**1. Introduction**

Malaria remains a devastating disease, particularly in the tropics. The annual incidence of malaria worldwide is estimated to be between 294 and 500 million clinical cases, while estimates of annual mortality from malaria, caused largely by the protozoan *Plasmodium falciparum*, range from 0.97 to 2.7 million worldwide (World Malaria Report 20101, World Health Organization; Snow et al., 2005). The emergence of resistance to conventional antimalarial drugs and insecticides means that new chemotherapeutic approaches with alternative targets are needed (Ridley, 2002). Better understandings of antimalarial drugs and the biology of the parasites are needed to allow the development of new medications.

A review of the impact of continuous cultures of *P. falciparum* underscores their significant contributions to malaria research (Trager & Jensen, 1997). 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.

In order to identify the factors controlling parasite development, and the effects of growthpromoting factors on the parasite, we initially investigated growth-promoting substances to formulate a chemically defined culture medium (CDM) suitable for sustaining the complete development and intraerythrocytic growth of *P. falciparum*. We also developed a simple and sensitive flow-cytometry-based assay for following each developmental stage of the parasite's erythrocytic growth. The distinct roles of the growth-promoting factors in the growth of *P. falciparum* were then investigated.

<sup>1</sup>http://www.who.int/malaria/world\_malaria\_report\_2010/en/index.htm

**I**ntraerythrocytic *Plasmodium falciparum* Growth in

Serum-Free Medium with an Emphasis on Growth-Promoting Factors 75

parasite culture, but could also provide critical clues about the parasite's biology and its requirements for proliferation at the erythrocyte stage. To establish a CDM for continuous, intraerythrocytic growth of *P. falciparum*, we initially characterized the ability of the various components of GFS to sustain parasite growth (Asahi et al., 2005). Based on these results, we subsequently determined the ability of structurally defined chemicals to sustain parasite

We investigated the components of GFS and related substances that have shown an ability to sustain parasite growth (Asahi et al., 2005). A simple total lipid fraction of GFS obtained by lipid extraction has been shown to sustain complete parasite development. However, specific proteins such as bovine and human albumin, as well as the simple total lipid fraction of GFS, have also been shown to be important (Figure 1a). The simple total lipid fraction of GFS contained phospholipids, diacylglycerides (DAG), cholesterol, monoglycerides, nonesterified fatty acids (NEFA) and cholesteryl esters. The components of the NEFA fraction were mainly *cis*-9-octadecenoic acid (C18:1-*cis*-9, 43%), hexadecanoic acid

Fig. 1. Abilities of fractions derived from GFS (a) and a total simple lipid fraction of GFS (b) to sustain growth of *P. falciparum.* Growth rate was estimated 4 days after inoculation.

growth, and formulated a CDM for *P. falciparum* growth (Asahi, 2009).

**3.1 The ability of GFS components to sustain parasite growth** 

## **2. Parasites and culture**

Cultures of the FCR3/FMG (ATCC Catalogue No. 30932, Gambia) strain of *P. falciparum*  were used in all experiments. 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 NaHCO3 (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% CO2, 5% O2, and 90% N2 at 37ºC. For subculture, 3-4 days after inoculation, infected RBC and uninfected RBC were washed. Parasitemia (% of infected RBC in total RBC) was adjusted to 0.1% (for subculture) or 0.4% (for growth tests), by adding uninfected RBC, and the hematocrit was adjusted to 2% by adding the appropriate volume of either GFSRPMI or the test medium. The cultures were synchronized at the ring stage by three successive exposures to 5% (w/v) D-sorbitol (Sigma-Aldrich) at 41- and 46-h intervals. After the third sorbitol treatment, residual schizonts and cell debris were removed by isopycnic density centrifugation on 63% Percoll PLUS (GE Healthcare Bio-Science Corp., USA). The current cultivation method remains essentially the same as initially described (Jensen, 2002), with a few refinements, particularly in terms of the culture medium. The growth experiments were performed by replacing GFSRPMI with test samples. Growth rate was estimated by dividing the parasitemia of the test sample after incubation by the initial parasitemia.

