PZQ treatment in in vitro cultured S. mansoni worms

Steps:

Day 1: Recovering S. mansoni worms from infected hamsters and starting the in vitro culture.

• Morning:

1. Put needed number of DMEM complete media aliquots in the incubator at 37ºC to warm them up for the in vitro culture.
2. Euthanize and dissect infected hamsters – See protocol “Hamster euthanasia and dissection: recovering adult worms and eggs”.
3. Recover adult worms in a small Petri dish containing DMEM complete media.
4. Separate male and female worms under a dissecting microscope.
5. Prepare your biosafety cabinet:
   1. Turn on the biosafety cabinet.
   2. Clean the bench surface and items inside the biosafety cabinet with 70% ethanol.
   3. Clean the following items with 70% ethanol when moving them into the biosafety cabinet:
      • Pouch containing sterile boats (for the DMEM complete media used to culture worms.) –.
      • Pouch containing new 96 well reservoir plate (The one that will received the complete media for worms culture).
      • 96 well plates with mesh filter insert (Never use the bottom of the plate– too deep and require 400 µL of media).
   4. Wait ~5-6 minutes before using safely the biosafety cabinet.
   5. Label your plates.
   6. Remove the mesh filter insert and keep it in your hand.
   7. Fill all the well of the reservoir plate with 250 µL of DMEM complete media and then put back the insert inside the filled plate. You can put a maximum of 60 worms/plate (no worms on the plate edges: these wells are filled with media only to limit evaporation in the inner wells) - See protocol “Long-term culture of S. mansoni worms”.
6. Aliquot male worms in 96 well plates (1 male worm/well – 60 worms total/plate, from B2 to G11)Version 0.0
7. Check under the microscope to be sure all the aliquoted worms are healthy (moving) and you have single worm per well.
8. Move the plates into the incubator (37ºC / 5% CO₂).

• Afternoon:

1. 1h30 – 2h after putting the plates in the incubator: inside the biosafety cabinet, replace media (only the wells containing worms) with fresh one (DMEM complete media – warmed at 37ºC).
2. Remove the excess of media in the insert by putting the insert briefly in contact with a kimwipe.
3. Don’t forget: in a well of a regular 96-well culture plate, add 250 µL of DMEM complete media from the same aliquot used to fill the plate containing the worms, as a “DMEM lactate background” for the lactate assay. You need to have a well per aliquot used.
4. Move all the plates back into the incubator (37ºC / 5% CO₂) for 24h, including the “DMEM lactate background” plate.
5. You can keep the remaining media at the end of each day, store it in sterile falcon at 4ºC and reuse only for the Day 5, step 5 (Afternoon).

Day 2: Recovering media from cultured worms (24h before PZQ treatment) for lactate assay and PZQ treatment of the worms.

• Morning:

1. Put the needed number of DMEM complete media aliquots in the incubator at 37ºC to have them ready for the afternoon.
2. Prepare PZQ stock solution (10 mg/mL): dilute 10 mg of PZQ powder (use a weighting paper and a precision scale) in 1 mL of DMSO.
3. Determine the volume of PZQ stock solution (10 mg/mL) to pipette to get the desired final concentration of PZQ in 30 mL of DMEM complete media.

• Afternoon:

1. Prepare the biosafety cabinet before removing the worms from the incubator:
   1. Turn on the biosafety cabinet.
   2. Clean the bench surface and items inside the biosafety cabinet with 70% ethanol.
   3. Clean the following items with 70% ethanol when moving them into the biosafety cabinet:
      • Pouch containing sterile boats (for the media containing PZQ and one for the media containing the same volume for DMSO only (controls of worm culture)).
      • Pouch containing fresh 96 well reservoir plate.
      • 96-well PCR plates for collecting media before PZQ treatment.
      • 96-well plates adhesive cover and rack to maintain the PCR plate.

             4. Wait ~5-6 minutes before using safely the biosafety cabinet.

     2. Prepare the media containing the PZQ drug by pipetting the exact volume of PZQ stock solution (10 mg/mL) to get the desired final concentration of PZQ in 30 mL of DMEM complete media.

     3. Prepare the media containing DMSO only (control of worm culture) by adding the same volume of DMSO as for the PZQ stock solution.

     4. Fill new 96 well reservoir plates with 250 µL of media containing PZQ drug and one new 96 well reservoir plate with 250 µL of media containing DMSO only (control).

     5. Remove the cover of the plates containing the worms and remove carefully the mesh filter insert (do not discard the reservoir plates containing the media: needed for L-lactate assay).

     6. Remove the excess of media in the insert by putting the insert briefly in contact with a kimwipe.

     7. Put the mesh insert containing the worms in the new 96 well reservoir plates containing PZQ drugs or DMSO control and put the cover back onto the plates.

     8. Move the plates back into the incubator (37ºC / 5% CO2) for 24h.

     9. In a new 96 well PCR plate, transfer 125 µL of the used media (t-1d before PZQ treatment) for further L-lactate quantification before PZQ treatment (to assess worm health before treatment). Add also 125 µL of DMEM complete media background and put it in the plate (e.g. H12 position).

   10. Seal the PCR plate using an adhesive seal and store at -80ºC until lactate quantification.

   11. Clean the biosafety cabinet using 70% ethanol and put UV lights for 5-10 min.

Day 3: Removing PZQ drug and washing treated worms.

