Cultrex 3d culture cell harvesting kit

For transcriptome analysis, 45 organoids were transferred to a glass-bottom dish (µ-Dish 35 mm, high Grid-500 Glass, Ibidi, Germany). The following day, medium was replaced by FluoroBrite™ DMEM (Gibco, United States), and laser-based ablation of ten neighboring cells within each organoid was conducted. Another dish with 45 organoids prepared analogously and kept under the same experimental conditions remained untreated as control. The organoids were incubated at 37°C, 5% CO₂ and a humidified atmosphere for 4.5 h after cell ablation, followed by organoid harvesting using Cultrex^® 3D Culture Cell Harvesting Kit (Trevigen, MD, United States) according to the manufacturer’s protocol. After final centrifugation for 5 min at 850 × g and 4°C, organoids were resuspended in 1× DNA/RNA Shield™ (Zymo Research, CA, United States) and stored at −80 °C until all samples were collected. RNA isolation was conducted using Quick-RNA™ Microprep Kit (R1050, Zymo Research, CA, United States) following the manufacturer’s instructions. Subsequent library generation, RNA sequencing run, raw data processing, and differential expression analysis were performed by the Genomic core facility of Hannover Medical School.

Longitudinal 3D-outgrowth quantification assays were carried out by plating cells (9000 cells/cm²) atop solidified cushions of Cultrex reconstituted basement extract (50 µl/well; 96 well plate; Trevigen), containing 3 mg/ml collagen type I (BD Biosciences) when specified as rigid. Cells were cultured in complete media supplemented with 5% Cultrex, as well as the p38 MAPK inhibitor, SB203580 (10 μM, Calbiochem), or doxycycline (1 μg/ml, Sigma-Aldrich), where indicated. Bioluminescent readings were obtained on designated days by addition of D-luciferin potassium salt (Gold Biotechnology; Cat. No. LUCK), followed by quantification via GloMax-Multi detection system (Promega). Initial bioluminescent reading to which subsequent cell growth was normalized was obtained 24 hr after cell plating, and complete media/Cultrex was replaced after every addition of D-luciferin. Longitudinal 3D-outgrowth assays for subsequent immunoblotting were carried out by plating cells (31,500 cells/cm²) atop solidified Cultrex cushion (600 µl/well; 6 well plate) and grown in complete media supplemented with 5% Cultrex. For protein and RNA isolation, cells were non-enzymatically isolated using Cultrex 3D-Culture Cell Harvesting Kit (Trevigen) and subsequently prepared for immunoblotting or real-time PCR or microarray analysis, as described below.

D2.OR cells were nonenzymatically isolated from 3D-culture using the Cultrex 3D-Culture Cell Harvesting Kit (Trevigen), and RNA was extracted using the RNeasy Mini Kit (QIAGEN). For patient-derived xenograft and tumor biopsy specimens, tissues were homogenized in TRIzol reagent (1 ml TRIzol/100 mg tissue), followed by RNA extraction and removal of DNA with DNase I treatment (Invitrogen). qRT-PCR was carried out as described (Gooding et al, 2017 (link)) using the primers listed in Table S2.