eoe sub articles

EoE Sub Articles

1. Organoid Processing for Histology: The Agarose Spin Down Method
NOTE: This protocol is adapted from a previous publication by Vlachogiannis et al. We have added a step involving agarose embedding to embed all populations of organoids successfully.
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	<li>Remove existing media from the well. Be careful not to aspirate the basement membrane domes.</li>
	<li>Add an equal (equal to the volume of media removed from step 1) volume of cell recovery s...

agarose spin down assay: a technique to embed organoids for histological analysis

Source: Lee, S. et al. <a href="https://www.jove.com/video/60367" target="_blank">Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts.</a> J. Vis. Exp. (2020)
In this video, we perform a spin-down assay to embed 3D prostate cancer organoids in agarose. These embedded organoids can be used for further histopathological analysis to study cancer growth and development.

<img alt="Figure 1" src="/files/ftp_upload/20388/20388_Figure1.jpg" /> 
Figure 1: A key step for successful agarose embedding. A process to detach the cell pellet from the wall of the 1.5 mL tube.

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

Source: Lee, S. et al. Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and ...

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.

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

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