Human egf

Endoscopic biopsy samples obtained from the terminal ileum were processed under a dissecting microscope to liberate intestinal crypts using EDTA chelation and mechanical disruption. Crypts were screened through a 100 μm filter, centrifuged, and suspended in ice-cold Matrigel. The suspension was plated on prewarmed cell culture plates. Following polymerization of Matrigel, propagation media (50% LWRN conditioned media from ATCC CRL-3276, supplemented with human EGF (Peprotech), A-83-01 (Tocris), SB202190 (Sigma), Gastrin (Sigma), Nicotinamide (Sigma), B27 (Life Technologies), N2 (Life Technologies), GlutaMax (Life Technologies), and HEPES (Sigma) in F12 Advanced DMEM (Life Technologies)) was added. For the first 48 h of culture, CHIR99021 (Stemgent) and thiazovin (Stemgent) were added to support stem cell growth. To induce differentiation, media was replaced with differentiation media (consisting of base culture media supplemented with 10% R-Spondin conditioned media, human EGF (Peprotech), human Noggin (Peprotech), A-83-01, Gastrin, NAC, B27, and N2).

Generation and culture of FT organoids was performed as described in Kessler et al^{13 (link)}. In brief, epithelial progenitors from human FT tissue samples were isolated by enzymatic digestion with collagenase I (Sigma). The retrieved primary cells were seeded in 2D culture for 5–7 days (ADF medium with 12 mM HEPES, 1% GlutaMAX^TM, 10 ng ml⁻¹ human EGF and 9 µM Y-27632) before seeding in Matrigel^TM (~30,000 cells/50 µl) for 3D organoid formation. Once the Matrigel^TM had set, cultures were overlaid with medium containing a specific growth factor cocktail to preserve stemness and support differentiation (ADF, 25% conditioned mouse Wnt3A-medium as described in Willert et al.^{39 (link)} and 25% conditioned mouse RSPO1 medium^{40 (link)}, supplemented with 12 mM HEPES, 1% GlutaMAX^TM, 2% B27, 1% N2, 10 ng ml⁻¹ human EGF (all from Invitrogen), 100  ng ml⁻¹ human noggin, 100 ng ml⁻¹ human FGF-10 (both from Peprotech), 1 mM nicotinamide, 9 µM ROCK inhibitor (Y-27632, both from Sigma) and 0.5 µM TGF-β RI Kinase Inhibitor IV (SB431542, Calbiochem)). For propagation, organoids were split every 2 to 3 weeks at a ratio of 1:2 to 1:3 using mechanical splitting with a syringe and needle (26 G gauge). Organoids were kept in a humidified incubator at 5% CO₂ and 37 °C, or 35 °C once infected.

The cells were plated in six-well ultra-low attachment plates (20 000 cells per well) (Corning, Corning, NY, USA) in serum-free DMEM/F12 medium, then added with 20 ng/ml human EGF, 10 ng/ml human bFGF, and 2% B27 (Invitrogen, Carlsbad, CA, USA). Cells were incubated at 37 °C with 5% CO₂. After 2 weeks, plates were analyzed for tumorsphere formation and counted by microscope.