In vitro Culturing of Dirofilaria immitis L3 - L4 Molt

Materials

  • NaHCO3
  • Millipore Water
  • NCTC-135 (Sigma N3262)
  • IMDM (Sigma I3390)
  • NCTC-135 Powder (Sigma N3262-10X1L)
  • Fetal Bovine Serum
  • Fungin (Invitrogen ANT-FN-1)
  • Pen/Strep 100X Stock (10,000 U/mL, Sigma P4333-100ML)
  • Gentamicin 100X Stock (0.4 mg/µL, Sigma G1272-100ML)
  • Ciprofloxacin (Sigma 17850)
  • 0.22 µm filter (500 mL, example: VWR 10040-436)

Protocol

Preparation of Culture Media

  1. In an autoclaved glass bottle, add the following in the exact order listed:

    Culture Media Component Volume/Quantity (250 mL)
    NCTC-135 Powder 1.16 g
    NaHCO3 0.28 g
    H2O 125 mL
    IMDM 125 mL
    Pen-Strep (10000 U/mL) 2.5 mL
    Gentamicin 2.5 mL
    Fungin 250 µL
    Ciprofloxacin 250 µL
    FBS 50 mL
  2. Replace cap on media bottle and shake to mix. Filter the media using a 0.22 µm filter.

  3. Label filtered media with: name of media, volume, date prepared, and initials. Store media at 4°C. Media is good for 1 month post-preparation.

Extraction of D. immitis L3 from Mosquitoes

Follow this Protocol

Preparing Parasites and Culture Plates (24-well bulk culture)

  1. Wash infective larvae:

    • Transfer infective larvae in minimal amount of RPMI-1640 to a 1.5 mL microcentrifuge tube.
    • Spin the microcentrifuge tube at 1000 x g for 10 minutes.
    • Pipette 100 μL from bottom of tube and transfer to new tube containing 500 μL RPMI.
    • To prevent worm loss, check wash from original tube by pipetting remaining solution into petri dish and examining under dissecting microscope. If a significant number of worms were left behind, pipette into the new tube with NI and spin again.
    • Repeat spin/washing infective larvae in media three times following the steps above.
  2. Under BSC, pipette 200 μL from bottom of microcentrifuge tube and transfer to new petri dish containing pre-warmed, sterile culture media

  3. Prepare 24-well cell culture plates with 800 µL of culture media under sterile conditions. Optional: Add a transwell to each well using a sterile forceps (Fisher Scientific 07-200-154).

  4. Transfer desired number of infective larvae (typically 25) in a volume of 200 µL from petri dish to each well in 24-well plates (total volume = 1 mL). Total of 600 larvae is typically used to fill an entire plate. Larvae are incubated at 37°C with 5% CO2.

  5. Change media under BSC every two days by transferring worms (in transwells) to new wells containing fresh culture media using sterile forceps.

  6. L3 molting should be monitored daily using a compound scope at 40x-200x total magnification.

Note: A majority of D. immitis L3 should molt within the first 2-3 days in culture.

Preparing Parasites and Culture Plates (96-well individual culture)

  1. Prepare a 96-well cell culture plate by adding 200 µL of molting culture media per well under sterile conditions.

  2. Wash freshly extracted L3, as described above.

  3. Transfer one L3 in a volume of 1 µL from petri dish containing culture media and L3 to each well of a 96-well plate.

  4. Place plate containing individually cultured L3 at 37°C with 5% CO2

  5. L3 molting should be monitored daily using a compound scope at 40x-200x total magnification.