Splitting Spheroids for Sub-culturing and Shipping: A Procedure for Propagating and Transferring Spheroids from Small Bowel Neuroendocrine Tumor
Published: April 30, 2023
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.
- Use a P1000 pipette to mechanically break the ECM and aspirate the ECM with SBNET spheroids to a sterile 1.5 mL tube.
- Centrifuge at 1,000 x g at 4 °C, remove all the supernatant and place the tube on ice.
- 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.
- Transfer 5-20 µL of the ECM and SBNET spheroids mixture to a new plate and allow ECM to solidify.
- Cover with new SBNET medium and transfer to the incubator. The recovery rate is approximately 95% to 100%.
- For shipping SBNET spheroids to another lab, transfer SBNET spheroids with new ECM to a T25 flask and allow ECM to solidify.
- Fill the T25 flask with SBNET spheroid medium, screw on the cap tightly, and prepare the shipment package.
- 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.
Declarações
The authors have nothing to disclose.
Materials
| 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 |
Tags
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Splitting Spheroids for Sub-culturing and Shipping: A Procedure for Propagating and Transferring Spheroids from Small Bowel Neuroendocrine Tumor. J. Vis. Exp. (Pending Publication), e20412, doi: (2023).