Preparation of in vitro schistosomules

 

Author Fred Lewis

Introduction

A number of methods are used to transform cercariae into the next developmental stage, the schistosomule. Two of the more commonly used laboratory methods are presented here.

 

A. Transformation of cercariae by vortexing

Equipment

Refrigerated centrifuge

Materials and reagents

S. mansoni cercariae

Dulbecco’s minimum essential medium (DMEM)

Percoll gradient suspension*

50 ml plastic, conical centrifuge tubes

250 ml tissue culture flasks

Procedure

·         Place cercariae in a 50 ml centrifuge tube and place on ice for 30 minutes.

·         Centrifuge the tube for 2 minutes at 100 x g and 4°C.

·         With a pipette, withdraw and discard all but the bottom 3 ml from the top of the pellet.

·         Add 3 ml DMEM.

·         Cap the tube and vortex 45 seconds at high speed.

·         Place tube on ice for 3 minutes, then vortex as before.

·         Gently pipette the cercarial suspension onto 40 ml of the pre-made Percoll suspension.

·         Centrifuge for 15 minutes at 500 x g and 4°C.

·         Withdraw and discard the top 40 ml of the suspension.

·         Resuspend the pellet, and add DMEM to 50 ml final volume.

·         Centrifuge for 5 minutes at 100 x g and 4°C.

·         Wash the final pellet twice, using the above procedures, with DMEM.

·         Place the resulting organisms into 250 ml tissue culture flasks in 100 ml DMEM at a density of approximately 500 organisms/ml, and incubate at 37°C in a 5% CO₂ incubator.

 

           *Formula for the Percoll medium

24 ml Percoll

4 ml 10X Eagle’s minimum essential medium (EMEM)

1.5 ml penicillin-streptomycin (10,000 U/ml penicillin and 10,000 µg /ml streptomycin)

1 ml of 1 M HEPES in 0.85% NaCl

9.5 ml distilled water

 

 

 

B. Transformation of cercariae by needle and syringe

 

Equipment

Refrigerated centrifuge

 

Materials and reagents

S. mansoni cercariae

DMEM

50 ml plastic, conical centrifuge tubes

10 ml plastic syringes with 22 gauge disposable hypodermic needles

Procedure

·         Place 10 ml of the cercarial suspension into a 50 ml plastic centrifuge tube

·         Using a 10 ml syringe with a 22 gauge needle, fill the syringe and repeatedly pass it through the needle (10-15 times)

·         Allow the cercariae to settle for 3 minutes, then withdraw and discard all but the lowest 3 ml of the suspension

·         Add DMEM to the resulting pellet and purify by Percoll gradient separation, as described above.

 

Follow-up comments/recommendations

Cercariae will begin the transformation into schistosomules following a number of stimuli, the most important of which is their placement into culture medium. The above two mechanical procedures, combined with Percoll separation, will yield a clean preparation by separating the cercarial tails from the bodies. Schistosomules prepared by the above procedures, and incubated at 37ºC will gradually undergo morphological and physiological changes. By 24 hours in culture, the organisms will resemble (in most respects) cercariae that have penetrated and resided in the skin for about 1 hour.

 

References

Colley, D.G. and Wikel, S.K. 1974. Schistosoma mansoni: simplified method for the production of schistosomules. Experimental Parasitology 35: 44-51.

 

Cousin, C.E., Stirewalt, M.S., and Dorsey, C.H. 1981. Schistosoma mansoni: ultrastructure of early transformation of skin- and shear-pressure-derived schistosomules. Experimental Parasitology 51: 341-365.

 

Lazdins, J.K., Stein, M.J., David, J.R., and Sher, A. 1982. Schistosoma mansoni: rapid isolation and purification of schistosomula of different developmental stages by centrifugation on discontinuous density gradients of percoll. Experimental Parasitology 53: 39-44.

 

Lewis, F. A. 1999. Schistosomiasis, in Current Protocols in Immunology, John Wiley and Sons, Inc., (R. Coico, Ed) Vol III, 19.1.1 – 19.1.28.