Cox Systems Biology

Author: Morgan Zych

Contents

• Materials
• Recovery of organoids from culture
• Embedding organoids in low melting point agar

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Materials

Recovery of organoids from culture

• Organoid Recovery Solution
• Phosphate Buffered Saline (PBS)(Stock)
• 10% Neutral Buffered Formalin
• 70% EtOH
• P1000 + P200 pipettes & tips
• 15mL conical tubes
• Glass/serological pipettes + pipettor

Embedding organoids in low melting point agar

• 2% Low Melting Point Agar (Invitrogen #16520-050)
• Heating block
• Histology Cassettes
  • + Foam Inserts

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Recovery of organoids from culture

All centrifugation steps done for 5 minutes @ 1600rpm, 4^oC unless otherwise stated.

1. Recover organoids from Matrigel domes.
   • In plates:
     • Remove media
     • Add 500mL room temperature PBS per well, swirl gently to wash.
     • Remove PBS and add 500mL organoid recovery solution per well
     • Disrupt each gel dome w/ pipette tip.
       • Disrupt dome w/ swirling/mixing motion.
     • Transfer contents into 15mL tube, pooling wells from each sample into corresponding tubes.
     • Add 500mL cold media to each well, scrape with pipette tip, and move to the same tube to ensure complete removal of well contents.
   • In 15mL tubes:
     • Top total volume of tube up to 10mL with cold media or PBS.
     • Centrifuge samples.
     • Remove supernatant with glass/serological pipette, leaving all gel in pellet.
     • Add 1mL of organoid recovery solution to each tube (unless its contents represent >4 wells, then add 2mL) and pipette up and down to resuspend gel into solution
     • Incubate organoids in organoid recovery solution for 75 minutes in fridge.
2. Move each sample and its organoid recovery solution into 15mL tubes if not in them already. Bring total volume of the tube up to 10mL with cold PBS.
3. Centrifuge samples.
4. Remove supernatant with glass/serological pipette and aspirator.
5. Resuspend organoids in 2mL of cold PBS with p1000 pipette.
6. Centrifuge samples.
7. Carefully remove as much supernatant as possible with p1000, followed by p200 pipette.
8. Add 1mL of 10% neutral buffered formalin per tube and incubate for 10 minutes at room temperature.
9. Top volume of each tube up to 10mL with room temperature PBS and centrifuge.
10. Quickly decant supernatant into formalin-specific waste stream by inverting the tube. Resuspend the organoids in 1mL of PBS with p1000, then top total volume up to 10mL with PBS. Centrifuge.
11. Repeat step 9.
12. Resuspend organoids in 1mL of 70% ethanol with p1000, then top total volume up to 3-5mL with 70% ethanol.
13. Allow organoids to settle to bottom of tubes.

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Embedding organoids in low melting point agar

1. If not already aliquoted, prepare 2% low melting point agar in water and put into 1.5mL snap cap tubes. Unused aliquots of 2% agar can be kept in the fridge for years
2. Warm a heating block to 65^oC. Heat the agar in the block until liquefied. In the meantime, prepare labelled snap cap tubes for all samples.
3. Once organoids are settled to the bottom of their tubes (in 70% EtOH), pipette excess ethanol away, leaving the organoids in approximately 100mL volume.
4. Transfer organoids in ethanol to corresponding labeled snap cap tubes.
5. QUICKLY add 300mL agar per tube & mix carefully.
   1. Avoid bubbles but mix thoroughly.
   2. Work quickly as agar polymerizes/solidifies rapidly.
6. Centrifuge tubes for 5 minutes at 4^oC at 500G to aggregate the organoids and allow the agar to polymerize around them.
   1. If unsatisfied w/ the organoids’ location within the agar, put the snap cap tube back on the heating block for ~10 minutes and centrifuge again.
7. Label histology cassettes for each sample using pencil
8. Add small amount of ethanol to each tube (containing organoids + agar) and flick the tube very hard to dislodge the contents.
9. Remove agar w/ organoids embedded from tubes.
   1. Gently grab the agar puck with forceps so as not to damage/break the agar; or
   2. tip the whole agar puck out of the tube once it is dislodged.
10. Make a transverse cut of the agar puck with bias towards the tip of the cone where the organoids are.
    1. Do not cut the portion of the puck containing the organoids too thin or it could curl once embedded in paraffin, leading to them occupying different planes within the block.
11. Place both halves of the cut agar puck inside the labelled cassette for the sample. Add a foam insert to the cassette if you think the sample could fall out of the cassette.
12. Place the entire cassette into a bucket of 70% ethanol and submit for paraffin embedding.

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Last updated: 02/2025