<table><caption>**Table 2.** *Cont.*</caption><thead><tr><th>Animal Sera <sup>1</sup></th><th>Parasite Growth <sup>2</sup></th><th>References</th></tr></thead><tbody><tr><td rowspan="5">Horse</td><td>very good (without adaptation)</td><td>[29,99]</td></tr><tr><td>poor</td><td>[156]</td></tr><tr><td>poor for 96 h; no growth beyond 96 h</td><td>[93,155]</td></tr><tr><td>fair to good up to 30 days</td><td>[169]</td></tr><tr><td>very good after adaptation</td><td>[175]</td></tr><tr><td rowspan="6">Sheep</td><td>poor to fair (data not shown)</td><td>[29]</td></tr><tr><td>poor</td><td>[156]</td></tr><tr><td>poor to no growth</td><td>[93,155]</td></tr><tr><td>poor during adaptation;<br/>fair to good after <sup>10</sup></td><td>[181]</td></tr><tr><td>poor to fair (parasite<br/>strain-dependent)</td><td>[169]</td></tr></tbody></table>

¹ Most experiments were performed with 10% (v/v) animal serum in the RPMI 1640 medium. ² Relative terms are used in this table: “very good” if parasite growth is similar to human serum, “good” if parasite growth is approximately ≥75% compared with human serum, “fair” for parasite growth 25–75%, and “poor” for growth <25%. Because different factors (days of culture, different serum batches, parasite strains) used by the investigators may influence the results, readers are advised to consult the original references for details. ³ To obtain parasite growth similar to human serum, additional supplements (hypoxanthine, neopeptone) may be required [155]. ⁴ These investigators used the medium 199 and 15–20% calf serum. The culture medium was changed twice daily. ⁵ Supplemented with human erythrocyte extract or proteose peptone. ⁶ After adaptation with a glucose peptone supplement. ⁷ In this experiment, a higher amount of rabbit serum (15%) was added to RPMI 1640 [179]. ⁸ The author reported that two *P. falciparum* strains were adapted to the medium supplemented with 5–10% goat serum in long-term cultivation. ⁹ Long-term cultivation failed, partly due to agglutination. ¹⁰ Poor during the adaptation period of primary isolate (first 57 days of cultivation); fair to good thereafter (culture terminated on day 74 of this study) [181].

Rabbit serum can replace human serum for long-term cultivation with Trager's method after the adaptation of the parasites [178]. For short-term (single to three cycles) cultivation and in vitro drug susceptibility assays using laboratory-adapted *P. falciparum* strains, 10–20% rabbit serum may replace 10% human serum to supplement either RPMI 1640 or the combination of RPMI 1640–NCTC–IMDM media (v/v/v 2:1:1) [99,177]. For fresh clinical isolates without prior adaptation to in vitro culture conditions, the addition of 10% rabbit serum to a whole-blood RPMI 1640 mixture (v/v 1:9) may enhance the ability of the parasites to undergo schizogony during the first intraerythrocytic cycle [183].

In several studies, parasite growth was unsatisfactory with swine, equine, ovine, and caprine sera for short-term and/or long-term cultivation [154–156]. In other studies, horse, goat, and sheep sera were found to sustain parasite growth for several weeks, with or without an adaptation period [29,99,169,175,181]. However, in one study using fresh clinical isolates cultivated for a single intraerythrocytic cycle, one batch of goat serum proved to be a suitable serum substitute, while horse serum did not support parasite growth [103]. In contrast, in another study using the reference *P. falciparum* clone 3D7, RPMI 1640–10% goat serum was unsuitable to sustain parasite growth for a short-term period (three cycles), while RPMI 1640–10% horse serum supported the parasite growth [99]. These contradictory results may have been due to batch-to-batch differences in the nutritional quality of animal sera. Another possible explanation may lie in anti-human erythrocyte antibodies in animal sera [184]. The latter can be removed by citration and adsorption with washed human erythrocytes, as described by these authors. Calf serum and horse serum treated with citrate and adsorbed with uninfected human erythrocytes before use for *P. falciparum* cultivation were reported to support parasite growth to a similar extent as human serum. This method has not been widely adopted by malaria researchers to date.

For short-term cultivation (one or two cycles), monkey serum (owl monkey (*Aotus trivirgatus*) or squirrel monkey (*Saimiri sciureus*)) has been shown to support the growth of laboratory-adapted *P. falciparum* strains to some extent, or as well as human serum, without any adaptation period [117,153,159,163]. However, for obvious reasons related to animal
<table><caption>**Table 2. Cont.**</caption><thead><tr><th>Animal Sera <sup>1</sup></th><th>Parasite Growth <sup>2</sup></th><th>References</th></tr></thead><tbody><tr><td rowspan="5">Horse</td><td>very good (without adaptation)</td><td>[29,99]</td></tr><tr><td>poor</td><td>[156]</td></tr><tr><td>poor for 96 h; no growth beyond 96 h</td><td>[93,155]</td></tr><tr><td>fair to good up to 30 days</td><td>[169]</td></tr><tr><td>very good after adaptation</td><td>[175]</td></tr><tr><td rowspan="6">Sheep</td><td>poor to fair (data not shown)</td><td>[29]</td></tr><tr><td>poor</td><td>[156]</td></tr><tr><td>poor to no growth</td><td>[93,155]</td></tr><tr><td>poor during adaptation;<br>fair to good after <sup>10</sup></td><td>[181]</td></tr><tr><td>poor to fair (parasite<br>strain-dependent)</td><td>[169]</td></tr></tbody></table>

¹ Most experiments were performed with 10% (v/v) animal serum in the RPMI 1640 medium. ² Relative terms are used in this table: “very good” if parasite growth is similar to human serum, “good” if parasite growth is approximately ≥75% compared with human serum, “fair” for parasite growth 25–75%, and “poor” for growth <25%. Because different factors (days of culture, different serum batches, parasite strains) used by the investigators may influence the results, readers are advised to consult the original references for details. ³ To obtain parasite growth similar to human serum, additional supplements (hypoxanthine, neopeptone) may be required [155]. ⁴ These investigators used the medium 199 and 15–20% calf serum. The culture medium was changed twice daily. ⁵ Supplemented with human erythrocyte extract or proteose peptone. ⁶ After adaptation with a glucose peptone supplement. ⁷ In this experiment, a higher amount of rabbit serum (15%) was added to RPMI 1640 [179]. ⁸ The author reported that two *P. falciparum* strains were adapted to the medium supplemented with 5–10% goat serum in long-term cultivation. ⁹ Long-term cultivation failed, partly due to agglutination. ¹⁰ Poor during the adaptation period of primary isolate (first 57 days of cultivation); fair to good thereafter (culture terminated on day 74 of this study) [181].

Rabbit serum can replace human serum for long-term cultivation with Trager's method after the adaptation of the parasites [178]. For short-term (single to three cycles) cultivation and in vitro drug susceptibility assays using laboratory-adapted *P. falciparum* strains, 10–20% rabbit serum may replace 10% human serum to supplement either RPMI 1640 or the combination of RPMI 1640–NCTC–IMDM media (v/v/v 2:1:1) [99,177]. For fresh clinical isolates without prior adaptation to in vitro culture conditions, the addition of 10% rabbit serum to a whole-blood RPMI 1640 mixture (v/v 1:9) may enhance the ability of the parasites to undergo schizogony during the first intraerythrocytic cycle [183].

In several studies, parasite growth was unsatisfactory with swine, equine, ovine, and caprine sera for short-term and/or long-term cultivation [154–156]. In other studies, horse, goat, and sheep sera were found to sustain parasite growth for several weeks, with or without an adaptation period [29,99,169,175,181]. However, in one study using fresh clinical isolates cultivated for a single intraerythrocytic cycle, one batch of goat serum proved to be a suitable serum substitute, while horse serum did not support parasite growth [103]. In contrast, in another study using the reference *P. falciparum* clone 3D7, RPMI 1640–10% goat serum was unsuitable to sustain parasite growth for a short-term period (three cycles), while RPMI 1640–10% horse serum supported the parasite growth [99]. These contradictory results may have been due to batch-to-batch differences in the nutritional quality of animal sera. Another possible explanation may lie in anti-human erythrocyte antibodies in animal sera [184]. The latter can be removed by citration and adsorption with washed human erythrocytes, as described by these authors. Calf serum and horse serum treated with citrate and adsorbed with uninfected human erythrocytes before use for *P. falciparum* cultivation were reported to support parasite growth to a similar extent as human serum. This method has not been widely adopted by malaria researchers to date.

For short-term cultivation (one or two cycles), monkey serum (owl monkey (*Aotus trivirgatus*) or squirrel monkey (*Saimiri sciureus*)) has been shown to support the growth of laboratory-adapted *P. falciparum* strains to some extent, or as well as human serum, without any adaptation period [117,153,159,163]. However, for obvious reasons related to animal
protection and rights and the availability of other serum sources, the use of monkey serum for routine cultivation of *P. falciparum* is not recommended.

In the experiments conducted by Sax and Rieckmann, a two-to-three-week adaptation period was required for three of four human serum-adapted *P. falciparum* strains to grow in a rabbit serum-supplemented RPMI 1640 medium at a similar rate as a human serum-supplemented RPMI 1640 medium [178]. One *P. falciparum* strain did not require any transition period for adaptation to rabbit serum. The RPMI 1640 medium containing 5 or 10% rabbit serum yielded satisfactory parasite growth, while lower (2.5%) or higher (15%) concentrations of rabbit serum were associated with lower parasite growth. For investigators planning to use rabbit serum instead of human serum, it is advisable to perform preliminary tests on different rabbit sera batches and use pooled serum samples.

Several, but not all, investigators have found that fetal calf serum is a suitable substitute for human serum in *P. falciparum* culture. Like human serum, batch-to-batch variation in its ability to support in vitro parasite cultivation exists. In the experience of Trager and Jensen, none of the three different lots of commercially available fetal calf serum supported parasite growth as much as human serum [115]. Moreover, the growth rates in fetal calf serum-supplemented RPMI 1640 diminished with time. This latter phenomenon was also reported by another research team [101]. Their observation is not in total agreement with the experience of some other researchers, who have obtained satisfactory long-term cultivation with fetal or newborn calf serum [26,30,98,155]. Another study has also demonstrated that a large majority of fresh clinical isolates undergo schizogony during the first intraerythrocytic cycle in RPMI 1640 supplemented with 10% fetal calf serum [106,182].

It may be concluded that among animal sera, fetal calf serum is probably the best substitute for human serum for the cultivation of intraerythrocytic stages of *P. falciparum*. All human blood types are compatible with fetal calf serum. Moreover, large quantities of fetal calf serum are available at a reasonable cost through reliable commercial sources that usually provide proof of mycoplasma-negative samples. As for human serum, batch-to-batch variation in the capacity of fetal calf serum to support in vitro propagation of *P. falciparum* exists. A preliminary evaluation of each batch of fetal calf serum is required to ensure optimal culture conditions.

### 6.4. *Serum-Free Culture*

Human serum can be replaced with serum substitutes, some of which are commercially available. There are obvious advantages of using serum replacements: limited batch-to-batch variation, which is a prerequisite for a standardized protocol; compatibility with any human blood type; production at industrial scale and availability through commercial sources, usually at lower costs than non-immune human serum; ease in handling and transport; limited risks of blood-borne pathogens, such as human immunodeficiency virus and hepatitis virus. Serum replacements with which in vitro cultivation of *P. falciparum* has been achieved are listed in Table 3 [54,99,101,122,155,156,167,174,185–199].

<table><caption>**Table 3.** Serum substitutes for *P. falciparum* cultivation.</caption><thead><tr><th>Substitute</th><th>Composition</th><th>Comments</th><th>References</th></tr></thead><tbody><tr><td rowspan="4">Bovine or human serum albumin</td><td>1 g/L fatty acid-free bovine or human serum albumin</td><td>Pre-Trager cultivation method; very good growth over 48 h</td><td>[185,186]</td></tr><tr><td>Fatty acid-free bovine or human serum albumin (concentration not cited)</td><td>Unsatisfactory growth with the candle jar method</td><td>[156]</td></tr><tr><td>5 g/L bovine serum albumin + 10% dialyzable human serum factors</td><td>No growth beyond 24 h</td><td>[187]</td></tr><tr><td>6 g/L fatty acid-free bovine albumin + 10 mg/L adenosine</td><td>Poor growth (culture terminated on day 13)</td><td>[188]</td></tr></tbody></table>
protection and rights and the availability of other serum sources, the use of monkey serum for routine cultivation of *P. falciparum* is not recommended.

