Continuous in vitro culture of Babesia divergens in a serum-free medium

Babesia divergens was cultivated in RPMI 1640 (25 mM HEPES) supplemented with 10 % human serum (RPMI-10 % HS) with a high percentage of parasitized erythrocytes (PPE) (greater than or equal to 40 %). Standardization of in vitro tests, purification of exoantigens, biochemical studies and the safety of the culture handler motivated the development of a serum-free defined medium. Removal of serum greatly reduced the PPE but, after a period of adaptation, the culture was continuous and the parasite was able to develop a 3 % routine PPE. Addition of vitamins or reduced glutathione in basal medium (RPMI) did not improve the PPE. The Supplementation of basal medium with lipidic carrier (Albumax I or bovine serum albumin-Cohn's fraction V) promoted the growth of B. divergens with high PPE (> 30 %) close to those obtained in RPMI-10 % HS. Neither protein nor lipid fractions alone were able to restore the growth of B. divergens. Nevertheless, the whole lipid fraction from serum or Albumax I added to delipidated albumin partially restored the growth (7 % PPE), indicating that the presentation of specific lipids by a carrier is crucial for the parasite. All the data indicate that Albumax I can replace human serum offering the advantages of safety, standardization for chemosensitivity tests, and exoantigen purification.