Leishmania spp: completely defined medium without serum and macromolecules (CDM/LP) for the continuous in vitro cultivation of infective promastigote forms.

Abstract

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 RPMI 1640 medium for all Leishmania 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. 1 A similar promastigote form develops when parasites are cultured in cell-free medium. 2

Isotopomer Profiling of Leishmania mexicana Promastigotes Reveals Important Roles for Succinate Fermentation and Aspartate Uptake in Tricarboxylic Acid Cycle (TCA) Anaplerosis, Glutamate Synthesis, and Growth

“…CDM has the following composition: glucose (6 mM), alanine (1.9 mM), arginine (0.4 mM), glycine (3.1 mM), histidine (0.04 mM), isoleucine (0.045 mM), leucine (0.61 mM), lysine (0.55 mM), methionine (0.13 mM), phenylalanine (0.18 mM), proline (0.26 mM), serine (0.76 mM), threonine (0.34 mM), tyrosine (0.033 mM), valine (1.1 mM), and tryptophan (0.15 mM), vitamins, heme, and 0.5% bovine serum albumin (BSA) containing bound fatty acids (32). For the labeling studies, unlabeled components were replaced with the following U- 13 Sampling and Metabolite Extraction-Parasite metabolism was quenched using the protocol described previously (33).…”

Effect of Human Urine on Cell Cycle and Infectivity of Leismania Species Promastigotes In Vitro

“…6 Completely defined serum-free medium was used for conservation of infectivity in vitro . 16 Attempts were made to increase infectivity of parasites by co-culture with J774 macrophages. 4 All of these studies showed that culture medium should contain factors that enable development of non-infective procyclic promastigotes into infective metacyclics.…”

Metabolomic-Based Strategies for Anti-Parasite Drug Discovery

“…17 As a result, efforts have been made to create minimal media that are as close as possible to the most relevant biological environment. [17][18][19][20] Metabolomics has recently been used to show that bloodstream-form T. brucei used only glucose, cysteine, glutamine, and aromatic amino acids from the HMI-11 classically used to culture bloodstream-form trypanosomes. 17 A minimal medium comprising these nutrients, salts, buffer, but still requiring serum was devised.…”