Splitting Spheroids for Sub-culturing and Shipping: A Procedure for Propagating and Transferring Spheroids from Small Bowel Neuroendocrine Tumor

Protocolo

1. Splitting SBNET Spheroids

NOTE: This is done for expansion and for sharing with other researchers.

1. Use a P1000 pipette to mechanically break the ECM and aspirate the ECM with SBNET spheroids to a sterile 1.5 mL tube.
2. Centrifuge at 1,000 x g at 4 °C, remove all the supernatant and place the tube on ice.
3. Add 2-4x the volume of the new ECM to the pellet. Mix the new ECM with the old ECM and SBNET spheroids by pipetting up and down 10x, avoiding introducing air bubbles.
4. Transfer 5-20 µL of the ECM and SBNET spheroids mixture to a new plate and allow ECM to solidify.
5. Cover with new SBNET medium and transfer to the incubator. The recovery rate is approximately 95% to 100%.
6. For shipping SBNET spheroids to another lab, transfer SBNET spheroids with new ECM to a T25 flask and allow ECM to solidify.
7. Fill the T25 flask with SBNET spheroid medium, screw on the cap tightly, and prepare the shipment package.
8. Upon receiving an SBNET spheroid culture, remove the culture medium and perform steps 1.1 to 1.5 to put SBNET spheroids back in culture.

Materiales

Name	Company	Catalog Number	Comments
DMEM	Gibco	11965-092	Medium for tissue preparation
DMEM/F-12	Gibco	11320-033	Medium for organoid cultures
FBS	Gibco	16000044	Reagent for culture media
Glutamine	Gibco	A2916801	Reagent for culture media
Insulin	Sigma	I0516	Reagent for culture media
Matrigel	Corning	356235	Matrix to embed and anchor organoids
Nicotinamide	Sigma	72340	Reagent for culture media
PEN/STREP	Gibco	15140-122	Reagent for culture media