In the experiments conducted by Sax and Rieckmann, a two-to-three-week adaptation period was required for three of four human serum-adapted *P. falciparum* strains to grow in a rabbit serum-supplemented RPMI 1640 medium at a similar rate as a human serum-supplemented RPMI 1640 medium [178]. One *P. falciparum* strain did not require any transition period for adaptation to rabbit serum. The RPMI 1640 medium containing 5 or 10% rabbit serum yielded satisfactory parasite growth, while lower (2.5%) or higher (15%) concentrations of rabbit serum were associated with lower parasite growth. For investigators planning to use rabbit serum instead of human serum, it is advisable to perform preliminary tests on different rabbit sera batches and use pooled serum samples.

Several, but not all, investigators have found that fetal calf serum is a suitable substitute for human serum in *P. falciparum* culture. Like human serum, batch-to-batch variation in its ability to support in vitro parasite cultivation exists. In the experience of Trager and Jensen, none of the three different lots of commercially available fetal calf serum supported parasite growth as much as human serum [115]. Moreover, the growth rates in fetal calf serum-supplemented RPMI 1640 diminished with time. This latter phenomenon was also reported by another research team [101]. Their observation is not in total agreement with the experience of some other researchers, who have obtained satisfactory long-term cultivation with fetal or newborn calf serum [26,30,98,155]. Another study has also demonstrated that a large majority of fresh clinical isolates undergo schizogony during the first intraerythrocytic cycle in RPMI 1640 supplemented with 10% fetal calf serum [106,182].

It may be concluded that among animal sera, fetal calf serum is probably the best substitute for human serum for the cultivation of intraerythrocytic stages of *P. falciparum*. All human blood types are compatible with fetal calf serum. Moreover, large quantities of fetal calf serum are available at a reasonable cost through reliable commercial sources that usually provide proof of mycoplasma-negative samples. As for human serum, batch-to-batch variation in the capacity of fetal calf serum to support in vitro propagation of *P. falciparum* exists. A preliminary evaluation of each batch of fetal calf serum is required to ensure optimal culture conditions.

### 6.4. *Serum-Free Culture*

Human serum can be replaced with serum substitutes, some of which are commercially available. There are obvious advantages of using serum replacements: limited batch-to-batch variation, which is a prerequisite for a standardized protocol; compatibility with any human blood type; production at industrial scale and availability through commercial sources, usually at lower costs than non-immune human serum; ease in handling and transport; limited risks of blood-borne pathogens, such as human immunodeficiency virus and hepatitis virus. Serum replacements with which in vitro cultivation of *P. falciparum* has been achieved are listed in Table 3 [54,99,101,122,155,156,167,174,185–199].

<table><caption>**Table 3.** Serum substitutes for *P. falciparum* cultivation.</caption><thead><tr><th>Substitute</th><th>Composition</th><th>Comments</th><th>References</th></tr></thead><tbody><tr><td rowspan="4">Bovine or human serum albumin</td><td>1 g/L fatty acid-free bovine or human serum albumin</td><td>Pre-Trager cultivation method; very good growth over 48 h</td><td>[185,186]</td></tr><tr><td>Fatty acid-free bovine or human serum albumin (concentration not cited)</td><td>Unsatisfactory growth with the candle jar method</td><td>[156]</td></tr><tr><td>5 g/L bovine serum albumin + 10% dialyzable human serum factors</td><td>No growth beyond 24 h</td><td>[187]</td></tr><tr><td>6 g/L fatty acid-free bovine albumin + 10 mg/L adenosine</td><td>Poor growth (culture terminated on day 13)</td><td>[188]</td></tr></tbody></table>
<table><caption>**Table 3. Cont.**</caption><thead><tr><th>Substitute</th><th>Composition</th><th>Comments</th><th>References</th></tr></thead><tbody><tr><td></td><td>5 g/L fatty acid-free bovine albumin or 5 g/L Cohn fraction V of bovine albumin</td><td>Good for short-term (3–5 days) culture; better growth with Cohn fraction V</td><td>[54]</td></tr><tr><td></td><td>0.4 or 4 g/L bovine serum albumin</td><td>Poor growth in the GIT medium <sup>4</sup></td><td>[101]</td></tr><tr><td></td><td>Stearic acid</td><td>Pre-Trager cultivation method; fair growth at 48 h</td><td>[185,186]</td></tr><tr><td></td><td>Stearic, palmitoleic, elaidic acids + bovine albumin</td><td>Poor growth with Trager's method</td><td>[188]</td></tr><tr><td rowspan="2">Lipid</td><td>Cis-vaccenic, oleic, and linoleic acids (10 µM), individually or in combination + bovine albumin</td><td>Fair growth (about 50% of that observed with human plasma); successful long-term cultivation</td><td>[188]</td></tr><tr><td>Lipid-cholesterol-rich mixture + Cohn fraction V of bovine albumin</td><td>Very good growth; suitable for long-term cultivation</td><td>[54]</td></tr><tr><td></td><td>Bacto-peptone, Yeastolate, lactalbumin hydrolysate, bovine insulin, polyvinylpyrrolidone</td><td>no growth</td><td>[156]</td></tr><tr><td rowspan="2">Peptones</td><td>0.15% Neopeptone® or Proteose-Peptone® no. 3</td><td>Fair growth after adaptation; good growth when combined with fetal calf serum</td><td>[155]</td></tr><tr><td>0.04% proteose peptone</td><td>Very good growth if supplemented with 10% calf serum</td><td>[174]</td></tr><tr><td rowspan="2">Human serum fractions</td><td>5 mg/mL bovine serum albumin + 10% dialyzable human serum factors + HDL or LDL</td><td>Good growth at 48 h with 0.5 mg/mL HDL or 1.8 mg/mL LDL; long-term culture not performed</td><td>[187]</td></tr><tr><td>0.25–0.5 mg/mL HDL fraction</td><td>Successful short-term culture comparable to 10% human serum</td><td>[189]</td></tr><tr><td>GF21</td><td>Ammonium sulfate fraction of adult bovine serum, insulin, transferrin, ethanolamine, sodium selenite</td><td>Very good growth in the GIT medium <sup>4</sup>; successful long-term culture</td><td>[101]</td></tr><tr><td rowspan="2">Nutridoma-SR® <sup>1</sup></td><td rowspan="2">Albumin, insulin, transferrin, cholesterol, organic and inorganic compounds</td><td>At 4% (v/v), growth comparable with serum-supplemented RPMI; may be strain-dependent</td><td>[190]</td></tr><tr><td>Nutridoma alone at 4%, poor growth; 1% Nutridoma + 0.25% or 0.5% Albumax®, very good growth</td><td>[191]</td></tr><tr><td rowspan="2">Ultroser® G <sup>2</sup></td><td rowspan="2">Binding proteins, fatty acids, phospholipids, adhesion factors, hormones, vitamins, growth factors</td><td>1–4% Ultroser, no growth; ≤0.5% Ultroser + ≤2% human serum, moderate growth for 4 days</td><td>[192]</td></tr><tr><td>2–4% Ultroser, no growth; 0.5–1% low growth on days 5–9</td><td>[193]</td></tr><tr><td rowspan="2">Albumax I or II <sup>3</sup></td><td rowspan="2">Lipid-enriched bovine serum albumin</td><td>No growth unless combined with 1% Nutridoma</td><td>[191]</td></tr><tr><td>Very good growth</td><td>[54,99,122,167,194–199]</td></tr></tbody></table>

The following relative terms were used to classify the extent of the suitability of serum substitutes: very good growth, comparable with medium supplemented with human plasma or serum; good growth, ≥75% of growth observed with medium supplemented with human plasma or serum; fair growth, 25–75% of growth observed with medium supplemented with human plasma or serum; poor growth, ≤25% of growth observed with medium supplemented with human plasma or serum. ¹ Boehringer (Mannheim, Germany). ² Invitrogen Gibco BRL. The exact composition of the Ultroser® G serum substitute is unknown. Its partial composition was determined by Blixt et al. [200]. ³ Invitrogen Gibco BRL. ⁴ The GIT medium is a 1:1 mixture of Iscove's and F-12 media supplemented with 10% GF21. HDL, high-density lipoprotein; LDL, low-density lipoprotein.
<table><caption>**Table 3. Cont.**</caption><thead><tr><th>Substitute</th><th>Composition</th><th>Comments</th><th>References</th></tr></thead><tbody><tr><td></td><td>5 g/L fatty acid-free bovine albumin or 5 g/L Cohn fraction V of bovine albumin</td><td>Good for short-term (3–5 days) culture; better growth with Cohn fraction V</td><td>[54]</td></tr><tr><td></td><td>0.4 or 4 g/L bovine serum albumin</td><td>Poor growth in the GIT medium <sup>4</sup></td><td>[101]</td></tr><tr><td></td><td>Stearic acid</td><td>Pre-Trager cultivation method; fair growth at 48 h</td><td>[185,186]</td></tr><tr><td></td><td>Stearic, palmitoleic, elaidic acids + bovine albumin</td><td>Poor growth with Trager's method</td><td>[188]</td></tr><tr><td rowspan="2">Lipid</td><td>Cis-vaccenic, oleic, and linoleic acids (10 µM), individually or in combination + bovine albumin</td><td>Fair growth (about 50% of that observed with human plasma); successful long-term cultivation</td><td>[188]</td></tr><tr><td>Lipid-cholesterol-rich mixture + Cohn fraction V of bovine albumin</td><td>Very good growth; suitable for long-term cultivation</td><td>[54]</td></tr><tr><td rowspan="3">Peptones</td><td>Bacto-peptone, Yeastolate, lactalbumin hydrolysate, bovine insulin, polyvinylpyrrolidone</td><td>no growth</td><td>[156]</td></tr><tr><td>0.15% Neopeptone® or Proteose-Peptone® no. 3</td><td>Fair growth after adaptation; good growth when combined with fetal calf serum</td><td>[155]</td></tr><tr><td>0.04% proteose peptone</td><td>Very good growth if supplemented with 10% calf serum</td><td>[174]</td></tr><tr><td rowspan="2">Human serum fractions</td><td>5 mg/mL bovine serum albumin + 10% dialyzable human serum factors + HDL or LDL</td><td>Good growth at 48 h with 0.5 mg/mL HDL or 1.8 mg/mL LDL; long-term culture not performed</td><td>[187]</td></tr><tr><td>0.25–0.5 mg/mL HDL fraction</td><td>Successful short-term culture comparable to 10% human serum</td><td>[189]</td></tr><tr><td>GF21</td><td>Ammonium sulfate fraction of adult bovine serum, insulin, transferrin, ethanolamine, sodium selenite</td><td>Very good growth in the GIT medium <sup>4</sup>; successful long-term culture</td><td>[101]</td></tr><tr><td rowspan="2">Nutridoma-SR® <sup>1</sup></td><td rowspan="2">Albumin, insulin, transferrin, cholesterol, organic and inorganic compounds</td><td>At 4% (v/v), growth comparable with serum-supplemented RPMI; may be strain-dependent</td><td>[190]</td></tr><tr><td>Nutridoma alone at 4%, poor growth; 1% Nutridoma + 0.25% or 0.5% Albumax®, very good growth</td><td>[191]</td></tr><tr><td rowspan="2">Ultroser® G <sup>2</sup></td><td rowspan="2">Binding proteins, fatty acids, phospholipids, adhesion factors, hormones, vitamins, growth factors</td><td>1–4% Ultroser, no growth; ≤0.5% Ultroser + ≤2% human serum, moderate growth for 4 days</td><td>[192]</td></tr><tr><td>2–4% Ultroser, no growth; 0.5–1% low growth on days 5–9</td><td>[193]</td></tr><tr><td rowspan="2">Albumax I or II <sup>3</sup></td><td rowspan="2">Lipid-enriched bovine serum albumin</td><td>No growth unless combined with 1% Nutridoma</td><td>[191]</td></tr><tr><td>Very good growth</td><td>[54,99,122,167,194–199]</td></tr></tbody></table>

The following relative terms were used to classify the extent of the suitability of serum substitutes: very good growth, comparable with medium supplemented with human plasma or serum; good growth, ≥75% of growth observed with medium supplemented with human plasma or serum; fair growth, 25–75% of growth observed with medium supplemented with human plasma or serum; poor growth, ≤25% of growth observed with medium supplemented with human plasma or serum. ¹ Boehringer (Mannheim, Germany). ² Invitrogen Gibco BRL. The exact composition of the Ultroser® G serum substitute is unknown. Its partial composition was determined by Blixt et al. [200]. ³ Invitrogen Gibco BRL. ⁴ The GIT medium is a 1:1 mixture of Iscove's and F-12 media supplemented with 10% GF21. HDL, high-density lipoprotein; LDL, low-density lipoprotein.
One of the earliest attempts to replace human plasma with fatty-acid-free bovine or human albumin or stearic acid suggested the suitability of bovine albumin, and to a lesser extent, human albumin and stearic acid, to support the in vitro development of *P. falciparum* and/or *P. knowlesi* for a single cycle [185,186,201]. These experiments were conducted with *P. knowlesi* maintained in *Macaca mulatta* or *P. falciparum* maintained in *Aotus trivirgatus* and were cultivated in vitro in the now outdated Harvard medium supplemented with plasma or serum substitutes. Jensen reported that the use of fatty-acid-free bovine or human serum albumin as a replacement for human serum in RPMI 1640 did not result in satisfactory parasite growth with the candle jar technique [156]. Another serum substitute, composed of Bacto-peptone, Yeastolate, lactalbumin hydrolysate, bovine insulin, and polyvinylpyrrolidone, resulted in poor parasite growth with the candle jar method.

