Immunocompetent Alveolus-on-Chip Model for Studying Alveolar Mucosal Immune Responses
Summary
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.
Abstract
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.
Introduction
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, 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.
Protocol
HUVEC cells are isolated from umbilical cords and used up to passage 4. Primary monocytes are isolated from healthy donors from whole blood. The study was approved by the ethics committee of the Jena University Hospital, Jena, Germany (3939-12/13). According to the Declaration of Helsinki, all individuals donating cells for the study gave their informed consent.
1. Day 1: Preparation of the biochip
- The biochips are available in different sizes and models. For experiments here, use the model BC002. Place the biochip in a glass Petri dish, fill the dish, and flush all cavities with sterile 70% ethanol (EthOH). Incubate for 45 min at room temperature. Afterwards, flush cavities 2x with sterile ddH2O. Remove the ddH2O.
- Coat the biochip membrane of both cavities with sterile collagen IV solution (0.5 mg/mL in 0.5 mM acetic acid; use 1:100 concentration in Dulbecco's phosphate buffered saline containing magnesium and calcium PBS (+/+)) by gently flushing the collagen IV solution through both cavities using a 1,000 µL pipet with blue tips. Incubate for 15 min at 37 °C and 5% CO2.
NOTE: Using a 1,000 µL pipet with blue filter (P1000) tips is recommended throughout the entire protocol. The diameter of the opening of the filter tip completely covers the opening of the ports connecting the channels, which avoids the insertion of air bubbles into the chip. - After 30 min incubation, flush the lower and upper chambers 2x with sterile PBS (+/+) to flush out the remaining collagen and acetic acid.
- Prepare EC medium by adding 1:100 (v/v) Penicillin/Streptomycin (10,000 U/mL). Fill the upper chamber of each cavity with 250 µL of EC medium and the lower chamber with 150 µL of EC medium.
- Harvest HUVEC from a confluent T25 flask: Wash with 5 mL of Dulbecco's phosphate buffered saline without magnesium and calcium PBS (-/-), add 1 mL of 0.25% Trypsin + 1 mM EDTA in PBS (-/-) and incubate for 3 min at 37 °C. Stop trypsin activity by adding 5 mL of PBS (+/+) + 5% FCS and centrifuge at 350 x g for 5 min at room temperature (RT) in a 50 mL conical tube. Remove the supernatant and resuspend the cell pellet in 1 mL of EC medium.
- Use a 1:10 serial dilution when performing cell counting. Add 10 µL of the resuspended cell pellet to 90 µL of cell medium. Combine the samples carefully. Use a microcentrifuge tube with 10 µL of trypan blue stain. Dilute the cell solution with trypan blue (1:1) using cell counting chamber slides. Count the cells either manually with a Neubauer Chamber or with an automated cell counter. Determine cell counts and adjust concentration to 0.4 x 106 cells per 200 µL.
- Replace the endothelial cell medium in the upper chamber of the biochip with 200 µL of EC medium containing suspended HUVECs. Pipette gently to avoid the formation of air bubbles in the channels or chambers. Place the glass Petri dish with the biochips at 37 °C and 5% CO2.
2. Day 2: Checking of the cell layer
- On Day 2, visually check for the confluency of the HUVEC cell layer in the upper chamber. Perform medium exchange in the upper chamber with 300 µL of EC medium. Pipette gently to avoid detachment of cells from the membrane. Perform media exchange daily after 24h. Place the glass Petri dish with the biochips back at 37 °C and 5% CO2.
3. Day 3: Preparation of lower chamber
- Visually check the confluency of the endothelial cells layer. At this point, determine the presence of a confluent layer and cobblestone-like morphology (Figure 1). Perform medium exchange in the upper chamber with 300 µL of EC medium.
- Next, prepare the lower chamber for seeding the cells forming the epithelial layer. Flush the lower chamber gently with 150 µL of the RPMI medium (add 1:100 (v/v) Penicillin/Streptomycin (10,000 U/mL), 5% fetal cow serum, 1 µM dexamethasone).
