advanced biochip perfusion and air-liquid interface establishment of human umbilical vein endothelial cells for tissue culture analysis

Etablering av en lung-on-chip-modell för studier av luftvägsinfektioner

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.

Författare i fokus: Utveckling av en mikrofluidisk lung-on-chip-modell för djupgående studier av mänskligt immunsvar och infektionsmekanismer

Immunokompetent alveol-on-chip-modell för att studera alveolära slemhinneimmunsvar

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.

Avancerad biochipperfusion och luft-vätskegränssnittsetablering av humana navelvensendotelceller för vävnadskulturanalys

Innan du ansluter den steriliserade slangen till biochipet, spola slangen först med 200 mikroliter PBS följt av 200 mikroliter EC-medium. Efter att ha spolat den övre och nedre kammaren med nypreparerat cellodlingsmedium, sätt in behållaren på utloppssidan av biochipet och anslut behållaren med slangen till biochipets inlopp. För cirkulär mediumöverflöd, anslut slangen till behållaren och biochipet.Fyll nu behållaren med 500 mikroliter EC-medium. Anslut biochipet till slangen för att starta överflödet med en flödeshastighet på 21 mikroliter per minut. Nästa dag, stoppa överflödet och töm reservoarerna.Under ett sterilt säkerhetsskåp pipetterar du 500 mikroliter EC-medium i behållarna och spolar försiktigt den nedre kammaren med 200 mikroliter RPMI plus-medium. Starta sedan om perfusionen med en flödeshastighet på 21 mikroliter per minut. För att upprätta ett luftvätskegränssnitt från dag 12 till dag 14, öppna pluggarna vid den nedre kammaren och sug försiktigt upp mediet tills en luftbubbla är synlig.Efter att ha säkerställt att all vätska från kanalen och kammaren har avlägsnats, stäng försiktigt portarna, fyll behållaren med nyberett vaskulärt EC-medium och fortsätt med profusionsodling endast i den övre kammaren fram till dag 14. Koppla bort biochipsen från flödessystemet. Efter att ha tagit bort slangen, undersök cellskikten från biochipet under ett mikroskop för cellsammanflöde.Tvätta biochipsens håligheter med PBS för att avlägsna cellodlingsmediet. Tillsätt 300 mikroliter 4% paraformaldehydlösning i PBS till den övre kammaren och 200 mikroliter till den nedre kammaren. Efter 10 minuters inkubation, tvätta kammaren tre gånger med PBS.För permeablisering, tillsätt 300 mikroliter 0,25% Triton X 100 i PBS och inkubera i 30 minuter, efter tvätt med PBS, pipettera 300 mikroliter 3%bovint serumalbumin i PBS i båda kamrarna och inkubera i en timme. För immunofluorescensfärgning, förbered 50 mikroliter antikroppslösningar med 3%BSA för varje membran med lämpliga spädningsförhållanden. För att komma åt cellerna i biochipet, gör ett exakt snitt över utsidan av kaviteten och ta bort bindningsfolien.Använd en skalpell för att skära ut membranet genom att ta bort kanterna som är förseglade mot biochipet. Efter att ha delat membranet i två delar, samla membranbitarna med en pincett och placera dem på ett mikroskopiskt objektglas för färgning, tillsätt 50 mikroliter antikroppslösning till varje membranbit och inkubera över natten vid fyra grader Celsius, vilket skyddar mot ljus. Efter att ha tvättat membranet tre gånger med PBS, tillsätt en droppe monteringsmedium på den märkta bilden och placera motsvarande membran.Tillsätt en droppe av monteringsmediet på toppen av membranet och placera ett täckglas. Immunofluorescensfärgning av humana endotelceller i navelvenen avslöjade morfologiska förändringar och uttryck av marcoprotein efter 14 dagars samodling. På den vaskulära sidan V föreslog koherent uttryck vid endotelcellgränser konfluens och endotelial barriärbildning.Epitelsidan utvärderades för uttryck av ecaherrin och surfaktant A, vilket indikerar alveolär epitelial integritet och funktion.

advanced-biochip-perfusion-air-liquid-interface-establishment-human

Research

JoVE Journal

Immunology and Infection

Advanced Biochip Perfusion and Air-Liquid Interface Establishment of Human Umbilical Vein Endothelial Cells for Tissue Culture Analysis

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

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 ...