*Plasmodium falciparum* strains were successfully maintained for more than one month in a serum-free medium with unsaturated C-18 fatty acids added to the RPMI-HEPES-NaHCO₃ medium containing 6 g/L bovine albumin and 10 mg/L adenosine [188]. Among the fatty acids tested, *cis*-vaccenic, oleic, and linoleic acids supported parasite growth at the concentration of 10 µM (approximately 3 mg/L). Lower (0.1–5 µM) or higher (up to 100 µM) concentrations resulted in either suboptimal growth or no growth. The growth rate in a medium supplemented with one of the three fatty acids was about half of the control cultures maintained in the medium supplemented with 10% human plasma. Various combinations of unsaturated C-18 fatty acids added to the medium did not improve the growth rate. It was confirmed in the study that, as in previous studies, bovine albumin, alone or in combination with stearic acid, did not support parasite growth beyond a few cycles. Furthermore, in their earlier work (unpublished data cited in [188]), saturated fatty acids from C-14 to C-24 did not support the in vitro growth for a single intraerythrocytic cycle.

An established strain initially adapted to the RPMI 1640 medium supplemented with 10% human serum can be gradually adapted to grow in a serum-free RPMI 1640 medium containing peptone additives (Neopeptone® or Proteose-Peptone® no. 3; 0.15% w/v final concentration) over a one-month period [155]. However, the parasite growth rate was much lower than in human serum-supplemented RPMI 1640. After adaptation, when 5–10% fetal calf serum was added to either Neopeptone® or Proteose-Peptone® no. 3, long-term cultivation with parasite growth comparable to, or better than, that observed with 10% human serum was possible. That study has also shown that human serum fractions II (gamma globulins), IV (α globulins), and V (albumin) used as a replacement for human serum do not improve parasite growth. Peptones can be replaced by 50 µM hypoxanthine [96].

One of the key steps necessary to attain the serum-free culture of *P. falciparum* is to identify human serum component(s) required to satisfy the parasites' nutritional needs. In short-term experiments with a laboratory-adapted strain grown in a "basic" RPMI 1640 medium (supplemented with fatty acid-free bovine serum albumin (5 mg/mL) and 10% dialyzable human serum factors), parasites did not grow even for 48 h in the absence of nondialyzable macromolecules [187]. The addition of seric lipoproteins (low-density lipoproteins (LDLs) and high-density lipoproteins (HDLs)) to the medium supported parasite growth, but not to the extent of the control medium with 10% human serum. The continuous culture was not performed with the lipoprotein-supplemented "basic" medium in that study. Further studies showed that the human HDL fraction is the major lipid source for *P. falciparum* during its intraerythrocytic cycle [189,202]. At the concentration of 0.25–0.5 mg/mL HDL, this lipoprotein fraction was shown to support parasite growth for two complete cycles in the RPMI 1640-HEPES-NaHCO₃ medium supplemented with 50 µg/mL hypoxanthine and 600 µg/mL reduced glutathione (2% starting parasitemia, 4% hematocrit, 6% O₂–3% CO₂ gas mixture) to a similar level as the culture medium containing 5% human serum. The other two lipoprotein fractions, LDLs and very-low-density lipoproteins (VLDLs), did not support parasite growth and induced hemolysis. Long-term cultivation was not performed in that study. In a more recent study [203], it was confirmed that human serum HDLs at a concentration of 0.75 mg/mL, but not LDLs, can
One of the earliest attempts to replace human plasma with fatty-acid-free bovine or human albumin or stearic acid suggested the suitability of bovine albumin, and to a lesser extent, human albumin and stearic acid, to support the in vitro development of *P. falciparum* and/or *P. knowlesi* for a single cycle [185,186,201]. These experiments were conducted with *P. knowlesi* maintained in *Macaca mulatta* or *P. falciparum* maintained in *Aotus trivirgatus* and were cultivated in vitro in the now outdated Harvard medium supplemented with plasma or serum substitutes. Jensen reported that the use of fatty-acid-free bovine or human serum albumin as a replacement for human serum in RPMI 1640 did not result in satisfactory parasite growth with the candle jar technique [156]. Another serum substitute, composed of Bacto-peptone, Yeastolate, lactalbumin hydrolysate, bovine insulin, and polyvinylpyrrolidone, resulted in poor parasite growth with the candle jar method.

*Plasmodium falciparum* strains were successfully maintained for more than one month in a serum-free medium with unsaturated C-18 fatty acids added to the RPMI-HEPES-NaHCO₃ medium containing 6 g/L bovine albumin and 10 mg/L adenosine [188]. Among the fatty acids tested, *cis*-vaccenic, oleic, and linoleic acids supported parasite growth at the concentration of 10 µM (approximately 3 mg/L). Lower (0.1–5 µM) or higher (up to 100 µM) concentrations resulted in either suboptimal growth or no growth. The growth rate in a medium supplemented with one of the three fatty acids was about half of the control cultures maintained in the medium supplemented with 10% human plasma. Various combinations of unsaturated C-18 fatty acids added to the medium did not improve the growth rate. It was confirmed in the study that, as in previous studies, bovine albumin, alone or in combination with stearic acid, did not support parasite growth beyond a few cycles. Furthermore, in their earlier work (unpublished data cited in [188]), saturated fatty acids from C-14 to C-24 did not support the in vitro growth for a single intraerythrocytic cycle.

An established strain initially adapted to the RPMI 1640 medium supplemented with 10% human serum can be gradually adapted to grow in a serum-free RPMI 1640 medium containing peptone additives (Neopeptone® or Proteose-Peptone® no. 3; 0.15% w/v final concentration) over a one-month period [155]. However, the parasite growth rate was much lower than in human serum-supplemented RPMI 1640. After adaptation, when 5–10% fetal calf serum was added to either Neopeptone® or Proteose-Peptone® no. 3, long-term cultivation with parasite growth comparable to, or better than, that observed with 10% human serum was possible. That study has also shown that human serum fractions II (gamma globulins), IV (α globulins), and V (albumin) used as a replacement for human serum do not improve parasite growth. Peptones can be replaced by 50 µM hypoxanthine [96].

One of the key steps necessary to attain the serum-free culture of *P. falciparum* is to identify human serum component(s) required to satisfy the parasites' nutritional needs. In short-term experiments with a laboratory-adapted strain grown in a "basic" RPMI 1640 medium (supplemented with fatty acid-free bovine serum albumin (5 mg/mL) and 10% dialyzable human serum factors), parasites did not grow even for 48 h in the absence of nondialyzable macromolecules [187]. The addition of seric lipoproteins (low-density lipoproteins (LDLs) and high-density lipoproteins (HDLs)) to the medium supported parasite growth, but not to the extent of the control medium with 10% human serum. The continuous culture was not performed with the lipoprotein-supplemented "basic" medium in that study. Further studies showed that the human HDL fraction is the major lipid source for *P. falciparum* during its intraerythrocytic cycle [189,202]. At the concentration of 0.25–0.5 mg/mL HDL, this lipoprotein fraction was shown to support parasite growth for two complete cycles in the RPMI 1640-HEPES-NaHCO₃ medium supplemented with 50 µg/mL hypoxanthine and 600 µg/mL reduced glutathione (2% starting parasitemia, 4% hematocrit, 6% O₂–3% CO₂ gas mixture) to a similar level as the culture medium containing 5% human serum. The other two lipoprotein fractions, LDLs and very-low-density lipoproteins (VLDLs), did not support parasite growth and induced hemolysis. Long-term cultivation was not performed in that study. In a more recent study [203], it was confirmed that human serum HDLs at a concentration of 0.75 mg/mL, but not LDLs, can
replace whole human serum for short-term (72 h) cultivation. At a higher concentration of 3 mg/mL, HDL was toxic.

A chemically defined fraction of adult bovine serum, commercialized as “Daigo’s GF21” (Wako Pure Chemical Industries Ltd., Osaka, Japan), has been used as a serum substitute for in vitro cultivation of various cell lines. This product has been reported to be a promising serum substitute for malaria cultivation [101]. It consists of a 55–70% ammonium sulfate fraction of adult bovine serum supplemented with insulin, transferrin, ethanolamine, and sodium selenite. For optimal cell growth, Daigo’s GF21 is diluted to 10% (v/v) in an appropriate culture medium (RPMI 1640 and others, depending on the cell culture), which contains an equivalent of 3 g of protein/L. For malaria cultivation, RPMI 1640 supplemented with Daigo’s GF21 results in suboptimal growth. The best result is obtained with Daigo’s T basal medium (a modified mixture of IMDM and the F-12 medium) supplemented with 10% (v/v) Daigo’s GF21 [101].

The same manufacturer also produces the GIT medium, a ready-to-use culture medium composed of an equal volume of IMDM and Ham’s F-12 medium, insulin, transferrin, ethanolamine, sodium selenite, and 10% (v/v) GF21. The composition of the GIT medium is almost identical to Daigo’s T basal medium supplemented with 10% (v/v) Daigo’s GF21. An addition of fetal bovine serum (0.1–3%) may further enhance the in vitro growth of certain cell lines. In short-term cultures of laboratory-adapted *P. falciparum* strains and field isolates, parasite growth was poor in RPMI 1640 supplemented with GF21. In contrast, parasite growth was similar in the GIT medium and RPMI 1640–10% human serum for laboratory strains and better in the GIT medium than in RPMI 1640–10% human serum for fresh clinical isolates [101,103]. Furthermore, the Japanese investigators reported successful continuous cultivation of two *P. falciparum* strains in an unsupplemented GIT medium for more than a year [101]. These laboratory strains readily adapted to the GIT medium. Among 12 lots of the GIT medium tested for malaria cultivation, there was no batch-to-batch variation in supporting parasite growth. This medium has also been used by another Japanese research team to maintain an additional reference clone of *P. falciparum* for long-term cultivation [204].

