Lung-tumor Tissue Explant Culture: A Procedure to Generate and Culture Precision-cut Lung Tumor Slices

1.Generation of Precision-cut Lung Tumor Slices

1. Transfer the tumor-bearing lungs in HBSS + P/S into a 10 cm tissue culture plate. Separate the lung lobes using sterile scissors and forceps, and select lobes with tumors on the surface for slicing.
   NOTE: Tumors >3 mm in size are suitable for slicing. Since normal lung tissue surrounding the big tumor compromises the slicing due to differences in tissue stiffness, tumor tissue undergoing slicing should be separated away from the normal tissue, such that a cleared tumor region faces the vibratome blade.
2. Place the selected lung lobe with a tumor tissue on a piece of filter paper to avoid slipping, and cut a part of the normal lung or additional tumor tissue that surrounds the tumor away with a sterile scalpel to generate a flat tissue piece surface.
   1. Dip the flat slide in a drop of cyanoacrylate adhesive. Mount this side onto the vibratome’s specimen holder so that the tumor faces the blade in an upright position. Let the glue dry for 2-3 min.
      NOTE: The normal lung tissue glued to the specimen holder does not interfere with tumor tissue slicing, and slicing is stopped before the normal tissue is reached. The tumor tissue sometimes bends due to the spongy texture of the normal lung tissue glued to the specimen holder, compromising its upright position. If this happens, glue a piece of additional normal lung support tissue next to the pre-mounted normal lung tissue to retain the tumor in an upright position (Figure 1A iii).
3. Place the specimen holder into the metal buffer tray and fill it with cold HBSS + P/S until the tissue is immersed in the buffer, and cover the buffer tray with the plexiglass lid that is provided with the instrument. Place the metal buffer tray onto the white ice bath and add ice so to keep the tissue cool while slicing.
4. Attach the white ice bath to the vibratome. Select suitable slicing settings: amplitude ranging between 2.5–2.8, slicing speed between 0.10-0.14 ms, and a cutting thickness ranging between 160–250 µm. Bring the vibratome blade to the slicing position, set the slicing window and start slicing.
   NOTE: Slicing settings need to be adjusted according to the hardness of the tissue. Hard tissue is easier to slice than softer tissue, and softer tissues requires slicing with lower speed (0.1–0.12 ms) and higher amplitude (2.6-2.8). A 4–5 mm large tumor typically provides 15–20 slices of 200 µM thickness. For short-term cultivation, murine tumors can be sliced under semi-sterile conditions outside the laminar hood. However, clinical tumors should always be sliced inside a class II biosafety laminar hood to avoid exposure to possible infectious agents in the human tissue.
5. Using sterile forceps, collect the slices in 1 mL of HBSS in separate wells of a 24-well plate held on ice, closely keeping track of the order in which slices are sliced. Mark each well of the 24-well plate according to the experimental plan. For example, mark sequential wells of a 24-well plate as culture time points or as 0 h, vehicle control (C), drug treated (T) (Figure 1B ii).
   NOTE: Do not disturb or pull the tumor tissue while collecting a slice, as this will alter the tumor’s orientation with respect to the angle of the blade, leading to inconsistencies in the thicknesses of the subsequent slices.
6. When all slices are collected, transfer them onto titanium grids (2-3 slices per grid) placed in a 6-well plate containing 2.5 mL of culture medium per well. Make sure that no air bubbles are formed between the titanium grid and the medium.
   1. To load a slice on to the grid, keep the 6-well plate in an angled position so that a portion of the medium covers the grid, and place the slice in the medium on the grid; use forceps to spread the slice if it curls. Load the 6-well plates onto the rotating incubation unit placed inside a humidified incubator maintained at 37 °C with 95% air and 5% CO₂, and start the rotation cycle (Figure 1C i-ii).
      NOTE: Metallic grids and 6-well plates need to be accurately weight balanced before turning on the rotating unit. It is important to position the slices in the middle of the grid so that they fully alternate between the air and liquid phases during the rotation cycles. Slices that are placed too low or high are not appropriately exposed to oxygen or nutrients (Figure 1C i), which can compromise tissue viability. It is important to follow the position of the slice on the grid during cultivation, as the slice can occasionally slide down. If this happens within 1–2 h of culture onset, correct its position and note down that this sample may be damaged. Slices that are mispositioned for an extended period of time should be discarded, as tissue viability is significantly affected by improper oxygenation and nutrient supply.
7. Collect a tissue slice adjacent to the cultured slices as a 0 h, uncultured, reference. Collect at least three reference slices to represent the top, middle and center of the tissue being sliced. Fix the 0 h slices immediately and process as needed.
   NOTE: If the number of slices is limited, e.g., if multiple compound treatments or technical replicates are done, comparisons of each treated sample with its neighboring 0 h sample can be difficult. In such cases, use the nearest 0 h slice (at least 400–600 µm apart) to assess relative tissue viability or expression of relevant markers at culture onset.
8. For long-term cultivation, replenish the culture medium every day. Lift the grid containing the tissue slices using sterile forceps, and place it in an empty well of the 6-well plate; replace 70% of the medium with fresh culture medium, and place the grid back in the medium. Continue the rotation cycle as explained in step 1.6.