Hi Felix,
I have bolded minor corrections (e.g. the need to abbreviate minutes to min and days to d or the need to write matrigel as Matrigel™ etc.
Tissue samples
The tissue used in this study was collected from patients recruited from the Dunedin Public Hospital. During either elective colonoscopy or resection, samples of the transverse colon were collected. In the event of a bowel resection, in which benign or malignant tissue is removed, ~2 mm2 samples were collected ~15 cm proximal or distal from the site of pathology.
- Two types of sample – large and biopsy?
-
Crypt Isolation
Tissue samples were transported to the laboratory in ice cold phosphate buffer (PBS, in mM 137 NaCl, 2.7 KCl, 4.3 Na2HPO4, 1.47 KH2PO4, pH 7.4, Life Technologies, USA) containing antibiotics, including Normacin (0.1 mg/ml, Integrated Science, NZ), Fungizone (2.5 µg ml-1, Life Technologies, USA) and Gentamycin (50 µg ml-1, Life Technologies, USA).
Samples were first washed in PBS to remove any faecal material. For resection samples the underlying connective tissue and muscle layers were removed via blunt dissection, isolating the epithelial layer which was subsequently cut into ~5 mm2 pieces. Both resection and biopsy samples were placed in a 15 ml tube containing 10 mM dithiothreitol (DTT, Sigma, USA) in PBS. Samples were mechanically washed until debris was no longer visible. Next, samples were incubated on ice in 8 mM, sterile, ice cold ethylenediaminetetraacetic acid (EDTA) in PBS. After incubation the EDTA was replaced with ice cold PBS, before the tissue was gently shaken to release the colonic crypts. Once the gross tissue settled, the crypt enriched supernatant was transferred to a 50 ml tube, at which point Foetal Bovine Serum (FBS, Gibco®, Thermo Fisher Scientific, NZ) was added to reach a final concentration of 5% (250 µl FBS/5 ml PBS). The gross tissue sample was then resuspended in ice cold PBS, and the above steps repeated six to eight times. Once the supernatant was clear, the crypt enriched supernatant was centrifuged at 40x g for 2 mins at 4 °C. The pellet was resuspended in Dulbecco modified Eagle medium (DMEM)/F12 containing penicillin/streptomycin (Pen/Strep, 100 1%, Life Technologies, USA), normacin (described above), Hepes (10 mM), Glutamax (5 µl/ml, Invitrogen, USA), N2 (1X, Invitrogen, USA), B27 (1X, Invitrogen, USA) + 5% FBS, spun down at 40x g for 2 mins at 4 °C, and the supernatant removed. In a 1.5 Eppendorf tube the pellet was resuspended in advanced DMEM/F12 (described above) + 5% FBS before a final centrifuge at 0.1 xg for 2 mins at 4 °C. The crypts were then suspended in Matrigel™. 1-2 drops of the Matrigel™ was pipetted into the required number of wells of a pre-warmed (37 °C) 24 well plate (Thermo Fisher Scientific, Walktham, MA, USA). The plate was incubated at 37 °C for 10 mins to polymerise the Matrigel™ before each well was overlaid in 500 µl of pre-warmed (37 °C) organoid growth media (Table X). Organoids were incubated at 37 °C in a 5% CO2/air mix.
Additive | Final Concentration | Supplier |
Advanced DMEM/F12 | ?? | Invitrogen, USA |
B27 | 1X | Invitrogen, USA |
Gastri | 1 µg/ml | Pharmaco |
Glutamax | 1X | Invitrogen, USA |
Hepes | 10 Mm | Sigma, USA |
Human Epidermal growth factor | 50 ng/ml | Invitrogen, USA |
LY 2157299 | 500 Nm | AxonMedChem, USA |
N2 | 1 µM | Invitrogen, USA |
N-acetyl-L-cysteine | 1 Mm | Sigma, USA |
Nicotinamide | 10 Mm | Sigma, USA |
Noggin | 100 ng/ml | PeproTech, USA |
Normacin | 0.1 mg/ml | Integrated Science, NZ |
Pen/Strep | 1% | Invitrogen, USA |
Prostaglandin E2 | 0.01 µM | Sigma, USA |
R-spondin conditioned media | 10% | - |
SB 202190 | 10 µM | Sigma, USA |
Wnt3A conditioned media | 50% | - |
Organoid culture
During establishment, organoids were incubated in organoid growth media described in Table X. The media was prepared in 50 ml aliquots, and stored at 4 °C until required. Organoid media was replaced every 2-3 d, with old media being removed, and replaced with 500 µl of pre-warmed organoid growth media.
