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1. Small bowel neuroendocrine tumor (SBNET) collection and cell dissociation
- Obtain resected patient SBNET samples after tumor tissue confirmation from the Surgical Pathology Core.
- Cut SBNETs into 5 mm cubes and store them in 25 mL of Dulbecco's Modified Eagle Medium (DMEM)/F12 medium in a conical tube for transportation to the laboratory.
- Transfer the tumors to DMEM containing 1% fetal bovine serum or FBS, 1% penicillin/streptomycin (Pen/Strep), and 1% glutamine (Wash Medium), and incubate in this Wash Medium for 15 min.
- Transfer the tumors to a new dish and mince them into pieces less than 1 mm in diameter using sterile curved scissors.
- Transfer the minced tissues to a new 50 mL tube containing 25 mL of Wash Medium.
- Centrifuge the sample at 500 x g for 15 min at 4 °C.
- Discard the supernatant and resuspend the pellets in 10 mL of Wash Medium containing collagenase (100 U/mL) and DNase (0.1 mg/mL).
- Allow the minced tumors to be digested in a 37 °C incubator with slow shaking (50 rpm) for 1.5 h.
2. Culture of SBNETs as tumor spheroids in Extra Cellular Matrix (ECM)
- After digestion is completed (step 1.8), centrifuge at 500 x g for 15 min at 4 °C, discard the supernatant and resuspend the pellet in 15 mL of Wash Medium.
- Place a 70 µm cell strainer on top of a new 50 mL tube and transfer 10 mL of the Wash Medium over the cell strainer. Swirl the Wash Medium to cover the side of the plastic tube to prevent NET cells from sticking to the side of the plastic tube.
- Filter the cell suspension through a cell strainer.
- Centrifuge at 500 x g for 15 min at 4 °C, discard the supernatant, resuspend the pellet in 200 µL of Wash Medium (total volume is ~ 250 µL), and place it on ice.
- Transfer 5 µL of cells to 500 µL of Wash Medium (1/100 dilution factor). Use the diluted cells for cell counting using a hemocytometer to obtain the number of cells per milliliter. Multiply by 100 to correct for the dilution factor.
- Multiply the number of cells per mL obtained in step 2.5 by the total volume of cells in suspension obtained in step 2.4 (~ 250 µL).
- Centrifuge at 500 x g for 15 min at 4 °C, discard the supernatant, and resuspend the cells in liquid ECM (1 x 106 cells/mL), and keep on ice.
- Transfer 5-20 µL of SBNET spheroids in ECM to a 96-well plate and allow the liquid ECM to solidify by placing the plate in a 37 °C incubator for 5 min.
- Add 200 µL of SBNET culture medium (DMEM/F12 + 10% FBS + 1% PEN/STREP + 1% Glutamine + 10 mM nicotinamide + 10 µg/mL insulin) to each well of the 96-well plate containing the SBNET spheroids in ECM.
- Alternatively, culture SBNET organoids in stem cell media similar to what has previously been described for growing either human liver or pancreatic organoids and listed in the Table of Materials.
- Change media every 5-7 days.
3. Characterization of SBNETS spheroids by immunofluorescence
- Transfer the organoids grown in ECM to a 1.5 mL tube using a P1000 pipette.
- Centrifuge at 1,500 x g for 1 min and remove the supernatant.
- Wash the organoid culture by adding 1 mL of phosphate buffer saline or PBS, mix, centrifuge at 1,500 x g for 1 min and remove the supernatant.
- Fix the organoids by adding 500 µL of 4% paraformaldehyde and incubate for 15 min.
- Wash the culture twice with 1 mL of PBS.
- Permeabilize the culture by adding 500 µL of PBS + 3% Bovine serum albumin or BSA + 0.1% Triton X 100 for 5 min.
- Then, wash three times with 1 mL of PBS + 3% BSA.
- Incubate for 1 h with primary antibodies against synaptophysin (SYP) at 1/600 dilution or chromogranin A (CgA) and somatostatin receptor 2 (SSTR2) at 1/400 dilution in antibody buffer (2.5% bovine serum albumin, 0.1% sodium azide, 25 mM Tris pH 7.4, 150 mM sodium chloride).
NOTE: Use 300 µL or more of antibody solution per tube. - Wash three times with 1 mL of PBS + 3% BSA.
- Incubate with secondary antibodies coupled to FITC at 1/500 dilution in antibody buffer for 1 h. Use 300 µL or more of antibody solution per tube.
- Wash 3 times with 1 mL of PBS + 3% BSA. Make sure to aspirate and discard all the supernatant.
- Add 5 µL of mounting medium containing the nuclear stain DAPI.
- Use a P20 pipette to transfer 5 µL of the SBNET spheroids from step 4.12 to a glass slide and seal with a cover slip.
- Take images using a fluorescent microscope using the 10x, 20x or 40x objectives.