Evaluating Cytotoxicity and Membrane Integrity in Lung Slices via Lactate Dehydrogenase Assay

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines

1. General Preparation of Human 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 HCV and 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.

1. Confirm the intactness of the human lung lobe.
   NOTE: A tear in the pleura prevents homogeneous filling of the tissue. Human lung material must be from the day of resection. Storage periods of about 2 h at room temperature (RT) prior to filling it with agarose on ice are tolerated. The tissue that we use in this protocol is obtained from patients undergoing resection. It is not from deceased patients. If the tissue has been stored on ice, pre-warm it to RT before filling it, otherwise, the agarose will polymerize immediately, and no homogeneous filling will be possible.
   Caution: Ensure that all persons in contact with native human material put on protective clothing consisting of a lab coat, two pairs of gloves, a cap, a face mask, and a pair of safety glasses. Human material is potentially infectious.
2. Weigh 7.5 g of low-gelling agarose and add it to 250 mL of bi-distilled water. Boil the agarose in a microwave until the agarose is dissolved. Cool it to approximately 40 °C, depending on the melting and gelling point of the agarose.
   NOTE: Several flasks are required, depending on the lobe size.
3. Pre-warm and keep the culture medium (Table of Materials) at 37 °C.
   NOTE: If the agarose is too hot, vitamins in the culture medium will lose effectiveness and cells will be damaged. If the temperature of the medium/agarose solution is below 37 °C, the gelling process will start and impair the homogeneous filling of the lung. Only a temperature range of 37 °C to 39 °C is recommended.
4. Place all the required materials within reach before starting: 5 – 10 clamps, a flexible catheter of approximately 1 m length, a suitable syringe (e.g., 50 mL) fitting the connection to the catheter, and an ice box filled with ice.
5. Cannulate the trachea/main bronchus by inserting the silicone tube and fixating it with a clamp oriented parallel to the tube, so that the clamp squeezes the tissue together alongside the silicone tube without pinching it off. Verify that the silicone tube is fixated inside and cannot slip out during the filling procedure. Close all other bronchi, blood vessels, and injuries with clamps, so that no agarose can leak out during the filling procedure.
6. Mix an equivalent volume of 3% low-gelling agarose with culture medium in a beaker. Instill the mixture into the lung using a 50-mL syringe. Prior to refilling the syringe with medium, clamp shut the catheter with fingers or a clamp to avoid air bubbles and agarose reflux. Fill the lung lobe until it is fully inflated. Carefully touch the lung pleura on the side; the pleura should be even and hard.
   NOTE: Depending on the size of the lung lobe, up to 2 or 3 L of agarose/medium solution can be required.
7. Repeat steps 1.5 – 1.6 for each bronchus in the case of several bronchi within a single specimen.
8. Put the lung on ice for polymerization of the agarose to gel for 20 – 40 min, depending on the amount of instilled agarose. Carefully touch the pleura on several sides to check whether it is hard and cool, indicating that polymerization is complete, otherwise, continue to wait. The pathologists will cut the lung into slices, take their samples, and store the lung material on ice for transportation.
9. Cut the human lung tissue into 3 – 5 cm slabs with a sharp knife.
   NOTE: Use a new blade for every lung to ensure sharpness.
10. Fill the tissue slicer with 400 mL ice-cold Earle's balanced salt solution (EBSS). Immediately cut cylindrical tissue cores out of the lung slabs using a semi-automated screwdriver with a coring tool of the preferred diameter (e.g., 8 mm; 10 mm).
    NOTE: This diameter must be equivalent to the diameter of the tissue holder inside the machine.
11. Adjust the thickness of the lung slices to the desired thickness value.
    NOTE: A thickness of approximately 250 µm is commonly used in PCLS experiments. The manufacturer of the tissue slicer recommends their Tissue Slice Thickness Gauge for verification of the slice thickness. Alternatively, whole-mount staining combined with confocal microscopy can be used to determine the slice thickness as described by Brismar et al.27
12. Transfer the tissue cores into the tissue holder of the tissue slicer. Put the weight (part of the tissue holder) on top of the tissue core. Set the arm speed and blade speed to 6 on the tissue slicer. Start slicing the tissue core into PCLS.
13. Supplement 500 mL of commercially available Dulbecco's Modified Eagle Medium (DMEM): Nutrient Mixture F-12 DMEM/F12 (1:1) with 5 mL of penicillin (10,000 units/mL) and streptomycin (10,000 µg/mL). Store the culture medium for several days under sterile conditions in a refrigerator. Pre-warm only the required volume of the medium.
    NOTE: Penicillin will be inactivated if kept at 37 °C for longer periods. Use serum-free and dye-free culture medium, as serum composition varies from batch to batch, and dyes, such as phenol-red, can interfere with assays.
14. Fill a Petri dish (100 x 15 mm) with 25 mL ice-cold culture medium. The medium should be drained out of the tissue slicer into a beaker by opening the clamp of the glass cylinder. Transfer the slices from a beaker into the Petri dish with culture medium by using an applicator (e.g., inoculation loop). Put the Petri dish into an incubator (37 °C, 5% CO2, 100% humidity). Allow the medium to warm up prior to washing steps.
    NOTE: All further steps are performed under sterile conditions.
15. Place a cell strainer into the Petri dish for washing the PCLS. Completely remove the culture medium with a 10-mL serological pipette through the cell strainer and add 25 mL of 37 °C pre-warmed fresh medium. Repeat this step 3 – 4 times every 30 min.
    NOTE: The cell strainer prevents the slices from being sucked into the pipette, to avoid any damage to the slices.
16. Transfer the lung slices carefully into a 24-well culture plate with a minimum of 500 µL culture medium for two slices per well. Expose the lung tissue to substances (see steps 3.1 – 3.4), e.g., for 24 h at 37 °C, 5% CO2.
    NOTE: For the transfer, preferably use an inoculation loop and let the slices float onto the loop in order to prevent tissue damage. No further separation of the slices is needed, as only a shortage of sufficient fresh medium will induce cell death in tissue slices. Slices can overlap or touch each other in the well: this has no influence on tissue viability.
17. Put all residual human material into a plastic flask with a fixative (e.g., 10% buffered formaldehyde) for at least 24 hours and burn this in the disposal process.
    Caution: Formaldehyde is toxic, perform this step under a hood.

