In vitro cultivation of Schistosoma mansoni

Abstract

The rate of growth and development of S. mansoni cultivated in vitro was assessed by comparison with the optimum rate of development in the white mouse. Development was divided into six stages recognized by precise criteria Clegg, 1959: Stage 1, lung form 7 days no cell division (colchicine-aceto-orcein squash technique; Smyth, 1956). Stage 2, gut development (15 days); gut ceca joined behind ventral sucker; 100-200 mitoses in males, 60-80 in females, after 3-hour incubation in colchicine (100 μg per milliliter). Stage 3, organogeny (21 days); males, 2 small testes; females, narrow uterus. Stage 4, gametogeny (28 days); males, 8 testes, anterior 2 with spermatozoa; females, small ovary; mating begins. Stage 5, shell protein (30 days); egg-shell protein in vitelline cells gives bright orange reaction with diazo salt Smyth and Clegg, 1959. Stage 6, oviposition (34-35 days); first eggs produced. Finely chopped lungs of mice exposed to 500 cercariae 7 days previously were incubated in Hank's saline containing 0.1 mg heparin per milliliter, and lung forms (stage 1) were recovered. Larvae were concentrated and freed from pieces of lung in a centrifuge tube containing a wire mesh filter. Lung forms were cultured in medium II containing equal parts of inactivated rabbit serum and Earle's saline, rabbit red cells (1%), lactalbumin hydrolyzate (0.5%), glucose (0.1%), penicillin (100 units per milliliter), streptomycin (100 μg per milliliter), at 37 °C and pH 7.4, obtained by gassing with 8.8% CO2 in air. Development to stage 5 occurred but required 42 days in vitro compared with 30 days in the mouse. Cercariae, axenized by washing in sterile aquarium water containing penicillin (200 units per milliliter) and streptomycin (200 μg per milliliter), did not develop in medium II. Schistosomules recovered from the skin of mice 30 minutes after penetration, by a modification of the method used to collect lung forms, began to grow on the 6th day in culture and developed to stage 4. Enrichment of medium II with 10% chick embryo extract, 10% rabbit liver extract, 0.1% purified liver extract Sigma Chemical Co., or additional glucose (0.4%), did not allow development of lung forms beyond stage 5. Diffusion of metabolites was assisted by culturing lung forms in 0.3 ml of defibrinated rabbit blood in a small cellulose sac held inside a special culture tube containing medium II (modified by the addition of 10% chick embryo extract and 0.4% glucose). The culture tube was agitated by spinning at 500 rpm for 10 seconds at intervals of 5 minutes. In one experiment a single female matured completely (stage 6) in 24 days, and in another a male worm reached stage 6 in 30 days. In many other experiments with this technique growth was severely inhibited. Continuous-flow culture with the apparatus described by Clegg 1961 did not improve development because even moderate flow rates (in excess of 0.4 ml per hour) removed the sedimented red cells on which the schistosomules feed. Chloromycetin (25 μg per milliliter) inhibited growth of lung forms (stage 1) in culture, and stage 2 was not reached. The contribution of culture techniques to the elucidation of schistosome physiology is discussed. © 1965.