Gibco™Advanced DMEM/F-12

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost in advance the cultrex growth factor reduced BME type 2 gel on ice or in fridge	If the cell pellet is red, lyse the erythrocytes Prewarm 24-well suspension culture plates Change medium every 4 days, passage of the organoids every 1-4 weeks Culture every line of organoids physically separated to prevent cross-contamination

Upstream tips
Defrost in advance the cultrex growth factor reduced BME type 2 gel on ice or in fridge
Protocol tips
If the cell pellet is red, lyse the erythrocytes Prewarm 24-well suspension culture plates Change medium every 4 days, passage of the organoids every 1-4 weeks Culture every line of organoids physically separated to prevent cross-contamination

Publication protocol

Upon arrival, tissues were photographed and cut into 1-3 mm3 pieces. Two random pieces were snap frozen and stored at −80°C for DNA isolation, two random pieces were fixed in formalin for histopathological analysis and immunohistochemistry, and the remainder was processed for the isolation of viable cells. The remaining tissue was minced, washed with 10 mL AdDF+++ (Advanced DMEM/F12 containing 1x Glutamax, 10 mM HEPES, and antibiotics) and digested in 10 mL BC organoid medium (Table S2) containing 1-2 mg·ml-1collagenase (Sigma, C9407) on an orbital shaker at 37°C for 1-2 h. The digested tissue suspension was sequentially sheared using 10 mL and 5 mL plastic and flamed glass Pasteur pipettes. After every shearing step the suspension was strained over a 100 μm filter with retained tissue pieces entering a subsequent shearing step with ∼10ml AdDF+++. 2% FCS were added to the strained suspension before centrifugation at 400 rcf. The pellet was resuspended in 10ml AdDF+++ and centrifuged again at 400 rcf. In case of a visible red pellet, erythrocytes were lysed in 2 mL red blood cell lysis buffer (Roche, 11814389001) for 5 min at room temperature before the addition of 10ml AdDF+++ and centrifugation at 400 rcf. Needle biopsies of metastatic BC lesions were processed as above following removal of macroscopically obvious non tumor tissue (e.g., liver). For organoid culture, the pellet was resuspended in 10 mg·ml-1 cold Cultrex growth factor reduced BME type 2 (Trevigen, 3533-010-02) and 40 μL drops of BME-cell suspension were allowed to solidify on prewarmed 24-well suspension culture plates (Greiner, M9312) at 37°C for 20 min. Upon completed gelation, 400 μL of BC organoid medium (Table S2) was added to each well and plates transferred to humidified 37°C / 5% CO2 incubators at either 2% or ambient O2. Medium was changed every 4 days and organoids were passaged every 1-4 weeks: cystic organoids were resuspended in 2 mL cold AdDF+++ and mechanically sheared through flamed glass Pasteur pipettes. Dense organoids were dissociated by resuspension in 2 mL TrypLE Express (Invitrogen, 12605036), incubation for 1-5 min at room temperature, and mechanical shearing through flamed glass Pasteur pipettes. Following the addition of 10 mL AdDF+++ and centrifugation at 300 rcf. or 400 rcf. respectively, organoid fragments were resuspended in cold BME and reseeded as above at ratios (1:1 to 1:6) allowing the formation of new organoids. Single cell suspensions were initially seeded at high density and reseeded at a lower density after ∼1 week. In order to prevent misidentification and/or cross-contamination of BC organoids, we cultured every line physically separate, implemented the use of a laboratory management system, and validated the identity of biobanked organoid lines by single-nucleotide polymorphism (SNP) based fingerprinting. All organoid lines tested negative in the MycoAlert mycoplasma detection kit (Lonza, LT07-318).

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