Lung viral infection modelling in a bioengineered whole-organ.

Lung infections are one of the leading causes of death worldwide, and this situation has been exacerbated by the emergence of COVID-19. Pre-clinical modelling of viral infections has relied on cell cultures that lack 3D structure and the context of lung extracellular matrices. Here, we propose a bioreactor-based, whole-organ lung model of viral infection. The bioreactor takes advantage of an automated system to achieve efficient decellularization of a whole rat lung, and recellularization of the scaffold using primary human bronchial cells. Automatization allowed for the dynamic culture of airway epithelial cells in a breathing-mimicking setup that led to an even distribution of lung epithelial cells throughout the distal regions. In the sealed bioreactor system, we demonstrate proof-of-concept for viral infection within the epithelialized lung by infecting primary human airway epithelial cells and subsequently injecting neutrophils. Moreover, to assess the possibility of drug screening in this model, we demonstrate the efficacy of the broad-spectrum antiviral remdesivir. This whole-organ scale lung infection model represents a step towards modelling viral infection of human cells in a 3D context, providing a powerful tool to investigate the mechanisms of the early stages of pathogenic infections and the development of effective treatment strategies for respiratory diseases.

Hydrogel-in-hydrogel live bioprinting for guidance and control of organoids and organotypic cultures.

Three-dimensional hydrogel-based organ-like cultures can be applied to study development, regeneration, and disease in vitro. However, the control of engineered hydrogel composition, mechanical properties and geometrical constraints tends to be restricted to the initial time of fabrication. Modulation of hydrogel characteristics over time and according to culture evolution is often not possible. Here, we overcome these limitations by developing a hydrogel-in-hydrogel live bioprinting approach that enables the dynamic fabrication of instructive hydrogel elements within pre-existing hydrogel-based organ-like cultures. This can be achieved by crosslinking photosensitive hydrogels via two-photon absorption at any time during culture. We show that instructive hydrogels guide neural axon directionality in growing organotypic spinal cords, and that hydrogel geometry and mechanical properties control differential cell migration in developing cancer organoids. Finally, we show that hydrogel constraints promote cell polarity in liver organoids, guide small intestinal organoid morphogenesis and control lung tip bifurcation according to the hydrogel composition and shape.

Mitochondrial fission links ECM mechanotransduction to metabolic redox homeostasis and metastatic chemotherapy resistance.

Metastatic breast cancer cells disseminate to organs with a soft microenvironment. Whether and how the mechanical properties of the local tissue influence their response to treatment remains unclear. Here we found that a soft extracellular matrix empowers redox homeostasis. Cells cultured on a soft extracellular matrix display increased peri-mitochondrial F-actin, promoted by Spire1C and Arp2/3 nucleation factors, and increased DRP1- and MIEF1/2-dependent mitochondrial fission. Changes in mitochondrial dynamics lead to increased production of mitochondrial reactive oxygen species and activate the NRF2 antioxidant transcriptional response, including increased cystine uptake and glutathione metabolism. This retrograde response endows cells with resistance to oxidative stress and reactive oxygen species-dependent chemotherapy drugs. This is relevant in a mouse model of metastatic breast cancer cells dormant in the lung soft tissue, where inhibition of DRP1 and NRF2 restored cisplatin sensitivity and prevented disseminated cancer-cell awakening. We propose that targeting this mitochondrial dynamics- and redox-based mechanotransduction pathway could open avenues to prevent metastatic relapse.

In vitro models of fetal lung development to enhance research into congenital lung diseases.

