Organotypic lung tissue culture as a preclinical model to study host- influenza A viral infection: A case for repurposing of nafamostat mesylate.

Reliable and effective models for recapitulation of host-pathogen interactions are imperative for the discovery of potential therapeutics. Ex vivo models can fulfill these requirements as the multicellular native environment in the tissue is preserved and be utilized for toxicology, vaccine, infection and drug efficacy studies due to the presence of immune cells. Drug repurposing involves the identification of new applications for already approved drugs that are not related to the prime medical indication and emerged as a strategy to cope with slow pace of drug discovery due to high costs and necessary phases to reach the patients. Within the scope of the study, broad-spectrum serine protease inhibitor nafamostat mesylate was repurposed to inhibit influenza A infection and evaluated by a translational ex vivo organotypic model, in which human organ-level responses can be achieved in preclinical safety studies of potential antiviral agents, along with in in vitro lung airway culture. The safe doses were determined as 10 µM for in vitro, whereas 22 µM for ex vivo to be applied for evaluation of host-pathogen interactions, which reduced virus infectivity, increased cell/tissue viability, and protected total protein content by reducing cell death with the inflammatory response. When the gene expression levels of specific pro-inflammatory, anti-inflammatory and cell surface markers involved in antiviral responses were examined, the significant inflammatory response represented by highly elevated mRNA gene expression levels of cytokines and chemokines combined with CDH5 downregulated by 5.1-fold supported the antiviral efficacy of NM and usability of ex vivo model as a preclinical infection model.

Lung carcinoma spheroids embedded in a microfluidic platform.

Three-dimensional (3D) spheroid cell cultures are excellent models used in cancer biology research and drug screening. The objective of this study was to develop a lung carcinoma spheroid based microfluidic platform with perfusion function to mimic lung cancer pathology and investigate the effect of a potential drug molecule, panaxatriol. Spheroids were successfully formed on agar microtissue molds at the end of 10 days, reaching an average diameter of about 317.18 ± 4.05 µm and subsequently transferred to 3D dynamic microfluidic system with perfusion function. While the size of the 3D spheroids embedded in the Matrigel matrix in the platform had gradually increased both in the static and dynamic control groups, the size of the spheroids were reduced and fragmented in the drug treated groups. Cell viability results showed that panaxatriol exhibited higher cytotoxic effect on cancer cells than healthy cells and the IC50 value was determined as 61.55 µM. Furthermore, panaxatriol has been more effective on single cells around the spheroid structure, whereas less in 3D spheroid tissues with a compact structure in static conditions compared to dynamic systems, where a flow rate of 2 µL/min leading to a shear stress of 0.002 dyne/cm2 was applied. Application of such dynamic systems will contribute to advancing basic research and increasing the predictive accuracy of potential drug molecules, which may accelerate the translation of novel therapeutics to the clinic, possibly decreasing the use of animal models. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10616-021-00470-7.

Erratum: "Human lung-on-chips: Advanced systems for respiratory virus models and assessment of immune response" [Biomicrofluidics 15, 021501 (2021)].

[This corrects the article DOI: 10.1063/5.0038924.].

Human lung-on-chips: Advanced systems for respiratory virus models and assessment of immune response.

Respiratory viral infections are leading causes of death worldwide. A number of human respiratory viruses circulate in all age groups and adapt to person-to-person transmission. It is vital to understand how these viruses infect the host and how the host responds to prevent infection and onset of disease. Although animal models have been widely used to study disease states, incisive arguments related to poor prediction of patient responses have led to the development of microfluidic organ-on-chip models, which aim to recapitulate organ-level physiology. Over the past decade, human lung chips have been shown to mimic many aspects of the lung function and its complex microenvironment. In this review, we address immunological responses to viral infections and elaborate on human lung airway and alveolus chips reported to model respiratory viral infections and therapeutic interventions. Advances in the field will expedite the development of therapeutics and vaccines for human welfare.

Effects of hydrostatic pressure and supercritical carbon dioxide on the viability of Botryococcus braunii algae cells.

In bio-based industries, Botryococcus braunii is identified as a potential resource for production of hydrocarbons having a wide range of applications in chemical and biopolymer industries. For a sustainable production platform, the algae cultivation should be integrated with downstream processes. Ideally the algae are not harvested, but the product is isolated while cultivation and growth is continued especially if the doubling time is slow. Consequently, hydrocarbons can be extracted while keeping the algae viable. In this study, the effects of pressure on the viability of B. braunii cells were tested hydrostatically and under supercritical CO2 conditions. Viability was determined by light microscopy, methylene blue uptake and by re-cultivation of the algae after treatments to follow the growth. It was concluded that supercritical CO2 was lethal to the algae, whereas hydrostatic pressure treatments up to 150 bar have not affected cell viability and recultivation was successful.

