This research has been supported in part by the American Thoracic Society Foundation Research Program, the New Jersey Health Foundation, and the National Science Foundation (CAREER Award 2143620) to J.K.; and the National Institutes of Health (P41 EB027062) to G.V.N.

The animal tissue protocol below has been approved by the animal welfare guideline and regulations of the Institute for Animal Care and Use Committee (IACUC) at Stevens Institute of Technology, and it complies with the National Institutes of Health (NIH) guidelines for the use of experimental animals.
1. Design and construction of imaging-guided rat trachea bioreactor
<ol>
	<li>Designing and fabrication of rat trachea bioreactor
	<ol>
		<li>Create a computer-aided design (CA...

<ol>
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In this work, we created an imaging-guided bioreactor that can allow (i) monitoring of the trachea lumen in situ after the cell removal and exogenous cell delivery and (ii) long-term in vitro culture of the cell-seeded trachea tissue. Using this custom-built bioreactor, we demonstrated (i) selective removal of the endogenous epithelial cells from the trachea lumen using detergent and vibration-assisted airway wash and (ii) uniform distribution of exogenous cells onto the luminal surface of the denuded t...

The luminal surface of the respiratory tract is lined by a layer of epithelium that mainly consists of multi-ciliated, club, goblet, and basal stem cells1,2. The epithelial layer serves as a primary defense mechanism of the lung, acting as a biophysical barrier that protects the underlying airway tissue against inhaled pathogens, particulates, or chemical gases. It protects the airway tissue via multiple mechanisms, including intercellular tight junction formation, mucociliary clearance, and antimicrobial and antioxidant secretion3,4. The defective airway epithelium is associated with devastating respiratory diseases, such as chronic obstructive pulmonary disease (COPD)5, primary ciliary dyskinesia (PCD)6, and cystic fibrosis (CF)7.
Advances in lung-on-chip (LOC) technology represent an opportunity to study human lung development, model various lung diseases, and develop new therapeutic materials in tightly regulated in vitro environments. For example, airway epithelium and endothelium can be cultured on opposite sides of a thin, porous membrane to mimic the gas exchanging lung tissue, allowing faithful disease modeling and drug testing8. Similarly, in vitro disease models have been created to model airway diseases in vitro, such as COPD9 and cystic fibrosis10. However, a major challenge of LOC devices is recapitulating the complex three-dimensional (3D) architecture of the lung tissue and dynamic cell-tissue matrix interactions in vitro11.
Recently, innovative tissue engineering methodologies have been developed that allow manipulation of ex vivo lung tissues12. Using these methodologies, denuded allogenic or xenogeneic tissue grafts can be prepared by removing the endogenous cells from the lung tissue via chemical, physical, and mechanical treatments13. In addition, the preserved native tissue extracellular matrix (ECM) in the decellularized lung scaffolds provide the physio-mimetic structural, biochemical, and biomechanical cues for implanted cells to attach, proliferate, and differentiate14,15.
Here, an imaging-guided bioreactor system created by combining LOC and tissue engineering technologies to allow in vitro tissue manipulation and culture of explanted rat tracheal tissues is reported. Using this airway tissue bioreactor, the protocol demonstrates selective removal of the endogenous epithelial cells without disrupting the underlying subepithelial cellular and biochemical components of the airway tissue. We next show the homogenous distribution and instantaneous deposition of the newly seeded exogenous cells, such as mesenchymal stem cells (MSCs), on the denuded airway lumen by instilling the cell-loaded collagen I pre-gel solution. In addition, by using the micro-optical imaging device integrated into the bioreactor, the visualization of the trachea lumen during epithelium removal and endogenous cell delivery is also done. Further, it is shown that the trachea and newly implanted cells can be cultured in the bioreactor without noticeable cell death and tissue degradation for 4 days. We envision that the imaging-enabled bioreactor platform, the thin film-based de-epithelialization technique, and the cell delivery method used in this study can be useful for generating airway tissues for in vitro disease modeling and drug screening.
The bioreactor includes a rectangular chamber connected to a programmable syringe pump, perfusion pump, and ventilator for culturing isolated rat trachea. The bioreactor features inlets and outlets connected to the trachea or the tissue culture chamber to separately supply reagents (e.g., culture media) to the internal and external spaces of the trachea (Figure 1). A custom-built imaging system can be used to visualize the interior of the in vitro-cultured rat trachea at the cellular level (Figure 2). The endogenous epithelium of the trachea is removed via the instillation of a detergent-based decellularization solution followed by vibration-assisted airway washing (Figure 3). Hydrogel solution, such as type I collagen, is used as a delivery vehicle for seeding exogenous cells uniformly and instantaneously across the denuded trachea lumen (Figure 4). All the materials used to construct the bioreactor and conduct the experiments are provided in the Table of Materials.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1&#215; PBS</td>