- For the lower chamber, seed 0.5 x 106 H441 cells suspended in 200 µL of RPMI medium (RPMI) per cavity.
- Harvesting H441 cells
- Wash the cells with 5 mL of PBS (-/-) once. Add 2 mL of 0.25% Trypsin + 1 mM EDTA in PBS (-/-).
- Incubate the cells for 5 min at 37 °C and 5% CO2. The incubation time varies depending on the confluency of the layer. Within 5-7 min, cells should detach completely.
- Stop the trypsin activity with 10 mL of PBS (+/+) + 5% FBS, collect the cells, and centrifuge at 200 x g for 4 min.
- After centrifugation, discard the supernatant, resuspend the pellet in 1 mL of RPMI, and count.
- Seed 0.5 x 106 cells per chamber by gently pipetting the cell solution into the lower chamber. Close all the ports and flip the biochip upside-down so the cells can attach to the PET membrane. Keep the biochip upside-down and incubate overnight in a cell culture incubator at 37 °C and 5% CO2.
4. Days 4 – Day7: Maintenance of cells and harvesting monocytes
- Perform daily medium exchange in the upper and lower chamber. For the upper chamber, perform the medium exchange with 300 µL of EC medium.
- Use 200 µL of RPMI+ (RPMI medium plus 1:100 (v/v) Penicillin/Streptomycin (10,000 U/mL), 10 ng/mL GM-CSF, 10% autologous human serum, 1 µM dexamethasone) for the lower chamber. On day 7, flush the lower chamber with RPMI+ to prepare the cavity for seeding monocytes.
- Seed 0.1 x 106 monocytes suspended in 200 µL RPMI+ in the lower chamber.
- Harvesting monocytes
- Wash the cells with 1 mL of PBS (-/-) and incubate for 7 min at 37 °C in pre-warmed PBS (-/-) containing 4 mg/mL Lidocaine + 5 mM EDTA at 37 °C and 5% CO2.
- Collect the cells and centrifuge at 350 x g for 7 min, resuspend the cells in 1mL of RPMI+, and count. Place the biochip ports facing downwards so the cells attach to the membrane.
5. Day 8: Biochip perfusion
- At this point, ensure the biochips are ready to be connected to the flow and perfused. Before starting the perfusion, prepare an empty incubator and place the peristaltic pump inside.
- Perfusion of the biochips
- Before attaching the sterilized tubing to the biochip, flush the tubing with 200 µL of PBS (+/+), followed by 200 µL of EC medium. The tubing has an inner diameter of 0.5 mm, consisting of longer (20 cm) and shorter (12 cm) sides divided by two peristaltic pump stoppers. Put the tubing aside and prepare the reservoirs for the medium.
- Perform medium exchange in the upper and lower chambers by flushing them with the respective freshly prepared cell culture medium.
- Insert the reservoir on the outlet side of the biochip and connect the reservoir with the tubing to the inlet of the biochip (Figure 2). Connect the tubing to the reservoir and the biochip so that there is a circular medium perfusion between the reservoir and the biochip.
- Once everything is connected to the ports, fill in the reservoir with a 500 µL of EC medium. Connect the biochip with the tubing on a perfusion with a flow rate of 21 µL/min (Figure 2). Observe closely during the first 15 min of perfusion for the insertion of bubbles. Perform medium exchange daily in both chambers.
6. Day 9 – Day 11: Establishing an air-liquid interface
- Stop the perfusion, empty the reservoirs, and refill with freshly prepared medium under a sterile safety cabinet. Pipette 500 µL of EC medium into the reservoirs and flush gently the lower chamber with 200 µL of RPMI+. Start the perfusion again with a perfusion rate of 21 µL/min.
- Air Liquid Interface (ALI)
- Establish ALI from day 12 until the end of the experiment (day 14). During ALI culture, expose the epithelial cells to air.
