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Immunology JoVE (Open Access) Citable · DOI

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

DOI: 10.3791/57042-v
What you'll learn
  • ✓ Prepare and culture human precision-cut lung slices for substance exposure testing
  • ✓ Measure cytotoxic effects using WST-1, LDH, and microscopic viability assays
  • ✓ Assess immunomodulatory responses to airborne substances in respiratory tissue models
Protocol

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

Difficulty
advanced
Total time
~3–5 days (tissue preparation, chemical exposure, assay incubation, and image acquisition)
Model organism
Human lung tissue (precision-cut lung slices)
Biosafety
BSL-1

Steps

1
Prepare human PCLS and expose to chemicals

Obtain and slice human lung tissue, culture precision-cut lung slices in appropriate media, and expose them to test substances via direct application or aerosol exposure.

▶ 01:26
2
Measure cytotoxicity with WST-1 assay

Apply WST-1 reagent to exposed PCLS samples and quantify cell viability/metabolic activity by colorimetric detection, indicating chemically-induced cytotoxic damage.

▶ 04:28
3
Quantify cell death using LDH assay

Measure lactate dehydrogenase release from damaged cells into culture supernatant as a marker of cytotoxicity and membrane integrity loss.

▶ 06:01
4
Assess viability by confocal microscopy

Use fluorescent stains and confocal laser scanning microscopy to visualize and quantify viable versus damaged cells in three-dimensional PCLS tissue sections.

▶ 07:29
5
Image and render three-dimensional PCLS staining

Acquire z-stack confocal images and generate 3D reconstructions to visualize spatial distribution of cell viability and immunomodulatory markers in intact lung tissue architecture.

▶ 09:49
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