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 (CAD) model of the bioreactor chamber with relevant design, such as inlets, outlets, and tissue culture chamber, using CAD generator software. For this study, use the geometry and dimensions presented in Figure 1A-C. The tutorial of CAD generator software can be found in16,17.</li>
		<li>Export the generated CAD model to a computer numerical control (CNC) controller software and cut the polytetrafluoroethylene (PTFE) plastic using a CNC machine to create the bioreactor chamber. The tutorial of the CNC controller software can be found in18. 
		NOTE: In addition to PTFE, other plastic materials, such as ultra-high molecular weight polyethylene (UHMWPE) and polyetherimide can be used to fabricate the culture chamber.</li>
		<li>Sterilize all bioreactor components, such as Luer adapters and screws, to avoid contamination of the tissues and cells cultured in the device. Assemble all bioreactor components to the main tissue culture chamber in a clean environment (Figure 1A).</li>
	</ol>
	</li>
	<li>Construction of gradient index (GRIN) lens-based imaging device
	<ol>
		<li>To create the in situ imaging device, insert a tube lens into a stackable lens tube and secure it using a retaining ring. Mount the lens-tube assembly onto a scientific CMOS camera via a C-mount adapter.</li>
		<li>Use software, such as Micro-Manager, to operate the camera and acquire photos and videos. Aim an object located at a long distance (e.g., 10 m from the camera) and adjust the distance between the tube lens and imaging sensor of the camera until a focused image of the object is formed on the computer screen by the imaging software being used.</li>
		<li>Mount a filter lens on a dual-edge super-resolution dichroic mirror and a laser to the device using optical cage system components, including assembly rod, threaded cage plate, and cage cube.</li>
		<li>Connect the objective lens (20x) to the device. Mount a GRIN lens (diameter = 500 &#181;m) at the distal end of the lens tube via the XY translator. Adjust the distance between the GRIN lens and the objective lens to form focused microscopic images (Figure 2A,B).</li>
	</ol>
	</li>
</ol>
2. Isolation of the rat trachea
<ol>
	<li>Sanitize the surgical area and sterilize the surgical instruments using an autoclave at 121 &#176;C for 30 min prior to the surgery.</li>
	<li>Place a rat in the induction chamber and deliver 2.5% of isoflurane for 15 min using a small vaporizer to anesthetize the animal. Assess the anesthesia depth by pedal reflex. To do this, firmly pinch the toe and confirm that the animal does not respond to the toe pinch.</li>
	<li>After anesthesia, remove the isoflurane from the chamber and administer 1 mL of 1,000 unit/mL heparin through the lateral tail vein to prevent blood clotting in the pulmonary vasculature.</li>
	<li>To euthanize the animal, expose the animal to 5% isoflurane for an additional 15-20 min. Remove the animal from the induction chamber and place it on the surgical board in a face-up supine position.</li>
	<li>Fix the rat on the surgical board by immobilizing the legs and tail using adhesive tape. Next, sterilize the rat&#39;s neck and chest regions using 70% isopropyl alcohol (IPA). Open the abdominal cavity by making a 3-4 cm incision using scissors in the skin. 
	NOTE: Be careful not to cut the skin deeply by pointing the tips of the scissors upward.</li>
	<li>Transect the inferior vena cava (IVC) using the scissors and confirm the euthanasia by exsanguination.</li>
	<li>Perform the tracheotomy by making an incision using scissors in the midline of the neck and exposing the trachea. Perform midline thoracotomy by making an incision in the chest wall and cutting between the ribs to reach the trachea end connected to the lung. Next, use a bicep and scissors to cut both ends of the trachea and isolate the trachea.</li>
	<li>After isolation, rinse the trachea using 20 mL of 1x phosphate-buffered saline (1x PBS). Transfer the trachea to the bioreactor using sterilized biceps. Secure the two ends of the trachea to the Luer connector using a 4-0 suture thread.</li>
	<li>Deliver 5 mL of culture medium to the bioreactor chamber using the programmable syringe pump through the tubing connected to the bioreactor chamber at a flow rate of 5 mL/min. 