- To establish the ALI culture, prepare a new vascular medium EC (add 1:100 (v/v) Penicillin/Streptomycin (10,000 U/mL), 10 ng/ml GM-CSF, 20% autologous human serum, 1µM dexamethasone) to maintain all cell types in the chip. At this point, the medium contains a higher concentration of AS (20%).
- Open the plugs at the lower chamber under a sterile safety cabinet and carefully soak up the medium until an air bubble is visible, as shown in Figure 3. Ensure the entire liquid from the channel and the chamber is removed and close the ports carefully.
- Fill in the reservoirs with EC (-/-) medium and continue perfusion culture with a flow rate of 21 µL/min. Exchange medium only at the upper chamber until day 14 and keep the epithelial cells in the lower chamber with air only.
7. Day 14: Tissue harvesting and analysis
- Disconnect the biochip from the flow. Plug out the tubing and check on the microscope for cell confluency. Due to the air in the lower chamber, the cell layers might appear fuzzy and difficult to visualize. At this point, the model is ready for further experimentation.
- Fixation and staining of tissue in biochip
- Wash the cavities with PBS (+/+) to remove the cell culture medium. Pipette 300 µL of 4% paraformaldehyde (PFA) dissolved in PBS (-/-) to the upper chamber and 200 µL in the lower chamber and incubate for 10 min at RT.
NOTE: When working with fixation chemicals such as PFA, use a fume hood. Collect and dispose of the waste accordingly. - Wash the chambers 3x with PBS (+/+). After fixation, store the biochips at 4 °C or use them directly for immunofluorescence staining of cells.
- For permeabilization, pipette 300 µL of 0.25% Triton X-100 in PBS (+/+). Incubate for 30 min at RT.
- After incubation, wash both chambers with PBS (+/+) and pipette 300 µL of 3% Bovine serum albumin (BSA) in PBS (+/+) in both chambers. Incubate for 1 h at RT.
- In the meantime, prepare the antibody panel for immunofluorescence staining. To prepare the antibody panel for immunofluorescence staining, a tube with a sufficient amount of 3% BSA was set up to dilute the antibodies. Use 50 µL of antibody dilutions for each membrane. Use a dilution ratio of 1:100 for primary antibody panels and 1:200 for secondary antibody panels. Additionally, for the secondary antibody panel, prepare DAPI at a dilution ratio of 1:2,000. For corresponding antibodies, see (Table 1).
- To directly access the cells within the biochip, the bonding foil must be removed. To open the microfluidic chambers, make an exact cut across the outside of the cavity and remove the bonding foil. Cut the membrane out by removing the edges sealed to the biochip with the scalpel.
NOTE: Work precisely and accurately during this step to avoid any membrane damage and injuries due to the sharpness of the scalpel. - After detaching the membrane from the biochip, divide the membrane into two pieces. Collect the membrane pieces with a tweezer and place them on a microscopic slide for staining.
- Pipette 50 µL of antibody solution per membrane piece. Incubate overnight at 4 ˚C, protected from light.
- After incubation, place the membrane in 500 µL of PBS (-/-) in a 24 well-plate and wash the membrane 3x for 5 min each in PBS (+/+). After washing, pipette 50 µL of secondary antibody solution and incubate for 1 h at RT protected from light.
- Wash the membrane with PBS (+/+) 3x for 5 min each. Meanwhile, prepare and label the microscopic slides for the experiment. Place a drop of mounting medium on the slide and place the corresponding membrane. Repeat this step for all membranes. At the end, place one more drop of the mounting medium on the top of the membrane and use a cover glass. Store at 4 ˚C.
- Wash the cavities with PBS (+/+) to remove the cell culture medium. Pipette 300 µL of 4% paraformaldehyde (PFA) dissolved in PBS (-/-) to the upper chamber and 200 µL in the lower chamber and incubate for 10 min at RT.
