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

Published: April 30, 2023

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 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
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.

開示

The authors have nothing to disclose.

Materials

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

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記事を引用
Agarose Spin Down Assay: A Technique to Embed Organoids for Histological Analysis. J. Vis. Exp. (Pending Publication), e20388, doi: (2023).

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