PURPOSE: This paper aims to build upon previous work to definitively establish in vitro models of murine pseudoglandular stage lung development. These can be easily translated to human fetal lung samples to allow the investigation of lung development in physiologic and pathologic conditions. METHODS: Lungs were harvested from mouse embryos at E12.5 and cultured in three different settings, i.e., whole lung culture, mesenchyme-free epithelium culture, and organoid culture. For the whole lung culture, extracted lungs were embedded in Matrigel and incubated on permeable filters. Separately, distal epithelial tips were isolated by firstly removing mesothelial and mesenchymal cells, and then severing the tips from the airway tubes. These were then cultured either in branch-promoting or self-renewing conditions. RESULTS: Cultured whole lungs underwent branching morphogenesis similarly to native lungs. Real-time qPCR analysis demonstrated expression of key genes essential for lung bud formation. The culture condition for epithelial tips was optimized by testing different concentrations of FGF10 and CHIR99021 and evaluating branching formation. The epithelial rudiments in self-renewing conditions formed spherical 3D structures with homogeneous Sox9 expression. CONCLUSION: We report efficient protocols for ex vivo culture systems of pseudoglandular stage mouse embryonic lungs. These models can be applied to human samples and could be useful to paediatric surgeons to investigate normal lung development, understand the pathogenesis of congenital lung diseases, and explore novel therapeutic strategies.

A Novel Microfluidic Platform for Biomechano-Stimulations on a Chip.

Mechanical stress has been proven to be an important factor interfering with many biological functions through mechano-sensitive elements within the cells. Despite the current interest in mechano-transduction, the development of suitable experimental tools is still characterized by the strife to design a compact device that allows high-magnification real-time imaging of the stretched cells, thus enabling to follow the dynamics of cellular response to mechanical stimulations. Here we present a microfluidic multi-layered chip that allows mechanical deformation of adherent cells maintaining a fixed focal plane, while allowing independent control of the soluble microenvironment. The device was optimized with the aid of FEM simulation and fully characterized in terms of mechanical deformation. Different cell lines were exposed to tunable mechanical strain, which results in continuous area deformation up to 20%. Thanks to the coupling of chemical glass etching, 2-dimensional deformation of a thin elastomeric membrane and microfluidic cell culture, the developed device allows a unique combination of cell mechanical stimulation, in line imaging and accurate control of cell culture microenvironment.

A mechanical checkpoint controls multicellular growth through YAP/TAZ regulation by actin-processing factors.

Key cellular decisions, such as proliferation or growth arrest, typically occur at spatially defined locations within tissues. Loss of this spatial control is a hallmark of many diseases, including cancer. Yet, how these patterns are established is incompletely understood. Here, we report that physical and architectural features of a multicellular sheet inform cells about their proliferative capacity through mechanical regulation of YAP and TAZ, known mediators of Hippo signaling and organ growth. YAP/TAZ activity is confined to cells exposed to mechanical stresses, such as stretching, location at edges/curvatures contouring an epithelial sheet, or stiffness of the surrounding extracellular matrix. We identify the F-actin-capping/severing proteins Cofilin, CapZ, and Gelsolin as essential gatekeepers that limit YAP/TAZ activity in cells experiencing low mechanical stresses, including contact inhibition of proliferation. We propose that mechanical forces are overarching regulators of YAP/TAZ in multicellular contexts, setting responsiveness to Hippo, WNT, and GPCR signaling.

Stochastic model-assisted development of efficient low-dose viral transduction in microfluidics.

Adenoviruses are commonly used in vitro as gene transfer vectors in multiple applications. Nevertheless, issues such as low infection efficiency and toxicity effects on host cells have not been resolved yet. This work aims at developing a new versatile tool to enhance the expression of transduced genes while working at low viral doses in a sequential manner. We developed a microfluidic platform with automatically controlled sequential perfusion stages, which includes 10 independent channels. In addition, we built a stochastic mathematical model, accounting for the discrete nature of cells and viruses, to predict not only the percentage of infected cells, but also the associated infecting-virus distribution in the cell population. Microfluidic system and mathematical model were coupled to define an efficient experimental strategy. We used human foreskin fibroblasts, infected by replication-incompetent adenoviruses carrying EGFP gene, as the testing system. Cell characterization was performed through fluorescence microscopy, followed by image analysis. We explored the effect of different aspects: perfusion, multiplicity of infection, and temporal patterns of infection. We demonstrated feasibility of performing efficient viral transduction at low doses, by repeated pulses of cell-virus contact. This procedure also enhanced the exogenous gene expression in the sequential microfluidic infection system compared to a single infection at a higher, nontoxic, viral dose.