Representative Results
An examination of morphological alterations and the expression of marker proteins could be performed using immunofluorescence staining. After co-culturing for 14 days, the vascular and epithelial sides are analyzed for expression of respective cell markers. This method is useful for studying the interactions and integrity of vascular and epithelial components, which is essential for disease modeling as a functional biological readout related to infection. Immunofluorescence staining could be supported by quantifying analysis, morphological assessment, and functional assays, including cytokine release, effluent analysis of cell death markers such as LDH release, and change in morphometrics of cells5. On the vascular side, the integrity and morphology of the endothelial cell junction proteins, such as VE-cadherin, can be studied to assess confluency and endothelial barrier formation6 (Figure 4A). After the perfusion, the endothelial cell layer should be confluent with a clearly structured expression of VE-cadherin at the endothelial cell borders. The bottom part of the membrane can be immunostained for epithelial markers such as E-cadherin and Surfactant Protein A (SP-A)7. The expression of SP-A, a key component of pulmonary surfactant and a complex mixture of lipids and proteins, reduces surface tension at the epithelial air-liquid interface and thereby prevents in vivo the collapse of alveoli during exhalation and maintaining proper lung function and gas exchange6,8. In our model, the epithelial cells express SP-A after the ALI treatment and can be visualized by immunofluorescence staining (Figure 4B). The confluency and integrity of the epithelial cell layer can be confirmed by immunofluorescence staining for E-cadherin, an important epithelial junctional marker of alveolar epithelial cells (Figure 4C). The presence of macrophages can be confirmed by immunofluorescence staining for the macrophage maker protein CD686 (Figure 4C). To study viral, bacterial, or fungal infection, please find details on infection models provided in Deinhardt-Emmer et al.6, Schicke et al.7, and Hoang et al.9.
Figure 1: Representative image of a confluent HUVECs layer in the biochip on day 3. The image shows brightfield microscopy with 10x magnification. Please click here to view a larger version of this figure.
Figure 2: Set up for the alveolus-on-chip and ALI induction. (A) Representative image of the perfusion set-up for the alveolus-on-chip model and (B) a representative image of inducing an ALI in the chamber by soaking up the medium and introducing air to the cells. The formation of an air bubble inside the chamber indicates that the cells are exposed to air. Please click here to view a larger version of this figure.
Figure 3: Schematic overview of the alveolus-on-chip. A detailed protocol for 14 days of culture indicates important steps for seeding and growing the cells within the biochip. Please click here to view a larger version of this figure.
Figure 4: Immunofluorescence staining of endothelial and epithelial cell layers. (A) HUVEC endothelial cells expressing VE-cadherin (orange), (B-C) H441 epithelial cells expressing (B) SP-A (red) and (C) E-cadherin (orange), SP-A (red), and CD68 (green) expression on the epithelial side. DAPI (blue) stains DNA and indicates the cell nuclei. The scale bar represents 50 µm. Please click here to view a larger version of this figure.
| Cavity | Cells |
| Upper Cavity | Endothelial (0.4 x 106) |
| Lower Cavity | Epithelial (0.5×106H441+ 1×105 monocytes) |
Table 1: Overview of the cell seeding in the biochip.
