Modelling bronchial epithelial-fibroblast cross-talk in idiopathic pulmonary fibrosis (IPF) using a human-derived in vitro air liquid interface (ALI) culture.

Idiopathic Pulmonary Fibrosis (IPF) is a devastating form of respiratory disease with a life expectancy of 3-4 years. Inflammation, epithelial injury and myofibroblast proliferation have been implicated in disease initiation and, recently, epithelial-fibroblastic crosstalk has been identified as a central driver. However, the ability to interrogate this crosstalk is limited due to the absence of in vitro models that mimic physiological conditions. To investigate IPF dysregulated cross-talk, primary normal human bronchial epithelial (NHBE) cells and primary normal human lung fibroblasts (NHLF) or diseased human lung fibroblasts (DHLF) from IPF patients, were co-cultured in direct contact at the air-liquid interface (ALI). Intercellular crosstalk was assessed by comparing cellular phenotypes of co-cultures to respective monocultures, through optical, biomolecular and electrical methods. A co-culture-dependent decrease in epithelium thickness, basal cell mRNA (P63, KRT5) and an increase in transepithelial electrical resistance (TEER) was observed. This effect was significantly enhanced in DHLF co-cultures and lead to the induction of epithelial to mesenchymal transition (EMT) and increased mRNA expression of TGFß-2, ZO-1 and DN12. When stimulated with exogenous TGFß, NHBE and NHLF monocultures showed a significant upregulation of EMT (COL1A1, FN1, VIM, ASMA) and senescence (P21) markers, respectively. In contrast, direct NHLF/NHBE co-culture indicated a protective role of epithelial-fibroblastic cross-talk against TGFß-induced EMT, fibroblast-to-myofibroblast transition (FMT) and inflammatory cytokine release (IL-6, IL-8, IL-13, IL-1ß, TNF-α). DHLF co-cultures showed no significant phenotypic transition upon stimulation, likely due to the constitutively high expression of TGFß isoforms prior to any exogenous stimulation. The model developed provides an alternative method to generate IPF-related bronchial epithelial phenotypes in vitro, through the direct co-culture of human lung fibroblasts with NHBEs. These findings highlight the importance of fibroblast TGFß signaling in EMT but that monocultures give rise to differential responses compared to co-cultures, when exposed to this pro-inflammatory stimulus. This holds implications for any translation conclusions drawn from monoculture studies and is an important step in development of more biomimetic models of IPF. In summary, we believe this in vitro system to study fibroblast-epithelial crosstalk, within the context of IPF, provides a platform which will aid in the identification and validation of novel targets.

A Conformable Organic Electronic Device for Monitoring Epithelial Integrity at the Air Liquid Interface.

Air liquid interfaced (ALI) epithelial barriers are essential for homeostatic functions such as nutrient transport and immunological protection. Dysfunction of such barriers are implicated in a variety of autoimmune and inflammatory disorders and, as such, sensors capable of monitoring barrier health are integral for disease modelling, diagnostics and drug screening applications. To date, gold-standard electrical methods for detecting barrier resistance require rigid electrodes bathed in an electrolyte, which limits compatibility with biological architectures and is non-physiological for ALI. This work presents a flexible all-planar electronic device capable of monitoring barrier formation and perturbations in human respiratory and intestinal cells at ALI. By interrogating patient samples with electrochemical impedance spectroscopy and simple equivalent circuit models, disease-specific and patient-specific signatures are uncovered. Device readouts are validated against commercially available chopstick electrodes and show greater conformability, sensitivity and biocompatibility. The effect of electrode size on sensing efficiency is investigated and a cut-off sensing area is established, which is one order of magnitude smaller than previously reported. This work provides the first steps in creating a physiologically relevant sensor capable of mapping local and real-time changes of epithelial barrier function at ALI, which will have broad applications in toxicology and drug screening applications.

Digesting Digestion: An Educational Laboratory to Teach Students about Enzymes and the Gastrointestinal Tract.

Digestion is a fundamentally important process for an individual's life. However, the physical process of digestion is hidden inside the body, making it challenging to understand and a particularly difficult topic for students to learn in the classroom. Traditional approaches to teaching body processes include a mixture of textbook teaching and visual learning. However, digestion is not particularly visual. This activity is designed to engage students using a combination of visual, inquiry-based, and experiential learning approaches and introduces the scientific method to students in secondary school. The laboratory simulates digestion, creating a "stomach" inside of a clear vial. Students fill the vials with a protease solution and visually observe the digestion of food. By making predictions about the types of biomolecules that will be digested, students begin to learn and understand basic biochemistry in a relatable context, while simultaneously understanding anatomical and physiological concepts. We trialled this activity at two schools, where we received positive feedback from teachers and students, indicating that the practical enhanced student understanding of the digestion process. We see this lab as a valuable learning activity that can be extended broadly across multiple classrooms around the world.