	NOTE: The culture medium is composed of Dulbecco&#39;s modified Eagle&#39;s medium (DMEM), recombinant human FGF-basic (0.1 ng/mL), fetal bovine serum (FBS, 10%), and antibiotic-antimycotic (1%).</li>
	<li>Tightly close the bioreactor lid with the acrylic plastic lid and screws. Close the bioreactor chamber tubing connections with the male/female Luer plugs to prevent the flow of the culture medium in the tubing.</li>
</ol>
3. Imaging-guided removal of the trachea epithelium
<ol>
	<li>To visualize the trachea lumen either in bright-field or fluorescent, place the bioreactor on the imaging stage (Figure 3A).</li>
	<li>Prepare carboxyfluorescein succinimidyl ester (CFSE) solution (concentration: 100 &#956;M) in the CFSE cell labeling kit by diluting the CFSE solution with 1x PBS. 
	NOTE: The concentration of the original CFSE solution is 10 mM (in dimethyl sulfoxide (DMSO)).</li>
	<li>Infuse 500 &#181;L of the CFSE solution through the trachea via the syringe pump through the tubing connected to the trachea cannula at a flow rate of 5 mL/min. Stop the pump when the CFSE solution fills the trachea. Wait for 10 min, and then wash the trachea lumen by infusion of 10 mL of 1x PBS using the syringe pump to remove the residual unincorporated CFSE reagent.</li>
	<li>Insert the distal imaging end of the GRIN lens into the trachea via the Luer connector attached to one end of the trachea. Then, gently move the GRIN lens inside the trachea until the trachea surface is focused and take photos and videos in bright-field or fluorescence at 20x magnification.</li>
	<li>For capturing bright-field images in the Micro-Manager software follow the steps below.
	<ol>
		<li>Introduce white light through the cage cube to illuminate the trachea lumen. Click on the Live icon to show the luminal surface of the trachea in real-time. Use Imaging Setting &#62; Exposure (ms) to change exposure time to the desired value. In this study, the exposure time used was in the range of 10 ms and 50 ms.</li>
		<li>To adjust the contrast and brightness of images, use the Histogram and Intensity Scaling window to move black and white arrows at the endpoint of the interactive histogram display. Alternatively, use the Auto Stretch option that allows the software to adjust the brightness and contrast automatically to optimal levels.</li>
		<li>Click on the Snap icon to freeze the image. Then, use Setting &#62; Export Images as Displaced to save the images in the desired format. Alternatively, use Setting &#62; ImageJ to directly export the image to the ImageJ software and save it.</li>
	</ol>
	</li>
	<li>To obtain photos and videos in fluorescent mode, illuminate the trachea lumen with CFSE-specific laser light (CFSE: excitation wavelength: 488 nm, emission wavelength: 515 nm) through the cage cube. Follow the steps 3.5.2-3.5.3 to acquire the images. After taking photos and videos, gently remove the GRIN lens from the trachea.</li>
	<li>Perform de-epithelialization as described below.
	<ol>
		<li>Prepare 2% sodium dodecyl sulfate (SDS) decellularization solution in distilled (DI) water. Instill 50 &#181;L of SDS through the trachea at a flow rate of 6.3 mL/min, by using the syringe pump through the trachea cannula to generate a thin film of the detergent solution on the trachea lumen.</li>
		<li>Close the bioreactor&#39;s tubing connections using male/female Luer plugs and transfer the bioreactor to an incubator. Allow the SDS to dwell within the trachea for 10 min at 37 &#176;C. Instill another 50 &#181;L of SDS solution through the trachea and incubate for 10 min.</li>
		<li>Remove lysed epithelium and SDS by irrigating the trachea lumen with 500 &#181;L of 1x PBS via syringe pump at a flow rate of 10 mL/min for 3x. Place the bioreactor on a shaker and mechanically vibrate the bioreactor at 20 Hz frequency and displacement amplitude of approximately 0.3 mm to physically promote detachment of SDS-treated epithelial cells from the trachea lumen. 
		NOTE: In this study, the shaker was custom-built by assembling a subwoofer speaker, a subwoofer plate amplifier, and an accelerometer. A sinusoidal waveform was generated by a computer and fed into the shaker via the amplifier, while the response of the shaker was monitored via the accelerometer (Figure 3B). In addition, conventional shakers, such as electromagnetic and inertia shakers, can be used to promote the detachment of the cells. To do this, set the frequency and acceleration of the shaker to 20 Hz and 0.5 g, respectively (equivalent to 0.3 mm displacement amplitude).</li>