• Morning:

1. Put the needed number of DMEM complete media aliquots in the incubator at 37ºC to have them ready for the afternoon.

• Afternoon:

1. Prepare the biosafety cabinet before removing the worms from the incubator (see Day 2 – step 4)
2. Remove the culture plates from the incubator and put them inside the biosafety cabinet.
3. Remove the 96-well tray from the reservoir plate: you now have only a big “one well tray” and pour 35 mL of warm DMEM complete media into this “one well tray”.
4. Remove the cover and then the mesh filter insert containing the worms from the 96-well tray.
5. Remove the excess of media containing PZQ drug in the insert by putting the insert briefly in contact with a kimwipe.
6. Place the insert into the “one well tray” filed with drug free media and put back the cover on the plate.
7. Proceed the same way with all the plates that need rinsing and then wait 5 min.
8. Repeat the rinsing steps (4 to 8) two times (total of 3 rinsing).
9. During the last 5 min rinsing with drug free media, prepare new reservoir plates by labeling them and fill them with 250 µL of warm DMEM complete media.
10. Place the insert in the new filled reservoir plates.
11. Move the plates back in the incubator (37ºC / 5% CO₂) for 24h.

Day 4: Changing media after 24h of culture.

• Morning:

1. Put the needed number of DMEM complete media aliquots in the incubator at 37ºC to have them ready for the afternoon.

• Afternoon:

1. Prepare the biosafety cabinet before removing the worms from the incubator (see Day 2 – step 4).
2. After 24h in culture, remove the culture plates from the incubator and put them inside the biosafety cabinet.
3. Remove the cover of the plate and carefully remove the mesh filter insert. Keep the mesh filter insert in your hand while you remove all the “old” media, only from wells used by the worms.
4. Replace with 250 µL of fresh DMEM complete media.
5. Don’t forget: in a well of a regular 96-well culture plate, add 250 µL of DMEM complete media from the same aliquot used to fill the plate containing the worms, as a “DMEM lactate background” for the lactate assay.
6. Remove the excess of media containing PZQ drug in the insert by putting the insert briefly in contact with a kimwipe.
7. Put back the insert in the plate containing fresh media and put the cover. Proceed with all the plates the same way.
8. Move all the plates back into the incubator (37ºC / 5% CO₂) for 24h, including the “DMEM lactate background” plate.

Day 5: Recovering media from cultured worms (72h after PZQ treatment) for lactate assay and worms freezing.

• Morning:

1. Put the needed number of DMEM complete media aliquots in the incubator at 37ºC to have them ready for the afternoon. For this last day, as the culture will end on this Day 5, you can reuse rest of DMEM media (if you have some) you have kept in the fridge from each previous day for the steps below.

• Afternoon:

1. Prepare the biosafety cabinet before removing the worms from the incubator (see Day 2 – step 4).
2. After 24h in culture, remove the culture plates from the incubator and put them inside the biosafety cabinet.
3. Remove the cover of the plate and carefully remove the mesh filter insert. Keep the mesh filter insert in your hand while you remove all the “old” media, only from wells used by the worms.
4. In a new 96 well PCR plate, transfer 125 µL of the used media (t+3d after PZQ treatment) for further L-lactate quantification after PZQ treatment (to assess worm recovery 3 days after treatment). Don’t forget to recover 125 µL of DMEM complete media background and put it in the plate (e.g. H12 position).
5. In the culture plate, add 125 µL of sterile water (or the remaining DMEM complete media you did not have used during the week but already have thawed) to replace the DMEM complete media withdrawn and place the insert containing the worms back in the plate.
6. Seal the plate using an adhesive seal and store at -80ºC until lactate quantification.
7. Proceed with all the plates the same way.
8. Tape the cover of the culture plates containing the mesh filter insert with the worms.
9. Store the culture plates with worms at -80ºC for further DNA analysis.

Materials:

• Biosafety cabinet
• Binocular
• Cell sieve to recover adult schistosome worms from perfused rodent 
• Incubator at 37°C
• Culture chamber
• Mix of gaz containing 5% CO₂
• Thin forceps
• Kimwipes
• Sterile disposable reservoirs (boats)
• Sterile petri dishes
• Sterile water
• Sterile 96 well culture plates (used for DMEM background)
• Millipore Multiscreen 96 well-plate with 100 µm mesh insert (for culturing 1 worm/well) (Millipore catalog number: MANM10010)
• Millipore 96 well-plate – reservoir plates (to use with the mesh insert) (Millipore catalog number: MAMCS9610)
• Micropipette P200 (to remove the media from the wells)
• Filtered tips P200
• 96 well PCR plates (recovering media for L-lactate assay)
• 96 well PCR plates adhesive covers
• 96 well plates racks
• Tape + sharpie

Products:

• DMEM supplemented media at 37°C (see Solution preparation)
• Ethanol 70%
• Praziquantel (anthelminthic – powder - Sigma-Aldrich product number: P4668)
• DMSO (to dilute Praziquantel - Sigma-Aldrich product number: D8418)

Solution preparation:

• DMEM supplemented media (aliquoted in 50 mL conicals and stored at -20ºC)

- DMEM high glucose (4.5g/L) – L-glutamine media (ThermoFisher catalog number: 11965092)

- 15% of heat inactivated Fetal Bovine Serum

- 100 UI penicillin and 100 µg/mL streptomycin (10 mL/L of the stock solution – 10,000 UI penicillin and 10 mg/mL streptomycin; Sigma-Aldrich product number: P4333)

Hygiene and security:

• Sterile culture conditions in biosafety cabinet

        Use lab coat and gloves spread with ethanol 70% - change gloves often.