Supported by NIH grant HD052670.

1. Coating of plates with ECM proteins
<ol>
	<li>Under sterile conditions, mix 120 &mu;g of laminin with 12 ml of cold sterile 1X PBS per plate.</li>
	<li>Take Bioflex untreated plates and add 2ml of the solution to each well (final concentration of laminin is 2 &mu;g/cm2). Alternatively, other ECM proteins could be used, such as collagen-1 [10 &mu;g/cm2], fibronectin [5 &mu;g/cm2], vitronectin [0.5 &mu;g/cm2] or elastin [10 &mu;g/cm2]. The coating technique is similar to what it is described for laminin-coating. Make sure the wells are completely covered by the solution.</li>
	<li>Wrap the plates with plastic and put at 4&deg;C on a flat surface overnight to allow the laminin to adsorb to the bottom of the wells. In these conditions, plates can be stored for at least one week while still retaining good ECM function.</li>
	<li>Next day, under sterile conditions, wash the wells 3 times with 1X PBS.</li>
	<li>Add 1ml of 1% BSA in 1X PBS to each well and incubate at 37&deg;C for 1h to block non-specific binding sites on the membranes.</li>
	<li>Rinse the plates 3 times with 1X PBS to remove unadsorbed proteins.</li>
	<li>Keep the plates at 37&deg;C in DMEM until cells are isolated from step 2.14.</li>
</ol>
2. Isolation of fetal rodent lung type II epithelial cells
The day before isolation have the screen cups with different size nylon meshes autoclaved and ready.
<ol>
	<li>Obtain fetal lungs from timed-pregnant mouse/rat on E17-19 of gestation. Transfer the tissue into a 50 ml conical tube containing DMEM medium and place it on ice.</li>
	<li>Make digestion buffer in a 50 ml conical tube: (10 ml). For this volume, lung tissue from approximately 20 fetuses from mouse/rat is used. 
	8.5 ml DMEM 
	0.5 ml 500mM pH 7.4 HEPES 
	5 mg Collagenase 1 
	5 mg Collagenase 1A 
	1 ml Chicken serum, heat inactivated</li>
	<li>Mix well until all components are dissolved and filter through a 0.2 micron syringe filter inside the sterile hood. Place the conical tube containing the digestion buffer in a water bath at 37&deg;C.</li>
	<li>Remove the DMEM medium from the conical tube from step 2.1 and transfer the tissue into a sterile Petri dish.</li>
	<li>Mince the lungs well with sterile scissors or razor blade to make tissue pieces less than 1 mm and transfer the tissue into the conical tube containing the pre-warmed digestion buffer from step 2.3.</li>
	<li>Digest the tissue by pipetting up and down: 
	100 times with 10 ml pipette 
	100 times with 5 ml pipette 
	100 times with Pasteur pipette</li>
	<li>Centrifuge the homogenate at 1,300 rpm for 5 minutes at room temperature.</li>
	<li>Carefully remove the supernatant by aspiration and re-suspend the pellet containing the cells in 15 ml of DMEM plus 20% FBS.</li>
	<li>Take out the screen cups with 100-, 30- and 15-micron nylon meshes and place them on sterile 150 ml beakers.</li>
	<li>Add cell homogenate from step 2.8 and sequentially filter through 100-, 30-, and 15-micron mesh screen cups.</li>
	<li>Collect clumped non-filtered cells from the 30- and 15-micron meshes after several washes with DMEM plus 10% FBS to facilitate the filtration of non-epithelial cells.</li>
	<li>Discard the filtrate from the 15-micron mesh which contains mostly fibroblasts.</li>
	<li>Purify further type II cells by incubating cells from step 2.11 in 75-cm2 flasks for 30 min (approximatelly10 ml of cell suspension per flask).</li>
	<li>Collect the supernatant and centrifuge at 1,300 rpm for 5 minutes at room temperature to pellet the cells.</li>
	<li>Carefully remove the supernatant by aspiration and re-suspend the pellet containing the cells in serum-free DMEM. The volume of resuspension depends on the amount of tissue processed. Approximately, we plate the equivalent of cells obtained from one fetus in each well (2 ml) in order to achieve between 60-70% confluency the following day.</li>
