The medium used in all organotypic 3D cultures was DMEM/F12 supplemented with 15% iFBS, 5% l -glutamine, 2.5% Pen-Strep, 1 μg/ml hydrocortisone, 0.2 U/ml insulin, 0.1 nmol/l cholera toxin and 25 ng/ml EGF (modified from [22 (link)]). In indicated experiments, the medium was further supplemented with Slit2 recombinant protein (0.5 µg/ml).
For imaging purposes and to perform cell viability assay, organotypic 3D cell culturing was performed primarily as described previously [23 (link)]. Briefly, cells were seeded as single cells between growth factor reduced Matrigel layers (Corning #356231) on 96-well angiogenesis µ-plates (Ibidi GmbH) in the density of 2000 cells/well. After Matrigel polymerization, medium was gently added on the top and replaced with fresh medium every 2–3 days. The formation of organoid-like structures was followed up to 12 days.
To extract RNA and protein lysates from organotypic 3D cell cultures, the cells were suspended in Matrigel to final concentration of 4 mg/ml and in the density of 250,000 cells/ml, and seeded on pre-heated tissue culture plates as drops (vol 80 µl). The dishes were first kept up-side down for 30 min in 37 °C until Matrigel was solidified. Subsequently, the medium was gently added to cover the drops and it was changed every 2–3 days during the 12 days culturing period.
For imaging purposes and to perform cell viability assay, organotypic 3D cell culturing was performed primarily as described previously [23 (link)]. Briefly, cells were seeded as single cells between growth factor reduced Matrigel layers (Corning #356231) on 96-well angiogenesis µ-plates (Ibidi GmbH) in the density of 2000 cells/well. After Matrigel polymerization, medium was gently added on the top and replaced with fresh medium every 2–3 days. The formation of organoid-like structures was followed up to 12 days.
To extract RNA and protein lysates from organotypic 3D cell cultures, the cells were suspended in Matrigel to final concentration of 4 mg/ml and in the density of 250,000 cells/ml, and seeded on pre-heated tissue culture plates as drops (vol 80 µl). The dishes were first kept up-side down for 30 min in 37 °C until Matrigel was solidified. Subsequently, the medium was gently added to cover the drops and it was changed every 2–3 days during the 12 days culturing period.
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