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

– Place freshly harvested tumor-bearing lungs on a tissue culture plate. Separate the lung lobes. Transfer a tumor-bearing lobe onto a filter paper to avoid slipping. Cut a part of the lobe surrounding the tumor to generate a flat surface. Immerse the flat surface in cyanoacrylate glue to facilitate adhesion.

Next, mount on a vibratome specimen holder, securing the tumor in an upright position. Transfer the specimen holder onto a buffer tray and fill the tray with a suitable buffer. Place the tray in an ice bath to prevent tissue damage from heat generated during slicing. Use a vibratome to obtain thin tumor tissue slices.

Simultaneously, assemble a customized titanium grid in a culture plate containing a suitable growth medium to support tissue slice culture. Angle the plate to allow a portion of the solution to cover the grid. Place the tissue slices in the medium. Transfer the plate into a rotating incubation unit. During rotating incubation, the tissue slices alternate between air and liquid phases, enabling proper exposure to nutrients and oxygen. In this protocol, we will demonstrate the preparation and subsequent culturing of lung tumor explants.

– Transfer the tumor-bearing lungs into a 10-centimeter tissue culture plate. Use sterile scissors and forceps to separate the lung lobes and select lobes with tumors on the surface for slicing. Place a lung lobe with a tumor tissue on a piece of filter paper to avoid slipping and cut out part of the normal lung or additional tumor tissue that surrounds the tumor using a sterile scalpel to generate a flat tissue piece surface.

Dip the flat side in a drop of cyanoacrylate adhesive and mount it onto the vibratome specimen holder so that the tumor faces the blade in an upright position and let it dry for two to three minutes. Place the vibratome specimen holder into the metal buffer tray. Fill the tray with cold HBSS supplemented with Pen-Strep until the tissue is immersed in the buffer.

Cover the buffer tray with the Plexiglas lid that is provided with the instrument and place the tray into a white ice bath and add ice to keep the tissue cool during slicing. Attach the white ice bath to the vibratome. Then select suitable slicing settings and start slicing. Bring the vibratome blade to the slicing position and set the slicing window. Use sterile forceps to collect the slices in a 24-well plate filled with 1 milliliter of HBSS supplemented with Pen-Strep per well and keep on ice.

While keeping track of the slicing order, mark each well of the 24-well plate according to the experimental plan. Collect a tissue slice adjacent to the cultured slices as a zero hours or uncultured reference. Collect at least three reference slices to represent the top, center, and bottom of the tissue and once slicing is completed, continue with fixing and processing.

After that, prepare a six-well plate containing 2.5 milliliters of culture medium per well by placing titanium grids in it. Make sure that no air bubbles are formed between the titanium grid and the medium. To load a slice onto the grid, keep the six-well plate in an angled position so that a portion of the medium covers the grid.

Then place the slice in the medium on the grid and use forceps to spread it. After placing the slices, load the six-well plates onto the rotating incubation unit placed inside a humidified incubator maintained at 37 degrees Celsius with 95% O2 and 5% CO2. Start the rotation cycle.

For long-term cultivation, replenish the culture medium every day by using sterile forceps to lift the grid containing the tissue slices and place it in an empty well of the six-well plate. Replace 70% of the medium with fresh culture medium and place the grid back in. Continue the rotation cycle as previously.

For drug treatment on the tumor slices, add 2.5 milliliters of media with the diluted drug or vehicle control into the six-well plate. Place the titanium grids into the wells, and then place the slices onto the grids as done previously. After performing the treatments for 24 hours, continue with fixing and processing.