Recombinant egf

Patient-derived organoids culture of colorectal cancer was performed as previously described.^{42 (link),43 (link)} Fresh tumor tissues collected from CRC patients were washed three times with ice-cold PBS containing penicillin-streptomycin, and minced into small pieces (3–5 mm) quickly, then digested with EDTA (5 mM) for 60 min on ice. When the cells were in clumps under microscopic observations, finished digestion. The cells were mixed with Matrigel (Corning) and then seeded into a 24-well plate. When Matrigel polymerisation (10 min at 37 °C), added 500 μL advanced DMEM/F12 medium containing 10 mM HEPES, 1× N2 (Life Technologies), 2 mM GlutaMAX, 1× B27, 10 nM gastrin I (Biogems), 50 ng/mL recombinant EGF, 500 nM A83-01 (Biogems), 100 ng/mL recombinant Noggin (Peprotech), 500 ng/mL R-spondin-1 (Peprotech), 10 μM SB202190 (Sigma), 10 μM Y-27632 (Abmole), 10 mM nicotinamide (Sigma), 1 mM N-acetylcysteine (Sigma) and 100 U/mL penicillin/streptomycin. 24 h later, the organoids were infected with a non-targeting hairpin or shCSN6 lentivirus using polybrene (10 μg/mL, Millipore, TR-1003-G) for 6 hours, then replaced the medium with fresh medium and renewed every three days. The organoid images were taken at day 0, 3, 6, 9. The size and number of organoids were quantified using Fiji.

Human CRC organoid culture was performed as previously described.^{28 (link)} Fresh CRC surgical specimens were washed with cold phosphate-buffered saline (PBS) and cut into small pieces, then digested with EDTA. After digesting into clumps of cells, the sample was seeded into Matrigel in 24-well plates. Following Matrigel polymerisation (10 min at 37 °C), 500 μl human culture media (Advanced DMEM/F12 containing, 1× N2 (Life Technologies), 10 mM HEPES, 2 mM GlutaMAX, 1× B27, 10 nM gastrin I (Biogems), 50 ng/ml recombinant EGF, 500 nM A83-01 (Biogems), 100 ng/ml recombinant Noggin (Peprotech), 500 ng/ml R-spondin-1 (Peprotech), 10 μM SB202190 (Sigma) 10 μM Y-27632 (Abmole), 10 mM nicotinamide (Sigma), 1 mM N-acetylcysteine (Sigma) and penicillin/streptomycin. The sample was transduced with 500 μl of virus supernatant (scrambled or CSN6-specific shRNA) in the presence of polybrene (Millipore, TR-1003-G) and incubated for 8 h, then changed the fresh culture medium and renewed the culture medium every 3 days. The organoid numbers in each well were quantified at day 12.