		<li>Instill 500 &#181;L of 1x PBS twice through the trachea lumen to remove residual SDS and cell debris while the trachea is mechanically vibrated. Following the epithelium removal procedure, evaluate the clearance of the epithelial layer by measuring the intensity of CFSE using the GRIN lens imaging device as in step 3.6 (Figure 3C).</li>
	</ol>
	</li>
</ol>
4. Trachea tissue preparation for further analyses
<ol>
	<li>To confirm the epithelium removal, perform further tissue analyses, such as hematoxylin and eosin (H&#38;E) staining, trichrome, pentachrome, and immunostaining. To do this, remove the trachea from the bioreactor, and fix it in 30 mL of 4% neutral buffered paraformaldehyde solution in 1x PBS (pH = 7.4) at 4 &#176;C overnight.</li>
	<li>Dehydrate and embed the fixed trachea tissue by following the steps below.
	<ol>
		<li>After fixation, wash the trachea tissue with 10 mL of 1x PBS, transfer the trachea to 30 mL of 70% alcohol, and keep it at 4 &#176;C overnight. Cut the trachea into small cylindrical sections (~5 mm) using a sharp blade and insert the tissue sections in the tissue embedding cassettes (length x width x height: 4 cm x 2.5 cm x 0.5 cm). Keep two trachea sections in each cassette.</li>
		<li>Dehydrate the sections in a series of isopropyl alcohol (IPA) solutions - 85%, 90%, 95%, 100% - for 1 h in 30 mL of each solution. Remove the previous solution before adding the next solution.</li>
		<li>Upon completion, submerge the cassettes in 30 mL of clearing agent (e.g., xylene) for 2 h to displace the IPA solution from the tissue sections completely. Perform this step in a fume hood with proper ventilation.</li>
		<li>Submerge the cassettes in paraffin for 2 h, and then embed them in paraffin at 4 &#176;C overnight. Next, cut the paraffin-embedded tissues into thin sections (5-8 &#956;m) using a microtome device for the H&#38;E, trichrome, pentachrome, and immunostaining.</li>
	</ol>
	</li>
	<li>To prepare the tissues for scanning electron microscopy (SEM) analysis, fix the trachea in 30 mL of 2.5% glutaraldehyde solution in 1x PBS (pH = 7.4) at 4 &#176;C overnight. Then, dehydrate the fixed trachea tissue for SEM by following the steps below.
	<ol>
		<li>After fixation, rinse the trachea tissue with 10 mL of 1x PBS. Cut the trachea tissue longitudinally into small semi-cylinder sections (length: ~5 mm) using scissors and insert the tissue sections in the cassettes.</li>
		<li>Dehydrate the sections in a series of IPA solutions - 35%, 50%, 70%, 85%, 95%, and 100% - for 10 min in 30 mL of each solution. Remove the previous solution before adding the next solution.</li>
		<li>Perform hexamethyldisilazane (HMDS)-based drying method by submerging the tissues in the following solutions: 100% IPA: HMDS (2:1; v/v) for 10 min, followed by 100% IPA: HMDS (1:2; v/v) for 10 min, and finally 100% HMDS for overnight. 
		NOTE: HMDS is toxic. Work under a fume hood during all drying steps.</li>
		<li>Remove the tissues from the HMDS solution and allow them to dry under the fume hood for 1 h. Mount the section on aluminum pin stubs using a carbon double-sided conductive tape or silver conductive paste for SEM imaging.</li>
	</ol>
	</li>
</ol>
5. Homogeneous distribution of exogenous cells onto the denuded tracheal lumen
<ol>
	<li>Prepare a de-epithelialized rat trachea using the protocol in step 3. Thaw frozen mesenchymal stem cells (MSCs) for 30 s in a 37 &#176;C water bath. 
	NOTE: In this study, we used mesenchymal stem cells (MSCs) as a model cell to show the distribution of exogenous cells onto the de-epithelialized trachea. Ideally, primary airway epithelial cells, basal cells, or induced-human pluripotent cells (iPSCs) can be used for epithelium regeneration purposes.</li>
	<li>Count the cells with a hemocytometer and prepare a cell solution with a concentration of 5 x 106 cells/mL. Label the cells fluorescently by incubating the cells with 2 mL of CFSE solution (concentration: 100 &#181;M) at room temperature for 15 min. Rinse the cells with 5 mL of 1x PBS for 3x and resuspend the cells in fresh culture medium at a final concentration of 3 x 107 cells/mL.</li>
