Embedding 3D Spheroids in Agarose Gel Drops: A Technique to Preserve Spheroids for Downstream Analysis

1. Embedding of 3D Spheroids

1. Prepare the agarose gel into which the spheroids are embedded (only necessary first time performing the protocol)
   1. Mix 1 g of bacto-agar in 50 mL of ddH₂O.
   2. Heat slowly in the microwave oven until the bacto-agar has dissolved and a homogeneous gel has formed. Do not allow the gel to boil.
   3. Keep the bacto-agar warm in a water bath at 60 °C.
   4. Keep at 4 °C between experiments.
2. Embedding of spheroids
   1. On day 1, for each condition, pool a minimum of 12 spheroids in a 1.5 mL tube.
   2. Wash once with 1 mL of ice-cold 1X PBS.
   3. To fix the spheroids, add 1 mL of 4% paraformaldehyde.
      NOTE: Handling of paraformaldehyde should be performed in a fume hood.
   4. Let them incubate for 24 h at RT.
   5. On day 2, heat the agarose gel carefully by placing it in a water-filled beaker in a microwave oven. Ensure that the gel does not boil. Keep warm on a benchtop heating plate, at 60 °C until use.
   6. Wash spheroids twice with 1 mL of ice-cold 1X PBS.
   7. Aspirate most of the 1X PBS (leaving approximately 100 µL at this point is practical for handling the spheroids).
   8. Prepare a 20 µL pipette by cutting the pipette tip at an incline to obtain a pointier tip with a larger hole (Figure 1).
      NOTE: The next part has to be done quickly to ensure optimal spheroid transfer and to avoid solidification of gel drop. If no heating block is available, it is recommended to first catch the spheroids and then make the agarose drop (i.e., switching the order of points 1.2.9 and 1.2.10).
   9. Make an agarose gel drop on a microscope slide. Place the slide on a warm heating block to prevent the agarose from solidifying.
   10. Using the modified pipette tip (step 1.2.8), catch as many spheroids as possible in a volume of 15–20 µL.
   11. Carefully inject the 15–20 µL spheroid-containing 1X PBS into the center of the agarose gel drop without touching the microscope slide.
       NOTE: This is a slightly difficult point. The spheroids will be lost if the pipette tip touches the microscope slide when injecting the spheroids into the gel drop. It is advisable to practice the whole process of making the agarose drop and injecting the spheroids by injecting a colored liquid into the drop. This will allow visualization of a potential penetration through the drop, as the colored liquid will be leaking out onto the slide.
   12. Let the agarose gel drop harden by incubating for 5–10 min at RT or at 4 °C. Once the gel drop has solidified somewhat (but is still rather soft), carefully push the gel drop from the microscope slide into a plastic tissue cassette with a scalpel.
   13. Cover the plastic tissue cassettes in 70% ethanol.
       NOTE: At this point, the spheroids can be used directly or stored for months.
   14. Embed the agarose-embedded spheroid in paraffin, section into 2–3 µm thick layer slides, and stain with hematoxylin and eosin or subject to immuno-histological staining.