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