Agarose-Based Preparation of Human Precision-Cut Lung Slices

Overview

In this video, we describe a procedure to inflate isolated human lungs with agarose and prepare human precision-cut lung slices. The prepared ex vivo lung slices can be used for further immunologic experiments.

Protocol

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. Let the pathologists 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.
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.
    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.

Materials

Name	Company	Catalog Number	Comments
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
Tissue culture dishes	Sigma (München, Germany)	Z666246-420EA
Inoculation loop	Copan Diagnostics (Murrieta, USA)	CD176S01
TPP Tissue culture plates 24wells	Sigma (München, Germany)	Z707791-126EA
Cassette	Thermo Scientific (Schwerte, Germany)	1000957
Nunc MaxiSorp flat-bottom	Fisher Scientific GmbH (Hannover, Germany)	44-2404-21
Nunc MicroWell 96-Well	Thermo Scientific (Schwerte, Germany)	260836
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