Transfer-Tattoo-Like Cell-Sheet Delivery Induced by Interfacial Cell Migration.

A direct transfer of a cell sheet from a culture surface to a target tissue is introduced. Commercially available, flexible parylene is used as the culture surface, and it is proposed that the UV-treated parylene offers adequate and intermediate levels of cell adhesiveness for both the stable cell attachment during culture and for the efficient cell transfer to a target surface. The versatility of this cell-transfer process is demonstrated with various cell types, including MRC-5, HDFn, HULEC-5a, MC3T3-E1, A549, C2C12 cells, and MDCK-II cells. The novel cell-sheet engineering is based on a mechanism of interfacial cell migration between two surfaces with different adhesion preferences. Monitoring of cytoskeletal dynamics and drug treatments during the cell-transfer process reveals that the interfacial cell migration occurs by utilizing the existing transmembrane proteins on the cell surface to bind to the targeted surface. The re-establishment and reversal of cell polarity after the transfer process are also identified. Its unique capabilities of 3D multilayer stacking, freeform design, and curved surface application are demonstrated. Finally, the therapeutic potential of the cell-sheet delivery system is demonstrated by applying it to cutaneous wound healing and skin-tissue regeneration in mice models.

In Vitro Models for Studying Respiratory Host-Pathogen Interactions.

Respiratory diseases and lower respiratory tract infections are among the leading cause of death worldwide and, especially given the recent severe acute respiratory syndrome coronavirus-2 pandemic, are of high and prevalent socio-economic importance. In vitro models, which accurately represent the lung microenvironment, are of increasing significance given the ethical concerns around animal work and the lack of translation to human disease, as well as the lengthy time to market and the attrition rates associated with clinical trials. This review gives an overview of the biological and immunological components involved in regulating the respiratory epithelium system in health, disease, and infection. The evolution from 2D to 3D cell biology and to more advanced technological integrated models for studying respiratory host-pathogen interactions are reviewed and provide a reference point for understanding the in vitro modeling requirements. Finally, the current limitations and future perspectives for advancing this field are presented.

Building Scaffolds for Tubular Tissue Engineering.

Hollow organs and tissue systems drive various functions in the body. Many of these hollow or tubular systems, such as vasculature, the intestines, and the trachea, are common targets for tissue engineering, given their relevance to numerous diseases and body functions. As the field of tissue engineering has developed, numerous benchtop models have been produced as platforms for basic science and drug testing. Production of tubular scaffolds for different tissue engineering applications possesses many commonalities, such as the necessity for producing an intact tubular opening and for formation of semi-permeable epithelia or endothelia. As such, the field has converged on a series of manufacturing techniques for producing these structures. In this review, we discuss some of the most common tissue engineered applications within the context of tubular tissues and the methods by which these structures can be produced. We provide an overview of the general structure and anatomy for these tissue systems along with a series of general design criteria for tubular tissue engineering. We categorize methods for manufacturing tubular scaffolds as follows: casting, electrospinning, rolling, 3D printing, and decellularization. We discuss state-of-the-art models within the context of vascular, intestinal, and tracheal tissue engineering. Finally, we conclude with a discussion of the future for these fields.

Self-assembled photoactive polyelectrolyte/perylene-diimide composites.

A new class of polyelectrolyte-surfactant (PE-surf) composites having potential applications as thin film organic semiconductors is introduced. These materials are comprised of cationic asymmetrically substituted perylene diimides and oppositely charged poly(acrylate) polyanions. Thin films of the composite materials are prepared by mixing and drop casting aqueous solutions of the two precursors onto appropriate substrates. The resulting materials yield photovoltages of >140 mV for approximately equal to 0.6 W/cm(2) illumination intensities, when incorporated in p-n heterojunction devices. Solution-phase spectra obtained from the PE-surf complexes exhibit excimer-like emission and evidence for formation of weakly coupled aggregates in the ground state. Wide-angle X-ray scattering data show the composite films are locally amorphous, while small-angle X-ray data are consistent with a mixture of polymorphic structures that incorporate planar PE-surf bilayers of 3.9-nm repeat distances. Images obtained by conventional far-field light microscopy and multiphoton-excited fluorescence microscopy (MPEFM) indicate that the films are heterogeneous, incorporating submicrometer sized clusters dispersed among much thinner film regions that also incorporate dye. Polarization-dependent MPEFM studies prove the clusters are semiorganized, yielding order parameters (s and P(4)) of 0.09 and 0.01 for in-plane alignment of the chromophores, consistent with a relatively high degree of disorder.