Cytotoxic responses of carnosic acid and doxorubicin on breast cancer cells in butterfly-shaped microchips in comparison to 2D and 3D culture.

Two dimensional (2D) cell culture systems lack the ability to mimic in vivo conditions resulting in limitations for preclinical cell-based drug and toxicity screening assays and modelling tumor biology. Alternatively, 3D cell culture systems mimic the specificity of native tissue with better physiological integrity. In this regard, microfluidic chips have gained wide applicability for in vitro 3D cancer cell studies. The aim of this research was to develop a 3D biomimetic model comprising culture of breast cancer cells in butterfly-shaped microchip to determine the cytotoxicity of carnosic acid and doxorubicin on both estrogen dependent (MCF-7) and independent (MDA-MB231) breast cancer cells along with healthy mammary epithelial cells (MCF-10A) in 2D, 3D Matrigel™ and butterfly-shaped microchip environment. According to the developed mimetic model, carnosic acid exhibited a higher cytotoxicity towards MDA-MB 231, while doxorubicin was more effective against MCF-7. Although the cell viabilities were higher in comparison to 2D and 3D cell culture systems, the responses of the investigated molecules were different in the microchips based on the molecular weight and structural complexity indicating the importance of biomimicry in a physiologically relevant matrix.

Formulation of organic and inorganic hydrogel matrices for immobilization of ß-glucosidase in microfluidic platform.

The aim of this study was to formulate silica and alginate hydrogels for immobilization of ß-glucosidase. For this purpose, enzyme kinetics in hydrogels were determined, activity of immobilized enzymes was compared with that of free enzyme, and structures of silica and alginate hydrogels were characterized in terms of surface area and pore size. The addition of polyethylene oxide improved the mechanical strength of the silica gels and 68% of the initial activity of the enzyme was preserved after immobilizing into tetraethyl orthosilicate-polyethylene oxide matrix where the relative activity in alginate beads was 87%. The immobilized ß-glucosidase was loaded into glass-silicon-glass microreactors and catalysis of 4-nitrophenyl ß-d-glucopyranoside was carried out at various retention times (5, 10, and 15 min) to compare the performance of silica and alginate hydrogels as immobilization matrices. The results indicated that alginate hydrogels exhibited slightly better properties than silica, which can be utilized for biocatalysis in microfluidic platforms.

Synthesis and characterization of cryogel structures for isolation of EPSs from Botryococcus braunii.

In this study, the objective was to separate exopolysaccharides (EPSs) released in the broth subsequent to outdoor cultivation of Botryococcus braunii. For this, poly(2-hydroxyethyl methacrylate) (PHEMA) cryogels were synthesized. After that, the surface was modified by coupling Concanavalin A. Box-Behnken statistical design was used to evaluate the effect of freezing temperature, Con A concentration and flow rate on Con A binding capacity. Optimum synthesis conditions were elicited as -14.48°C freezing temperature, 1.00mg/ml Con A concentration and 0.30ml/min flow rate yielding 3.18mg Con A/g cryogel, whereas -16°C, 1.00mg/ml and 0.30ml/min yielded the highest (3.38mg) binding capacity in experimental cryogel preparation. The EPS adsorption capacity of the optimum cryogel column was found as 3.26mg EPS/g cryogel corresponding to adsorption yield of 80%. Besides; swelling test, elemental analysis, Micro-CT, SEM and FTIR analysis were carried out for characterization of the synthesized cryogels.

Diffusion phenomena of cells and biomolecules in microfluidic devices.

Biomicrofluidics is an emerging field at the cross roads of microfluidics and life sciences which requires intensive research efforts in terms of introducing appropriate designs, production techniques, and analysis. The ultimate goal is to deliver innovative and cost-effective microfluidic devices to biotech, biomedical, and pharmaceutical industries. Therefore, creating an in-depth understanding of the transport phenomena of cells and biomolecules becomes vital and concurrently poses significant challenges. The present article outlines the recent advancements in diffusion phenomena of cells and biomolecules by highlighting transport principles from an engineering perspective, cell responses in microfluidic devices with emphases on diffusion- and flow-based microfluidic gradient platforms, macroscopic and microscopic approaches for investigating the diffusion phenomena of biomolecules, microfluidic platforms for the delivery of these molecules, as well as the state of the art in biological applications of mammalian cell responses and diffusion of biomolecules.