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

Overview

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 a pipette tip. Collect the dissociated dome and cell recovery solution in a 1.5 mL tube.
4. Centrifuge at 300 x g and 4 °C for 5 min.
5. Remove the supernatant (cell recovery solution). Save all supernatants in separate tubes until the end, when the presence of organoids is confirmed in the final pelleting step.
6. Add desired volume (Table 1) of cold PBS and gently pipette up and down to disturb the pellet mechanically.
7. Centrifuge at 300 x g and 4 °C for 5 min.
8. Remove the supernatant (PBS).
9. Fix the pellet in a matched volume (e.g., 500 µL for one pellet from the 24-well plate culture condition, Table 1) of 4% PFA for 60 min at room temperature.
10. Following fixation, centrifuge at 300 x g and 4 °C for 5 min.
11. Remove the supernatant (PFA).
12. Wash with matched volume (e.g., 500 µL for one pellet from the 24-well plate culture condition, Table 1) of PBS and centrifuge at 300 x g and 4 °C for 5 min.
13. Prepare warm agarose (2% agarose in PBS).
    NOTE: Here, cell pellets for frozen sections can be directly resuspended in 200 µL of OCT compound.
14. Resuspend the cell pellet in 200 µL of agarose (2% in PBS).
    1. Immediately after adding agarose, gently detach the cell pellet from the wall of the 1.5 mL tube using the 25 G needle attached to the 1 mL syringe. As shown in Figure 1, if the cell pellet is not physically detached from the wall of the 1.5 mL tube, then there is a risk of losing all or part of the cell pellet during the agarose embedding process.
15. Wait until the 2% agarose in PBS is completely solidified.
16. Detach the solidified agarose block from the 1.5 mL tube using a 25 G needle attached to the 1 mL syringe.
17. Transfer the detached agarose block containing the cell pellet to a new 1.5 mL tube.
18. Fill the tube with 70% EtOH and proceed further using the conventional protocol for tissue dehydration and paraffin embedding.

Table 1: Seeding density, basement membrane volume, and medium volume needed for one dome

Culture Plate	Seeding Density (cells)	Basement Membrane Volume (μL)	Medium (μL)
48 well	25,000-50,000	20	250
24 well	50,000-250,000	40	500
6 well	50,000-250,000	40	2,000

Representative 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.

Materials

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