Agarose Spin Down Assay: A Technique to Embed Organoids for Histological Analysis

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.

To process the organoids for histology, remove existing media from the well taking care not to aspirate the basement membrane domes. Add an equal volume of cell recovery solution and incubate the plate for 60 minutes at 4 degrees Celsius.

After the incubation, dislodge the basement membrane dome using a pipette and crush it with the pipette tip. Collect the dissociated dome and the cell recovery solution into a 1.5-milliliter tube. Centrifuge the tube at 300 x g and 4 degrees Celsius for 5 minutes, then remove the supernatant and set it aside.

Save all the supernatant until the final step when the presence of organoids is confirmed. Add the desired volume of cold PBS and gently pipet up and down to mechanically dislodge the pellet without disrupting the organoids. Repeat the centrifugation and remove the supernatant.

Fix the pellet in a matched volume of 4% PFA for 60 minutes at room temperature. After fixation, repeat the centrifugation step and PBS wash. Then, slowly add 200 microliters of warm 2% agarose and immediately but gently detach the cell pellet from the tube wall without disrupting it using a 25 gauge needle attached to a 1-milliliter syringe.

For the agarose spin down method, it is critical to detach the cell pellet from the wall of the tube right after adding agarose using a 25 gauge needle.

Once the agarose has completely solidified, detach it from the tube with a 25 gauge needle attached to a 1-milliliter syringe and transfer it to a new 1.5-milliliter tube. Fill the tube with 70% ethanol and proceed with the conventional protocol for tissue dehydration and paraffin embedding.

• Spin-down - A process to separate substances of different densities by centrifugation.
• Organoid paraffin embedding - A technique to preserve biological tissues in paraffin wax.
• Cell recovery solution - A solution used to dissociate cells from a matrix for further analysis.
• Agar embedding - A method to preserve tissue samples in an agarose gel block.
• Agarose embedding protocol - A detailed procedure for encasing biological samples in agarose.

• Introduce Spin-down – A method utilized to separate different-density particles by centrifugal force (e.g., spin-down).
• Key Concepts – Overview of tissue sample preservation techniques such as paraffin and agar embedding (e.g., organoid paraffin embedding).
• Underlying Mechanisms – Description of the procedure to detach and process organoids (e.g., cell recovery solution).
• Connect to Experiment – Explanation of the importance of proper organoid and agarose handling in the experiment.

• What is the spin-down technique and how is it applied in the procedure?
• How does organoid paraffin embedding contribute to tissue preservation?
• What role does cell recovery solution play during the dissociation of organoids?

• Practical Applications – Spin-down and embedding techniques in biological and medical research (e.g., spin-down).
• Industry Impact – Areas like pathology and histology benefit from these methods (e.g., organoid paraffin embedding).
• Societal Importance – Advancement in disease diagnosis and treatment (e.g., cell recovery solution).
• Link to Scientific Advancements – Development of improved tissue preservation and analysis methods.