EFFECTS OF SELECT MEDIUM SUPPLEMENTS ON IN-VITRO DEVELOPMENT OF CRYPTOSPORIDIUM-PARVUM IN HCT-8 CELLS

Surface-sterilized oocysts of Cryptosporidium parvum were applied to s ubconfluent monolayers of human adenocarcinoma (HCT-8) cells grown on coverslips in six-well cluster plates, Parasite-infected cultures were then incubated in RPMI 1640 with 10% fetal bovine serum, 15 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer, and anti biotics at 37 degrees C in a 5% CO2-95% air incubator for 2 h to allow sporozoites to excyst and enter cells. After cultures were washed fre e of debris, fresh cell culture media containing select supplements we re added and cultures were reincubated. Parasite growth was assessed 6 6 h later by counting the number of parasite developmental stages in 2 5 random x100 oil fields by Nomarski interference-contrast microscopy. Four vitamin supplements, calcium pantothenate, L-ascorbic acid, foli c acid, and 4-(para)-aminobenzoic acid, each resulted in a significant increase in parasite numbers in vitro, The addition of insulin and th e sugars glucose, galactose, and maltose also had a positive effect on parasite growth, although the effect was less pronounced than with an y of the vitamins, Using the above information, we developed a supplem ental medium formulation consisting of RPMI 1640 with 10% fetal bovine serum, 15 mM HEPES, 50 mM glucose, and 35 mu g of ascorbic acid, 1.0 mu g of folic acid, 4.0 mu g of 4-aminobenzoic acid, 2.0 mu g of calci um pantothenate, 0.1 U of insulin, 100 U of penicillin G, 100 mu g of streptomycin, and 0.25 mu g of amphotericin B (Fungizone) per ml (pH 7 .4). The growth of C, parvum in this medium was found to be enhanced a pproximately 10-fold compared with that in control medium without addi tional glucose, insulin, or vitamins.