True-scale biomimetic multi-generation airway platforms of the human bronchial epithelium for in vitro cytotoxicity screening

Lung exposure to inhaled particulate matter may injure the epithelial tissue and lead to a loss of function in affected regions via inflammation for example. Screening for the critical contaminate concentrations may provide essential information towards damage assessment and epithelial healing. To date, most approaches have typically relied on traditional in vitro well plate assays or alternatively in vivo animal experiments. Yet, such methods manifest some outstanding disadvantages such as the inability to capture physiological flow and aerosol deposition characteristics as well as significant differences in anatomy, immune system and inflammatory responses compared to humans. The advent of organ-on-chip platforms has shown promising results to reconcile many such drawbacks. In an attempt to provide an attractive in vitro gateway to monitor airway health, we discuss here a novel biomimetic platform which emulates the bronchial epithelium of a human upper airway, allowing to study organ-level characteristics in a homeostatic cellular microenvironment. This device reconstitutes a multi-generation pulmonary epithelial airway environment, capturing realistic respiratory transport phenomena and critical cellular barrier functions at an air-liquid interface (ALI), in analogy to the bronchial lumen. As a proof of concept, we demonstrate its feasibility for in vitro based assays by exposing the device to cytotoxic aerosolized particles under respiratory flow conditions. Subsequently, we investigate the cytotoxic effects of these particles including cellular viability, cytokine and mucus secretion as a function of local particle deposition patterns. Ultimately, our bronchial airway models are intended to provide off-the-shelf in vitro kits geared for the end-user interested in a wide range of broader biological assays that may be attractive for cytotoxicity and drug screening. Please click Additional Files below to see the full abstract

Microfluidic acini-on-chip platforms as a tool to study bacterial lung exposure

Bacterial invasion of the respiratory system leads to complex immune responses involving many cell types. In the alveolar regions, the first line of defense includes the alveolar epithelium, secreted surfactant, alveolar lining fluid and alveolar macrophages. The epithelium consists of alveolar type I and type II cells. Both cell types are known to have immuno-modulatory functions characterized by the secretion of pro-inflammatory cytokines. Epithelial in vitro models offer attractive platforms to investigate biological functionality, but have typically relied on traditional well plate assays that come short of mimicking the complexity of the airway environment and do not capture physiological flows or relevant anatomical features. In the last decade, microfluidics have gained significant momentum in laying the foundations for constructing in vitro models that mimic physiologically-relevant organ functions. Here we propose to use acinus-on-chip platforms that mimic more closely native acinar microflows at true scale in a multi-generation alveolated tree. Acinar chips are cultured with human Alveolar Epithelial Lentivirus immortalized (hAELVi) cells at an air-liquid interface (ALI); such cells show alveolar type I like characteristics and maintained barrier function, leading to high trans-epithelial electrical resistance (TEER) in analogy to primary cells harvested from human tissue. To model bacterial infection, i.e. a strong stimulator of the innate arm of the immune system, lipopolysaccharides (LPS) will be used. LPS is a major outer surface membrane protein expressed on Gram-negative bacteria. The alveolar epithelium is exposed to LPS-laden aerosols and cell response is monitored mainly by secretion of pro-inflammatory cytokines. Our acinus-on-chip allows quantitative on-line measurements of alveolar barrier function, absorption kinetics and immunologically relevant responses, giving further insight to the role played by type I alveolar cells in lung immunity. Please click Additional Files below to see the full abstract

Electroconductive Hydrogel Based on Functional Poly(Ethylenedioxy Thiophene).

Poly(ethylene dioxythiophene) with functional pendant groups bearing double bonds is synthesized and employed for the fabrication of electroactive hydrogels with advantageous characteristics: covalently cross-linked porous 3D scaffolds with notable swelling ratio, appropriate mechanical properties, electroactivity in physiological conditions, and suitability for proliferation and differentiation of C2C12 cells. This is a new approach for the fabrication of conductive engineered constructs

Advancing human pulmonary disease models in preclinical research: opportunities for lung-on-chips..