	<li>Prepare hydrogel pre-gel solution as a vehicle for cell delivery. For this study, use collagen I as a delivery vehicle for MSCs cells and follow the manufacturer&#39;s instructions to prepare the pre-gel. For example, to obtain 3.6 mg/mL collagen pre-gel, mix one part of the chilled neutralization solution with nine parts of the rat tail collagen in a 1.5 mL sterile tube. Then, gently pipet the mixture up and down for adequate mixing. 
	NOTE: Other biocompatible hydrogels may be used instead of collagen I according to the study and user needs.</li>
	<li>Once the hydrogel solution is prepared, add the cells quickly to the solution with desired concentration (e.g., 5 x 106 cells/mL). To obtain a uniform cell-hydrogel mixture, mix the cells and gel solution by gently pipetting with a micropipette.</li>
	<li>Attach one end of the trachea within the bioreactor to a programmable syringe pump through a Luer connector. Deliver 5 mL of fresh culture medium at 37 &#176;C into the bioreactor chamber to cover the exterior surface of the trachea using the syringe pump at a flow rate of 5 mL/min.</li>
	<li>Administer a 10 &#181;L bolus of the cell-hydrogel mixture into the de-epithelialized trachea within the bioreactor using the syringe pump at a flow rate of 5 mL/min to generate a cell-hydrogel layer on the trachea lumen (Figure 4).</li>
	<li>After cell injection, place the bioreactor in a sterile cell culture incubator at 37 &#176;C and 5% CO2 for gelation. For collagen I, the gelation occurs in 30 min.</li>
	<li>To visualize the distribution of implanted cells, sterilize the GRIN lens by wiping with 70% IPA or ethanol and place the bioreactor on the imaging stage. Take photos and videos in both bright-field and fluorescent modes as needed.</li>
	<li>After 30 min of cell seeding, infuse 1 mL of culture medium into the trachea lumen using a syringe pump at a flow rate of 1 mL/min.</li>
	<li>Culture the cell-seeded trachea within the bioreactor in an incubator at 37 &#176;C for the desired time. For long-term culture, refresh the culture medium in the trachea lumen and the bioreactor chamber every 24 h. During the cell culture, keep the media inside the lumen static and change it every 24 h, while the media outside the trachea is continuously perfused via a unidirectional flow at 5 mL/min.</li>
	<li>After culturing the cells for a certain time period (e.g., 1 and 4 days in this study), remove the trachea from the bioreactor. Use scissors to cut the trachea longitudinally into two semi-cylinder sections (i.e., upper and lower sections) on days 1 and 4 of in vitro culture to expose the inner surfaces for monitoring cell growth and calculating cell density. Use a conventional fluorescent microscope to visualize the cells on the inner surfaces.</li>
	<li>To acquire the images using Micro-Manager software, follow steps 3.5 and 3.6. Use a fluorescein isothiocyanate (FITC) filter to visualize the CFSE-labeled cells in fluorescence mode. Analyze the images taken by the fluorescent microscope and calculate cell densities on upper and lower sections using the ImageJ software. To calculate the number of cells and the cell density, follow the steps below.
	<ol>
		<li>Open an image in ImageJ software and convert the image to a binary image (16-bit) using Image &#62; Type &#62; 16-bit. Set the image scale with the scale bar using Analyze &#62; Set Scale.</li>
		<li>Adjust the threshold of the image to highlight the structures of the cells to count. Use Image &#62; Adjust &#62; Threshold. Use Analyze &#62; Analyze Particles to count the number of the cells. Calculate the density of the cells by dividing the number of cells by the surface area of the image.</li>
	</ol>
	</li>
	<li>To assess the viability after cell seeding, use a cell viability kit. For this study, incubate the trachea tissue and the cells in the bioreactor at 37 &#176;C for 6 h and stain the cells with 4 mM calcein-AM and 2 mM ethidium homodimer-1 in 1 mL of 1x PBS at room temperature for 15 min.</li>
	<li>After rinsing with 1x PBS, visualize the cells using a fluorescent microscope. Count the live and dead cells using the steps described in step 5.12. Calculate cell viability as the percentage of live cells (stained with calcein-AM) per total cells (both live and dead cells).</li>
</ol>

