Membrane-free culture and real-time barrier integrity assessment of perfused intestinal epithelium tubes

In vitro models that better reflect in vivo epithelial barrier (patho-)physiology are urgently required to predict adverse drug effects. Here we introduce extracellular matrix-supported intestinal tubules in perfused microfluidic devices, exhibiting tissue polarization and transporter expression. Forty leak-tight tubules are cultured in parallel on a single plate and their response to pharmacological stimuli is recorded over 125 h using automated imaging techniques. A study comprising 357 gut tubes is performed, of which 93% are leak tight before exposure. EC50-time curves could be extracted that provide insight into both concentration and exposure time response. Full compatibility with standard equipment and user-friendly operation make this Organ-on-a-Chip platform readily applicable in routine laboratories

Electrostatic charge characteristics of jet nebulized aerosols

Background: Liquid droplets can be spontaneously charged in the absence of applied electric fields by spraying. It has been shown by computational simulation that charges may influence particle deposition in the airways. The electrostatic properties of jet nebulized aerosols and their potential effects on lung deposition have hardly been studied. A modified electrical low pressure impactor (ELPI) was employed to characterize the aerosol charges generated from jet nebulized commercial products. Methods: The charge and size measurements were conducted at 50% RH and 22°C with a modified ELPI. Ventolin®, Bricanyl®, and Atrovent® were nebulized using PARI LC® Plus jet nebulizers coupled to a DeVilbiss Pulmo-Aide® compressor. The aerosols were sampled in 30-sec durations. The drug deposits on the impactor stages were assayed chemically using high-performance liquid chromatography (HPLC). The charges of nebulized deionized water, isotonic saline, and the three commercial products diluted with saline were also measured to analyze the contributions of the major nebule ingredients on charging. No mass assays were performed on these runs. Results: All three commercial nebules generated net negative charges. The magnitude of the charges reduced over the period of nebulization. Ventolin® and Bricanyl® yielded similar charge profiles. Highly variable charges were produced from deionized water. On the other hand, nebulized saline reproducibly generated net positive charges. Diluted commercial nebules showed charge polarity inversion. The charge profiles of diluted salbutamol and terbutaline solutions resembled those of saline, while the charges from diluted ipratropium solutions fluctuated near neutrality. Conclusions: The charge profiles were shown to be influenced by the concentration and physicochemical properties of the drugs, as well as the history of nebulization. The drugs may have unique isoelectric concentrations in saline at which the nebulized droplets would carry near-zero charges. According to results from computational simulation models in the literature, the numbers of elementary charges per droplet estimated from the data were not high enough to potentially affect lung deposition. © Copyright 2010, Mary Ann Liebert, Inc.link_to_subscribed_fulltex

Capillary pinning assisted patterning of cell-laden hydrogel microarrays in microchips

We present a capillary pinning technique that gives complete control on the local patterning of hydrogel structures in closed microchips. The technique relies on selective trapping of liquids at predefined locations in a microchip using capillary barriers. In selective patterning, the abrupt expansion in the cross-sectional geometry of a microchannel at capillary barriers results in a confined advancement of the liquid–air meniscus. This protocol describes a detailed procedure to design and fabricate microarrays of different hydrogel types, fabricated with photopolymerization or thermogelation. The process can be subdivided into two parts. First, a PDMS microchip containing microfeatures with customized patterns is fabricated. Second, the microchip is filled with a hydrogel precursor to be cross-linked by either photopolymerization or thermogelation. The production of the microchip takes approximately 2 days, depending on the substrate selection. Preparation of the hydrogel solutions takes 1–2 h, whereas the patterning and reaction to cross-link the hydrogels is completed in a few minutes

96 perfusable blood vessels to study vascular permeability in vitro

Abstract Current in vitro models to test the barrier function of vasculature are based on flat, two-dimensional monolayers. These monolayers do not have the tubular morphology of vasculature found in vivo and lack important environmental cues from the cellular microenvironment, such as interaction with an extracellular matrix (ECM) and exposure to flow. To increase the physiological relevance of in vitro models of the vasculature, it is crucial to implement these cues and better mimic the native three-dimensional vascular architecture. We established a robust, high-throughput method to culture endothelial cells as 96 three-dimensional and perfusable microvessels and developed a quantitative, real-time permeability assay to assess their barrier function. Culture conditions were optimized for microvessel formation in 7 days and were viable for over 60 days. The microvessels exhibited a permeability to 20 kDa dextran but not to 150 kDa dextran, which mimics the functionality of vasculature in vivo. Also, a dose-dependent effect of VEGF, TNFα and several cytokines confirmed a physiologically relevant response. The throughput and robustness of this method and assay will allow end-users in vascular biology to make the transition from two-dimensional to three-dimensional culture methods to study vasculature