Nutridoma-SR (Boehringer Mannheim, Mannheim, Germany; now Roche Diagnostics) is a biochemically defined supplement designed to replace fetal calf serum for cell cultures. It is composed of human proteins, insulin, transferrin, cholesterol, and organic and inorganic compounds. In the initial study designed to induce gametocyte production in a serum-free medium, Lingnau et al. observed that a laboratory strain of *P. falciparum* grows readily without any adaptation period in the serum-free RPMI 1640-HEPES-NaHCO₃ medium supplemented with 50 mg/L hypoxanthine and Nutridoma-SR (10% hematocrit, 3% O₂-5% CO₂) [193]. Parasite growth in the Nutridoma-SR-supplemented RPMI 1640 medium was concentration-dependent within the range of 0.5% and 4% Nutridoma-SR. Using 4% Nutridoma-SR, the asexual parasites grew to a similar extent in RPMI 1640 supplemented with 10% human serum during the 14-day cultivation period with daily medium change (fresh erythrocytes were not added in this study to enhance gametocyte production). Lower concentrations of Nutridoma-SR resulted in suboptimal growth. In the follow-up study designed to evaluate the suitability of Nutridoma-SR as a serum substitute for asexual parasite propagation, several *P. falciparum* strains grew readily in RPMI 1640 supplemented with 50 mg/L hypoxanthine and 4% Nutridoma-SR (8–10% hematocrit, 3% O₂-5% CO₂) [190]. Compared with the growth in the standard RPMI 1640-HEPES-NaHCO₃ medium supplemented with 10% human serum, the growth in the Nutridoma-supplemented RPMI 1640 was similar or better in three of four parasite strains over two to three weeks of continuous culture. One strain developed in both media, but the growth rate was much higher in RPMI 1640 supplemented with human serum.

These findings were not fully confirmed by another group. Under similar experimental conditions (but at lower hematocrit (2%) and 5% O₂-5% CO₂ gas mixture), Flores et al. were unable to cultivate three different laboratory-adapted *P. falciparum* strains beyond a few intraerythrocytic cycles using the RPMI 1640-HEPES-NaHCO₃ medium supplemented
replace whole human serum for short-term (72 h) cultivation. At a higher concentration of 3 mg/mL, HDL was toxic.

A chemically defined fraction of adult bovine serum, commercialized as “Daigo’s GF21” (Wako Pure Chemical Industries Ltd., Osaka, Japan), has been used as a serum substitute for in vitro cultivation of various cell lines. This product has been reported to be a promising serum substitute for malaria cultivation [101]. It consists of a 55–70% ammonium sulfate fraction of adult bovine serum supplemented with insulin, transferrin, ethanolamine, and sodium selenite. For optimal cell growth, Daigo’s GF21 is diluted to 10% (v/v) in an appropriate culture medium (RPMI 1640 and others, depending on the cell culture), which contains an equivalent of 3 g of protein/L. For malaria cultivation, RPMI 1640 supplemented with Daigo’s GF21 results in suboptimal growth. The best result is obtained with Daigo’s T basal medium (a modified mixture of IMDM and the F-12 medium) supplemented with 10% (v/v) Daigo’s GF21 [101].

The same manufacturer also produces the GIT medium, a ready-to-use culture medium composed of an equal volume of IMDM and Ham’s F-12 medium, insulin, transferrin, ethanolamine, sodium selenite, and 10% (v/v) GF21. The composition of the GIT medium is almost identical to Daigo’s T basal medium supplemented with 10% (v/v) Daigo’s GF21. An addition of fetal bovine serum (0.1–3%) may further enhance the in vitro growth of certain cell lines. In short-term cultures of laboratory-adapted *P. falciparum* strains and field isolates, parasite growth was poor in RPMI 1640 supplemented with GF21. In contrast, parasite growth was similar in the GIT medium and RPMI 1640–10% human serum for laboratory strains and better in the GIT medium than in RPMI 1640–10% human serum for fresh clinical isolates [101,103]. Furthermore, the Japanese investigators reported successful continuous cultivation of two *P. falciparum* strains in an unsupplemented GIT medium for more than a year [101]. These laboratory strains readily adapted to the GIT medium. Among 12 lots of the GIT medium tested for malaria cultivation, there was no batch-to-batch variation in supporting parasite growth. This medium has also been used by another Japanese research team to maintain an additional reference clone of *P. falciparum* for long-term cultivation [204].

Nutridoma-SR (Boehringer Mannheim, Mannheim, Germany; now Roche Diagnostics) is a biochemically defined supplement designed to replace fetal calf serum for cell cultures. It is composed of human proteins, insulin, transferrin, cholesterol, and organic and inorganic compounds. In the initial study designed to induce gametocyte production in a serum-free medium, Lingnau et al. observed that a laboratory strain of *P. falciparum* grows readily without any adaptation period in the serum-free RPMI 1640–HEPES-NaHCO₃ medium supplemented with 50 mg/L hypoxanthine and Nutridoma-SR (10% hematocrit, 3% O₂–5% CO₂) [193]. Parasite growth in the Nutridoma-SR-supplemented RPMI 1640 medium was concentration-dependent within the range of 0.5% and 4% Nutridoma-SR. Using 4% Nutridoma-SR, the asexual parasites grew to a similar extent in RPMI 1640 supplemented with 10% human serum during the 14-day cultivation period with daily medium change (fresh erythrocytes were not added in this study to enhance gametocyte production). Lower concentrations of Nutridoma-SR resulted in suboptimal growth. In the follow-up study designed to evaluate the suitability of Nutridoma-SR as a serum substitute for asexual parasite propagation, several *P. falciparum* strains grew readily in RPMI 1640 supplemented with 50 mg/L hypoxanthine and 4% Nutridoma-SR (8–10% hematocrit, 3% O₂–5% CO₂) [190]. Compared with the growth in the standard RPMI 1640–HEPES-NaHCO₃ medium supplemented with 10% human serum, the growth in the Nutridoma-supplemented RPMI 1640 was similar or better in three of four parasite strains over two to three weeks of continuous culture. One strain developed in both media, but the growth rate was much higher in RPMI 1640 supplemented with human serum.

These findings were not fully confirmed by another group. Under similar experimental conditions (but at lower hematocrit (2%) and 5% O₂–5% CO₂ gas mixture), Flores et al. were unable to cultivate three different laboratory-adapted *P. falciparum* strains beyond a few intraerythrocytic cycles using the RPMI 1640–HEPES-NaHCO₃ medium supplemented
with 0.2% D-glucose and either 4% Nutridoma-SR or Albumax® I [191]. A sustained in vitro growth was obtained by combining Nutridoma-SR and Albumax® I with an optimal growth rate observed with 1% (v/v) Nutridoma/0.25% (w/v) or 0.5% Albumax® I. These combinations of serum supplements allowed a continuous culture for more than one month. No further studies seem to have been performed with Nutridoma as a serum substitute for malaria cultivation.

Some other serum substitutes have been assessed for their suitability to support *P. falciparum* cultures. Ultroser® (Invitrogen Gibco Life Technologies) is a serum replacement containing binding proteins, fatty acids, phospholipids, adhesion factors, hormones, vitamins, and growth factors [200]. The manufacturer recommends 2–4% Ultroser® for serum-free cultures of mammalian cells. In a study designed to assess parasite growth for two complete cycles, Mohapatra and Hommel found that 1–4% Ultroser® G in RPMI 1640, as well as 1–2% Ultroser® combined with 1–5% human serum, did not support parasite growth [192]. The addition of low Ultroser® concentrations ≤0.5% together with a low concentration of human serum (≤2%) led to satisfactory parasite growth for 4 days. In another study, RPMI 1640 supplemented with 0.5–4% Ultroser® G also did not support asexual parasite growth for the first three days [193]. The use of 0.5–1% Ultroser® G, but not 2–4% Ultroser® G, seemed to support the growth of asexual parasites when cultivation was pursued beyond 5 days. Ultroser® SF, which is identical to Ultroser® G except for the omission of steroids, also supported parasite growth to a similar extent. Ultroser® HY did not support parasite growth [106,193]. It is necessary to reemphasize that the experiments conducted by Lingnau et al. [193] were designed to induce gametocytogenesis and cultivate gametocytes. The study was, therefore, not designed for the optimal growth of asexual parasites (i.e., fresh uninfected erythrocytes were not added during the 15-day cultivation period). Further attempts to replace human or animal serum with Ultroser® serum substitute for the cultivation of asexual *P. falciparum* parasites do not seem to have been made.

Ofulla et al. reported that the growth rate of a laboratory-adapted strain cultivated in the RPMI 1640-HEPES-NaHCO₃ medium supplemented with 5 g/L of fatty acid-free bovine albumin with daily medium change for five days (initial parasitemia 0.1%, hematocrit 5%) attained almost 90% of that with 10% human serum [54]. Long-term cultures were not performed with this medium preparation. Growth rates were much lower or nil (<60%) at higher (10–20 g/L) or lower (0.2–4 g/L) concentrations of fatty acid-free bovine albumin. The use of a 5 g/L Cohn fraction V of bovine albumin, which contains fatty acids, improved the short-term parasite growth compared with fatty-acid-free bovine albumin. An even better growth of the parasites that is comparable with that in 10% human serum was observed with the medium supplemented with a 5 g/L Cohn fraction V of bovine albumin and a lipid-cholesterol-rich mixture. The latter mixture, which contains fatty acids, cholesterol, lipoproteins, and phospholipids derived from bovine serum, supports parasite growth better than 3 mg/L *cis*-vaccenic, oleic, and linoleic acids, individually or in combination, which were previously tested by Willet and Canfield [188]. The RPMI 1640-HEPES-NaHCO₃ medium supplemented with 2 g/L extra D-glucose, 60 mg/L cold hypoxanthine, a 5 g/L Cohn fraction V of bovine albumin, and a 10 mL/L lipid-cholesterol-rich mixture was successfully used for long-term cultivation of several *P. falciparum* strains. The findings of Ofulla et al. paved the way for the use of a similar supplement derived from bovine serum, Albumax®, which is available through a commercial source (Invitrogen Gibco Life Technologies, Paisley, United Kingdom) [54].

Most of the serum replacements mentioned in this section have been evaluated in individual laboratories. The exception is Albumax® (available as Albumax® I and Albumax® II; the difference is the percentage of residual immunoglobulins), which has gained popularity among malaria researchers, both for continuous culture and in vitro drug susceptibility assays. The use of Albumax® as a replacement for human serum eliminates the potential problem of ABO compatibility between erythrocytes and serum and is cheaper than type AB human serum. It is readily available through an international supplier, may be
with 0.2% D-glucose and either 4% Nutridoma-SR or Albumax® I [191]. A sustained in vitro growth was obtained by combining Nutridoma-SR and Albumax® I with an optimal growth rate observed with 1% (v/v) Nutridoma/0.25% (w/v) or 0.5% Albumax® I. These combinations of serum supplements allowed a continuous culture for more than one month. No further studies seem to have been performed with Nutridoma as a serum substitute for malaria cultivation.

Some other serum substitutes have been assessed for their suitability to support *P. falciparum* cultures. Ultroser® (Invitrogen Gibco Life Technologies) is a serum replacement containing binding proteins, fatty acids, phospholipids, adhesion factors, hormones, vitamins, and growth factors [200]. The manufacturer recommends 2–4% Ultroser® for serum-free cultures of mammalian cells. In a study designed to assess parasite growth for two complete cycles, Mohapatra and Hommel found that 1–4% Ultroser® G in RPMI 1640, as well as 1–2% Ultroser® combined with 1–5% human serum, did not support parasite growth [192]. The addition of low Ultroser® concentrations ≤0.5% together with a low concentration of human serum (≤2%) led to satisfactory parasite growth for 4 days. In another study, RPMI 1640 supplemented with 0.5–4% Ultroser® G also did not support asexual parasite growth for the first three days [193]. The use of 0.5–1% Ultroser® G, but not 2–4% Ultroser® G, seemed to support the growth of asexual parasites when cultivation was pursued beyond 5 days. Ultroser® SF, which is identical to Ultroser® G except for the omission of steroids, also supported parasite growth to a similar extent. Ultroser® HY did not support parasite growth [106,193]. It is necessary to reemphasize that the experiments conducted by Lingnau et al. [193] were designed to induce gametocytogenesis and cultivate gametocytes. The study was, therefore, not designed for the optimal growth of asexual parasites (i.e., fresh uninfected erythrocytes were not added during the 15-day cultivation period). Further attempts to replace human or animal serum with Ultroser® serum substitute for the cultivation of asexual *P. falciparum* parasites do not seem to have been made.

