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

  • N. GRANDE (a1), E. PRECIGOUT (a1), M. L. ANCELIN (a2), K. MOUBRI (a1), B. CARCY (a1), J. L. LEMESRE (a3), H. VIAL (a2) and A. GORENFLOT (a1)...

Abstract

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) ([ges ]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.

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Laboratoire de Biologie Cellulaire et Moléculaire, UPRES No. 699, UFR des Sciences Pharmaceutiques et Biologiques, 15 Avenue Charles Flahault, F-34060 Montpellier Cedex 02, France. Tel: +33 04 67 54 64 81. Fax: +33 04 67 54 66 21. E-mail: agorenf@pharma.univ-montpl.fr.

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