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Hollow Nanoneedle Array and Its Utilization for Repeated Administration of Biomolecules to the Same Cells

We present a novel hollow nanoneedle array (NNA) device capable of simultaneously delivering diverse cargo into a group of cells in a culture over prolonged periods. The silica needles are fed by a common reservoir whose content can be replenished and modified in real time while maintaining contact with the same cells. The NNA, albeit its submicrometer features, is fabricated in a silicon-on-insulator wafer using conventional, large scale, silicon technology. 3T3-NIH fibroblast cells and HEK293 human embryonic kidney cells are shown to grow and proliferate successfully on the NNAs. Cargo delivery from the reservoir through the needles to a group of HEK293 cells in the culture is demonstrated by repeated administration of fluorescently labeled dextran to the same cells and transfection with DNA coding for red fluorescent protein. The capabilities demonstrated by the NNA device open the door to large scale studies of the effect of selected cells on their environment as encountered, for instance, in the study of cell-fate decisions, the role of cell-autonomous <i>versus</i> nonautonomous mechanisms in developmental biology, and in the study of excitable cell-networks

PerfuPul-A Versatile Perfusable Platform to Assess Permeability and Barrier Function of Air Exposed Pulmonary Epithelia.

Complex in vitro models, especially those based on human cells and tissues, may successfully reduce or even replace animal models within pre-clinical development of orally inhaled drug products. Microfluidic lung-on-chips are regarded as especially promising models since they allow the culture of lung specific cell types under physiological stimuli including perfusion and air-liquid interface (ALI) conditions within a precisely controlled in vitro environment. Currently, though, such models are not available to a broad user community given their need for sophisticated microfabrication techniques. They further require systematic comparison to well-based filter supports, in analogy to traditional Transwells®. We here present a versatile perfusable platform that combines the advantages of well-based filter supports with the benefits of perfusion, to assess barrier permeability of and aerosol deposition on ALI cultured pulmonary epithelial cells. The platform as well as the required technical accessories can be reproduced via a detailed step-by-step protocol and implemented in typical bio-/pharmaceutical laboratories without specific expertise in microfabrication methods nor the need to buy costly specialized equipment. Calu-3 cells cultured under liquid covered conditions (LCC) inside the platform showed similar development of transepithelial electrical resistance (TEER) over a period of 14 days as cells cultured on a traditional Transwell®. By using a customized deposition chamber, fluorescein sodium was nebulized via a clinically relevant Aerogen® Solo nebulizer onto Calu-3 cells cultured under ALI conditions within the platform. This not only allowed to analyze the transport of fluorescein sodium after ALI deposition under perfusion, but also to compare it to transport under traditional static conditions

Advanced in vitro lung-on-chip platforms for inhalation assays: From prospect to pipeline

With rapid advances in micro-fabrication processes and the availability of biologically-relevant lung cells, the development of lung-on-chip platforms is offering novel avenues for more realistic inhalation assays in pharmaceutical research, and thereby an opportunity to depart from traditional in vitro lung assays. As advanced models capturing the cellular pulmonary make-up at an air-liquid interface (ALI), lung-on-chips emulate both morphological features and biological functionality of the airway barrier with the ability to integrate respiratory breathing motions and ensuing tissue strains. Such in vitro systems allow importantly to mimic more realistic physiological respiratory flow conditions, with the opportunity to integrate physically-relevant transport determinants of aerosol inhalation therapy, i.e. recapitulating the pathway from airborne flight to deposition on the airway lumen. In this short opinion, we discuss such points and describe how these attributes are paving new avenues for exploring improved drug carrier designs (e.g. shape, size, etc.) and targeting strategies (e.g. conductive vs. respiratory regions) amongst other. We argue that while technical challenges still lie along the way in rendering in vitro lung-on-chip platforms more widespread across the general pharmaceutical research community, significant momentum is steadily underway in accelerating the prospect of establishing these as in vitro "gold standards

Alcune osservazioni sul gerundio in italiano e tedesco

HIV-1 protease is a key enzyme in the life cycle of HIV/AIDS, as it is responsible for the formation of the mature virus particle. We demonstrate here that phage-display peptides raised against this enzyme can be used as peptide sensors for the detection of HIV-1 protease in a simple, one-pot assay. The presence of the enzyme is detected through an energy transfer between two peptide sensors when simultaneously complexed with the target protein. The multivalent nature of this assay increases the specificity of the detection by requiring all molecules to be interacting in order for there to be a FRET signal. We also perform molecular dynamics simulations to explore the interaction between the protease and the peptides in order to guide the design of these peptide sensors and to understand the mechanisms which cause these simultaneous binding events. This approach aims to facilitate the development of new assays for enzymes that are not dependent on the cleavage of a substrate and do not require multiple washing steps