Ofulla et al. reported that the growth rate of a laboratory-adapted strain cultivated in the RPMI 1640-HEPES-NaHCO₃ medium supplemented with 5 g/L of fatty acid-free bovine albumin with daily medium change for five days (initial parasitemia 0.1%, hematocrit 5%) attained almost 90% of that with 10% human serum [54]. Long-term cultures were not performed with this medium preparation. Growth rates were much lower or nil (<60%) at higher (10–20 g/L) or lower (0.2–4 g/L) concentrations of fatty acid-free bovine albumin. The use of a 5 g/L Cohn fraction V of bovine albumin, which contains fatty acids, improved the short-term parasite growth compared with fatty-acid-free bovine albumin. An even better growth of the parasites that is comparable with that in 10% human serum was observed with the medium supplemented with a 5 g/L Cohn fraction V of bovine albumin and a lipid-cholesterol-rich mixture. The latter mixture, which contains fatty acids, cholesterol, lipoproteins, and phospholipids derived from bovine serum, supports parasite growth better than 3 mg/L *cis*-vaccenic, oleic, and linoleic acids, individually or in combination, which were previously tested by Willet and Canfield [188]. The RPMI 1640-HEPES-NaHCO₃ medium supplemented with 2 g/L extra D-glucose, 60 mg/L cold hypoxanthine, a 5 g/L Cohn fraction V of bovine albumin, and a 10 mL/L lipid-cholesterol-rich mixture was successfully used for long-term cultivation of several *P. falciparum* strains. The findings of Ofulla et al. paved the way for the use of a similar supplement derived from bovine serum, Albumax®, which is available through a commercial source (Invitrogen Gibco Life Technologies, Paisley, United Kingdom) [54].

Most of the serum replacements mentioned in this section have been evaluated in individual laboratories. The exception is Albumax® (available as Albumax® I and Albumax® II; the difference is the percentage of residual immunoglobulins), which has gained popularity among malaria researchers, both for continuous culture and in vitro drug susceptibility assays. The use of Albumax® as a replacement for human serum eliminates the potential problem of ABO compatibility between erythrocytes and serum and is cheaper than type AB human serum. It is readily available through an international supplier, may be
transported to the field at ambient temperature, and is stable. Some of its disadvantages include the unknown exact composition, possible batch-to-batch differences in supporting parasite growth observed by some investigators, and differences in drug-protein binding properties between bovine albumin (and other proteins) and human serum, leading to widely different in vitro responses for some antimalarial drugs. Albumax® is usually diluted to a final concentration (w/v) of 0.5% in the RPMI 1640 medium. The concentration of Albumax® is based on the observation that human serum contains approximately 50 g/L albumin, which is diluted to 5 g/L (=0.5%) in the RPMI medium with 10% serum. The RPMI 1640-Albumax® liquid mixture can be stored at +4 °C for up to 2 months [194].

Several research groups have demonstrated that various laboratory-adapted *P. falciparum* strains initially adapted to the RPMI 1640/human serum medium can be adapted with ease to the RPMI 1640/Albumax® medium. Although one of the first attempts to adapt several strains to RPMI 1640/0.5% Albumax® I was a failure without additional supplements (i.e., Nutridoma-SR) [191], in subsequent experimental studies performed by other research groups, it was reported that several *P. falciparum* strains, including reference clones (HB3, 3D7, W2, Dd2), were maintained in a continuous culture in the RPMI 1640/0.5% Albumax® I medium, but technical details were not provided [195,196]. The growth of more than 10 different *P. falciparum* strains in RPMI 1640/0.5% Albumax® was reported to be comparable to RPMI 1640/10% human serum, especially when the former medium was supplemented with hypoxanthine [194]. For short-term cultivation (i.e., up to 7 days; starting parasitemia, 0.5%; hematocrit 6%; replacement of culture media every 48 h) of a laboratory-adapted strain (3D7 clone), RPMI 1640 supplemented with Albumax® II at low concentrations (0.01%, 0.05%, and 0.1%) supported parasite growth to a similar extent as RPMI 1640/0.5% Albumax® II [99]. Another laboratory-adapted strain (D6/Sierra Leone) was easily adapted to grow continuously for more than two months in the RPMI 1640/0.5% Albumax® I medium and yielded higher parasitemia than the control parasites grown in RPMI 1640/10% human serum [197]. Fresh isolates of *P. falciparum* have also been adapted to grow in RPMI 1640/0.15% Albumax® (one isolate "BINH/Kenya" was maintained for more than 2 years), RPMI 1640/2.5% Albumax® I/0.5 g/L hypoxanthine (5 of 38 Malian isolates for 3-4 weeks), or RPMI 1640/0.5% Albumax®/0.05% hypoxanthine (21 of 33 Indian isolates for up to 35 days; 10 of 12 cryopreserved Indian isolates for up to 45 days) in a continuous culture [167,198,199].

RPMI 1640 supplemented with 0.5% Albumax® has also been used successfully to perform in vitro (i.e., with laboratory-adapted strains) or ex vivo (i.e., with fresh clinical isolates without prior adaptation to in vitro culture) drug susceptibility assays (i.e., 48-72 h short-term cultivation) [106,168,197,205-208]. Some authors have reported that $IC_{50}$ values for different antimalarial drugs obtained with RPMI 1640/human serum and RPMI 1640/Albumax® media may be significantly different, probably due to differences in drug binding properties of albumin present in human serum and Albumax® [106,168,205]. The aforementioned studies suggest that most, if not all, strains already adapted to continuous culture in RPMI 1640/human serum, as well as fresh isolates that can be adapted to in vitro culture conditions with RPMI 1640/human serum and can probably be also adapted to the RPMI 1640/Albumax® medium.

Human serum contains 40–50 g/L of albumin [197], and compared to Albumax® (lipid-enriched bovine serum albumin), has more phospholipids and cholesterol, but less fatty acid [209]. The composition of human serum is also much more complex (it contains a variety of amino acids, proteins, lipids, carbohydrates, and electrolytes) and is probably more nutritive, but there is a wide interindividual variation. The combined supplement of human serum and Albumax® to RPMI 1640 increases the protein content to support parasite growth, especially freshly isolated parasites that are adapted to in vitro cultivation during the initial intraerythrocytic cycles. For continuous cultivation under optimal conditions and/or for rapid adaptation to serum-free conditions, some investigators recommend the addition of hypoxanthine (40–200 µM) [167,194]. Binh et al. observed that, compared with the parasite growth in Albumax®-supplemented RPMI 1640, a two-to-five-fold greater
transported to the field at ambient temperature, and is stable. Some of its disadvantages include the unknown exact composition, possible batch-to-batch differences in supporting parasite growth observed by some investigators, and differences in drug-protein binding properties between bovine albumin (and other proteins) and human serum, leading to widely different in vitro responses for some antimalarial drugs. Albumax® is usually diluted to a final concentration (w/v) of 0.5% in the RPMI 1640 medium. The concentration of Albumax® is based on the observation that human serum contains approximately 50 g/L albumin, which is diluted to 5 g/L (=0.5%) in the RPMI medium with 10% serum. The RPMI 1640-Albumax® liquid mixture can be stored at +4 °C for up to 2 months [194].

Several research groups have demonstrated that various laboratory-adapted *P. falciparum* strains initially adapted to the RPMI 1640/human serum medium can be adapted with ease to the RPMI 1640/Albumax® medium. Although one of the first attempts to adapt several strains to RPMI 1640/0.5% Albumax® I was a failure without additional supplements (i.e., Nutridoma-SR) [191], in subsequent experimental studies performed by other research groups, it was reported that several *P. falciparum* strains, including reference clones (HB3, 3D7, W2, Dd2), were maintained in a continuous culture in the RPMI 1640/0.5% Albumax® I medium, but technical details were not provided [195,196]. The growth of more than 10 different *P. falciparum* strains in RPMI 1640/0.5% Albumax was reported to be comparable to RPMI 1640/10% human serum, especially when the former medium was supplemented with hypoxanthine [194]. For short-term cultivation (i.e., up to 7 days; starting parasitemia, 0.5%; hematocrit 6%; replacement of culture media every 48 h) of a laboratory-adapted strain (3D7 clone), RPMI 1640 supplemented with Albumax® II at low concentrations (0.01%, 0.05%, and 0.1%) supported parasite growth to a similar extent as RPMI 1640/0.5% Albumax® II [99]. Another laboratory-adapted strain (D6/Sierra Leone) was easily adapted to grow continuously for more than two months in the RPMI 1640/0.5% Albumax® I medium and yielded higher parasitemia than the control parasites grown in RPMI 1640/10% human serum [197]. Fresh isolates of *P. falciparum* have also been adapted to grow in RPMI 1640/0.15% Albumax® (one isolate "BINH/Kenya" was maintained for more than 2 years), RPMI 1640/2.5% Albumax® I/0.5 g/L hypoxanthine (5 of 38 Malian isolates for 3-4 weeks), or RPMI 1640/0.5% Albumax®/0.05% hypoxanthine (21 of 33 Indian isolates for up to 35 days; 10 of 12 cryopreserved Indian isolates for up to 45 days) in a continuous culture [167,198,199].

RPMI 1640 supplemented with 0.5% Albumax® has also been used successfully to perform in vitro (i.e., with laboratory-adapted strains) or ex vivo (i.e., with fresh clinical isolates without prior adaptation to in vitro culture) drug susceptibility assays (i.e., 48-72 h short-term cultivation) [106,168,197,205-208]. Some authors have reported that $IC_{50}$ values for different antimalarial drugs obtained with RPMI 1640/human serum and RPMI 1640/Albumax® media may be significantly different, probably due to differences in drug binding properties of albumin present in human serum and Albumax® [106,168,205]. The aforementioned studies suggest that most, if not all, strains already adapted to continuous culture in RPMI 1640/human serum, as well as fresh isolates that can be adapted to in vitro culture conditions with RPMI 1640/human serum and can probably be also adapted to the RPMI 1640/Albumax® medium.

Human serum contains 40–50 g/L of albumin [197], and compared to Albumax® (lipid-enriched bovine serum albumin), has more phospholipids and cholesterol, but less fatty acid [209]. The composition of human serum is also much more complex (it contains a variety of amino acids, proteins, lipids, carbohydrates, and electrolytes) and is probably more nutritive, but there is a wide interindividual variation. The combined supplement of human serum and Albumax® to RPMI 1640 increases the protein content to support parasite growth, especially freshly isolated parasites that are adapted to in vitro cultivation during the initial intraerythrocytic cycles. For continuous cultivation under optimal conditions and/or for rapid adaptation to serum-free conditions, some investigators recommend the addition of hypoxanthine (40–200 µM) [167,194]. Binh et al. observed that, compared with the parasite growth in Albumax®-supplemented RPMI 1640, a two-to-five-fold greater
growth rate is obtained when 10% non-immune human serum or 10% autologous serum from patients with acute malaria is added (i.e., 10% human serum + 1.5% Albumax®) [167]. For short-term cultivation (72 h), a combined supplement of 10–15% human serum, 0.5% Albumax® II, and hypoxanthine supported parasite growth twice as much as Albumax® II alone, but much less than 15% human serum [210]. Another research group recently reported that a 1:1 mixture of human serum and Albumax® II supports parasite growth (based on the multiplication factor) better than, or equivalent to, either 10% human serum alone or Albumax® II alone supplemented to the standard culture medium (RPMI 1640, 25 mM HEPES, 25 mM NaHCO₃) [211]. The mixed supplement (Albumax® + human serum) may not necessarily be beneficial. Depending on the parasite strain and how it was adapted to different culture media, different results may be obtained. For example, Singh et al. observed that once D6/Sierre Leone clone was adapted to a culture medium with only 0.5% Albumax after 2 months of continuous cultivation, parasite growth was better (i.e., with two-fold higher parasitemia) with RPMI 1640 supplemented with 0.5% Albumax® I than RPMI 1640 supplemented with 10% human serum [197].

The use of serum-free culture media in experiments has raised some concerns about the
possible altered biology of malaria parasites. In one study, transcriptome analysis using mi-
croarrays in a reference strain grown in RPMI 1640/human serum or RPMI 1640/Albumax®
did not reveal any major difference in the gene expression profile [197]. Those authors
found 43 genes with increased expression in parasites cultivated with Albumax®. This
result was not confirmed in another study in which transcriptome analysis was performed
by RNA sequencing [212]. In the latter study, cultivation in RPMI 1640 supplemented with
Albumax® resulted in an increased expression of more than 500 genes and parasites with
fewer knobs and decreased cytoadherence. These results tend to be supported by another
series of experiments carried out by Ribacke et al., who recommended that, based on their
findings, *P. falciparum* parasites should be grown in RPMI 1640/10% human serum rather
than RPMI 1640/0.5% Albumax® for optimal conditions in experiments on cytoadherence
and rosetting [213]. Other authors have reported that the replacement of human serum with
Albumax® to cultivate the reference clone 3D7 results in a decreased rate of erythrocyte
invasion, increased duration of the life cycle (40 h with 5% human serum + 0.25% Albumax®
II versus 45 h with 0.5% Albumax® II alone), and altered drug response [214,215].

