anchor block. Subsequently, the PGE-coated PS is UV-irradiated for covalent photoimmobilization of the polymer on the PS substrate. Online monitoring of the adsorption process via QCM-D measurements and detailed characterization of the resulting coatings via AFM, ellipsometry, and water contact angle (CA) measurements revealed the formation of an ultrathin PGE layer with an average dry thickness of 0.7 ± 0.1 nm. Adhesion and proliferation of human dermal fibroblasts on PGE-coated PS and tissue culture PS (TCPS) were comparable. For temperature-triggered detachment, fibroblasts were cultured in PGE-coated PS culture dishes at 37 °C for 24 h until they reached confluency. Intact cell sheets could be harvested from the thermoresponsive substrates within 51 ± 17 min upon cooling to 20 °C, whereas sheets could not be harvested from uncoated PS and TCPS control dishes. Live/dead staining and flow cytometry affirmed a high viability of the fibroblasts within the cell sheets. Hence, ultrathin layers of thermoresponsive poly(glycidyl ether)s on hydrophobic PS substrates are functional coatings for cell sheet fabrication.

A human in vitro model that mimics the renal proximal tubule.

Human in vitro-manufactured tissue and organ models can serve as powerful enabling tools for the exploration of fundamental questions regarding cell, matrix, and developmental biology in addition to the study of drug delivery dynamics and kinetics. To date, the development of a human model of the renal proximal tubule (PT) has been hindered by the lack of an appropriate cell source and scaffolds that allow epithelial monolayer formation and maintenance. Using extracellular matrices or matrix proteins, an in vivo-mimicking environment can be created that allows epithelial cells to exhibit their typical phenotype and functionality. Here, we describe an in vitro-engineered PT model. We isolated highly proliferative cells from cadaveric human kidneys (human kidney-derived cells [hKDCs]), which express markers that are associated with renal progenitor cells. Seeded on small intestinal submucosa (SIS), hKDCs formed a confluent monolayer and displayed the typical phenotype of PT epithelial cells. PT markers, including N-cadherin, were detected throughout the hKDC culture on the SIS, whereas markers of later tubule segments were weak (E-cadherin) or not (aquaporin-2) expressed. Basement membrane and microvilli formation demonstrated a strong polarization. We conclude that the combination of hKDCs and SIS is a suitable cell-scaffold composite to mimic the human PT in vitro.