preparation and maintenance of human umbilical vein endothelial cells in biochips for establishing an air-liquid interface

Establishing a Lung-on-Chip Model for Respiratory Infection Studies

The human lung has a remarkable role in respiration and immune defense, with complex interactions between the immune responses of the alveolar mucosa1. The ability of the alveoli to create an efficient immune response is vital for preventing lung infections and securing pulmonary health. Since the lungs are constantly exposed to a wide range of potential risks, including bacteria, viruses, fungi, allergies, and particulate matter,&#160;understanding the complexities of alveolar mucosal immune responses is critical for discovering the mechanisms behind respiratory infections, inflammatory disorders and treating pulmonary diseases1.
To study infection and inflammation-related processes of the respiratory tract in vitro, models that could faithfully mimic the alveolar milieu and the immune responses are required. 2D cell culture and animal modules have been used for decades as essential tools for biomedical research on lung immune response. However, they often have limitations in their translational potential to human situations. Lung-on-chip models can contribute to filling the gap between traditional in vitro and in vivo models and provide a novel approach to studying human-specific immune responses2,3. Lung-on-chip models can mimic the air-liquid interface, which is required for lung cells to recapitulate physiological conditions of the respiratory tract and to develop a more accurate and robust tissue model. This culture technique enables a precise examination of cell differentiation, functioning, and responses to drugs or disease-related stimuli in vitro2.
In this study, we present a microfluidic-based model of the human alveolus as an effective tool to recapitulate the human alveolar milieu by applying perfusion to mimic blood flow and biomechanical stimulation of endothelial cells and incorporating an air-liquid interface with epithelial cells exposed towards an air phase4. We have developed a microfluidic perfused alveolus-on-chip that mimics the physical structure and biological interactions of the human alveolus, with a particular focus on the air-liquid interface. This interface plays a crucial role in the differentiation of respiratory epithelial cells, which is essential for accurately modeling the pulmonary environment. The model uses human distal lung epithelial cells (H441) and human umbilical vein endothelial cells (HUVECs), separated by porous polyethylene terephthalate (PET) membranes, with primary monocyte-derived macrophages positioned between the cell layers. This setup replicates the intricate cellular arrangement of the alveolus and is critical for accurately simulating the air-liquid interface, which is a significant factor in the physiological function of lung tissue.
The rationale behind the model development extends to integrating both circulating and tissue-resident immune cells. This approach is designed to accurately mimic the inflammatory host response to human respiratory infections, providing a dynamic environment to study pathogen-host interactions. The presence of macrophages allows for the examination of immediate immune responses and their interaction with pathogens, reflecting the first line of defense against respiratory infections. Furthermore, the design of the biochip platform facilitates convenient and precise manipulation of both biophysical and biochemical cues, which is crucial for replicating alveolus function in vitro. This flexibility is instrumental in dissecting the contributing factors to human infections, enabling researchers to adjust conditions to reflect various disease states or to test potential therapeutic interventions. The compatibility of the platform with multiple readout technologies, including advanced microscopy, microbiological analyses, and biochemical effluent analysis, enhances its utility. These capabilities allow for a comprehensive assessment of the tissue response to infections, including evaluating cellular behavior, pathogen proliferation, and the effectiveness of immune responses.
We present a detailed protocol and techniques to create and utilize a human alveolus-on-chip model focused on replicating the air-liquid interface and integrating immune cells to study human infections in vitro.

Author Spotlight: Developing a Microfluidic Lung-on-Chip Model for In-Depth Study of Human Immune Response and Infection Mechanisms

Immunocompetent Alveolus-on-Chip Model for Studying Alveolar Mucosal Immune Responses