The genetic basis of alteration in parasite biology in different culture conditions was re-
cently explored. Several gene candidates (e.g., aspartate transaminase, erythrocyte-binding
antigen-140, cysteine protease autophagy-related 4) in three loci have been associated with
differential growth rates in two laboratory-adapted *P. falciparum* clones in the standard
RPMI 1640 medium supplemented with either 10% human serum or 0.5% Albumax®
II [209]. These observations suggest that biological fitness, defined as the capacity to pro-
liferate competitively (i.e., the capacity to outgrow the other parasites when placed in
the presence of other parasite populations or strains), may be at least partly dependent
on culture conditions, especially serum supplements or serum substitutes. These recent
experimental studies may be the first step to pave the way to identify the genes associated
with biological fitness and nutritional requirements of the parasites maintained under
different culture conditions.

The fractionation of serum components and the reconstitution of lipid-free serum albumin and various combinations of fatty acids have led to the discovery that the key components provided by human or animal serum and Albumax® may be serum albumin, oleic acid, and palmitic acid [216]. These three components, at a concentration of 30 µM each, supported the growth of a laboratory-adapted strain for over one month, at a growth rate that is 20% less than the control culture maintained with 10% human serum. The optimal ratio of serum albumin and two fatty acids is still undetermined. These essential fatty acids are metabolized into lipids (phosphatidylcholine, phosphatidylethanolamine, diacylglycerol, triacylglycerol) that are major constituents of membranes in the parasite. In a follow-up study, six combinations of human serum-derived fatty acids that can replace whole human serum for a continuous culture for over six months of a reference *P. falciparum*
growth rate is obtained when 10% non-immune human serum or 10% autologous serum from patients with acute malaria is added (i.e., 10% human serum + 1.5% Albumax®) [167]. For short-term cultivation (72 h), a combined supplement of 10–15% human serum, 0.5% Albumax® II, and hypoxanthine supported parasite growth twice as much as Albumax® II alone, but much less than 15% human serum [210]. Another research group recently reported that a 1:1 mixture of human serum and Albumax® II supports parasite growth (based on the multiplication factor) better than, or equivalent to, either 10% human serum alone or Albumax® II alone supplemented to the standard culture medium (RPMI 1640, 25 mM HEPES, 25 mM NaHCO₃) [211]. The mixed supplement (Albumax® + human serum) may not necessarily be beneficial. Depending on the parasite strain and how it was adapted to different culture media, different results may be obtained. For example, Singh et al. observed that once D6/Sierre Leone clone was adapted to a culture medium with only 0.5% Albumax after 2 months of continuous cultivation, parasite growth was better (i.e., with two-fold higher parasitemia) with RPMI 1640 supplemented with 0.5% Albumax® I than RPMI 1640 supplemented with 10% human serum [197].

The use of serum-free culture media in experiments has raised some concerns about the
possible altered biology of malaria parasites. In one study, transcriptome analysis using mi-
croarrays in a reference strain grown in RPMI 1640/human serum or RPMI 1640/Albumax®
did not reveal any major difference in the gene expression profile [197]. Those authors
found 43 genes with increased expression in parasites cultivated with Albumax®. This
result was not confirmed in another study in which transcriptome analysis was performed
by RNA sequencing [212]. In the latter study, cultivation in RPMI 1640 supplemented with
Albumax® resulted in an increased expression of more than 500 genes and parasites with
fewer knobs and decreased cytoadherence. These results tend to be supported by another
series of experiments carried out by Ribacke et al., who recommended that, based on their
findings, *P. falciparum* parasites should be grown in RPMI 1640/10% human serum rather
than RPMI 1640/0.5% Albumax® for optimal conditions in experiments on cytoadherence
and rosetting [213]. Other authors have reported that the replacement of human serum with
Albumax® to cultivate the reference clone 3D7 results in a decreased rate of erythrocyte
invasion, increased duration of the life cycle (40 h with 5% human serum + 0.25% Albumax®
II versus 45 h with 0.5% Albumax® II alone), and altered drug response [214,215].

The genetic basis of alteration in parasite biology in different culture conditions was re-
cently explored. Several gene candidates (e.g., aspartate transaminase, erythrocyte-binding
antigen-140, cysteine protease autophagy-related 4) in three loci have been associated with
differential growth rates in two laboratory-adapted *P. falciparum* clones in the standard
RPMI 1640 medium supplemented with either 10% human serum or 0.5% Albumax®
II [209]. These observations suggest that biological fitness, defined as the capacity to pro-
liferate competitively (i.e., the capacity to outgrow the other parasites when placed in
the presence of other parasite populations or strains), may be at least partly dependent
on culture conditions, especially serum supplements or serum substitutes. These recent
experimental studies may be the first step to pave the way to identify the genes associated
with biological fitness and nutritional requirements of the parasites maintained under
different culture conditions.

The fractionation of serum components and the reconstitution of lipid-free serum albumin and various combinations of fatty acids have led to the discovery that the key components provided by human or animal serum and Albumax® may be serum albumin, oleic acid, and palmitic acid [216]. These three components, at a concentration of 30 µM each, supported the growth of a laboratory-adapted strain for over one month, at a growth rate that is 20% less than the control culture maintained with 10% human serum. The optimal ratio of serum albumin and two fatty acids is still undetermined. These essential fatty acids are metabolized into lipids (phosphatidylcholine, phosphatidylethanolamine, diacylglycerol, triacylglycerol) that are major constituents of membranes in the parasite. In a follow-up study, six combinations of human serum-derived fatty acids that can replace whole human serum for a continuous culture for over six months of a reference *P. falciparum*
strain were identified [217]. These findings help to understand which components of the human serum are required for the development of *P. falciparum* parasite and, hopefully, will be a step forward toward the development of a chemically defined, serum-free medium for optimal malaria cultivation of various parasite strains and fresh clinical isolates.

## 7. Hypoxanthine

Hypoxanthine, a purine base precursor of adenine and guanine that readily enters intraerythrocytic malaria parasites, has been identified as an important serum component necessary to enhance in vitro growth [218–221]. Although other purines can replace hypoxanthine, the RPMI 1640 medium does not contain purines. The addition of human serum and human erythrocytes was thus the only source of hypoxanthine during the early phase of the cultivation method developed by Trager and Jensen [27]. In the alternative cultivation method developed by Haynes et al. [26], the medium that they used, medium 199 with Earle's salts, contains 0.354 mg/L of hypoxanthine.

Human serum or plasma dialyzed through membrane pore size corresponding to low molecular mass <6000–8000 (which eliminates purines, pyrimidines, peptides, and mineral salts) results in poor parasite growth when it is used to supplement RPMI 1640 [93,156]. By contrast, media containing hypoxanthine, such as Ham's F12 (4.77 mg/L), support parasite growth even when they are supplemented with exhaustively dialyzed human serum. By adding different components that are present in Ham's F12, but not in RPMI 1640, Divo and Jensen demonstrated that the key dialyzable low molecular weight component that is not supplied by RPMI 1640 but is necessary for enhanced parasite growth is hypoxanthine [93]. These findings were confirmed in other experiments [222]. The optimal concentration of hypoxanthine to support parasite growth in RPMI 1640 supplemented with pooled adult bovine serum (which contains $6 \times 10^{-7}$ M [0.6 µM] of hypoxanthine) was $3–12 \times 10^{-5}$ M (30–120 µM) [93]. In the experiments conducted by Asahi et al. [222], the optimal hypoxanthine concentration was estimated to lie between 15 and 120 µM. In other experiments, the lowest concentration of exogenous hypoxanthine required to complete one intraerythrocytic cycle (in RPMI 1640 with presumably 10% human serum) was determined to be 2 µM; optimal growth of 3D7 clone required $\ge 5$ µM of hypoxanthine [223]. Other purines, such as inosine, adenosine, and, to a lesser extent, adenine, also support parasite growth in RPMI 1640 supplemented with 10% pooled adult bovine serum. In comparison, the concentration of hypoxanthine in RPMI 1640 supplemented with human serum is $1.5–2.0 \times 10^{-5}$ M (15–20 µM) [224]. The concentrations of hypoxanthine in human plasma and erythrocytes have been estimated to be $1840 \pm 1320$ nM (1.84 ± 1.32 µM) and $11,100 \pm 7600$ nM (11.1 ± 7.6 µM), respectively [225]. Human erythrocytes contain, on average, a six-fold (up to 10-to-15-fold) higher concentration of hypoxanthine compared to the plasma level.

The effect of hypoxanthine added to RPMI-TES-NaHCO$_3$ with high glucose concentration (4 g/L) on the growth of laboratory-adapted strains in suspension culture (10% erythrocyte, 0.8–1.5% starting parasitemia) was studied over four days [51]. The growth of a reference *P. falciparum* clone increased with increasing hypoxanthine concentration between 0.5 and 5 µg/mL, remained constant at concentrations between 5 and 50 µg/mL, and decreased at higher hypoxanthine concentrations of 50–100 µg/mL. However, the growth rates of three uncloned Thai isolates were not affected by the addition of hypoxanthine. Another research group studied the effect of low hypoxanthine ("hypoxanthine-deprived medium" with 0.5 µM hypoxanthine in RPMI 1640/0.5% Albumax® II versus 90 µM hypoxanthine in the control parasites) on the growth of a laboratory-adapted strain (NF54$^{attB}$) over three intraerythrocytic cycles [226]. During the first 24 h in a hypoxanthine-deprived medium, parasitemia and morphology under an optical microscope remained comparable to the control parasites. Cultivation for 40 h in a hypoxanthine-deprived medium resulted in a decrease in parasitemia by about 50% (when assessed at 72 h), delayed schizogony, and abnormal morphology (presence of vacuole-like structures). The parasites cultivated in a hypoxanthine-deprived medium for 40–72 h, but not more than 72 h, were able to
strain were identified [217]. These findings help to understand which components of the human serum are required for the development of *P. falciparum* parasite and, hopefully, will be a step forward toward the development of a chemically defined, serum-free medium for optimal malaria cultivation of various parasite strains and fresh clinical isolates.

## 7. Hypoxanthine

Hypoxanthine, a purine base precursor of adenine and guanine that readily enters intraerythrocytic malaria parasites, has been identified as an important serum component necessary to enhance in vitro growth [218–221]. Although other purines can replace hypoxanthine, the RPMI 1640 medium does not contain purines. The addition of human serum and human erythrocytes was thus the only source of hypoxanthine during the early phase of the cultivation method developed by Trager and Jensen [27]. In the alternative cultivation method developed by Haynes et al. [26], the medium that they used, medium 199 with Earle's salts, contains 0.354 mg/L of hypoxanthine.

Human serum or plasma dialyzed through membrane pore size corresponding to low molecular mass <6000–8000 (which eliminates purines, pyrimidines, peptides, and mineral salts) results in poor parasite growth when it is used to supplement RPMI 1640 [93,156]. By contrast, media containing hypoxanthine, such as Ham's F12 (4.77 mg/L), support parasite growth even when they are supplemented with exhaustively dialyzed human serum. By adding different components that are present in Ham's F12, but not in RPMI 1640, Divo and Jensen demonstrated that the key dialyzable low molecular weight component that is not supplied by RPMI 1640 but is necessary for enhanced parasite growth is hypoxanthine [93]. These findings were confirmed in other experiments [222]. The optimal concentration of hypoxanthine to support parasite growth in RPMI 1640 supplemented with pooled adult bovine serum (which contains $6 \times 10^{-7}$ M [0.6 µM] of hypoxanthine) was $3–12 \times 10^{-5}$ M (30–120 µM) [93]. In the experiments conducted by Asahi et al. [222], the optimal hypoxanthine concentration was estimated to lie between 15 and 120 µM. In other experiments, the lowest concentration of exogenous hypoxanthine required to complete one intraerythrocytic cycle (in RPMI 1640 with presumably 10% human serum) was determined to be 2 µM; optimal growth of 3D7 clone required $\ge 5$ µM of hypoxanthine [223]. Other purines, such as inosine, adenosine, and, to a lesser extent, adenine, also support parasite growth in RPMI 1640 supplemented with 10% pooled adult bovine serum. In comparison, the concentration of hypoxanthine in RPMI 1640 supplemented with human serum is $1.5–2.0 \times 10^{-5}$ M (15–20 µM) [224]. The concentrations of hypoxanthine in human plasma and erythrocytes have been estimated to be $1840 \pm 1320$ nM ($1.84 \pm 1.32$ µM) and $11,100 \pm 7600$ nM ($11.1 \pm 7.6$ µM), respectively [225]. Human erythrocytes contain, on average, a six-fold (up to 10-to-15-fold) higher concentration of hypoxanthine compared to the plasma level.

