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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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

  1. Designing and fabrication of rat trachea bioreactor
    1. Create a computer-aided design (CA.......

Representative Results

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 .......

Discussion

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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
1× PBSGibco, Thermo Fisher Scientific10-010-031
3-port connectorWorld Precision Instruments14048-20
4-port connectorWorld Precision Instruments14047-10
AccelerometerSTMicroelectronicsIIS3DWBTR
Achromatic doubletThorlabsAC254-150-A-ML
Aluminum pin stubTED PELLA16111
Antibiotic-antimycoticThermo Fisher Scientific15240062
Assembly rodThorlabsER1
Button head screwsMcMaster-Carr91255A274
Cage cubeThorlabsC4W
Carbon double-sided conductive tapeTED PELLA16073
CFSE labelling kitAbcamab113853
Citrisolv (clearing agent)Decon1061
C-mount adapterThorlabsSM1A9
Collagen IAdvanced BioMatrix5153
Conductive liquid silver paintTED PELLA16034
Dichroic mirrorSemrockDI03-R488Reflected laser wavelengths:  473.0 +- 2 nm 488.0 +3/-2 nm
Dulbecco's modified Eagle’s mediumGibco, Thermo Fisher Scientific11965118
Female luer bulkhead to hose barb adapterCole-ParmerEW-45501-30
Female luer to tubing barbCole-ParmerEW-45508-03
Female to male luer connectorCole-ParmerZY-45508-80
Fetal bovine serumGibco, Thermo Fisher Scientific10082147
Filter lensChroma Technology CorpET535/50m
Fluorescent microscopeNikonEclipse E1000 - D
Fusion 360Autodesk
Hex nutMcMaster-Carr91813A160
Hexamethyldisilazane (HMDS)Fisher ScientifcAC120585000
Imaging fiberSELFOC, NSG groupGRIN lens
LaserOpto EngineMDL-D-488-150mW
Lens tubesThorlabsSM1L40
LIVE/DEAD Viability/Cytotoxicity Kit (Invitrogen)Thermo Fisher ScientificL3224
MACH 3 CNC Control SoftwareNewfangled Solutions
Objective lensOlympusUCPLFLN20X
Peristaltic PumpCole ParmerL/S standard digital pump system
Recombinant human FGF-basicPeproTech100-18B
Retaining ringThorlabsSM1RR
Scientific CMOS cameraPCO PandaPCO Panda 4.2
Sodium dodecyl sulfateVWR97064-472
Solidworks (2019)Dassault Systèmes
Stackable lens tubeThorlabsSM1L10
Subwoofer plate amplifierDayton AudioSPA250DSP
Subwoofer speakerDayton AudioRSS21OHO-4Diaphragm diameter: 21 cm
Syringe PumpWorld Precision InstrumentsAL-4000
Threaded cage plateThorlabsCP33
Threaded luer adapterCole-ParmerEW-45513-81
Tube lensThorlabsAC254-150-A-ML
Tygon TubingCole-Parmer13-200-110
XY TranslatorThorlabsCXY1

References

  1. Rackley, C. R., Stripp, B. R. Building and maintaining the epithelium of the lung. The Journal of Clinical Investigation. 122 (8), 2724-2730 (2012).
  2. Rayner, R. E., Makena, P., Prasad, G. L., Cormet-Boyaka, E.

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