Discussion
The alveolus-on-chip model represents a multilayered tissue model of the human alveolus, integrating essential cell types of the lower respiratory tract, including lung epithelial cells, endothelial cells, and macrophages, cultured in an organotypic arrangement at an ALI with medium perfusion of the endothelial lining. Cells of different layers express specific cell marker proteins such as E-cadherin, a calcium-dependent adhesion molecule of lung epithelial cells, which is central in establishing intercellular epithelial barriers5,10. E-cadherin forms a physical barrier element and controls the immunological response to harmful pathogens and substances in the environment10,11. H441 epithelial cells further express surfactant protein A, which is also crucial in the first line of host defense against infection by mediating immunomodulatory and antibacterial activity8. Integrating immune cells in the alveolus model increases its cellular complexity, thereby better replicating the immune response to inflammatory triggers, i.e., during infection. In this context, the model was used to study the co-infection of influenza virus A and human opportunistic pathogen Staphylococcus aureus (S. aureus)5. In the study, a mechanism of co-infection in pneumonia was revealed in which viral infection of epithelial cells triggers a significant inflammatory response, which causes considerable damage to endothelial cells, leading to a loss of barrier function. In another study, the model was used to demonstrate that S. aureus infection reduces the extracellular levels of surfactant protein-A (SP-A), a crucial immune defense component in the lungs, likely due to bacterial proteases6. While the expression of SP-A is not directly affected by either S. aureus or influenza virus A, it is significantly down-regulated by TNF-α, a cytokine highly produced in response to bacterial infections, especially in the presence of macrophages. These results suggest that bacterial mono- and super-infections can lower SP-A levels in the lung, potentially worsening the outcome of bacterial pneumonia6. Further, the alveolus model has been used to study fungal infection of the alveolus with Aspergillus fumigatus and the efficacy of antifungal drugs by leveraging high-resolution microscopy and algorithm-based image analysis in combination with molecular biological assays, including immunofluorescence staining and cytokine profiling7. Modeling of invasive pulmonary aspergillosis in the alveolus-on-chip revealed key functions of human macrophages that could partially inhibit the growth of the fungus. Macrophages were central in releasing proinflammatory cytokines and chemokines, which correlated with an increased number of invasive fungal hyphae. Additionally, the study confirmed that the fungistatic drug caspofungin effectively limits fungal growth and induces morphological changes in the fungal structure, aligning with findings from other studies. These studies confirm the potential of the model platform for identifying cellular targets of infection and testing drugs at clinically relevant concentrations.
There are several aspects that need to be considered during the establishment of the alveolus-on-chip. Precise cell seeding is critical for achieving uniform cell distribution and layer formation. Make sure cells are seeded at the appropriate density to achieve confluence without overgrowing each other, which may compromise the cell layers’ integrity and functionality. During the ALI establishment process, carefully remove the medium from the apical side and close the ports to prevent contamination. Maintaining optimal co-culture conditions, such as temperature, CO2 levels, and medium composition, is critical for the survival and interaction of both endothelial and epithelial cells. Always perform medium changes carefully to avoid disturbing the cell layers or introducing air bubbles. Pipette gently and ensure that all used equipment is sterile to minimize the risk of contamination. To prevent air bubbles, pipette slowly and tilt the chip during medium changes to allow bubbles to escape. Work in a clean, sterile, and controlled environment to minimize contamination. Consider the complexity and precision required to establish and maintain the 3D model for subsequent experiments with the model. If cell layers are not forming correctly, consider optimizing cell density and cell batches, as differences in cell growth can occur depending on the donor and batch.
While the model offers significant improvements over existing methods, it is also essential to acknowledge that some limitations must be considered. One such limitation is the complexity and cost associated with the operation of the microfluidic device. Running organ-on-chip models requires specialized equipment and expertise in microfluidics to adopt the alveolus-on-chip model. Additionally, while this model can successfully replicate many of the key features of the alveolar environment, it cannot fully replicate all of the cellular interactions and mechanical forces found in vivo. For instance, the model does not have a comprehensive immune system or the full range of cell types in the human lung, which could impact the accuracy of pathogen-host interaction studies.
Despite these limitations, the lung-on-chip model offers advancements compared to traditional 2D cell cultures such as Transwell systems. By incorporating an air-liquid interface, endothelial and epithelial cells, and immune components in a multilayered tissue within a dynamic, perfused environment, the model represents a more physiologically relevant approach for studying lung physiology, disease, and treatment responses in vitro. This is crucial for advancing our understanding of human respiratory infections and developing more effective therapies. The model’s ability to precisely manipulate biophysical and biochemical cues represents an important feature for dissecting the mechanisms of disease progression and drug action. Using human cell material can further reduce interspecies-related differences seen in animal experiments. The integration of multiple biomarker analyses additionally further enhances the depth and breadth of data obtainable from a single experiment, which contributes to reducing the costs of in vitro experimentation. While acknowledging its limitations, the lung-on-chip model represents a significant step forward in respiratory disease research, offering a more accurate, ethical, and efficient alternative to traditional methods.
