Culturing schistosomula

Introduction
Infectious cercariae are transformed in vitro into schistosomula. Schistosomula can be cultured in vitro in a complex medium. These in vitro cultured schistosomula do not grow at the same rate as those in a permissive host, nor will they become patent adults, but in our hands about 50% of them will mature with fully formed guts, and 10% will develop into sexually distinct male and female worms. Hundreds of thousands of worms can be easily maintained, providing a vast amount of parasite material, allowing for genetic manipulation through RNAi and transgenesis. The percentage of schistosomula forming guts and growing properly can be increased (50% versus 20%) by supplementing the media with conditioned media during the first week of culture.

Equipment
37 °C/5% CO2 incubator
Biosafety cabinet/Tissue culture hood

Reagents
Schistosomulum Wash (SW)
500 ml RPMI (Cellgro, 15-040)
5 ml Hepes Buffer (Cellgro, 25-060-CI)
10 ml Antibiotic/Antimycotic (Invitrogen, 15240-062)

Schistosomulum Wash plus Tween (SWAT)
SW + 0.5% Tween 20

Schistosomulum Medium (SM)
20X Lactalbumin hydrolysate/glucose-
2.5 g lactalbumin hydrolysate (Sigma, L9010)
2.5 g glucose (Sigma, G5400)
250 ml Basal Medium Eagle (Gibco, 21010046)
• Filter sterilize
• Store at 4 °C

For 1 liter of SM:
50 ml 20X Lactalbumin hydrolysate/glucose
0.5 ml Hypoxanthine (1 mM) (-20 °C) (Sigma, H9377)
1 ml Serotonin (1 mM) (-20 °C) (Sigma, H9523)
1 ml Insulin (8mg/ml) (4 °C) (Sigma, I0516)
1 ml Hydrocortisone (1 mM) (-20 °C) (Sigma, H0888)
1 ml Triiodothyronine (0.2 mM) (-20 °C) (Calbiochem, 64245)
5 ml MEM Vitamins (100X) (-20 °C) (Invitrogen, 11120-052)
50 ml Schneiders Medium (Drosophila) (+4 °C) (Invitrogen, 11720067)
10 ml Hepes Buffer (+4 °C) Triiodothyronine
100 ml Human Serum (thaw at 37 °C prior to use) (Gemini, 100-512)
20 ml Antibiotic/Antimycotic (Invitrogen, 15240-062)
1 L Basal Medium Eagle (Gibco, 21010046)
• Store at 4°C

Procedure
1. Start with 10,000-50,000 schistosomula in a single well of a six well plate, with 4 ml of medium. We use tissue-cultured treated plates. We do not add red blood cells at this stage.
2. Two days after the initial culture is set-up, tilt the 6 well plate at an angle and take out at least 3 ml of the “conditioned medium” being sure to leave the schistosomula behind. We syringe-filter the conditioned medium to avoid fungus contamination problems with the cultures.
3. Add 3 ml of SWAT and gently pipet the schistosomula in the SWAT using a P1000. Overly vigorous pipeting will cause schistosomula to float on the surface of the liquid.
4. Let the schistosomula settle (1-2 minutes) and remove as much of the added SWAT as possible, being sure to leave the schistosomula behind.
5. Add 3 ml of fresh medium plus 1 ml of conditioned medium.
6. Supplement with ~50 μl of packed mouse red blood cells. Red blood cells should be washed with SW once prior to use (this is done in a 15 ml conical tube, using approximately 1 ml of heparinized mouse blood plus 10 ml of SW, followed by centrifugation at 3000 rpm for 5 minutes).
7. Continue to feed and wash the schistosomula every other day (Monday, Wednesday, and Friday seems to work well). Supplement 3 ml of fresh medium with 1 ml of conditioned medium for as long as the conditioned medium lasts (~3-4 media changes). Once you run out of conditioned medium, add 4 ml of fresh medium and ~50 μl of packed mouse red blood cells to each well after you remove the SWAT from the wash.

Comments
Schistosomula can be cultured this way for at least two months. Approximately 50% of the schistosomula will not grow and very few (~10%) will grow to sexually distinct adults (mostly males). The biggest obstacle to success is fungal/bacterial contamination, probably a reflection of the fact that the parasites originate from non-sterile snails; this is the reason that we use more than the recommended concentration of Antibiotic/Antimycotic. This does not appear to affect parasite growth.

References
1. Basch PF. 1981. Cultivation of Schistosoma mansoni in vitro. I. Establishment of cultures from cercariae and development until pairing. Journal of Parasitology 67(2):179-85.
2. Tucker, M. S., Karunaratne, L. B., Lewis, F. A., Frietas, T. C., and Liang, Y-S. 2013. Schistosomiasis, in Current Protocols in Immunology 19.1.1-19.1.57, John Wiley and Sons, Inc., (R. Coico, Ed). Published online November 2013 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/0471142735.im1901s103.