LEISHMANIA SPF?: COMPLETELY DEFINED MEDIUM WITHOUT SERUM AND MACROMOLECULES (CDM/LP) FOR THE CONTINUOUS ZN VZTRO CULTIVATION OF INFECTIVE PROMASTIGOTE FORMS
Abstract. The elimination of serum or of serum-derived macromolecules that supplant the fetal calf serum requirement from Leishmania culture media could decrease costs and improve the feasibility of large-scale production of well-defined parasite material. We report a completely defined medium, without serum-derived protein and/or macromolecules as a serum substitute, of common, available, and inexpensive constituents that can be used in place of serum-supplemented media for the continuous in vitro cultivation of promastigote forms of various Leishmania species. Typical promastigote morphology was observed in Giemsa-stained smears, regardless of the strain analyzed. Electrophoretic analysis showed that the proteinase patterns of aserically grown promastigote forms were similar to those obtained in serum-supplemented RE" 1640 medium for all Leish?nania studied. Similar antigenic profiles were recognized in immunoblots by sera from hosts with visceral or cutaneous leishmaniasis after growing promastigotes in the two different culture media. For parasites causing both cutaneous and visceral leishmaniasis, the absence of serum and macromolecules in the culture medium did not markedly change their in vitro infectivity for resident mouse macrophages and their virulence in animals compared with parasites cultivated in nondefined medium. Serum-free technology will be increasingly important in providing stability and reproducibility as research using promastigote moves closer to therapeutic applications. Parasites from the genus Leishmania cause a variety of disease states in humans and other mammals in tropical and subtropical areas, which include, cutaneous, mucocutaneous, and visceral leishmaniasis. The parasite undergoes a digenic life cycle between a nonmotile intracellular amastigote stage parasitizing the mammalian phagocytic cells and a flagellated, motile promastigote stage in the midgut of its sandfly vector.' A similar promastigote form develops when parasites are cultured in cell-free medium.2 In vitro standardized cultivation of the members of the Leishmania genus is a useful approach for yielding amount of parasites suitable for diagnosis purposes to provide a better knowledge of host-parasite relationships and for the determination of biologic and immunologic characteristics of the parasite. One of the primary goals of culturists has been to achieve the long-term maintenance of active and dividing populations of different Leishmania species. The different media developed over the past 90 years can be classified in two major categories: semi-solid biphasic media and liquid monophasic media. Leishmania promastigotes were first grown on diphasic blood agar (NNN),3.4 which was later enriched with bacteriologic additives such as brain heart infusion: and is used today with various modifications of the liquid phase added to the solid These nondefined diphasic media are still used today for adaptation and cultivation of Leishmania strains directly isolated from both vertebrate and invertebrate hosts. However, they have some disadvantages. For example, they are complex to prepare and difficult to standardize. Moreover, they contained blood and bacteriologic additives as important factors for parasite replication, which complicate biologic and immunologic studies. Furthe; progress has been made with the use of serumenriched liquid monophasic media. Nondefined glucose-lactalbumin-serum-hemoglobin medium, originally developed for African trypanosomes,lo.ll liyer infusion tryptose (LIT) medium,12 Panmede medium,13 modified NIH medium using I ---hemolyzed rabbit blood,14 and semi-defined HOMEN's medium15 have been reported. Serum-emiched tissue culture media, initially formulated for the cultivation of mammalian or insect cells, have been successfully used to cultivate several Old and New World Leishmania species. These include 199H medium, RPMI 1640 medium, Dulbecco's minimal essential medium,5.16 Schneider's Drosophila medium, and Grace's insect tissue culture medium.16-18 However, all of these tissue culture media require addition of high concentrations of fetal bovine serum (10-30%) as an essential factor for long-term growth of Leishmania promastigotes. Defined media were developed for the cultivation of certain Leishmania. Leishmania tarentolae has been cultured in a chemically defined medium;lg however, this medium could not support the growth of other Leishmania species. Attempts to replace serum by bovine serum albuminzo.21 or mixture of purine bases, vitamins, ;utd bovine albumin fraction IVz2 were also made for cultivating L. donovani promastigote forms. An easily prepared, nearly defìned medium containing salts, glucose, and tryptose (i.e., an LIT medium), to which was added concentrated RPMI 1640 and 199 media, was shown to be adequate for the cultivation of L. chagasi and L. amazonensis proma~tigotes~~ and other Leishmania species.z4 Media developed for serum-free growth of mammalian cells were adapted for the serial cultivation of different Leishmania species. These media were enriched with large concentrations of certain amino acids, vitamins, bovine albumin, hormones, peptide supplement~,2~-~~ or more recently, with human urine.32.33 In the present paper, we report a completely chemically defined culture medium free of serum, macromolecules, proteins, and peptides that readily supports the growth and maintenance of promastigote forms of various Leishmania species without compromising parasite growth rates. Morphologic, biochemical, immunologic, and biologic properties of asencally grown promastigote forms are presented. 41 Fonds Docu meritai re O RSTGP