			<td>Gibco, Thermo Fisher Scientific</td>
			<td>10-010-031</td>
			<td></td>
		</tr>
		<tr>
			<td>3-port connector</td>
			<td>World Precision Instruments</td>
			<td>14048-20</td>
			<td></td>
		</tr>
		<tr>
			<td>4-port connector</td>
			<td>World Precision Instruments</td>
			<td>14047-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Accelerometer</td>
			<td>STMicroelectronics</td>
			<td>IIS3DWBTR</td>
			<td></td>
		</tr>
		<tr>
			<td>Achromatic doublet</td>
			<td>Thorlabs</td>
			<td>AC254-150-A-ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum pin stub</td>
			<td>TED PELLA</td>
			<td>16111</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-antimycotic</td>
			<td>Thermo Fisher Scientific</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Assembly rod</td>
			<td>Thorlabs</td>
			<td>ER1</td>
			<td></td>
		</tr>
		<tr>
			<td>Button head screws</td>
			<td>McMaster-Carr</td>
			<td>91255A274</td>
			<td></td>
		</tr>
		<tr>
			<td>Cage cube</td>
			<td>Thorlabs</td>
			<td>C4W</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon double-sided conductive tape</td>
			<td>TED PELLA</td>
			<td>16073</td>
			<td></td>
		</tr>
		<tr>
			<td>CFSE labelling kit</td>
			<td>Abcam</td>
			<td>ab113853</td>
			<td></td>
		</tr>
		<tr>
			<td>Citrisolv (clearing agent)</td>
			<td>Decon</td>
			<td>1061</td>
			<td></td>
		</tr>
		<tr>
			<td>C-mount adapter</td>
			<td>Thorlabs</td>
			<td>SM1A9</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen I</td>
			<td>Advanced BioMatrix</td>
			<td>5153</td>
			<td></td>
		</tr>
		<tr>
			<td>Conductive liquid silver paint</td>
			<td>TED PELLA</td>
			<td>16034</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichroic mirror</td>
			<td>Semrock</td>
			<td>DI03-R488</td>
			<td>Reflected laser wavelengths:&#160; 473.0 +- 2 nm 488.0 +3/-2 nm</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#8217;s medium</td>
			<td>Gibco, Thermo Fisher Scientific</td>
			<td>11965118</td>
			<td></td>
		</tr>
		<tr>
			<td>Female luer bulkhead to hose barb adapter</td>
			<td>Cole-Parmer</td>
			<td>EW-45501-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Female luer to tubing barb</td>
			<td>Cole-Parmer</td>
			<td>EW-45508-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Female to male luer connector</td>
			<td>Cole-Parmer</td>
			<td>ZY-45508-80</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco, Thermo Fisher Scientific</td>
			<td>10082147</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter lens</td>
			<td>Chroma Technology Corp</td>
			<td>ET535/50m</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Nikon</td>
			<td>Eclipse E1000 - D</td>
			<td></td>
		</tr>
		<tr>
			<td>Fusion 360</td>
			<td>Autodesk</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hex nut</td>
			<td>McMaster-Carr</td>
			<td>91813A160</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexamethyldisilazane (HMDS)</td>
			<td>Fisher Scientifc</td>
			<td>AC120585000</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging fiber</td>
			<td>SELFOC, NSG group</td>
			<td>GRIN lens</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser</td>
			<td>Opto Engine</td>
			<td>MDL-D-488-150mW</td>
			<td></td>
		</tr>
		<tr>
			<td>Lens tubes</td>
			<td>Thorlabs</td>
			<td>SM1L40</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Viability/Cytotoxicity Kit (Invitrogen)</td>
			<td>Thermo Fisher Scientific</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr>
			<td>MACH 3 CNC Control Software</td>
			<td>Newfangled Solutions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Objective lens</td>
			<td>Olympus</td>
			<td>UCPLFLN20X</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic Pump</td>
			<td>Cole Parmer</td>
			<td>L/S standard digital pump system</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human FGF-basic</td>
			<td>PeproTech</td>
			<td>100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>Retaining ring</td>
			<td>Thorlabs</td>
			<td>SM1RR</td>
			<td></td>
		</tr>
		<tr>
			<td>Scientific CMOS camera</td>
			<td>PCO Panda</td>
			<td>PCO Panda 4.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>VWR</td>
			<td>97064-472</td>
			<td></td>
		</tr>
		<tr>
			<td>Solidworks (2019)</td>
			<td>Dassault Syst&#232;mes</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stackable lens tube</td>
			<td>Thorlabs</td>
			<td>SM1L10</td>
			<td></td>
		</tr>
		<tr>
			<td>Subwoofer plate amplifier</td>
			<td>Dayton Audio</td>
			<td>SPA250DSP</td>
			<td></td>
		</tr>
		<tr>
			<td>Subwoofer speaker</td>
			<td>Dayton Audio</td>
			<td>RSS21OHO-4</td>
			<td>Diaphragm diameter: 21 cm</td>
		</tr>
		<tr>
			<td>Syringe Pump</td>
			<td>World Precision Instruments</td>
			<td>AL-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Threaded cage plate</td>
			<td>Thorlabs</td>
			<td>CP33</td>
			<td></td>
		</tr>
		<tr>
			<td>Threaded luer adapter</td>
			<td>Cole-Parmer</td>
			<td>EW-45513-81</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube lens</td>
			<td>Thorlabs</td>
			<td>AC254-150-A-ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Tygon Tubing</td>
			<td>Cole-Parmer</td>
			<td>13-200-110</td>
			<td></td>
		</tr>
		<tr>
			<td>XY Translator</td>
			<td>Thorlabs</td>
			<td>CXY1</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging-guided bioreactor for generating bioengineered airway tissue

