Name: imaging-guided bioreactor for generating bioengineered airway tissue
Uploaded: 2022-04-06T13:50:00
Description: Watch this Scientific Journal Video about Imaging-Guided Bioreactor for Generating Bioengineered Airway Tissue at JoVE.com

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 ...

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 ...

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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Research

JoVE Journal

Bioengineering

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  ...

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 ...

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 ...

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 ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

Cardiac Spheroids as in vitro Bioengineered Heart Tissues to Study Human Heart Pathophysiology

Despite several advances in cardiac tissue engineering, one of the major challenges to overcome remains the generation of a fully functional vascular network comprising several levels of complexity to provide oxygen and nutrients within bioengineered heart tissues. Our laboratory has developed a three-dimensional in vitro model of the human heart, known as the "cardiac spheroid" or "CS". This presents biochemical, physiological, and pharmacological features typical of the human heart and is generated by co-culturing its three major cell types, such as cardiac myocytes, endothelial cells, and fibroblasts. Human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs or iCMs) are co-cultured at ratios approximating the ones found in vivo with human cardiac fibroblasts (HCFs) and human coronary artery endothelial cells (HCAECs) in hanging drop culture plates for three to four days. The confocal analysis of CSs stained with antibodies against cardiac Troponin T, CD31 and vimentin (markers for cardiac myocytes, endothelial cells and fibroblasts, respectively) shows that CSs present a complex endothelial cell network, resembling the native one found in the human heart. This is confirmed by the 3D rendering analysis of these confocal images. CSs also present extracellular matrix (ECM) proteins typical of the human heart, such as collagen type IV, laminin and fibronectin. Finally, CSs present a contractile activity measured as syncytial contractility closer to the one typical of the human heart compared to CSs that contain iCMs only. When treated with a cardiotoxic anti-cancer agent, such as doxorubicin (DOX, used to treat leukemia, lymphoma and breast cancer), the viability of DOX-treated CSs is significantly reduced at 10 &#181;M genetic and chemical inhibition of endothelial nitric oxide synthase, a downstream target of DOX in HCFs and HCAECs, reduced its toxicity in CSs. Given these unique features, CSs are currently used as in vitro models to study heart biochemistry, pathophysiology, and pharmacology.

This protocol aims to fabricate 3D cardiac spheroids (CSs) by co-culturing cells in hanging drops.&#160;Collagen-embedded CSs are treated with doxorubicin (DOX, a cardiotoxic agent) at physiological concentrations to model heart failure. In vitro testing using DOX-treated CSs may be used to identify novel therapies for heart failure patients.

Despite several advances in cardiac tissue engineering, one of the major challenges to overcome remains the generation of a fully functional vascular ...

Multi-Stream Perfusion Bioreactor Integrated with Outlet Fractionation for Dynamic Cell Culture

Certain cell and tissue functions operate within the dynamic time scale of minutes to hours that are poorly resolved by conventional culture systems. This work has developed a low-cost perfusion bioreactor system that allows culture medium to be continuously perfused into a cell culture module and fractionated in a downstream module to measure dynamics on this scale. The system is constructed almost entirely from commercially available parts and can be parallelized to conduct independent experiments in conventional multi-well cell culture plates simultaneously. This video article demonstrates how to assemble the base setup, which requires only a single multichannel syringe pump and a modified fraction collector to perfuse up to six cultures in parallel. Useful variants on the modular design are also presented that allow for controlled stimulation dynamics, such as solute pulses or pharmacokinetic-like profiles. Importantly, as solute signals travel through the system, they are distorted due to solute dispersion. Furthermore, a method for measuring the residence time distributions (RTDs) of the components of the perfusion setup with a tracer using MATLAB is described. RTDs are useful to calculate how solute signals are distorted by the flow in the multi-compartment system. This system is highly robust and reproducible, so basic researchers can easily adopt it without the need for specialized fabrication facilities.

This paper presents a method to construct and operate a low-cost, multichannel perfusion cell culture system for measuring the dynamics of secretion and absorption rates of solutes in cellular processes. The system can also expose cells to dynamic stimulus profiles.

Certain cell and tissue functions operate within the dynamic time scale of minutes to hours that are poorly resolved by conventional culture systems. This ...