	<li>Plate the cell suspension on Bioflex six-well plates precoated with laminin or fibronectin.</li>
</ol>
3. Isolation of fetal rodent lung fibroblasts
<ol>
	<li>Follow the same steps described above for type II cells isolation up to 2.11.</li>
	<li>Take the filtrate from the 15-micron mesh (without previous washing; approximate volume is 10-15 ml) and plate onto 75-cm2 at 37&deg;C for 30-60 min to allow fibroblasts to adhere.</li>
	<li>Aspirate the supernantant and replace with serum-free DMEM and incubate overnight.</li>
	<li>Next day, harvest the cells with 0.25% (wt/vol) trypsin in 0.4 mM EDTA and plate them on Bioflex plates precoated with fibronectin.</li>
</ol>
4. Experimental system to provide mechanical stimulation to lung cells
<ol>
	<li>Seed the cells (around 50% confluent) on Bioflex culture plates in serum-free DMEM (2 ml/well) and let them attach and spread for at least 6h before experiments. In general our laboratory maintains cells in culture for around 24h before applying mechanical strain. If a specific number of cells are required for the experiments, after isolation, type II cells are maintained in serum-free DMEM 75-cm2 flasks and the next day, cells are trypsinized, counted and then seeded.</li>
	<li>Next day, media are replaced with fresh serum-free DMEM (2 ml/well) and bioflex plates are mounted in a Flexcell FX-5000 Strain Unit. Monolayers should be no greater than 80% confluent before the initiation of the experiments.</li>
	<li>Equibiaxial strain is applied to the membranes. The regimen of strain varies depending on simulation of mechanical forces in vivo (see discussion).</li>
	<li>Cells grown on non-stretched membranes cultured in parallel are used as controls.</li>
	<li>At the end of the experiments, monolayers can be processed to analyze changes on gene expression (for example surfactant protein C) by real time-PCR, protein abundance by Western blot, etc. Also, monolayers can be fixed for immunocytochemistry experiments. For this technique, after fixation, silastic membranes are cut out from the plates and mounted on glass slides using 10-20 &mu;l of water as a mounting agent before permeabilization and incubation with antibodies. Supernatant can also be used to investigate the presence of growth factors, cytokines, etc.</li>
</ol>
5. Representative Results
Figure 1 and Figure 2 show representative phase-contrast photographs of E18 fetal mouse type II cells isolated using the technique described in this manuscript.
Figure 3 demonstrates that mechanical strain induces differentiation of fetal type II epithelial cells using surfactant protein-C as a marker.
<img src="/files/ftp_upload/3543/3543fig1.jpg" /> 
Figure 1. Representative phase-contrast photograph taken right after isolation showing the clumped appearance of fetal type II cells.
<img src="/files/ftp_upload/3543/3543fig2.jpg" /> 
Figure 2. E18 fetal type II epithelial cells were isolated as described here and plated on bioflex plates coated with laminin. Photograph was taken the following day after non-stretched cells were fixed in paraformaldehyde. The purity of the cells was determined to be 90 &plusmn; 5% by microscopic analysis of epithelial cell morphology and immunostaining for SP-C.
<img src="/files/ftp_upload/3543/3543fig3.jpg" /> 
Figure 3. Fetal type II epithelial cells were exposed to 5% cyclic strain at 40 cycles/min for 16h. A) Northern blot of surfactant protein C (SP-C) mRNA expression showing that strain induces type II cell differentiation using different ECM substrates. +/- signs represent exposure or not to strain, respectively. Data are presented as mean +/- SEM, n=3; * P&lt;0.05. B) Fluorescence immunocytochemistry images demonstrating SP-C protein levels (green) in fetal type II cells exposed or not (control) to mechanical strain . Nuclei were counterstained with dapi (blue). Bar, 10 &mu;m. C) Western blot results from three experiments showing that mechanical stretch increases SP-C protein. N=3; * P&lt;0.05.

