Source: Neuhaus, V. et al., Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices. J. Vis. Exp. (2018)
This video illustrates a technique for evaluating cytotoxicity in human precision-cut lung slices through the WST-1 assay. Human precision-cut lung slices are subjected to incremental concentrations of a cytotoxic chemical to quantify their cytotoxicity. A decrease in color intensity serves as confirmation of the chemical's detrimental impact on lung cells.
All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.
1. General Preparation of Human precision-cut lung slices (PCLS) and Subsequent Exposure to Chemicals
NOTE: Two persons are required to fill a lung. An up-to-date vaccination record for hepatitis A and B is recommended. Patients are routinely screened for hepatitis C virus (HCV) and human immunodeficiency virus (HIV) prior to lung transplantation. If an active infection with Mycobacterium tuberculosis is diagnosed or suspected, the lung should be rejected. Nevertheless, all fresh human lung tissue and samples derived from it must be treated as potentially infectious, and corresponding protective measures must be taken (particle filter masks (FFP2), protective eyewear, gloves) to ensure the occupational safety of the staff. The procedure takes 60 – 90 min.
2. Preparation of Solutions for Substances
NOTE: Prepare working solutions and controls immediately before use.
CAUTION: Handle substances according to safety instructions or, if unknown, as potentially harmful and follow routine safety precautions.
3. Positive and Negative References for Cytotoxicity Assays
4. Measurement of Chemically-induced Cytotoxicity in Human PCLS by water-soluble tetrazolium salt (WST-1) Assay
NOTE: The WST-1 assay is performed in a 24-well plate with two PCLS per well. Preferably, use duplicates for each parameter and pool the results of these duplicates after measurement.
The authors have nothing to disclose.
Silicon hose 3.0 x 5.0 mm | A. Hartenstein (Leipzig, Germany) | SS04 | |
Syringe | Faust Lab Science (Klettgau, Germany) | 9.410 050 | |
Coring tools | custom-made | custom-made | |
Trimming Blade Handle (FEATHER) | pfm medical ag (Cologne, Germany) | 205530001 | |
Trimming Blades (FEATHER) | pfm medical ag (Cologne, Germany) | 205500000 | |
Microtome: Tissue Slicer | Alabama R&D (Bad Homburg, Germany) | 303400-ADPT | Krumdieck Tissue Slicer (MD6000) |
Microtome blade | Wilkinson Sword (Solingen, Germany) | ENR-4027800011506 | |
Cell strainer filter (100 µm Nylon) | Becton Dickinson (Heidelberg, Germany) | BD352360 | |
Inoculation loop | Copan Diagnostics (Murrieta, USA) | CD176S01 | |
TPP Tissue culture plates 24wells | Sigma (München, Germany) | Z707791-126EA | |
Nunc MaxiSorp flat-bottom | Fisher Scientific GmbH (Hannover, Germany) | 44-2404-21 | |
Nunc MicroWell 96-Well | Thermo Scientific (Schwerte, Germany) | 260836 | |
Multiwell-Reader | Tecan Group Ltd. (Männedorf, Switzerland) | Tecan infinite F200Pro | Plate Reader |
Plate shaker | Edmund Buehler GmbH (Hechingen, Germany) | KM-2 Akku | |
Assays | |||
Cell Proliferation Reagent WST-1 | Roche (Basel, Switzerland) | 11644807001 | |
Reagents | |||
Agarose, low gelling temperature | Sigma (München, Germany) | A9414-100G | |
Balanced Salt Mixtures and Solutions, Cell Culture, Classic Media and Salts, Earle's Balanced Salts (EBSS) | Sigma (München, Germany) | E2888-500ML | |
Penicillin and streptomycin | Lonza (Verviers, Belgium) | 17-602E | |
Dulbecco´s Modified Eagle´s Medium Nutrient Mixture F-12 Ham (DMEM F-12) | Gibco (Darmstadt, Germany) | 11039-047 | Culture medium |
Dulbecco´s Phosphate with Ca and Mg (DPBS) | Lonza (Verviers, Belgium) | BE17-513F | Buffer solution |
Detergent | Sigma (Saint Louis, USA) | X100-100ML | Triton X-100 |