Home › Cell Biology › Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke
Cell Biology JoVE (Open Access) Citable · DOI

Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke

DOI: 10.3791/52325-v
What you'll learn
  • ✓ Culture and maintain human organotypic 3D bronchial and nasal tissue models
  • ✓ Expose tissue models to mainstream cigarette smoke at air-liquid interface
  • ✓ Measure ciliary beating, TEER, and CYP1A1/1B1 activity post-exposure
  • ✓ Perform RNA extraction and microarray analysis for systems-level toxicity assessment
Protocol

The aim of this protocol is to expose human organotypic 3D bronchial and nasal tissue models to mainstream cigarette smoke (CS) at the air-liquid interface. The impact of CS on the tissues is then investigated using a cytochrome P450 activity assay, a cilia beating measurement, and a systems biology approach.

Difficulty
advanced
Total time
~5–7 days (tissue culture preparation, repeated CS exposures, downstream assays)
Model organism
Human organotypic 3D tissue (bronchial and nasal epithelium)
Biosafety
BSL-1

Steps

1
Culture organotypic bronchial tissue models

Establish and maintain human 3D bronchial tissue cultures under standard conditions prior to smoke exposure experiments.

▶ 01:28
2
Expose tissue to mainstream cigarette smoke

Deliver whole cigarette smoke to organotypic tissue models at the air-liquid interface using standardized exposure apparatus.

▶ 02:04
3
Measure ciliary beating and transepithelial electrical resistance

Quantify ciliary beat frequency and TEER across tissue samples to assess epithelial integrity and ciliary function post-exposure.

▶ 03:22
4
Assay cytochrome P450 1A1/1B1 activity

Measure CYP1A1/1B1 enzymatic activity as a biomarker of xenobiotic metabolism and oxidative stress response.

▶ 04:20
5
Extract RNA from exposed tissue samples

Isolate total RNA from cigarette smoke-exposed and control tissues for downstream gene expression analysis.

▶ 05:02
6
Perform microarray analysis and data processing

Conduct microarray hybridization, washing, and staining to generate genome-wide expression profiles of smoke-exposed tissues.

▶ 06:35
7
Evaluate applicability of organotypic tissue models

Interpret combined results from cytotoxicity, functional, and molecular assays to validate organotypic models for toxicological screening.

▶ 08:07
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