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No conflicts of interest declared.

In this manuscript, we describe a method to isolate fetal type II epithelial cells and fibroblasts and to expose them to mechanical stimulation using the Flexcell Strain Apparatus. We have used this technique to assess differentiation of epithelial cells 1,2 and to study receptor and signaling pathways activated by stretch 3-9. In addition, this method can also be used to investigate cell responses induced by mechanical injury 10,11. The procedure is based on digestion of the lung tissue ...

<table><tbody><tr><td>DMEM</td><td>Sigma-Aldrich</td><td>D5648</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Collagenase 1</td><td>Sigma-Aldrich</td><td>C0130</td><td></td></tr><tr><td>Collagenase 1A</td><td>Sigma-Aldrich</td><td>C9891</td><td></td></tr><tr><td>Chicken serum</td><td>Sigma-Aldrich</td><td>C5405</td><td></td></tr><tr><td>Screen cups</td><td>Sigma-Aldrich</td><td>CD1-1KT</td><td></td></tr><tr><td>Syringe filters</td><td>Fisher Scientific</td><td>09-754-25</td><td></td></tr><tr><td>100 micron nylon mesh</td><td>Small Parts, Inc.</td><td>CMN-100-D</td><td></td></tr><tr><td>30 micron mesh</td><td>Small Parts, Inc.</td><td>CMN-30-D</td><td></td></tr><tr><td>15 micron mesh</td><td>Dynamic Aqua-Supply Ltd.</td><td>NTX15</td><td></td></tr><tr><td>Laminin</td><td>Sigma-Aldrich</td><td>L2020</td><td></td></tr><tr><td>Collagen-1</td><td>Collagen Biomed</td><td>PC0701</td><td></td></tr><tr><td>Fibronectin</td><td>Sigma-Aldrich</td><td>F1141</td><td></td></tr><tr><td>Vitronectin</td><td>Sigma-Aldrich</td><td>V-0132</td><td></td></tr><tr><td>Elastin</td><td>Sigma-Aldrich</td><td>E-6402</td><td></td></tr><tr><td>Bioflex plate</td><td>Flexcell International</td><td>BF-3001U</td><td>Uncoated</td></tr><tr><td>Flexcell Strain Unit</td><td>Flexcell International</td><td>FX-5000</td><td></td></tr></tbody></table>

an experimental system to study mechanotransduction in fetal lung cells

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key component of lung development is the differentiation of alveolar type II epithelial cells, the major source of pulmonary surfactant. These cells also participate in fluid homeostasis in the alveolar lumen, host defense, and injury repair. In addition, distal lung parenchyma cells can be directly exposed to exaggerated stretch during mechanical ventilation after birth. However, the precise molecular and cellular mechanisms by which lung cells sense mechanical stimuli to influence lung development and to promote lung injury are not completely understood. Here, we provide a simple and high purity method to isolate type II cells and fibroblasts from rodent fetal lungs. Then, we describe an in vitro system, The Flexcell Strain Unit, to provide mechanical stimulation to fetal cells, simulating mechanical forces in fetal lung development or lung injury. This experimental system provides an excellent tool to investigate molecular and cellular mechanisms in fetal lung cells exposed to stretch. Using this approach, our laboratory has identified several receptors and signaling proteins that participate in mechanotransduction in fetal lung development and lung injury.

Watch this Scientific Journal Video about An Experimental System to Study Mechanotransduction in Fetal Lung Cells at JoVE.com

An Experimental System to Study Mechanotransduction in Fetal Lung Cells

Mechanical forces play a key role in lung development and lung injury. Here, we describe a method to isolate rodent fetal lung type II epithelial cells and fibroblasts and to expose them to mechanical stimulation using an in vitro system.

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key ...

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12.9K Views.  Alpert Medical School of Brown University. Mechanical forces play a key role in lung development and lung injury. Here, we describe a method to isolate rodent fetal lung type II epithelial cells and fibroblasts and to expose them to mechanical stimulation using an in vitro system.