Passaging
Unless being used for an experiment, or if growth was insufficient, organoids were amplified every 5-7 d via passage. In a sterile environment, media was removed from each well before washing the organoids in 500 µl ice cold PBS. The PBS was carefully removed, before a second PBS wash was carried out. This PBS was gently aspirated up and down to dislodge the Matrigel™, allowing transport to a 1.5 ml Eppendorf tube. The well was then washed with a further 500 µl of PBS to remove any remaining Matrigel™. This step was carried out until all residue was collected. The Matrigel™ was then incubated on ice for 30 mins to dissolve the Matrigel™. Once dissolved, the Matrigel™ was spun down in a pre-cooled centrifuge (4 °C) at 900x g for 2 mins. The supernatant was then removed, and the Matrigel™ resuspended in ice-cold PBS to reach a final volume of 1 ml. Using a 1ml syringe, organoids were carefully sucked through a 25 gauge needle to mechanically break up the organoids into fragments. This was done at least 6 times to ensure sufficient organoid disruption. The fragments were then centrifuged at 900 xg for 2.5 mins (4 °C). The supernatant was carefully removed, and the fragments resuspended in Matrigel™ (45 µl/well). The Matrigel™ was then pipetted into the required number of wells in a pre-warmed (37 °C) 24-well plate before being overlaid in 500 µl of pre-warmed (37 °C) organoid growth media. During the first addition of media after the first 3 passages, the Rock inhibitor Y-27623 (final concentration 10 mM, Merek Millipore, USA) was added to the organoid growth media to reach a final concentration of 1% (5 µl Y-27627/495 µl organoid growth media). Organoids were cultured in this media for 2 d, before it was replaced with normal organoid growth media, which was subsequently replaced every 2-3 d.
Varying Wnt media
Organoids were passaged and cultured for 4 d before experimental treatment began. On day four photos were taken with a using a camera mounted on a dissecting microscope (Leica DM2700 M, Leica Biosystems, Germany), before each well was washed twice for five mins with PBS. The Matrigel™ was then incubated in organoid media containing either 0, 5, 10, 25 or 50% wnt3A conditioned media at 37°C for 11 d. Wnt3A media was replaced with appropriate volumes of DMEM/F12. The media was replaced with organoid media containing appropriate levels of wnt3A conditioned media every two d. Although the absolute concentration of wnt3A present in the wnt3A conditioned media is unknown, the same batch of wnt3A conditioned media was used for all experiments. At day 15, a further set of photos were taken before the organoids were processed for histochemistry (See XX).
Notch inhibition
Organoids were passaged and cultured for 4 d before experimental treatment began. On day four photos were taken with a dissecting microscope (Leica DM2700 M, Leica Biosystems, Germany), before each well was washed twice for five mins with PBS. The Matrigel™ was then incubated in organoid media containing either 5% wnt conditioned media ± N-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine 1,1-dimethylethyl ester (DAPT, a Notch inhibitor, Tocris, UK), 10% wnt conditioned media ± DAPT or 50% wnt conditioned media ± DAPT. DAPT was added to the respective media to reach a final concentration of 0.05% (0.25 µl DAPT/500 µl varying wnt media). At day 15, a further set of photos were taken before the organoids were processed for either histochemistry or qPCR (See XX).