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

Abstract

Source: Ear, P. H. et al. Establishment and Characterization of Small Bowel Neuroendocrine Tumor Spheroids. J. Vis. Exp. (2019)

This video demonstrates a strategy to split and propagate spheroids developed from small bowel neuroendocrine tumor cells. These spheroids have small bowel neuroendocrine tumor markers and can be propagated and further used to test anti-cancer drugs.

Protocol

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 plat....

Materials

List of materials used in this article

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