Video: An Experimental System to Study Mechanotransduction in Fetal Lung Cells

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop within the uterus. A newly developed ex vivo upright droplet culture method provides an attractive alternative to studies performed in utero. The ex vivo droplet culture provides the ability to examine and manipulate cellular interactions and diverse signaling pathways through use of various blocking and activating compounds; additionally, the effects of various pharmacological reagents on the development of specific organs can be studied without unwanted side effects of systemic drug delivery in utero. As compared to other in vitro systems, the droplet culture not only allows for the ability to study three-dimensional morphogenesis and cell-cell interactions, which cannot be reproduced in mammalian cell lines, but also requires significantly less reagents than other ex vivo and in vitro protocols. This paper demonstrates proper mouse fetal organ dissection and upright droplet culture techniques, followed by whole organ immunofluorescence to demonstrate the effectiveness of the method. The ex vivo droplet culture method allows the formation of organ architecture comparable to what is observed in vivo and can be utilized to study otherwise difficult-to-study processes due to embryonic lethality in in vivo models. As a model application system, a small-molecule inhibitor will be utilized to probe the role of vascularization in testicular morphogenesis. This ex vivo droplet culture method is expandable to other fetal organ systems, such as lung and potentially others, although each organ must be extensively studied to determine any organ-specific modifications to the protocol. This organ culture system provides flexibility in experimentation with fetal organs, and results obtained using this technique will help researchers gain insights into fetal development.

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

The&#160;ex vivo&#160;upright droplet culture is an alternative to current&#160;in vitro&#160;and&#160;in vivo&#160;experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop ...

Developmental Biology

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
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In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
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We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

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Medicine

Live Cell Imaging during Mechanical Stretch

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in their capability for measuring responses in real time in live cells or viable tissue. A protocol was developed with the use of a cell actuator to distend live cells grown on or tissues attached to an elastic substrate while imaging with confocal and atomic force microscopy (AFM). Preliminary studies show that tonic stretching of human bronchial epithelial cells caused a significant increase in the production of mitochondrial superoxide. Moreover, using this protocol, alveolar epithelial cells were stretched and imaged, which showed direct damage to the epithelial cells by overdistention simulating one form of lung injury in vitro. A protocol to conduct AFM nano-indentation on stretched cells is also provided.

A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy.

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Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

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Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

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Biology

Generation of 3D Whole Lung Organoids from Induced Pluripotent Stem Cells for Modeling Lung Developmental Biology and Disease

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Differentiation and Mechanical Stimulation of hiPSC-Derived Endothelial Cells

In Vitro Model of Fetal Human Vessel On-chip to Study Developmental Mechanobiology

The heart is the first organ to be functionally established during development, thus initiating blood circulation very early in gestation. Besides transporting oxygen and nutrients to ensure fetal growth, fetal circulation controls many crucial developmental events taking place within the endothelial layer through mechanical cues. Biomechanical signals induce blood vessel structural changes, establish arteriovenous specification, and control the development of hematopoietic stem cells. The inaccessibility of the developing tissues limits the understanding of the role of circulation in early human development; therefore, in vitro models are pivotal tools for the study of vessel mechanobiology. This paper describes a protocol to differentiate endothelial cells from human induced pluripotent stem cells and their subsequent seeding into a fluidic device to study their response to mechanical cues. This approach allows for long-term culture of endothelial cells under mechanical stimulation followed by&#160;retrieval of the endothelial cells for phenotypical and functional characterization. The in vitro model established here will be instrumental to elucidate&#160;the intracellular molecular mechanisms that transduce the signaling mediated by mechanical cues, which ultimately orchestrate vessel development during human fetal life.

Author Spotlight: Studying hiPSC-Derived Endothelial Cells Cultured Under Fluidic-Mediated Mechanical Stimulation 

Described here is a simple workflow to differentiate endothelial cells from human pluripotent stem cells followed by a detailed protocol for their mechanical stimulation. This allows for the study of the developmental mechanobiology of endothelial cells. This approach is compatible with downstream assays of live cells collected from the culture chip after mechanical stimulation.

The heart is the first organ to be functionally established during development, thus initiating blood circulation very early in gestation. Besides ...