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.

1. Dissolve water-soluble substances directly in the culture medium. For insoluble or poorly soluble chemicals, first dissolve the substance in the appropriate solvents depending on substance solubility. Substances with limited water solubility (<0.1 mg/mL) can be dissolved, for example, in DMSO or ethanol. Non-toxic solvent concentrations should be determined by titration beforehand. Make sure the substances do not precipitate out of the solution when diluted into the medium.
   NOTE: HClPt and sodium laureth sulfate (SLS) solutions are prepared.
2. Prepare substance stock solutions at 100-fold of the desired highest concentration in the culture medium or solvent. Weigh 12.5 mg HClPt and 34.4 mg SLS and prepare stock solutions by dissolving the chemicals in 1 mL culture medium.
   NOTE: No solvent is required for these chemicals.
3. Prepare a final dilution of 1:100 in a pre-warmed medium. In the case of prior use of solvent, this approach results in the same final solvent concentration for all substance concentrations.
4. Use the final solvent concentration (e.g., 1% as described in step 2.3) for reference treatment of PCLS. No solvent control was required for the chemicals mentioned in the results section.

3. Positive and Negative References for Cytotoxicity Assays

1. For all viability assays, prepare the following positive and negative controls:
   1. Tissue control: incubate PCLS in a culture medium only as a reference for untreated PCLS for 24 h at cell culture conditions (37 °C, 5% CO₂, 100% humidity).
   2. Vehicle control (if necessary): incubate PCLS with the final solvent concentration as a reference for PCLS treated with vehicle only (see step 1.4) for 24 h at cell culture conditions (37 °C, 5% CO₂, 100% humidity).
   3. Positive control: incubate PCLS with 1% detergent in buffer solution for 1 h at 4 °C.
      NOTE: If PCLS becomes colorless, the tissue is dead. Total L-lactate dehydrogenase (LDH) is determined in the supernatant, with an absorption of approximately 1.9 – 2.3 (see steps 4.1 – 4.4).

4. LDH Assay

Note: The LDH assay is performed in a 96-well plate with 100 µL culture supernatant in total after the post-incubation period.

1. After incubation of the PCLS with or without the test agents, take 50 µL of supernatant from step 1.16 and transfer it in duplicates into a new 96-well plate. This generates duplicates from each treated well of a 24-well plate.
2. Prepare the working solution of the LDH reagent immediately before the assay. The working solution consists of the catalyst solution (lyophilisate dissolved in 1 mL of bidistilled water) and the dye solution supplied by the manufacturer. For one 96-well plate, thoroughly mix 125 µL of catalyst solution with 6.25 mL of dye solution.
   Caution: Do not expose the solution to direct light.
3. Pipet 50 µL of the working solution into each well already containing 50 µL of supernatant and incubate the plate for 20 min at RT in the dark. No further mixing is required.
4. Measure the absorption of each well at 492 nm (reference: 630 nm) using a microplate reader. Subtract the absorption at 630 nm from 492 nm. These values will be used for the calculation in step 3.5.
   NOTE: Reliable data for cytotoxicity assessment can be obtained only from viable tissue. Absorption of the detergent-treated control should be over 1.
5. For analysis, set the absorption value of the positive control from step 2.1 as 100% and calculate the LDH release in treated samples in relation to the positive control (i.e., maximum LDH release).