We develop physiologically relevant in vitro models to answer mechanistic question in human infections. Our focus is on the host immune response. When we use this model to dissect the complex interaction of the pathogen to the host to identify molecular and cellular targets for therapeutic options in human infections.Working on a chip model like lung-on-chip model, balanced by logical complexity with specific research needs, offering insights into the human-host response regulation. This system mimic in vivo cellular composition and 3D structure, providing more defined conditions and high throughput than the animal experiments. In lung-on-chip technology, one of the primary challenges is selecting the right cell types that mimics lung's complex functionality for infection study results.Moreover, extending the timeframe of the experiment beyond traditional in vitro models is crucial. Leveraging is the cheap ability to maintain cell viability with continuous nutrient flow for longer observation, the experiment is also necessary. We developed the microfluidic base model of human alveolus as an effective tool to mimic human alveolar environment.It was achieved by applying perfusion to mimic blood flow, and also by a mechanical stimulation to the endothelial cells. And also integrating air-liquid interface for the epithelial cells by exposing them to the air. The next step is to extend this model to a more advanced platform, such as induced pluripotent stem cell-based model.By doing so, we aim to bring personalized medicine closer to application, particularly in the context of antiviral drug testing, and as a tool for biomedical research and pharmaceutical development.

Preparation and Maintenance of Human Umbilical Vein Endothelial Cells in Biochips for Establishing an Air-Liquid Interface

preparation-maintenance-human-umbilical-vein-endothelial-cells

Research

JoVE Journal

Immunology and Infection

46 vues.  Jena University Hospital. Pour commencer, placez les Biochips modèle BC002 dans une boîte de Pétri en verre et rincez toutes les cavités avec de l’éthanol stérile à 70 % pendant 45 minutes. Ensuite, rincez les cavités deux fois avec de l’eau stérile doublement distillée. À l’aide d’une pipette de 1 000 microlitres avec des pointes bleues, enduire la membrane de la biopuce de la cavité supérieure avec une solution stérile de collagène IV.Incuber la puce pendant 30 minutes à 37 degrés Cel...

Vidéo: Préparation et maintenance de cellules endothéliales de la veine ombilicale humaine dans des biopuces pour l’établissement d’une interface air-liquide

We introduce an advanced immunocompetent lung-on-chip model designed to replicate the human alveolar structure and function. This innovative model employs a microfluidic-perfused biochip that supports an air-liquid interface mimicking the environment in the human alveoli. Tissue engineering is used to integrate key cellular components, including endothelial cells, macrophages, and epithelial cells, to create a representative tissue model of the alveolus. The model facilitates in-depth examinations of the mucosal immune responses to various pathogens, including viruses, bacteria, and fungi, thereby advancing our understanding of lung immunity. The primary goal of this protocol is to provide details for establishing this alveolus-on-chip model as a robust in vitro platform for infection studies, enabling researchers to closely observe and analyze the complex interactions between pathogens and the host's immune system within the pulmonary environment. This is achieved through the application of microfluidic-based techniques to simulate key physiological conditions of the human alveoli, including blood flow and biomechanical stimulation of endothelial cells, alongside maintaining an air-liquid interface crucial for the realistic exposure of epithelial cells to air. The model system is compatible with a range of standardized assays, such as immunofluorescence staining, cytokine profiling, and colony-forming unit (CFU)/plaque analysis, allowing for comprehensive insights into immune dynamics during infection. The Alveolus-on-chip is composed of essential cell types, including human distal lung epithelial cells (H441) and human umbilical vein endothelial cells (HUVECs) separated by porous polyethylene terephthalate (PET) membranes, with primary monocyte-derived macrophages strategically positioned between the epithelial and endothelial layers. The tissue model enhances the ability to dissect and analyze the nuanced factors involved in pulmonary immune responses in vitro. As a valuable tool, it should contribute to the advancement of lung research, providing a more accurate and dynamic in vitro model for studying the pathogenesis of respiratory infections and testing potential therapeutic interventions.

Lung-on-chip models surpass traditional 2D cultures by mimicking the air-liquid interface and endothelial cell perfusion, simulating blood flow and nutrient exchange crucial for lung physiology studies. This enhances lung research relevance, offering a dynamic, physiologically accurate environment to advance the understanding and treatment of respiratory infections.

We introduce an advanced immunocompetent lung-on-chip model designed to replicate the human alveolar structure and function. This innovative model employs ...