Repeated injury to airway tissue can impair lung function and cause chronic lung disease, such as chronic obstructive pulmonary disease. Advances in regenerative medicine and bioreactor technologies offer opportunities to produce lab-grown functional tissue and organ constructs that can be used to screen drugs, model disease, and engineer tissue replacements. Here, a miniaturized bioreactor coupled with an imaging modality that allows in situ visualization of the inner lumen of explanted rat trachea during in vitro tissue manipulation and&#160;culture is described. Using this bioreactor, the protocol demonstrates imaging-guided selective removal of endogenous cellular components while preserving the intrinsic biochemical features and ultrastructure of the airway tissue matrix. Furthermore, the delivery, uniform distribution, and subsequent prolonged culture of exogenous cells on the decellularized airway lumen with optical monitoring in situ are shown. The results highlight that the imaging-guided bioreactor can potentially be used to facilitate the generation of functional in vitro airway tissues.

The GRIN lens-based in situ imaging modality can allow visualization of the tracheal inner lumen in situ (Figure 5A). Using this imaging method, both bright-field and fluorescent images of the native and de-epithelialized tracheas can be obtained (Figure 5B,C). No fluorescent signal was observed from the native trachea prior to CFSE labeling (Figure 5Bii). However, when the tracheal epithelium was ...

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Imaging-Guided Bioreactor for Generating Bioengineered Airway Tissue

The protocol describes an imaging-enabled bioreactor that allows the selective removal of the endogenous epithelium from the rat trachea and homogenous distribution of exogenous cells on the lumen surface, followed by long-term in vitro culture of the cell-tissue construct.

