Pleural effusion fluid was obtained immediately at the time of thoracentesis and was spun at 1000 RPM to create a cell pellet. The pellet was washed two to three times in Red Blood Cell lysis buffer (Biolegend Catalog number 420301), washed once in basal culture media [ (Advanced DMEM/F12 (Thermo Fisher 12634028), supplemented with 1% penicillin streptomycin, 1X Glutamax (Life Technologies Catalog number 35050061), and 1% HEPES (Life Technologies Catalog number 15630080)], and the cell pellet was allocated for WES, histology, and organoid generation. For solid tumors, the tumor was acquired immediately during the surgery. Some fresh tumor was snap frozen for later WES or fixed immediately in 10% formalin for later histologic analysis. Remaining tumor tissue was transported to the laboratory in basal culture media. The tumor was diced into approximately 2 mm sections and then crushed with the butt of a syringe. This homogenate was then poured into basal culture media containing Type II Collagenase (Life Technologies # 17101015) at a final concentration of 2.5mg/mL. The homogenate was shaken at 37°C for no longer than 30 minutes. The homogenate was then diluted 1:1 with basal culture media and filtered through a 70uM filter (Falcon Catalog number 352350). The cell suspension was then spun at 1000RPM to create a cell pellet. The pellet was washed with red blood cell lysis buffer two to three times, and then washed once with basal culture media.
For general culture, once a pellet of either liquid or solid tumor cells was obtained, the cells were mixed with growth factor reduced Matrigel (Corning Catalog number CB-40230C), with the final concentration of Matrigel at 75%, and there were approximately 10,000 or more cells/cell groups per 10uL droplet of Matrigel. The suspension was then rapidly plated into a 48-well plate with 15uL of suspension per well. Once the Matrigel was solidified, 250uL of general culture medium was added to each well. General culture medium was composed of Advanced DMEM/F12, supplemented with 1% penicillin streptomycin, 1X Glutamax, 1% HEPES, 100 ng/mL R-spondin 1 (Peprotech catalog number 120–38), 100 ng/mL Noggin (Peprotech catalog number 120–10C), 50 ng/mL EGF (Peprotech catalog number 100–15), 10 ng/mL FGF-10 (Peprotech catalog number 100–26), 10 ng/mL FGF2 (Peprotech catalog number 100–18B), 1× B27 (Life Technologies Catalog number 17504044), 10 mM Nicotinamide (Sigma Aldrich Catalog Number N0636), 1.25mM N-acetylcysteine (Sigma Aldrich catalog number A9165), 1uM Prostaglandin E2 (Catalog number R&D Systems Catalog number 2296), 10uM SB202190 (Sigma Aldrich Catalog number S7076), and 500nm A83–01 (Sigma Aldrich catalog number SML0788). Y-27632 dihydrochloride (AbMole Bioscience Catalog number M1817) was initially tested in early passage organoid cultures at a concentration of 10uM but eventually deemed unnecessary for culture maintenance. Overall tissue digestion and cell plating were similar to that described in Drost, et al 2016 (14 (link)).
For general culture, once a pellet of either liquid or solid tumor cells was obtained, the cells were mixed with growth factor reduced Matrigel (Corning Catalog number CB-40230C), with the final concentration of Matrigel at 75%, and there were approximately 10,000 or more cells/cell groups per 10uL droplet of Matrigel. The suspension was then rapidly plated into a 48-well plate with 15uL of suspension per well. Once the Matrigel was solidified, 250uL of general culture medium was added to each well. General culture medium was composed of Advanced DMEM/F12, supplemented with 1% penicillin streptomycin, 1X Glutamax, 1% HEPES, 100 ng/mL R-spondin 1 (Peprotech catalog number 120–38), 100 ng/mL Noggin (Peprotech catalog number 120–10C), 50 ng/mL EGF (Peprotech catalog number 100–15), 10 ng/mL FGF-10 (Peprotech catalog number 100–26), 10 ng/mL FGF2 (Peprotech catalog number 100–18B), 1× B27 (Life Technologies Catalog number 17504044), 10 mM Nicotinamide (Sigma Aldrich Catalog Number N0636), 1.25mM N-acetylcysteine (Sigma Aldrich catalog number A9165), 1uM Prostaglandin E2 (Catalog number R&D Systems Catalog number 2296), 10uM SB202190 (Sigma Aldrich Catalog number S7076), and 500nm A83–01 (Sigma Aldrich catalog number SML0788). Y-27632 dihydrochloride (AbMole Bioscience Catalog number M1817) was initially tested in early passage organoid cultures at a concentration of 10uM but eventually deemed unnecessary for culture maintenance. Overall tissue digestion and cell plating were similar to that described in Drost, et al 2016 (14 (link)).
