Agarose Spin Down Assay: A Technique to Embed Organoids for Histological Analysis
Transcription
3D organoid cultures of cancer cells recapitulate many pathophysiological features of human cancers. Thus, these in vitro cultures form an excellent model for cancer research.
For processing these organoids, begin with a culture of pre-formed 3D organoids grown in a suitable basement membrane matrix. Add a desired cell recovery medium and incubate. The medium facilitates the depolymerization of the matrix gel, releasing the 3D organoids into the solution.
Centrifuge this mixture to pelletize the organoids. The recovery media containing the matrix components remains in the supernatant. Discard the supernatant. Next, add paraformaldehyde solution to the organoid pellet. The chemical enters the organoid cells and binds to the amino acids, causing protein crosslinking and sample fixation. This step is vital for any downstream histological applications.
Pelletize the organoids to separate and remove the paraformaldehyde-containing supernatant. Add warm melted agarose to the organoids. Use a needle to detach and disperse the pelleted organoids into the liquid agarose. Allow the agarose to solidify and embed the organoids. Use the organoid-containing agarose plug for downstream histological assays.