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
H.K. and A.S.M. acknowledge funding from the Leibniz Science-Campus InfectoOptics Jena, financed by the funding line Strategic Networking of the Leibniz Association. M.A. and A.S.M. were supported by the IGF project IMPROVE funded by the Federal Ministry for Economic Affairs and Energy on the basis of a resolution of the German Bundestag. A.S.M further acknowledges financial support by the Cluster of Excellence Balance of the Microverse under Germany's Excellence Strategy – EXC 2051 – Project-ID 690 390713860.
Materials
| Consumables | ||
| Cellcounting chamber slides (Countess) | Invitrogen | C10283 |
| Cell culture Multiwell Plates, 24 Well, steril | Greiner Bio-One | 662 160 |
| Cell culture Multiwell Plates, 6 Well, steril | Greiner Bio-One | 657 160 |
| Coverslips (24x40mm; #1.5) | Menzel-Gläser | 15747592 |
| Eco wipes | Dr. Schuhmacher | 00-915-REW10003-01 |
| Eppies 2.0 | Sarstedt | 72.691 |
| Eppis 0.5 | Sarstedt | 72.699 |
| Eppis 1.5 | Sarstedt | 72.690.001 |
| Falcons 15mL | Greiner Bio-One | 188 271-TRI |
| Falcons 50mL | Greiner Bio-One | 227 261-TRI |
| Gauze swab | Noba | PZN 2417767 |
| Gloves Nitril 3000 | Meditrade | 1280 |
| Microscope slides | Menzel-Gläser | AAAA000001##12E |
| Multiwell Plates 24 Well, sterile | Greiner Bio-One | 662 160 |
| Pasteur pipettes (glass) 150mm | Assistent | 40567001 |
| Pasteur pipettes (glass) 230mm | Assistent | 40567002 |
| Round-bottom tubes (PS, 5mL) | Falcon | 352052 |
| Safety-Multifly-Set, 20G, 200mm | Sarstedt | 85.1637.235 |
| Scalpels | Dahlhausen | 11.000.00.715 |
| Serological pipettes 10mL | Greiner Bio-One | 607 160-TRI |
| Serological pipettes 25mL | Greiner Bio-One | 760 160-TRI |
| Serological pipettes 2mL | Greiner Bio-One | 710 160-TRI |
| Serological pipettes 50mL | Greiner Bio-One | 768 160-TRI |
| Serological pipettes 5mL | Greiner Bio-One | 606 160-TRI |
| S-Monovette, 7,5ml Z-Gel | Sarstedt | 1.1602 |
| S-Monovette, 9,0ml K3E | Sarstedt | 02.1066.001 |
| Softasept N | Braun | 3887138 |
| T25 flask | Greiner Bio-One | 690 960 |
| Tips sterile 10µL | Greiner Bio-One | 771 261 |
| Tips sterile 1250µL | Greiner Bio-One | 750 261 |
| Tips sterile 300µL | Greiner Bio-One | 738 261 |
| Tips unsterile 10µL | Greiner Bio-One | 765 290 |
| Tips unsterile 1000µL | Greiner Bio-One | 739 291 |
| Tips unsterile 200µL | Greiner Bio-One | 686 290 |
| Tweezers (Präzisionspinzette DUMONT abgewinkelt Inox08, 5/45, 0,06 mm) | Roth | K343.1 |
| Chemicals | ||
| Descosept AF | Dr. Schuhmacher | N-20338 |
| Ethanol 96% | Nordbrand-Nordhausen | 410 |
| Fluorescein isothiocyanate (FITC)-dextran (3-5kDa) | Sigma Aldrich | FD4-100MG |
| Fluorescent Mounting Medium | Dako | S3023 |
| Methanol | VWR | 20847.295 |
| Saponin | Fluka | 47036 |
| Tergazyme | Alconox | 1304-1 |
| Cell culture | ||
| Collagen IV | Sigma-Aldrich | C5533-5MG |
| Dexametason | Sigma-Aldrich | D4902 |