Mechanostimulation of Multicellular Organisms Through a High-Throughput Microfluidic Compression System

During embryogenesis, coordinated cell movement generates mechanical forces that regulate gene expression and activity. To study this process, tools such as aspiration or coverslip compression have been used to mechanically stimulate whole embryos. These approaches limit experimental design as they are imprecise, require manual handling, and can process only a couple of embryos simultaneously. Microfluidic systems have great potential for automating such experimental tasks while increasing throughput and precision. This article describes a microfluidic system developed to precisely compress whole Drosophila melanogaster (fruit fly) embryos. This system features microchannels with pneumatically actuated deformable sidewalls and enables embryo alignment, immobilization, compression, and post-stimulation collection. By parallelizing these microchannels into seven lanes, steady or dynamic compression patterns can be applied to hundreds of Drosophila embryos simultaneously. Fabricating this system on a glass coverslip facilitates the simultaneous mechanical stimulation and imaging of samples with high-resolution microscopes. Moreover, the utilization of biocompatible materials, like PDMS, and the ability to flow fluid through the system make this device capable of long-term experiments with media-dependent samples. This approach also eliminates the requirement for manual mounting which mechanically stresses samples. Furthermore, the ability to quickly collect samples from the microchannels enables post-stimulation analyses, including -omics assays which require large sample numbers unattainable using traditional mechanical stimulation approaches. The geometry of this system is readily scalable to different biological systems, enabling numerous fields to benefit from the functional features described herein including high sample throughput, mechanical stimulation or immobilization, and automated alignment.

The present protocol describes the design, fabrication, and characterization of a microfluidic system capable of aligning, immobilizing, and precisely compressing hundreds of Drosophila melanogaster embryos with minimal user intervention. This system enables high-resolution imaging and recovery of samples for post-stimulation analysis and can be scaled to accommodate other multicellular biological systems.

During embryogenesis, coordinated cell movement generates mechanical forces that regulate gene expression and activity. To study this process, tools such ...

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity greatly impacts cell behavior to regulate normal and pathologic processes including cell proliferation, differentiation, adhesion signaling and directional migration. In this regard, the innate ability of certain cell types to migrate towards a stiffer, or less compliant matrix substrate is referred to as durotaxis. This phenomenon plays an important role during embryonic development, wound repair and cancer cell invasion. Here, we describe a straightforward assay to study durotaxis, in vitro, using polydimethylsiloxane (PDMS) substrates. Preparation of the described durotaxis chambers creates a rigidity interface between the relatively soft PDMS gel and a rigid glass coverslip. In the example provided, we have used these durotaxis chambers to demonstrate a role for the cdc42/Rac1 GTPase activating protein, cdGAP, in mechanosensing and durotaxis regulation in human U2OS osteosarcoma cells. This assay is readily adaptable to other cell types and/or knockdown of other proteins of interests to explore their respective roles in mechanosignaling and durotaxis.

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity ...

Real-Time Monitoring of Cellular Dynamics in Mouse Lung Transplantation

Intravital Two-Photon Microscopy of the Transplanted Mouse Lung

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by crosstalk between donor and recipient cells. A better understanding of these processes relies on the use of preclinical animal models and is aided by an ability to study intra-graft immune cell trafficking in real-time. Intravital two-photon microscopy can be used to image tissues and organs for depths up to several hundred microns with minimal photodamage, which affords a great advantage over single-photon confocal microscopy. Selective use of transgenic mice with promoter-specific fluorescent protein expression and/or adoptive transfer of fluorescent dye-labeled cells during intravital two-photon microscopy allows for the dynamic study of single cells within their physiologic environment. Our group has developed a technique to stabilize mouse lungs, which has enabled us to image cellular dynamics in na&#239;ve lungs and orthotopically transplanted pulmonary grafts. This technique allows for detailed assessment of cellular behavior within the vasculature and in the interstitium, as well as for examination of interactions between various cell populations. This procedure can be readily learned and adapted to study immune mechanisms that regulate inflammatory and tolerogenic responses after lung transplantation. It can also be expanded to the study of other pathogenic pulmonary conditions.

Author Spotlight: Advancing Lung Transplant Immunology Through Intravital Imaging

Here, we present a protocol to intravitally image the transplanted mouse left lung using two-photon microscopy. This represents a valuable tool for studying cellular dynamics and interactions in real-time following murine lung transplantation.

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by ...

An Experimental System to Study Mechanotransduction in Fetal Lung Cells

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Mechanostimulation of Multicellular Organisms Through a High-Throughput Microfluidic Compression System

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Intravital Two-Photon Microscopy of the Transplanted Mouse Lung