Generation and Expansion of Primary, Malignant Pleural Mesothelioma Tumor Lines

This protocol provides a basis for tumor cell expansion that, in turn, allows for an in-depth study of tumor cell function and genomic states. In addition, it provides an in vitro model system to assess the impact of the tumor on the immune response. The main advantage of this technique is it provides a based protocol for establishing mesothelial tumor lines from patient tissue.

There's currently no standardized protocol for this model system. The most difficult aspect of this technique is the presence of fibroblast contamination in early passage cultures. It is critical to have a solid understanding of fibroblasts versus tumor cell morphology to determine whether fibroblast starvation attempts are working.

Begin by transferring 10 milliliters of tumor digesting enzymatic cocktail to a dissociation tube. Transfer the piece of tumor tissue to a sterile six-well plate lid using a sterile scalpel. Remove all fatty necrotic tissues and blood clots using a new sterile scalpel and forceps.

Cut the entire tumor tissue into one cubic millimeter small fragments. To ensure the tissue does not dry out, add 500 microliters of sterile Hank's Balanced Salt solution, or HBSS, to the tumor tissue. Transfer tumor fragments into the dissociation tube containing 10 milliliters of tumor digesting enzymatic cocktail.

Close the tube tightly. Place it cap side down on the tissue dissociator and supply heat to the dissociator. Select the pre-programmed setting on the dissociator to the 37 degree Celsius human tumor dissociation kit setting 1 and check the status after 10 minutes to ensure no clogging error is indicated by the flashing red light.

After one hour, remove the dissociation tube and place it in a laminar flow hood. Filter the digested tumor by placing a 70 micrometer cell strainer on a 50 milliliter conical tube and pipetting the digested tumor using a 10 milliliter pipette onto the filter. If the filter clogs, switch to a new filter.

Rinse the dissociation tube with 10 milliliters of fresh tumor digestion media and wash through the filter. Remove and discard the filter. Bring the total volume to 40 milliliters with the tumor digestion medium.

Centrifuge at 500 x g for five minutes at room temperature. Using a two milliliter aspirating pipette attached to a vacuum source, aspirate the supernatant without disturbing the pellet and resuspend the cells using 10 milliliters of sterile tumor medium. Then, again, centrifuge the tubes and aspirate the supernatant as demonstrated.

Next, resuspend the cells in three milliliters of sterile tumor medium and transfer the cell suspension to one well of a sterile six-well plate. Keep the plate in an incubator at 37 degrees with 5%carbon dioxide. After 24 hours, transfer the used medium from the digested tumor in well one to a new well in the same six-well plate, then add three milliliters of sterile tumor medium to well one.

Using an inverted phase microscope, determine the percentage of fibroblasts contamination in the early passage culture. If the fibroblasts contamination is greater than 20%of the cells in the early passage culture, continue culturing after replacing the tumor medium with the reduced serum medium. If the fibroblast population is not reduced to 10%or less, gently rinse the well with PBS to remove the medium containing serum.

Add one milliliter of trypsin per well in a six-well plate. Place the six-well plate or flask in an incubator at 37 degrees Celsius for one minute. Remove the plate or flask from the incubator and check the adhesion of the cells using an inverted microscope.

When the cells are partially lifting or floating, gently remove the suspended cells and trypsin only. Place the cells in a 15 milliliter conical tube containing an equal or greater volume of complete tumor medium. Repeat trypsinizing until there are three to four fractions removed.

Centrifuge all independent fractions at 500 x g for five minutes at room temperature. Using a two milliliter aspirating pipette attached to a vacuum source, aspirate the supernatant without disturbing the pellet. Resuspend the cells using five milliliters of sterile reduced serum medium.

Plate the cells in a T-25 flask for each fraction and place the flasks in an incubator at 37 degrees Celsius. For cryopreservation of early passage cells, thaw one tube of aliquoted freeze medium. Collect the cells by trypsinization by first washing the plate with PBS and adding trypsin as demonstrated previously.

Place the flask in an incubator at 37 degree Celsius for three minutes. Remove the flask from the incubator and check the adhesion of the cells using an inverted microscope. If the cells are lifting or floating, gently remove the suspended cells and trypsin.

Place the cells in a 15 milliliter conical tube containing an equal or greater volume of complete tumor medium. Mix the contents of the tube by pipetting gently. Remove 20 microliters of cell suspension for counting.

Then centrifuge the remaining cell suspension at 500 x g for five minutes at room temperature. When the cells are spinning, count using a lab specific counting protocol, such as trypan blue or acridine orange/propidium iodide. Resuspend the cells following centrifugation in freeze medium with a minimum of 5 x 10 to the sixth cells per milliliter of freeze medium and transfer one milliliter of this cell suspension to pre-labeled 1.5 milliliter of cryovials.

Place the cryovials in a controlled rate freeze chamber and transfer them immediately to minus 80 degrees Celsius. The figure shows increasing fiberblast contamination of 80%50%and 30%compared to a culture with no fibroblasts contamination. This figure shows representative flow cytometry surface staining of four primary mesothelioma tumor lines, MESO171, MESO176, NCIH2452, MS TO-211H and a melanoma tumor line, MEL526 as a negative control.

These cell lines can express both mesothelin and N-cadherin. The importance of testing the impact of enzymatic detachment on surface protein marker expression is also shown as both trypsin and the protease collagenase mixture resulted in the loss of surface expression of N-cadherin, while CD90 was not impacted. Important steps include proper processing of the tumor including removal of blood and fat before digestion, identifying fibroblast contamination, and determining how to limit fibroblast overgrowth is vital.

An important method following this procedure includes cytotoxic lymphocyte assays to test the ability of autologous T cells to recognize patient-derived tumor cells. This technique will identify new anti-tumor-specific T-cell receptors that could be of therapeutic importance. In addition, it will allow us to study the interaction between tumor and paired tumor infiltrating lymphocytes and immune dysfunction.