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V., Bacchetta, M., Vunjak-Novakovic, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+delivery+of+liquid+microvolumes+into+the+lung.">Targeted delivery of liquid microvolumes into the lung.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (37), 11530-11535 (2015).</li><li>Kim, J., O'Neill, J. D., Vunjak-Novakovic, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+retraction+of+microvolume+aqueous+plugs+traveling+in+a+wettable+capillary.">Rapid retraction of microvolume aqueous plugs traveling in a wettable capillary.</a> Applied Physics Letters. 107 (14), 144101 (2015).</li><li>O'Neill, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularization+of+human+and+porcine+lung+tissues+for+pulmonary+tissue+engineering.">Decellularization of human and porcine lung tissues for pulmonary tissue engineering.</a> The Annals of Thoracic Surgery. 96 (3), 1046-1056 (2013).</li><li>Sengyoku, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+hydroxide+based+non-detergent+decellularizing+solution+for+rat+lung.">Sodium hydroxide based non-detergent decellularizing solution for rat lung.</a> Organogenesis. 14 (2), 94-106 (2018).</li><li>Walters, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+human+airway+epithelium+derived+basal+cell+line+with+multipotent+differentiation+capacity.">Generation of a human airway epithelium derived basal cell line with multipotent differentiation capacity.</a> Respiratory Research. 14 (1), 135 (2013).</li><li>O'Neill, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-circulation+for+extracorporeal+support+and+recovery+of+the+lung.">Cross-circulation for extracorporeal support and recovery of the lung.</a> Nature Biomedical Engineering. 1 (3), 0037 (2017).</li><li>Guenthart, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+severely+damaged+lungs+using+an+interventional+cross-circulation+platform.">Regeneration of severely damaged lungs using an interventional cross-circulation platform.</a> Nature Communications. 10 (1), 1985 (2019).</li><li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-destructive+vacuum-assisted+measurement+of+lung+elastic+modulus.">Non-destructive vacuum-assisted measurement of lung elastic modulus.</a> Acta Biomaterialia. 131, 370-380 (2021).</li><li>Dorrello, N. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+vascularized+lung+grafts+for+lung+bioengineering.">Functional vascularized lung grafts for lung bioengineering.</a> Science Advances. 3 (8), 1700521 (2017).</li></ol>