The effect of hypoxanthine added to RPMI-TES-NaHCO$_3$ with high glucose concentration (4 g/L) on the growth of laboratory-adapted strains in suspension culture (10% erythrocyte, 0.8–1.5% starting parasitemia) was studied over four days [51]. The growth of a reference *P. falciparum* clone increased with increasing hypoxanthine concentration between 0.5 and 5 µg/mL, remained constant at concentrations between 5 and 50 µg/mL, and decreased at higher hypoxanthine concentrations of 50–100 µg/mL. However, the growth rates of three uncloned Thai isolates were not affected by the addition of hypoxanthine. Another research group studied the effect of low hypoxanthine ("hypoxanthine-deprived medium" with 0.5 µM hypoxanthine in RPMI 1640/0.5% Albumax® II versus 90 µM hypoxanthine in the control parasites) on the growth of a laboratory-adapted strain (NF54$^{attB}$) over three intraerythrocytic cycles [226]. During the first 24 h in a hypoxanthine-deprived medium, parasitemia and morphology under an optical microscope remained comparable to the control parasites. Cultivation for 40 h in a hypoxanthine-deprived medium resulted in a decrease in parasitemia by about 50% (when assessed at 72 h), delayed schizogony, and abnormal morphology (presence of vacuole-like structures). The parasites cultivated in a hypoxanthine-deprived medium for 40–72 h, but not more than 72 h, were able to
complete the second intraerythrocytic cycle if placed in the control RPMI 1640 with 90 µM of hypoxanthine.

When the standard RPMI 1640-HEPES-NaHCO₃-10% human serum (or plasma) mixture is supplemented with L-glutamine (5 mM) and hypoxanthine (50 mg/L), it is possible to maintain the continuous culture of a laboratory-adapted strain for up to three days without medium change, provided that the initial parasitemia (≤0.5%) and hematocrit (2.5%) are sufficiently low [227]. Furthermore, cultures in hypoxanthine-supplemented (0.37 mM) RPMI 1640/10% human plasma at 2.5% hematocrit and 0.3% initial parasitemia without medium change for 72 h grow to a similar extent as the control cultures (without hypoxanthine supplement) with daily medium replacement [170]. The addition of hypoxanthine may not be indispensable for cultures left for several days without daily medium replacement, provided that 10% human plasma or serum is used. In one study, cultures of a laboratory-adapted strain (5% hematocrit and 0.5% initial parasitemia) grew without medium change over the weekend (i.e., three days) to the same extent as the parallel cultures with once daily medium change using the standard medium supplemented with L-glutamine, 2 g/L extra glucose (final concentration, 4 g/L), and 10% human plasma, even without hypoxanthine supplement [125].

Based on the observations of several investigators, it may be stated that, in general, the addition of hypoxanthine leads to improved growth rates [51,54,93,101,194]. Despite these observations, hypoxanthine supplementation does not seem to be an absolute requirement for a successful culture, as short-term and long-term cultivation is possible without additional hypoxanthine as long as RPMI 1640 is supplemented with 10% human serum or plasma. It may be hypothesized that purine stores in the host erythrocyte and serum (or plasma) may provide a sufficient source of hypoxanthine. Nonetheless, for the routine cultivation of *P. falciparum* in RPMI 1640/10% human plasma or serum, an addition of hypoxanthine is beneficial to maintain a high growth rate. The situation is different if RPMI 1640 is supplemented with Albumax®. In this latter case, it is highly recommended to add hypoxanthine to the culture medium.

It should be noted that the presence of a "cold" hypoxanthine supplement in the culture medium may interfere with the incorporation of [³H]hypoxanthine in radioisotope-based drug susceptibility assays [49,50]. The usual practice is to provide a wash-out period of at least one complete intraerythrocytic cycle during which the parasites are cultivated in RPMI 1640 without hypoxanthine supplement, or the infected erythrocytes are thoroughly washed in the hypoxanthine-free medium before performing isotopic drug susceptibility assays.

## 8. Feeder Cells

Feeder cells refer to a monolayer of adherent animal cells that secrete proteins and other metabolites that supplement the culture medium to enhance the growth of a co-cultivated cell line. Feeder cells are first grown to confluency and irradiated to halt their proliferation. Another cell line, which is usually difficult to cultivate without extensive supplementation of the medium, is added to the feeder layer for co-cultivation.

This specialized technique has been applied to malaria cultivation. Early studies performed during the 1980s showed that when laboratory-adapted strains or fresh clinical isolates are cultivated on monolayers of murine hepatocytes, fibroblasts, or macrophages, or co-cultivated with human peripheral blood mononuclear cells, it was observed that parasites grow at a higher rate, attain higher parasitemia, and adapt better to in vitro culture conditions [157,158,228–230]. Instead of co-cultivation, another approach is to cultivate mouse hepatocytes separately and add the supernatant (v/v 5–15%) to *P. falciparum* cultures (RPMI 1640/10% human serum) [231]. The authors reported that the growth rate over a single intraerythrocytic cycle increased significantly (a mean of 1.4–1.5-fold) with the addition of 10–15% (v/v) supernatant in both laboratory-adapted strains and freshly collected isolates. More recently, the co-cultivation method of B lymphocytes and *P. falciparum* has been further refined [232]. The authors reported that activated B
complete the second intraerythrocytic cycle if placed in the control RPMI 1640 with 90 µM of hypoxanthine.

When the standard RPMI 1640-HEPES-NaHCO₃-10% human serum (or plasma) mixture is supplemented with L-glutamine (5 mM) and hypoxanthine (50 mg/L), it is possible to maintain the continuous culture of a laboratory-adapted strain for up to three days without medium change, provided that the initial parasitemia (≤0.5%) and hematocrit (2.5%) are sufficiently low [227]. Furthermore, cultures in hypoxanthine-supplemented (0.37 mM) RPMI 1640/10% human plasma at 2.5% hematocrit and 0.3% initial parasitemia without medium change for 72 h grow to a similar extent as the control cultures (without hypoxanthine supplement) with daily medium replacement [170]. The addition of hypoxanthine may not be indispensable for cultures left for several days without daily medium replacement, provided that 10% human plasma or serum is used. In one study, cultures of a laboratory-adapted strain (5% hematocrit and 0.5% initial parasitemia) grew without medium change over the weekend (i.e., three days) to the same extent as the parallel cultures with once daily medium change using the standard medium supplemented with L-glutamine, 2 g/L extra glucose (final concentration, 4 g/L), and 10% human plasma, even without hypoxanthine supplement [125].

Based on the observations of several investigators, it may be stated that, in general, the addition of hypoxanthine leads to improved growth rates [51,54,93,101,194]. Despite these observations, hypoxanthine supplementation does not seem to be an absolute requirement for a successful culture, as short-term and long-term cultivation is possible without additional hypoxanthine as long as RPMI 1640 is supplemented with 10% human serum or plasma. It may be hypothesized that purine stores in the host erythrocyte and serum (or plasma) may provide a sufficient source of hypoxanthine. Nonetheless, for the routine cultivation of *P. falciparum* in RPMI 1640/10% human plasma or serum, an addition of hypoxanthine is beneficial to maintain a high growth rate. The situation is different if RPMI 1640 is supplemented with Albumax®. In this latter case, it is highly recommended to add hypoxanthine to the culture medium.

It should be noted that the presence of a "cold" hypoxanthine supplement in the culture medium may interfere with the incorporation of [³H]hypoxanthine in radioisotope-based drug susceptibility assays [49,50]. The usual practice is to provide a wash-out period of at least one complete intraerythrocytic cycle during which the parasites are cultivated in RPMI 1640 without hypoxanthine supplement, or the infected erythrocytes are thoroughly washed in the hypoxanthine-free medium before performing isotopic drug susceptibility assays.

## 8. Feeder Cells

Feeder cells refer to a monolayer of adherent animal cells that secrete proteins and other metabolites that supplement the culture medium to enhance the growth of a co-cultivated cell line. Feeder cells are first grown to confluency and irradiated to halt their proliferation. Another cell line, which is usually difficult to cultivate without extensive supplementation of the medium, is added to the feeder layer for co-cultivation.

This specialized technique has been applied to malaria cultivation. Early studies performed during the 1980s showed that when laboratory-adapted strains or fresh clinical isolates are cultivated on monolayers of murine hepatocytes, fibroblasts, or macrophages, or co-cultivated with human peripheral blood mononuclear cells, it was observed that parasites grow at a higher rate, attain higher parasitemia, and adapt better to in vitro culture conditions [157,158,228–230]. Instead of co-cultivation, another approach is to cultivate mouse hepatocytes separately and add the supernatant (v/v 5–15%) to *P. falciparum* cultures (RPMI 1640/10% human serum) [231]. The authors reported that the growth rate over a single intraerythrocytic cycle increased significantly (a mean of 1.4–1.5-fold) with the addition of 10–15% (v/v) supernatant in both laboratory-adapted strains and freshly collected isolates. More recently, the co-cultivation method of B lymphocytes and *P. falciparum* has been further refined [232]. The authors reported that activated B
lymphocytes and laboratory-adapted *P. falciparum* strains can be co-cultured in direct contact in IMDM and, to a lesser extent, in RPMI 1640 and DMEM, supplemented with 5% human serum and 5% fetal bovine serum for up to 10 days. Co-culture enhanced cell growth mutually, with higher parasitemia and more rapid growth of *P. falciparum* and enhanced B-cell proliferation.

The underlying mechanism leading to improved growth conditions in co-cultures is not well understood. Feeder cells may provide additional nutrients that are either absent or insufficient in the RPMI-10% serum-erythrocyte mixture. These "sophisticated" techniques are not commonly used in most malaria research laboratories. For routine cultures, the use of feeder cells is not required.

## 9. Static versus Suspension Culture

In malaria blood cell cultivation, the term "static culture" means malaria-infected erythrocytes cultivated without agitation. A thin layer of erythrocytes is deposited on the flat-bottomed surface of the culture vessel, plates, flasks, or Petri dish, without any form of adhesion to the substrate. The term "suspension culture" refers to a constant or intermittent agitation of malaria-infected erythrocytes so that the cells remain suspended in the culture medium.

The rocker-dilution method, which involves a constant agitation of infected erythrocytes suspended in the culture medium, was used in early experiments on short-term cultures. Based on the observation that, in vivo, *P. falciparum*-infected erythrocytes remain attached to the endothelium of microvessels in various organs only during the more mature growth stages of the developing schizont, Trager and Jensen reasoned that alternative methods are more appropriate for the long-term cultivation of *P. falciparum* [27]. Two methods have been introduced: the more cumbersome, continuous flow method using a special flow-vial which injects a slow flow of medium over a settled layer of erythrocytes and the more practical, Petri dish-candle jar method, which is stationary. The current standard procedure involves a settled layer of erythrocytes under static conditions during the entire incubation period. The only moment when the erythrocytes are agitated into suspension is during the medium change in Petri dishes or culture flasks, once the spent medium is aspirated and the fresh medium is added.

In the human host, erythrocytes infected by *P. falciparum* rings are in constant circulation in the bloodstream (i.e., in suspension), whereas erythrocytes infected by mature trophozoites and schizonts are stationary due to cytoadherence in deep organs. In vivo, it may be hypothesized that a slow, constant flow of blood over stationary schizont-infected erythrocytes may be more favorable for the dispersion of merozoites devoid of motile organelles to initiate a new intraerythrocytic cycle in a higher number of erythrocytes. In contrast, merozoites released in a completely stationary environment, such as Petri dishes, tend to agglomerate and invade the same uninfected erythrocyte; multiple infections indicate that many merozoites penetrate close to the point of schizont rupture. Erythrocytes infected with two or more rings and trophozoites are more commonly observed in the stationary culture compared with the suspension culture [233].

