Gibco™Advanced DMEM/F-12

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost the BME in advance on ice or in fridge		Possibility to generate stocks of organoids to culture in a future

Upstream tips
Defrost the BME in advance on ice or in fridge
Downstream tips
Possibility to generate stocks of organoids to culture in a future

Publication protocol

Primary 3D cultures generated from surgical BC samples: cancer tissue-originated spheroids/organoids: Human bladder tumor specimens were collected from patients diagnosed with BC and treated with transurethral resection at the Department of Urology at Stony Brook. Because our current organoid lines were established from transurothelial resection of bladder tumor specimens, they were enriched for nonmuscle-invasive bladder tumors, which represent the majority of urothelial cancers. In this study, we used five organoid cultures developed from low-grade nonmuscle-invasive tumors of stage Ta or T1. S1, S2, S3 and S4 cell lines were tested at passage 1–2. The organoid line S2-1 was started from a previously frozen S2 sample (at passage 2). This study was approved by SUNY at Stony Brook Institutional Review Board committee and written informed consent was obtained from all patients involved. All tissue samples were examined by a dedicated uropathologist. Cancer tissue-originated spheroid (CTOS) preparation and culture were performed as described previously, with some modifications [13]. The approach was based on the partial preservation of intercellular junctions between urothelial cells during mild mechanical and enzymatic digestion with Liberase™ DH (dispase high). Briefly, primary tumors were minced finely in the tube using a pair of small scissors and digested enzymatically for 2 h using Liberase DH (Roche Applied Science, WI, USA) in advanced Dulbecco’s modified Eagle medium/Ham’s F-12 (Thermo Fisher). ROCK inhibitor (Y-27632, 10 μM) was always added to the starting culture and markedly increased culture survival, as indicated [14]. After passing the resulting mixture through filters and cell strainers, the cell fractions retained on the strainers with mesh 40 μm – termed the organoid fraction – were collected. The organoids were cultured with CTOS medium (advanced Dulbecco’s modified Eagle medium/Ham’s F-12, B27 [2% Thermo Fisher 17504001], A83-01 [5 μM], N-acetylcysteine [1.25 mM] and nicotinamide [10 mM]) [3]. For CTOS maintenance we add the combination of FGF10 (100 ng/ml of PeproTech 100-26), FGF7 (25 ng/ml of PeproTech 100-19), FGF2 (12.5 ng/ml of PeproTech 100-18B) and HER3 100 ng/ml from PeproTech (NJ, USA). For growth and cytotoxicity assays, CTOSs were further digested with TrypsinLE and additionally mechanically disrupted by pipetting. Single cells were resuspended in 20 μl of BME and placed into wells of a 96-well plate. When the BME was solidified, CTOS medium was added. Organoids were frozen in freezing medium (50% fetal bovine serum, 10% DMSO) and 40% organoid medium and could be recovered efficiently as described [3].

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing 3D Cell Culture Media Human bladder cancer organoid using Gibco™Advanced DMEM/F-12 from Thermo Fisher Scientific.

Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for Gibco™Advanced DMEM/F-12 below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing 3D Cell Culture Media Human bladder cancer organoid using Gibco™Advanced DMEM/F-12 from Thermo Fisher Scientific. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.