Gibco™Advanced DMEM/F-12

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost the Matrigel in advance on ice or in fridge	Seed 2000 cells per well, in 24-well plates. Grow organoids for 11 days	Fix recovered organoids (overnight in 4.0% paraformaldehyde), embedd them (2.0% Bacto-Agar: 2.5% gelatin for paraffin embedding) and subjected to histologic analyses

Upstream tips
Defrost the Matrigel in advance on ice or in fridge
Protocol tips
Seed 2000 cells per well, in 24-well plates. Grow organoids for 11 days
Downstream tips
Fix recovered organoids (overnight in 4.0% paraformaldehyde), embedd them (2.0% Bacto-Agar: 2.5% gelatin for paraffin embedding) and subjected to histologic analyses

Publication protocol

Organoids from human esophageal biopsies were established as described in Figure 1. Briefly, endoscopic biopsy pieces were preserved in KSFM medium and kept on ice until further processing. KSFM was then replaced with 1 mL 10 U/mL Dispase (Corning, Corning, NY) and incubated for 10 minutes at room temperature. After rinsing with 1 mL phosphate-buffered saline to remove Dispase, the biopsy pieces were incubated in 0.5 mL 0.05% trypsin-EDTA at 37°C for 10 minutes and mixed concurrently at 700∼800 rpm using ThermoMixer C (Eppendorf, Hamburg, DE). Using the rubber plunger head taken from a tuberculin syringe, dissociated cells were gently forced through a 70-μm cell strainer and collected in a 50-mL conical tube containing 2 mL 250 μg/mL soybean trypsin inhibitor (Sigma-Aldrich). Cells were filtrated again into a 5-mL round bottom polystyrene tube with a 35-μm cell strainer snap cap (BD Bioscience, Franklin Lakes, NJ). Following centrifugation at 1500 rpm at 4°C for 5 minutes, cells were resuspended in 50–100 μL KSFM and counted by the Countess II FL Automated Cell Counter (Invitrogen, Carlsbad, CA) where cell viability was determined by Trypan Blue dye exclusion test. When EPC2-hTERT and derivatives were used to generate 3D organoids, monolayer cultures were trypsinized to prepare cell suspensions as described.23 Using 24-well plates, 2000 cells were seeded per well in 50 μL of ice cold 1:1 mixture of Matrigel basement membrane matrix (Corning) and KSFMC, and incubated at 37°C for 30 minutes. After solidification, 500 μL of KSFMC containing 10 uM Y27632 (Sigma-Aldrich) was added to each well on the first day, and then wells were replenished with KSFMC without Y27632 every other day. When indicated, the previously described fully supplemented aDMEM/F12 was used instead of KSFMC. Alternatively, KSFM was supplemented with the growth supplements used in aDMEM/F12.27 When indicated, both monolayer and 3D organoid cultures were grown in the presence or absence of 1 μM Compound E, a GSI or 1–40 ng/mL of TNF-α, 1–10 ng/mL interleukin (IL)-4, IL5, or IL13 (R&D) as described.8, 28, 29, 30 We first established nonlethal stimulatory concentration of cytokines by performing dose response (data not shown). 3D organoids were grown for 11 days, unless otherwise noted, and phase-contrast images were acquired to determine their number and size using a Nikon Eclipse E600 microscope (Nikon, Tokyo, Japan). Organoid formation rate (OFR) was defined as the average number of ≥50 μm spherical structures at Day 11 divided by the total number of cells seeded in each well at Day 0. 3D organoids were recovered by digesting Matrigel with Dispase (10 U/mL), dissociated into single cell suspensions by incubation with 0.05% Trypsin-EDTA at 37°C for 10 minutes, and reseeded for secondary and subsequent passages. When indicated, 3D organoid structures recovered from Matrigel were fixed overnight in 4.0% paraformaldehyde and embedded in 2.0% Bacto-Agar: 2.5% gelatin for paraffin embedding and subjected to histologic analyses.

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