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.
