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

呼吸器感染症研究のための肺オンチップモデルの確立

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.

著者スポットライト:ヒトの免疫応答と感染メカニズムの詳細な研究のためのマイクロ流体肺オンチップモデルの開発

肺胞粘膜免疫応答を研究するための免疫適格肺胞オンチップモデル

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.

組織培養解析のためのヒト臍帯静脈内皮細胞の高度バイオチップ灌流と気液界面の確立

滅菌したチューブをバイオチップに接続する前に、まずチューブを200マイクロリットルのPBSで洗い流し、次に200マイクロリットルのEC培地で洗い流します。新たに調製した細胞培養培地で上部チャンバーと下部チャンバーを洗い流した後、バイオチップの出口側にリザーバーを挿入し、チューブ付きのリザーバーをバイオチップの入口に接続します。円形の培地を増殖させるには、チューブをリザーバーとバイオチップに接続します。次に、リザーバーに500マイクロリットルのEC培地を入れます。バイオチップをチューブに接続して、毎分21マイクロリットルの流量で増殖を開始します。翌日、氾濫を止めて貯水池を空にします。滅菌済みの安全キャビネットの下で、500マイクロリットルのEC培地をリザーバーにピペットで入れ、200マイクロリットルのRPMIプラス培地で下部チャンバーを静かに洗い流します。その後、毎分21マイクロリットルの流量で灌流を再開します。12日目から14日目にかけて気液界面を確立するには、下部チャンバーのプラグを開き、気泡が見えるまで培地を慎重に吸収します。チャネルとチャンバーからすべての液体が除去されたことを確認した後、ポートを慎重に閉じ、新たに調製した血管EC培地をリザーバーに充填し、14日目まで上部チャンバーでのみ増殖培養を続けます。バイオチップをフローシステムから外します。チューブを取り外した後、顕微鏡でバイオチップの細胞層を細胞の密度を調べます。バイオチップの空洞をPBSで洗浄し、細胞培養培地を取り出します。PBS中の4%パラホルムアルデヒド溶液を300マイクロリットルを上部チャンバーに、200マイクロリットルを下部チャンバーに加えます。10分間のインキュベーション後、チャンバーをPBSで3回洗浄します。透過化のために、PBS中の0.25%Triton X 100の300マイクロリットルを加え、30分間インキュベートし、PBSで洗浄した後、PBS中の300マイクロリットルの3%ウシ血清アルブミンを両チャンバー内でピペットで掴み、1時間インキュベートする。免疫蛍光染色には、適切な希釈比で各メンブレンに3%BSAを使用して50マイクロリットルの抗体溶液を調製します。バイオチップ内の細胞にアクセスするには、キャビティの外側を正確に切り込み、ボンディングホイルを取り外します。メスを使用して、バイオチップに密封されたエッジを取り除き、メンブレンを切り取ります。メンブレンを2つに分割した後、メンブレンピースをピンセットで集め、顕微鏡のスライドに載せて染色し、各メンブレンピースに50マイクロリットルの抗体溶液を加え、光から保護するために摂氏4度で一晩インキュベートします。メンブレンをPBSで3回洗浄した後、ラベル付けされたスライド上に封入剤を一滴加え、対応するメンブレンを配置します。メンブレンの上部に封入剤を一滴加え、カバーガラスを置きます。ヒト臍帯静脈内皮細胞の免疫蛍光染色により、14日間の共培養後に形態学的変化とマルコタンパク質の発現が明らかになりました。血管側では、内皮細胞境界でのVコヒーレント発現は、コンフルエンシーと内皮バリア形成を示唆していました。上皮側は、エカヘリンとサーファクタントタンパク質Aの発現について評価され、肺胞上皮の完全性と機能を示しました。

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 ...