Made by cells for cells - extracellular vesicles as next-generation mainstream medicines.

Current medicine has only taken us so far in reducing disease and tissue damage. Extracellular vesicles (EVs), which are membranous nanostructures produced naturally by cells, have been hailed as a next-generation medicine. EVs deliver various biomolecules, including proteins, lipids and nucleic acids, which can influence the behaviour of specific target cells. Since EVs not only mirror composition of their parent cells but also modify the recipient cells, they can be used in three key areas of medicine: regenerative medicine, disease detection and drug delivery. In this Review, we discuss the transformational and translational progress witnessed in EV-based medicine to date, focusing on two key elements: the mechanisms by which EVs aid tissue repair (for example, skin and bone tissue regeneration) and the potential of EVs to detect diseases at an early stage with high sensitivity and specificity (for example, detection of glioblastoma). Furthermore, we describe the progress and results of clinical trials of EVs and demonstrate the benefits of EVs when compared with traditional medicine, including cell therapy in regenerative medicine and solid biopsy in disease detection. Finally, we present the challenges, opportunities and regulatory framework confronting the clinical application of EV-based products.

The acid injury and repair (AIR) model: A novel ex-vivo tool to understand lung repair.

Research into mechanisms underlying lung injury and subsequent repair responses is currently of paramount importance. There is a paucity of models that bridge the gap between in vitro and in vivo research. Such intermediate models are critical for researchers to decipher the mechanisms that drive repair and to test potential new treatments for lung repair and regeneration. Here we report the establishment of a new tool, the Acid Injury and Repair (AIR) model, that will facilitate studies of lung tissue repair. In this model, injury is applied to a restricted area of a precision-cut lung slice using hydrochloric acid, a clinically relevant driver. The surrounding area remains uninjured, thus mimicking the heterogeneous pattern of injury frequently observed in lung diseases. We show that in response to injury, the percentage of progenitor cells (pro surfactant protein C, proSP-C and TM4SF1 positive) significantly increases in the injured region. Whereas in the uninjured area, the percentage of proSP-C/TM4SF1 cells remains unchanged but proliferating cells (Ki67 positive) increase. These effects are modified in the presence of inhibitors of proliferation (Cytochalasin D) and Wnt secretion (C59) demonstrating that the AIR model is an important new tool for research into lung disease pathogenesis and potential regenerative medicine strategies.

An Ex Vivo Acid Injury and Repair (AIR) Model Using Precision-Cut Lung Slices to Understand Lung Injury and Repair.

Recent advances in cell culture models like air-liquid interface culture and ex vivo models such as organoids have advanced studies of lung biology; however, gaps exist between these models and tools that represent the complexity of the three-dimensional environment of the lung. Precision-cut lung slices (PCLS) mimic the in vivo environment and bridge the gap between in vitro and in vivo models. We have established the acid injury and repair (AIR) model where a spatially restricted area of tissue is injured using drops of HCl combined with Pluronic gel. Injury and repair are assessed by immunofluorescence using robust markers, including Ki67 for cell proliferation and prosurfactant protein C for alveolar type 2/progenitor cells. Importantly, the AIR model enables the study of injury and repair in mouse lung tissue without the need for an initial in vivo injury, and the results are highly reproducible. Here, we present detailed protocols for the generation of PCLS and the AIR model. We also describe methods to analyze and quantify injury in AIR-PCLS by immunostaining with established early repair markers and fluorescence imaging. This novel ex vivo model is a versatile tool for studying lung cell biology in acute lung injury and for semi-high-throughput screening of potential therapeutics. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation of precision-cut lung slices Basic Protocol 2: The acid injury and repair model Basic Protocol 3: Analysis of AIR-PCLS: Immunostaining and imaging.

Placenta Stem/Stromal Cell-Derived Extracellular Vesicles for Potential Use in Lung Repair.

Many acute and chronic lung injuries are incurable and rank as the fourth leading cause of death globally. While stem cell treatment for lung injuries is a promising approach, there is growing evidence that the therapeutic efficacy of stem cells originates from secreted extracellular vesicles (EVs). Consequently, EVs are emerging as next-generation therapeutics. While EVs are extensively researched for diagnostic applications, their therapeutic potential to promote tissue repair is not fully elucidated. By housing and delivering tissue-repairing cargo, EVs refine the cellular microenvironment, modulate inflammation, and ultimately repair injury. Here, the potential use of EVs derived from two placental mesenchymal stem/stromal cell (MSC) lines is presented; a chorionic MSC line (CMSC29) and a decidual MSC cell line (DMSC23) for applications in lung diseases. Functional analyses using in vitro models of injury demonstrate that these EVs have a role in ameliorating injuries caused to lung cells. It is also shown that EVs promote repair of lung epithelial cells. This study is fundamental to advancing the field of EVs and to unlock the full potential of EVs in regenerative medicine.

Isolation and Characterization of Extracellular Vesicles from Mesenchymal Stromal Cells.

Extracellular vesicles (EVs) have received immense attention in the past decade for their diverse use in diagnosis and therapeutics. Enhancing our understanding of EVs and increasing the reliability and reproducibility of EV research demands the use of standard isolation procedures and multiple characterization methods. Here we describe the most commonly used EV isolation method involving ultracentrifugation, and various characterization methods that include nanoparticle tracking analysis, atomic force microscopy and electron microscopy, which measure the size, concentration, and morphology of EVs.