| DPBS (-/-) | Lonza | BE17-516F |
| DPBS (+/+) | Lonza | BE17-513F |
| EDTA solution | Sigma-Aldrich | E788S |
| Endothelial Cell Growth Medium | Promocell | C-22020 |
| Endothelial Cell Growth Medium supplement mix | Promocell | C-39225 |
| Fetal bovine Serum | Sigma-Aldrich | E2129-10g |
| H441 | ATCC | |
| Human recombinant GM-CSF | Peprotech | 300-30 |
| Lidocain | Sigma-Aldrich | L5647-15G |
| Penicillin-Streptomycin (10,000 U/mL) | Gibco | 15140-122 /-163 |
| RPMI | Gibco | 72400047 |
| Trypane blue stain 0.4% | Invitrogen | T10282 |
| Trypsin | Gibco | 15090-046 |
| Primary antibodies | ||
| Cadherin-5 / VE-Cadherin (goat) | BD | 610252 |
| CD68 (rabbit) | CellSignaling | 76437 |
| E-Cadherin (goat) | R&D | AF748 |
| SP-A (mouse) | Abcam | ab51891 |
| Secondary antibodies | ||
| AF488 (donkey anti mouse) | Invitrogen | R37114 |
| AF647 (donkey anti mouse) | invitrogen | A31571 |
| AF647 (donkey anti rabbit) | Invitrogen | A31573 |
| Cy3 (donkey anti goat) | jackson research | 705-165-147 |
| DAPI (4',6-Diamidino-2-Phenylindole, Dilactate) | Invitrogen | D3571 |
| Microfluidic | ||
| Chip | Dynamic 42 | BC002 |
| Male Luer Lock (small) | ChipShop | 09-0503-0270-09 |
| Male mini luer plugs, row of four,PP, green | Microfluidic chipshop | 09-0558-0336-11 |
| Male mini luer plugs, row of four,PP, opaque | Microfluidic chipshop | 09-0556-0336-09 |
| Male mini luer plugs, row of four,PP, red | Microfluidic chipshop | 09-0557-0336-10 |
| Plugs | Cole Parmer | GZ-45555-56 |
| Reservoir 4.5mL | ChipShop | 16-0613-0233-09 |
| Tubing | Dynamic 42 | ST001 |
| Equipment | ||
| Autoclave | Tuttnauer | 5075 ELV |
| Centrifuge | Eppendorf | 5424 |
| CO2 Incubator | Heracell | 150i |
| Countess automated cell counter | Invitrogen | C10227 |
| Flowcytometer | BD | FACS Canto II |
| Freezer (-20 °C) | Liebherr | LCexv 4010 |
| Freezer (-80 °C) | Heraeus | Herafreeze HFU 686 |
| Fridge | Liebherr | LCexv 4010 |
| Heraeus Multifuge | Thermo Scientific | X3R |
| Microscope | Leica | DM IL LED |
| Orbital shaker | Heidolph | Reax2000 |
| Peristaltic pump | REGLO Digital MS-4/12 | ISM597D |
| Pipettes 10µL | Eppendorf Research plus | 3123000020 |
| Pipettes 100µL | Eppendorf Research plus | 3123000047 |
| Pipettes 1000µL | Eppendorf Research plus | 3123000063 |
| Pipettes 2.5µL | Eppendorf Research plus | 3123000012 |
| Pipettes 20µL | Eppendorf Research plus | 3123000039 |
| Pipettes 200µL | Eppendorf Research plus | 3123000055 |
| Scale | Sartorius | 6101 |
| Scale | Sartorius | TE1245 |
| Sterile bench | Kojair | Biowizard SL-130 |
| Waterbath | Julabo | SW-20C |
| Fluorescence Microscope Setup | ||
| Apotome.2 | Zeiss | |
| Illumination device | Zeiss | HXP 120 C |
| Microscope | Zeiss | Axio Observer 5 |
| Optical Sectioning | Zeiss | ApoTome |
| Power Supply Microscope | Zeiss | Eplax Vp232 |
| Software | ||
| ZEN Blue Edition | Zeiss |
Riferimenti
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