### **3. CDM for continuous intraerythrocytic growth of** *P. falciparum* **using lipids**

The *P. falciparum* parasite develops through three distinct stages within RBC during its cycle of approximately 48 h: ring, trophozoite, and schizont (Bannister et al., 2000). 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., 1996, 2005; Cranmer 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. We previously reported that GFS supported intraerythrocytic growth of *P. falciparum* (Asahi and Kanazawa, 1994; Asahi et. al., 1996; Asahi et al., 2005). GFS contains lipid-rich albumin as a major component. Similarly, Cranmer et al. (1997) described a commercially available lipidenriched bovine albumin (Albumax II; Invitrogen) that could replace human serum for the *in vitro* cultivation of *P. falciparum*. These serum substitutes are currently widely employed to maintain parasite cultures. However, there is still insufficient information on these indispensable additives to allow direct identification of the functional components required for the growth of *P. falciparum.* The replacement of human serum or GFS in culture medium with chemically- or functionally-defined substances could not only be advantageous for

Cultures of the FCR3/FMG (ATCC Catalogue No. 30932, Gambia) strain of *P. falciparum*  were used in all experiments. 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 NaHCO3 (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% CO2, 5% O2, and 90% N2 at 37ºC. For subculture, 3-4 days after inoculation, infected RBC and uninfected RBC were washed. Parasitemia (% of infected RBC in total RBC) was adjusted to 0.1% (for subculture) or 0.4% (for growth tests), by adding uninfected RBC, and the hematocrit was adjusted to 2% by adding the appropriate volume of either GFSRPMI or the test medium. The cultures were synchronized at the ring stage by three successive exposures to 5% (w/v) D-sorbitol (Sigma-Aldrich) at 41- and 46-h intervals. After the third sorbitol treatment, residual schizonts and cell debris were removed by isopycnic density centrifugation on 63% Percoll PLUS (GE Healthcare Bio-Science Corp., USA). The current cultivation method remains essentially the same as initially described (Jensen, 2002), with a few refinements, particularly in terms of the culture medium. The growth experiments were performed by replacing GFSRPMI with test samples. Growth rate was estimated by dividing the parasitemia of the test sample after incubation by the initial

**3. CDM for continuous intraerythrocytic growth of** *P. falciparum* **using lipids**  The *P. falciparum* parasite develops through three distinct stages within RBC during its cycle of approximately 48 h: ring, trophozoite, and schizont (Bannister et al., 2000). 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., 1996, 2005; Cranmer 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. We previously reported that GFS supported intraerythrocytic growth of *P. falciparum* (Asahi and Kanazawa, 1994; Asahi et. al., 1996; Asahi et al., 2005). GFS contains lipid-rich albumin as a major component. Similarly, Cranmer et al. (1997) described a commercially available lipidenriched bovine albumin (Albumax II; Invitrogen) that could replace human serum for the *in vitro* cultivation of *P. falciparum*. These serum substitutes are currently widely employed to maintain parasite cultures. However, there is still insufficient information on these indispensable additives to allow direct identification of the functional components required for the growth of *P. falciparum.* The replacement of human serum or GFS in culture medium with chemically- or functionally-defined substances could not only be advantageous for

**2. Parasites and culture** 

parasitemia.

parasite culture, but could also provide critical clues about the parasite's biology and its requirements for proliferation at the erythrocyte stage. To establish a CDM for continuous, intraerythrocytic growth of *P. falciparum*, we initially characterized the ability of the various components of GFS to sustain parasite growth (Asahi et al., 2005). Based on these results, we subsequently determined the ability of structurally defined chemicals to sustain parasite growth, and formulated a CDM for *P. falciparum* growth (Asahi, 2009).