High-fidelity probing of the structure and heterogeneity of extracellular vesicles by resonance-enhanced atomic force microscopy infrared spectroscopy.

Extracellular vesicles (EVs) are highly specialized nanoscale assemblies that deliver complex biological cargos to mediate intercellular communication. EVs are heterogeneous, and characterization of this heterogeneity is paramount to understanding EV biogenesis and activity, as well as to associating them with biological responses and pathologies. Traditional approaches to studying EV composition generally lack the resolution and/or sensitivity to characterize individual EVs, and therefore the assessment of EV heterogeneity has remained challenging. We have recently developed an atomic force microscope IR spectroscopy (AFM-IR) approach to probe the structural composition of single EVs with nanoscale resolution. Here, we provide a step-by-step procedure for our approach and show its power to reveal heterogeneity across individual EVs, within the same population of EVs and between different EV populations. Our approach is label free and able to detect lipids, proteins and nucleic acids within individual EVs. After isolation of EVs from cell culture medium, the protocol involves incubation of the EV sample on a suitable substrate, setup of the AFM-IR instrument and collection of nano-IR spectra and nano-IR images. Data acquisition and analyses can be completed within 24 h, and require only a basic knowledge of spectroscopy and chemistry. We anticipate that new understanding of EV composition and structure through AFM-IR will contribute to our biological understanding of EV biology and could find application in disease diagnosis and the development of EV therapies.

Greater cellular stiffness in fibroblasts from patients with idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease involving degenerative breathing capacity. Fibrotic disease is driven by dysregulation in mechanical forces at the organ, tissue, and cellular level. While it is known that, in certain pathologies, diseased cells are stiffer than healthy cells, it is not known if fibroblasts derived from patients with IPF are stiffer than their normal counterparts. Using IPF patient-derived cell cultures, we measured the stiffness of individual lung fibroblasts via high-resolution force maps using atomic force microscopy. Fibroblasts from patients with IPF were stiffer and had an augmented cytoskeletal response to transforming growth factor-ß1 compared with fibroblasts from donors without IPF. The results from this novel study indicate that the increased stiffness of lung fibroblasts of IPF patients may contribute to the increased rigidity of fibrotic lung tissue.

Atomized Human Amniotic Mesenchymal Stromal Cells for Direct Delivery to the Airway for Treatment of Lung Injury.

BACKGROUND: Current treatment regimens for inhalation injury are mainly supportive and rely on self-regeneration processes for recovery. Cell therapy with mesenchymal stromal cells (MSCs) is increasingly being investigated for the treatment of inhalation injury. Human amniotic MSCs (hAMSCs) were used in this study due to their potential use in inflammatory and fibrotic conditions of the lung. This study aimed at demonstrating that hAMSCs can be atomized with high viability, for the purpose of achieving a more uniform distribution of cells throughout the lung. Another aim of this study was to set ground for future application to healthy and diseased lungs by demonstrating that hAMSCs were able to survive after being sprayed onto substrates with different stiffness. METHODS: Two methods of atomization were evaluated, and the LMA MAD780 device was selected for atomizing hAMSCs for optimized delivery. To mimic the stiffness of healthy and diseased lungs, gelatin gel (10% w/v) and tissue culture plastic were used as preliminary models. Poly-l-lysine (PLL) and collagen I coatings were used as substrates on which the hAMSCs were cultured after being sprayed. RESULTS: The feasibility of atomizing hAMSCs was demonstrated with high cell viability (81 ± 3.1% and 79 ± 11.6% for cells sprayed onto plastic and gelatin, respectively, compared with 85 ± 4.8% for control/nonsprayed cells) that was unaffected by the different stiffness of substrates. The presence of the collagen I coating on which the sprayed cells were cultured yielded higher cell proliferation compared with both PLL and no coating. The morphology of sprayed cells was minimally compromised in the presence of the collagen I coating. CONCLUSIONS: This study demonstrated that hAMSCs are able to survive after being sprayed onto substrates with different stiffness, especially in the presence of collagen I. Further studies may advance the effectiveness of cell therapy for lung regeneration.

Formulation of Biologically-Inspired Silk-Based Drug Carriers for Pulmonary Delivery Targeted for Lung Cancer.

The benefits of using silk fibroin, a major protein in silk, are widely established in many biomedical applications including tissue regeneration, bioactive coating and in vitro tissue models. The properties of silk such as biocompatibility and controlled degradation are utilized in this study to formulate for the first time as carriers for pulmonary drug delivery. Silk fibroin particles are spray dried or spray-freeze-dried to enable the delivery to the airways via dry powder inhalers. The addition of excipients such as mannitol is optimized for both the stabilization of protein during the spray-freezing process as well as for efficient dispersion using an in vitro aerosolisation impactor. Cisplatin is incorporated into the silk-based formulations with or without cross-linking, which show different release profiles. The particles show high aerosolisation performance through the measurement of in vitro lung deposition, which is at the level of commercially available dry powder inhalers. The silk-based particles are shown to be cytocompatible with A549 human lung epithelial cell line. The cytotoxicity of cisplatin is demonstrated to be enhanced when delivered using the cross-linked silk-based particles. These novel inhalable silk-based drug carriers have the potential to be used as anti-cancer drug delivery systems targeted for the lungs.