The authors declare no competing financial interests.

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 ...

Watch this Scientific Journal Video about Imaging-Guided Bioreactor for Generating Bioengineered Airway Tissue at JoVE.com

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 ...

imaging-guided-bioreactor-for-generating-bioengineered-airway-tissue

Research

JoVE Journal

Bioengineering

2.5K Views.  Stevens Institute of Technology. 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.

Video: Imaging-Guided Bioreactor for Generating Bioengineered Airway Tissue

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

Operation of a Benchtop Bioreactor

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to carefully control temperature, pH, and dissolved oxygen concentrations in particular makes them essential to efficient large scale growth and expression of fermentation products. This video will briefly describe the advantages of the fermentor over the shake flask. It will also identify key components of a typical benchtop fermentation system and give basic instruction on setup of the vessel and calibration of its probes. The viewer will be familiarized with the sterilization process and shown how to inoculate the growth medium in the vessel with culture. Basic concepts of operation, sampling, and harvesting will also be demonstrated. Simple data analysis and system cleanup will also be discussed.

Fermentors are used to increase culture yield and productivity of bioengineered cells. After screening multiple microbial or animal cell culture candidates in shake flasks, the next logical step is to increase the selected culture&#x2019;s biomass with the fermentor. This video demonstrates the setup and operation of a typical benchtop bioreactor system.

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to ...

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ&#39;s natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use.
The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Whole-organ decellularization produces natural biological scaffolds that may be used for regenerative medicine. The description of a nonhuman primate model of lung regeneration in which whole lungs are decellularized and then seeded with adult stem cells and endothelial cells in a bioreactor that facilitates vascular circulation and liquid media ventilation is presented.

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an ...

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid&#160;interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid&#160;interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow important mechanistic studies to evaluate pathways that could then be utilized to develop novel therapeutic agents.

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized medicine innovations. However, translating these advances into therapeutic applications will require in vitro systems that allow for robust, flexible, and cost effective bioreactor systems. There are several bioreactor systems currently utilized in research and commercial settings; however, many of these systems are not optimal for establishing, expanding, and monitoring the growth of different cell types. The culture parameters most challenging to control in these systems include, minimizing hydrodynamic shear, preventing nutrient gradient formation, establishing uniform culture medium aeration, preventing microbial contamination, and monitoring and adjusting culture conditions in real-time. Using a pneumatic single-use bioreactor system, we demonstrate the assembly and operation of this novel bioreactor for mammalian cells grown on micro-carriers. This bioreactor system eliminates many of the challenges associated with currently available systems by minimizing hydrodynamic shear and nutrient gradient formation, and allowing for uniform culture medium aeration. Moreover, the bioreactor&#8217;s software allows for remote real-time monitoring and adjusting of the bioreactor run parameters. This bioreactor system also has tremendous potential for scale-up of adherent and suspension mammalian cells for production of a variety therapeutic proteins, monoclonal antibodies, stem cells, biosimilars, and vaccines.

Using a pneumatic bioreactor, we demonstrate the assembly, operation, and performance of this single-use bioreactor system for the growth of mammalian cells.

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past studies have evaluated the performance of the reactor for the removal of COD1 and nitrogen species2-4 by heterotrophic and chemoautotrophic bacteria, respectively. The HDBR design eliminates the requirement for external flocculation/sedimentation processes while still yielding effluent containing low suspended solids. In this study, the HDBR is applied as a photobioreactor (PBR) in order to characterize the nitrogen removal characteristics of an algae-based photosynthetic microbial community. As previously reported for this HDBR design, a stable biomass zone was established with a clear delineation between the biologically active portion of the reactor and the recycling reactor fluid, which resulted in a low suspended solid effluent. The algal community in the HDBR was observed to remove 18.4% of total nitrogen species in the influent. Varying NH4+ and NO3- concentrations in the feed did not have an effect on NH4+ removal (n=44, p=0.993 and n=44, p=0.610 respectively) while NH4+ feed concentration was found to be negatively related with NO3- removal (n=44, p=0.000) and NO3- feed concentration was found to be positively correlated with NO3- removal (n=44, p=0.000). Consistent removal of NH4+, combined with the accumulation of oxidized nitrogen species at high NH4+ fluxes indicates the presence of ammonia- and nitrite-oxidizing bacteria within the microbial community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past ...

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...