DMEM:F12 with HEPES, Glucose and L-Glutamine

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost the Matrigel in advance on ice or in fridge	Use pre-warmed six-well culture plates

Upstream tips
Defrost the Matrigel in advance on ice or in fridge
Protocol tips
Use pre-warmed six-well culture plates

Publication protocol

Tissue preparation and culture of 2D-LC cells, LCOs and NBOs: Parts of the human samples (5–6 pieces of 1 mm3 tissue) were separated and transported to the laboratory on ice within 1 h of removal from the patients in cold Hank’s balanced salt solution (HBSS) with antibiotics (Lonza, Basel, Switzerland). Samples were washed three times with cold HBSS with antibiotics and were sectioned with sterile blades. Approximately two-thirds of the sectioned samples were used to establish PDX models, and the rest were incubated with 0.001% DNase (Sigma-Aldrich, MO, USA), 1 mg ml−1 collagenase/dispase (Roche, IN, USA), 200 U ml−1 penicillin, 200 mg ml−1 streptomycin, and 0.5 mg ml−1 amphotericin B (2% antibiotics, Sigma) in DMEM/F12 medium (Lonza) at 37 °C for 2 h with intermittent agitation. After incubation, the suspensions were repeatedly triturated by pipetting and passed through 70-μm cell strainers (BD Falcon, CA, USA). The strained cells were centrifuged at 112 × g for 3 min, and the pellet was resuspended in 100 μl MBM (serum-free medium (DMEM/F12; Lonza) supplemented with 20 ng ml−1 of bFGF (Invitrogen, CA, USA), 50 ng ml−1 human EGF (Invitrogen), N2 (Invitrogen), B27 (Invitrogen), 10 μM ROCK inhibitor (Enzo Life Sciences, NY, USA), and 1% penicillin/streptomycin (Gibco, OK, USA).
To evaluate tissue quality, 5 μl of the 100 μl cell suspension was put on a slide. After drying the medium in room temperature (RT), the cells were fixed in 100% ethanol followed by standard H&E staining. In parallel, 50 μl of the 100 μl cell suspension was seeded onto collagen-I-coated dishes (Corning, NY, USA) and cultured as 2D-LC cells in MBM for 5–7 days. One hundred microliters Matrigel (Corning) was added to the remaining 50 μl suspension for establishing LCOs, and the resulting cell suspension was allowed to solidify on pre-warmed six-well culture plates (Corning) at 37 °C for 10 min. After gelation, 3 ml MBM was added to the well. The medium was changed every 4 days, and the organoids were passaged after 1–3 weeks. For passaging, a solidified Matrigel drop containing the LCOs was harvested using cold DPBS and then centrifuged at 112 × g for 3 min at 4 °C. The pellets were washed with cold DPBS and centrifuged at 250 rcf for 15 min at 4 °C. The organoids were resuspended in 2 ml TrypLE Express (Invitrogen) and incubated for 10 min at 37 °C for dissociation. Afterwards, 10 ml DMEM/F12 containing 10% FBS was added and centrifuged at 112 × g for 3 min. The pellets were washed with DPBS and centrifuged at 112 × gfor 3 min. The pellets were resuspended in MBM + Matrigel (1:3) and reseeded at 1:3–1:4 ratios to allow the formation of new organoids. For single cell analysis, dissociated cells from organoids were seeded as single cells with Matrigel in in-house developed micro-well device. Only the wells containing a single cell were monitored. To culture NBOs, normal bronchial samples were used and the same steps as described above for LCO cultures were followed, except that the NBOs were cultured in MBM + WNA (MBM supplemented with 30% Wnt 3A conditioned medium, 100 ng ml−1 Noggin (Peprotech, NJ, USA), and 500 nM A83-01 (Peprotech).
Organoids successfully cultivated during passage 3 were regarded as success in organoid formation. Three-fourth were cryopreserved in three vials and one-fourth was cultivated additional one passage (passage 4) to expand organoids amount, and cryopreserved in six vials. More specifically, after the dimeter of organoids reached up 100–150 μm, organoids (>passage 3) were banked. To stock organoids, organoids were harvested using cold DPBS and then centrifuged at 112 × g for 3 min at 4 °C. The pellets were washed with cold DPBS and centrifuged at 250 rcf for 15 min at 4 °C. Supernatant was removed and organoid pellet was resuspended in freezing media; Culture Media 7: ES grade FBS (Gibco) 2: DMSO (Sigma) 1. The stock vials were stored in gas nitrogen tank.



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 lung cancer organoids using DMEM:F12 with HEPES, Glucose and L-Glutamine from Lonza .

Manufacturer protocol

Download the product protocol from Lonza for DMEM:F12 with HEPES, Glucose and L-Glutamine below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing 3D Cell Culture Media Human lung cancer organoids using DMEM:F12 with HEPES, Glucose and L-Glutamine from Lonza . Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.