Repeated injury to airway tissue can impair lung function and cause chronic lung disease, such as chronic obstructive pulmonary disease. Advances in ...

This protocol allows in vitro cultivation and direct microscopic visualization of the isolated airway tissues during different tissue manipulation procedures, such as de-epithelization and airway tissue regeneration. The in situ imaging proposed in this study allows rapid and non-destructive monitoring of the tracheal lumen during imaging-guided controlled removal of the endogenous epithelium and delivery of the exogenous cells. The imaging-guided bioreactive platform and the tissue manipulation protocols can be used to generate bioengineered airway tissue for disease modeling and drug screening.The construction and usage of the imaging-enabled airway tissue bioreactor will be demonstrated by two PhD students from our research group, Mohammad Mir, and Jiawen Chen. To begin creating in situ imaging device, insert a tube lens into a stackable lens tube and secure it using a retaining ring. Then, mount the lens table assembly onto a scientific CMOS camera via a C-mount adapter.Use Micromanager software to operate the camera and acquire photos and videos. While aiming for an object located at 10 meters from the camera, adjust the distance between the tube lens and the imaging sensor of the camera until a focused image of the object is formed on the computer screen. Next, use optical cage system components, such as assembly rod, threaded cage plate, and cage cube to mount a filter lens on a dual-edge, super-resolution, dichroic mirror and a laser to the device.Connect a 20X objective lens to the device. Then, mount a green lens with a diameter of 500 micrometers at the distal end of the lens tube via the X-Y translator. Adjust the distance between the green and the objective lenses to form the focused microscopic images.To visualize the isolated rat trachea lumen in bright-field or fluorescent, place the bioreactor on the imaging stage. Next, infuse 500 microliters of the freshly prepared carboxyfluorescein succinimidyl ester, or CFSE solution, at a flow rate of five milliliters per minute through the trachea via the syringe pump connected to the tubing of the trachea cannula. Once the CFSE solution fills the trachea, stop the pump.After 10 minutes, wash the trachea lumen by infusing 10 milliliters of PBS using the syringe pump to remove the residual unincorporated CFSE reagents. After the wash, insert the distal imaging end of the green lens into the trachea via the luer connector attached to one end of the trachea. Then, gently move the green lens inside the trachea until the trachea surface is focused.To capture the bright-field images in the micromanager software, illuminate the trachea lumen with white light through the cage cube. Then, click on the Live icon to show the lumenal surface of the trachea in real time. Use the Imaging setting and Exposure tabs to change the exposure time to the desired value.To adjust the contrast and brightness of images, use the histogram and intensity scaling window to move black and white arrows at the end point of the interactive histogram display. Click Stop Live to activate the Snap icon, then click on the Snap icon to freeze the image. Next, use the setting Export then Images as Displaced tabs to save the images in the desired format.To obtain photos and videos in a fluorescent mode, illuminate the trachea lumen with CFSE-specific laser light through the cage cube and acquire the images in real time by moving the imaging probe back and forth. Once the photos and videos are obtained, gently remove the green lens from the trachea. For the de-epithelialization of the trachea, instill 50 microliters of the freshly prepared 2%sodium dodecyl sulfate through the trachea cannula to generate a thin film of the detergent solution on the trachea lumen.After the instillation, close the bioreactor's tubing connections using male or female luer plugs and transfer the bioreactor to a 37-degree-Celsius incubator for 10 minutes to allow the SDS to dwell within the trachea. Repeat the instillation once. After the second instillation, remove the lysed epithelium and SDS by irrigating the trachea lumen thrice with 500 microliters of PBS via syringe pump at a flow rate of 10 milliliters per minute.Then, place the bioreactor on a shaker to mechanically vibrate at a frequency of 20 Hertz and a displacement amplitude of 0.3 millimeters to physically promote detachment of SDS-treated epithelial cells from the trachea lumen. While the trachea is mechanically vibrated, instill 500 microliters of PBS twice through the trachea lumen to remove the residual SDS and cell debris. Following the epithelium removal, evaluate the clearance of the epithelial layer by measuring