Continuous cultivation of intraerythrocytic Plasmodium falciparum in a serum-free medium with the use of a growth-promoting factor

Abstract

Serum-free media were used to culture Plasmodium falciparum. A commercial preparation, Daigo's GF21 developed as a growth-promoting factor for many kinds of mammalian cells, and consisting of the 55-70% ammonium sulphate fraction of adult bovine serum, insulin, transferrin, ethanolamine and sodium selenite, was found to sustain growth of the parasite when Daigo's T was employed as a basal medium. The optimal Daigo's GF21 concentration for parasite growth was between 5 and 20% (v/v), with the best results at 10%. Differential counts indicated that Daigo's GF21 is essential for schizogony. Established serum-free medium, GIT, consisting of Daigo's T basal medium and Daigo's GF21, yielded good parasite growth without any supplementation. Growth-promoting factor derived from adult bovine serum in Daigo's GF21 was shown to be crucial to parasite growth. The results presented here will not only be of practical value, but will provide important information about the developmental requirements for the parasite.

Intraerythrocytic Plasmodium falciparum Growth in Serum-Free Medium with an Emphasis on Growth-Promoting Factors

“…1982;Lingnau et al, 1994;Mi-Ichi et al, 2006;Nivet et al, 1983;Ofulla et al, 1993;Willet and Canfield, 1984), the establishment of a fully-defined culture medium for the parasite has represented a major challenge. We previously reported that GFS supported intraerythrocytic growth of P. falciparum (Asahi and Kanazawa, 1994;Asahi et. al., 1996;Asahi et al, 2005).…”

“…The parasites were routinely maintained using in vitro culture techniques. The culture medium was devoid of whole serum, and consisted of basal medium supplemented with 10% GFS (Daigo's GF21; Wako Pure Chemical Industries, Japan), as previously reported (Asahi, 2009;Asahi & Kanazawa, 1994;Asahi et al, 1996;Asahi et al, 2005;Asahi et al, 2011). This complete medium is referred to as GFSRPMI.…”

“…However, the development of P. falciparum requires the presence of currently unknown factors present in human serum (Jensen, 1979). Although numerous studies have attempted to identify the factors and substances able to sustain parasite growth (Asahi & Kanazawa, 1994;Asahi et al, 1996Asahi et al, , 2005Cranmer et al, 1997;Divo and Jensen. 1982;Lingnau et al, 1994;Mi-Ichi et al, 2006;Nivet et al, 1983;Ofulla et al, 1993;Willet and Canfield, 1984), the establishment of a fully-defined culture medium for the parasite has represented a major challenge.…”

“…The mechanisms responsible for the growth of the parasite, however, remain largely unknown. Culture media for P. falciparum require human serum, a growth-promoting fraction derived from adult bovine plasma (GFS), or lipid-enriched bovine albumin (Asahi & Kanazawa, 1994;Asahi et al, 1996;Cranmer et al 1997;Jensen, 1979). Elucidation of the factors able to induce the growth of P. falciparum could be of help, not only for successful culture of the parasite, but also for providing critical clues to understanding the biology of parasite proliferation during the erythrocytic phase.…”

Comparative studies of serum-free media and detection techniques for <i>in vitro</i> drug sensitivity assessment of <i>Plasmodium falciparum</i>

“…To avoid the obstacles associated with the dependence on human serum, including unpredictable risks with infectious agents and difficulties in securing normal human sera, particularly in malaria endemic areas, and in obtaining a particular blood group, human serum substitutes such as a growth-promoting fraction derived from adult bovine plasma (GFS; Wako Pure Chemical Industries Ltd., Japan) and lipid-enriched bovine albumin (AlbuMAX I; Life Technologies, Japan) have been exploited success-fully [4,5], and have been employed widely for in vitro cultivation of P. falciparum. However, sufficient information on the constituents of the serum substitutes is still lacking.…”