Insulin reduces the requirement for serum in Plasmodium falciparum culture

Abstract

Insulin added to Plasmodium falciparum cultures (0.2 IU /tn l) reduced the requirem ent fo r hum an serum fro m ten to fiv e percent. This represents an obvious advantage by its serum -sparing effect and by reducing the chances o f using contam inated serum in cultures. The growth-promoting ability o f insulin was observed eitherin cultureadapted P. falciparum or in newly-isolated samples.Keywords: P lasm odium falciparum . M alaria. Insulin. Culture medium.The possibility to keep a parasite in culture represents an im portant step towards a better knowledge of its biology. It also facilitates the study of the action of chemotherapeutic agents and immune factors in vitro, and constitutes a practical way to obtain parasite antigens. W ith the development by T rager and Jensen in 19768 o fa simple andeffective system, ithas been possible to keep Plasm odium falciparum in continuous culture. Parasitized erythrocytes are maintained in H EPE S-buffered R PM I 1640 medium supplemented with human serum (lOOml per liter of medium) for optimal parasite growth. This requi rement is an im portant limitation of the method, since it is not always easy to obtain fresh human serum in the required amount. Also, serum it can carry dangerous pathogens, or antimalarial antibodies and drugs which interfere with the growth of the parasite. The present investigation was carried out in an attem pt fínd a suitable way to replace or reduce the requirem ent for human serum in P. falcipa rum cultures. M A T E R IA L S A N D M E T H O D SP lasm odium falciparum isolates: Two P. falciparum isolates were used: F 83/2 isolatedin 1980 from an infected patient from Ituxi River, State of Amazonas, and kept in continuous culture for twelve months, and F 66/1 newly isolated from a patient from the locality of Em baúba, State of Rondonia. Culture conditions: Cultures were initiated by diluting P. fa lcip a ru m -parasitized erythrocytes with non-parasitized 0 + outdated erythrocytes from blood donors to a parasitaem ia o f0 .1 % . Erythrocytes were suspended in R P M I 1640 medium containing 25 mM of H E P E S , 0 .2 g /l of sodium bicarbonate, 25m g/l of gentamycin, and either 5 or 10% human type O + serum, or 5% human serum plus bovine crystalline insulin or N P H zinc insulin (manufactured by Eli Lilly and supplied by C E M E ) at different concentrations. Cultures were kept in 50ml tightly closed culture flasks, gassed with a mixture of 5% C 0 2 in air, and incubated at 37°C. Culture medium was changed daily till parasitaem ia reached 4% , when changes occurred twice a day. Parasitaem ia was assessed by examining 200 erythrocytes in Giem sa-stained smears. R E SU LT SAlthough insulin was not able to replace human serum in P. falciparum cultures, it allowed a reduction to half the am ount of serum required for optimum growth of the parasite. From the different concentra tions of insulin used (0 .0 5 ,0 .1 ,0 .2 and 0.5 IU/m l), the best results were achieved with 0.2 units per milliliter of medium. T he increase of the concentration of glucose (...