Hypoxanthine: a low molecular weight factor essential for growth of erythrocyticPlasmodium falciparumin a serum-free medium

Abstract

A low molecular weight factor in a basal medium essential for erythrocytic Plasmodium falciparum development in a serum-free medium using a cell growth-promoting factor derived from adult bovine serum was detected. The factor was hypoxanthine. The optimal hypoxanthine concentration for parasite growth was between 15 and 120 microM. The contribution of hypoxanthine to increased parasite growth was clearly evident in cultures on day 4. Among various low molecular weight supplements tested, adenine, adenosine, AMP, ATP, cyclic AMP, guanine, guanosine, inosine, inosine monophosphate, xanthine, NAD, NADH, NADP, NADPH and deoxyguanosine triphosphate showed a similar effect to that of hypoxanthine in the serum-free culture system. On the other hand, the addition of uric acid, FAD, thymidine, uridine, orotic acid, deoxythymidine triphosphate, deoxycytidine triphosphate, deoxyadenosine triphosphate, ribose-1-phosphate, or ethanolamine was not beneficial to the parasite growth. The results presented here will not only be of practical value, but will provide important information about the developmental requirements of 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.…”

“…The basal medium consisted of RPMI-1640 containing 2 mM glutamine, 25 mM 4-(2-hydroxylethyl)-piperazine ethanesulfonic acid, 24 mM NaHCO 3 (Invitrogen Ltd., USA), 25 µg/ml gentamycin (Sigma-Aldrich Corp., USA) and 150 µM hypoxanthine (Sigma-Aldrich). Briefly, erythrocytes (RBC) were preserved in Alsever's solution (Sigma-Aldrich; Asahi et al, 1996) for 3-30 days. They were then washed, dispensed into 24-well culture plates at a hematocrit (% of packed RBC in medium) of 2% (1 ml of suspension/well), and cultured in a humidified atmosphere of 5% CO 2 , 5% O 2 , and 90% N 2 at 37ºC.…”

“…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.…”