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

Abstract

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

Protocol

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.

1. Remove existing media from the well. Be careful not to aspirate the basement membrane domes.
2. Add an equal (equal to the volume of media removed from step 1) volume of cell recovery solution and incubate for 60 min at 4 °C.
3. Dislodge the basement membrane dome using a pipette and crush the basement membrane dome using....

Results

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.

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 mL Pipettman	Gilson	F123602
1 mL Syringe	BD Syringe	329654
1.5 mL tube	Spectrum Lab Products	941-11326-ATP083
25G Needle	BD PrecisionGlide Needle	305122
4% Paraformaldehyde (PFA)	Alfa Aesar	J61899
70% Ethanol (EtOH)	VWR	BDH1164-4LP
Agarose	Lonza	50000
Cell Culture Plate - 24 well	Costar	3524
Cell Culture Plate - 48 well	Costar	3548
Cell Culture Plate - 6 well	Costar	3516
Cell Dissociation Solution, Accumax	Innovative Cell Technologies, Inc.	AM105
Cell Recovery Solution	Corning	354253
Cell Scraper	Sarstedt	83.18
dPBS	Corning/Cellgro	21-031-CV
Pipette tips for 1 mL (Blue Tips)	Fisherbrand Redi-Tip	21-197-85
Small Table Top Centrifuge	ThermoFisher Scientific	75002426
OCT Compound	Tissue-Tek	4583
Water Bath	Fisher Sc	2320