the intensity of the CFSE using the green lens imaging device.After preparing a de-epithelialized rat trachea, thaw frozen mesenchymal stem cells, or MSCs, for 30 seconds in a 37-degree-Celsius water bath. Then, count the cells with a hemocytometer, followed by preparing a cell solution with a concentration of five times 10 to the six cells per milliliter. Then, label the cells fluorescently by incubating them with two milliliters of 100-micromolar CFSE solution at room temperature.After 15 minutes, rinse the cells with five milliliters of PBS three times before resuspending them in a fresh DMEM culture medium at a final concentration of three times 10 to the seven cells per milliliter. Immediately after the preparation of collagen I hydrogel, as described in the manuscript, add the cells to the hydrogel solution with the desired concentration. Then, mix the cells and gel solution with a micropipette to obtain a uniform cell-hydrogel mixture.Next, attach one end of the trachea within the bioreactor to a programmable syringe pump through a luer connector and deliver five milliliters of fresh culture medium into the bioreactor chamber at 37 degrees Celsius to cover the exterior surface of the trachea. Then, administer 10 microliters bolus of the cell-hydrogel mixture into the de-epithelialized trachea within the bioreactor to generate a cell-hydrogel layer on the trachea lumen. After cell injection, place the bioreactor in a sterile cell culture incubator at 37 degrees Celsius and 5%carbon dioxide for gellation for 30 minutes to allow gellation.To visualize the distribution of implanted cells, sterilize the green lens with 70%isopropyl alcohol or ethanol and place the bioreactor on the imaging stage to obtain the photos and videos in both bright-field and fluorescent modes. After 30 minutes of cell seeding, infuse one milliliter of culture medium into the trachea lumen at a flow rate of one milliliter per minute. Finally, culture the cell-seeded trachea within the bioreactor in an incubator at 37 degrees Celsius for the desired time.During the cell culture, keep the media inside the lumen static, while media outside the trachea is continuously perfused via a unidirectional flow. In the bright-field and fluorescent images of the de-epithelialized trachea, no fluorescent signal was observed prior to the CFSE labeling. With the CFSE, a uniform fluorescent signal was observed throughout the epithelium.Following de-epithelialization, the fluorescent intensity was decreased significantly, indicating ablation of the epithelium. The hematoxylin and eosin staining of the de-epithelialized trachea illustrated the removal of the pseudo-stratified epithelium from the trachea lumen, and the preservation of cells and extracellular matrix, or ECM micro-structures, in underlying tissue layers. Moreover, the pentachrome and trichrome staining confirmed the maintenance of trachea tissue architecture and ECM components, such as collagen and proteoglycans.The immunofluorescence of epithelial cells and collagen I revealed complete removal of the epithelium and preservation of collagen I within the sub-epithelial tissue. The scanning electron micrographs indicated that the native trachea was populated with multicilliated and goblet cells. In the de-epithelialized trachea, the basement membrane was exposed, as indicated by a mesh network of extracellular matrix fibers and the absence of epithelial cells.The fluorescence images of the de-epithelialized trachea, upon seeding with PBS and collagen, are shown here. Compared to the cells seeded via culture medium, the fluorescently-labeled cells delivered via hydrogel remained adhered more uniformly across the lumen. The cell viability study demonstrated that viability was not affected by the cell delivery procedure, and over 90%of cells remained viable.Creating a thin film of detergent in the de-epithelization process and using hydrogel as a delivery vehicle for cells are essential steps for the success of this protocol. Our next research goal is to implant lab-grown airway primary or stem cell onto the de-epithelialized airway tissue to investigate whether functional airway tissue can be prepared.

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JoVE Journal

Bioengineering

2.5K visualizações.  Stevens Institute of Technology. Este protocolo permite o cultivo in vitro e a visualização microscópica direta dos tecidos isolados das vias aéreas durante diferentes procedimentos de manipulação tecidual, como deselitelite e regeneração tecidual das vias aéreas. A imagem in situ proposta neste estudo permite o monitoramento rápido e não destrutivo do lúmen traqueal durante a remoção controlada guiada por imagens do epitélio endógeno e a entrega das células exógenas. A plataforma bioreativa guiada por imag...