During early attempts to develop malaria cultivation methods, most cultures had been performed in Erlenmeyer flasks in an atmosphere of 5% CO₂ in the air and placed on a rocking table in an incubator [16]. The rocker flask method does not yield satisfactory parasite growth. In the method developed by Trager and Jensen [27], a stationary, settled thin layer of infected erythrocytes is cultivated on the flat-bottomed surface of either a flow vial or a Petri dish. In later works performed in the late-1970s and 1980s, several investigators compared static and suspension cultures of established *P. falciparum* strains and concluded that cultures on an orbital shaking device or flask shaker led to a higher yield of parasites [29,51,53,109,233,234]. With suspension cultures at 10% erythrocyte suspension (v/v), the yield may be a two-fold increase over static cultures during short-term (two to three intraerythrocytic cycles) and long-term (one month) cultures [51,233]. In suspension cultures maintained at a lower erythrocyte suspension (v/v, 1%) and containing high
lymphocytes and laboratory-adapted *P. falciparum* strains can be co-cultured in direct contact in IMDM and, to a lesser extent, in RPMI 1640 and DMEM, supplemented with 5% human serum and 5% fetal bovine serum for up to 10 days. Co-culture enhanced cell growth mutually, with higher parasitemia and more rapid growth of *P. falciparum* and enhanced B-cell proliferation.

The underlying mechanism leading to improved growth conditions in co-cultures is not well understood. Feeder cells may provide additional nutrients that are either absent or insufficient in the RPMI-10% serum-erythrocyte mixture. These "sophisticated" techniques are not commonly used in most malaria research laboratories. For routine cultures, the use of feeder cells is not required.

## 9. Static versus Suspension Culture

In malaria blood cell cultivation, the term "static culture" means malaria-infected erythrocytes cultivated without agitation. A thin layer of erythrocytes is deposited on the flat-bottomed surface of the culture vessel, plates, flasks, or Petri dish, without any form of adhesion to the substrate. The term "suspension culture" refers to a constant or intermittent agitation of malaria-infected erythrocytes so that the cells remain suspended in the culture medium.

The rocker-dilution method, which involves a constant agitation of infected erythrocytes suspended in the culture medium, was used in early experiments on short-term cultures. Based on the observation that, in vivo, *P. falciparum*-infected erythrocytes remain attached to the endothelium of microvessels in various organs only during the more mature growth stages of the developing schizont, Trager and Jensen reasoned that alternative methods are more appropriate for the long-term cultivation of *P. falciparum* [27]. Two methods have been introduced: the more cumbersome, continuous flow method using a special flow-vial which injects a slow flow of medium over a settled layer of erythrocytes and the more practical, Petri dish-candle jar method, which is stationary. The current standard procedure involves a settled layer of erythrocytes under static conditions during the entire incubation period. The only moment when the erythrocytes are agitated into suspension is during the medium change in Petri dishes or culture flasks, once the spent medium is aspirated and the fresh medium is added.

In the human host, erythrocytes infected by *P. falciparum* rings are in constant circulation in the bloodstream (i.e., in suspension), whereas erythrocytes infected by mature trophozoites and schizonts are stationary due to cytoadherence in deep organs. In vivo, it may be hypothesized that a slow, constant flow of blood over stationary schizont-infected erythrocytes may be more favorable for the dispersion of merozoites devoid of motile organelles to initiate a new intraerythrocytic cycle in a higher number of erythrocytes. In contrast, merozoites released in a completely stationary environment, such as Petri dishes, tend to agglomerate and invade the same uninfected erythrocyte; multiple infections indicate that many merozoites penetrate close to the point of schizont rupture. Erythrocytes infected with two or more rings and trophozoites are more commonly observed in the stationary culture compared with the suspension culture [233].

During early attempts to develop malaria cultivation methods, most cultures had been performed in Erlenmeyer flasks in an atmosphere of 5% CO₂ in the air and placed on a rocking table in an incubator [16]. The rocker flask method does not yield satisfactory parasite growth. In the method developed by Trager and Jensen [27], a stationary, settled thin layer of infected erythrocytes is cultivated on the flat-bottomed surface of either a flow vial or a Petri dish. In later works performed in the late-1970s and 1980s, several investigators compared static and suspension cultures of established *P. falciparum* strains and concluded that cultures on an orbital shaking device or flask shaker led to a higher yield of parasites [29,51,53,109,233,234]. With suspension cultures at 10% erythrocyte suspension (v/v), the yield may be a two-fold increase over static cultures during short-term (two to three intraerythrocytic cycles) and long-term (one month) cultures [51,233]. In suspension cultures maintained at a lower erythrocyte suspension (v/v, 1%) and containing high
glucose concentration (extra 2 g/L in addition to 2 g/L in RPMI 1640; 40 mM TES), higher
parasitemia can be obtained compared to static cultures, without changing the medium
for 3–4 days [53]. These early studies support the observation that the suspension culture
generally results in a higher yield of parasites. In one study based on the comparison of the
in vitro growth of three parasite strains maintained in static and suspension cultures, the
benefit brought about by the suspension culture was not consistently observed, leading the
author to wonder whether adaptation to the suspension culture is strain-dependent [235].

Despite these initial doubts, subsequent studies have largely confirmed that suspen-
sion cultures yield not only a higher growth rate but also a lower number of multiply
infected erythrocytes (i.e., the presence of two or more asexual parasites in the same
erythrocyte) [236,237]. Allen et al. showed that a continuous suspension culture of syn-
chronized parasites (reference clone 3D7) also resulted in longer maintenance of synchrony
for three cycles compared to the static culture [237]. Furthermore, the suspension culture
to initiate the cultivation of cryopreserved *P. falciparum* isolates was reported to result in
higher multiplication rates than the static culture in 10 isolates [213]. A similar result (a
four-fold reduction in the number of multiply parasitized erythrocytes) was reported using
the rotation method (i.e., cultures in 15 mL polypropylene tubes that were tightly recapped
after gassing with 3% O₂, 5% CO₂, and 92% N₂ mixtures and rotated at 14 rotations per
minute (rpm) at 37 °C in a hybridization incubator or oven [238].

Because erythrocytes are not in a monolayer but stacked in multiple layers in static cultures, the exchange of dissolved gases, nutrients, micro- and macromolecules, and waste metabolites between the culture environment and the cells may also be somewhat suboptimal compared to the suspension cultures. In spite of these disadvantages of static cultures, supported by experimental observations, static cultures, rather than suspension cultures, have become a common practice in most malaria research laboratories for practical reasons. Suspension cultures require suitable culture vessels, such as culture flasks or tubes that can be tightly recapped. An appropriate gas mixture may need to be flushed into the vessels before tight closure. Petri dishes are not suitable or at least may be difficult to handle on a shaker. A horizontal orbital (flask) shaker placed in an incubator occupies space. An alternative method developed by Trager and Jensen, the continuous flow method, shares some features of both suspension (constant flow of culture medium) and stationary (settled erythrocyte layer) methods, although the erythrocytes remain stationary [27]. Flow vials are not commercially available; only a few specialized laboratories in the world still use the flow vial method or its variants. The growth rate of laboratory-adapted parasites in static cultures is generally satisfactory for routine cultivation. Based on these practical considerations, static cultures have been preferred over suspension cultures for routine purposes.

Investigators requiring a large number of parasites for their experiments (for example,
protein purification) may resort to the agitation of culture flasks to increase parasite yield.
With suspension culture, care must be taken to minimize the physical damage to erythro-
cytes due to constant agitation. An addition of methylcellulose (1 mg/mL) to the culture
medium has been suggested to protect the erythrocytes from mechanical damage, reducing
hemolysis in suspension cultures [51]. Moreover, the type of medium displacement used
is important. Vibratory suspension involving the minimum medium displacement was
suggested to be optimal in early studies [53]. Presumably, this prevents the washing away
of the released merozoites from the areas of cell concentration.

Although one of the basic principles of in vitro cultivation of malaria parasites in-
volves a thin layer of erythrocytes bathed in a shallow volume of medium in a stationary
set-up [27], “deep cultures” have been shown to be an alternative procedure for maintain-
ing and producing a large number of parasites. Moloney et al. successfully cultivated
laboratory-adapted strains in 5–15 L glass bottles containing a 3–8 L blood–medium–human
serum mixture [239]. These deep cultures were maintained in constant suspension by a
magnetic stirrer. To optimize parasite yield, the stirring speed needs to be increased with
glucose concentration (extra 2 g/L in addition to 2 g/L in RPMI 1640; 40 mM TES), higher
parasitemia can be obtained compared to static cultures, without changing the medium
for 3–4 days [53]. These early studies support the observation that the suspension culture
generally results in a higher yield of parasites. In one study based on the comparison of the
in vitro growth of three parasite strains maintained in static and suspension cultures, the
benefit brought about by the suspension culture was not consistently observed, leading the
author to wonder whether adaptation to the suspension culture is strain-dependent [235].

Despite these initial doubts, subsequent studies have largely confirmed that suspen-
sion cultures yield not only a higher growth rate but also a lower number of multiply
infected erythrocytes (i.e., the presence of two or more asexual parasites in the same
erythrocyte) [236,237]. Allen et al. showed that a continuous suspension culture of syn-
chronized parasites (reference clone 3D7) also resulted in longer maintenance of synchrony
for three cycles compared to the static culture [237]. Furthermore, the suspension culture
to initiate the cultivation of cryopreserved *P. falciparum* isolates was reported to result in
higher multiplication rates than the static culture in 10 isolates [213]. A similar result (a
four-fold reduction in the number of multiply parasitized erythrocytes) was reported using
the rotation method (i.e., cultures in 15 mL polypropylene tubes that were tightly recapped
after gassing with 3% O₂, 5% CO₂, and 92% N₂ mixtures and rotated at 14 rotations per
minute (rpm) at 37 °C in a hybridization incubator or oven [238].

Because erythrocytes are not in a monolayer but stacked in multiple layers in static cultures, the exchange of dissolved gases, nutrients, micro- and macromolecules, and waste metabolites between the culture environment and the cells may also be somewhat suboptimal compared to the suspension cultures. In spite of these disadvantages of static cultures, supported by experimental observations, static cultures, rather than suspension cultures, have become a common practice in most malaria research laboratories for practical reasons. Suspension cultures require suitable culture vessels, such as culture flasks or tubes that can be tightly recapped. An appropriate gas mixture may need to be flushed into the vessels before tight closure. Petri dishes are not suitable or at least may be difficult to handle on a shaker. A horizontal orbital (flask) shaker placed in an incubator occupies space. An alternative method developed by Trager and Jensen, the continuous flow method, shares some features of both suspension (constant flow of culture medium) and stationary (settled erythrocyte layer) methods, although the erythrocytes remain stationary [27]. Flow vials are not commercially available; only a few specialized laboratories in the world still use the flow vial method or its variants. The growth rate of laboratory-adapted parasites in static cultures is generally satisfactory for routine cultivation. Based on these practical considerations, static cultures have been preferred over suspension cultures for routine purposes.

Investigators requiring a large number of parasites for their experiments (for example,
protein purification) may resort to the agitation of culture flasks to increase parasite yield.
With suspension culture, care must be taken to minimize the physical damage to erythro-
cytes due to constant agitation. An addition of methylcellulose (1 mg/mL) to the culture
medium has been suggested to protect the erythrocytes from mechanical damage, reducing
hemolysis in suspension cultures [51]. Moreover, the type of medium displacement used
is important. Vibratory suspension involving the minimum medium displacement was
suggested to be optimal in early studies [53]. Presumably, this prevents the washing away
of the released merozoites from the areas of cell concentration.

Although one of the basic principles of in vitro cultivation of malaria parasites in-
volves a thin layer of erythrocytes bathed in a shallow volume of medium in a stationary
set-up [27], “deep cultures” have been shown to be an alternative procedure for maintain-
ing and producing a large number of parasites. Moloney et al. successfully cultivated
laboratory-adapted strains in 5–15 L glass bottles containing a 3–8 L blood–medium–human
serum mixture [239]. These deep cultures were maintained in constant suspension by a
magnetic stirrer. To optimize parasite yield, the stirring speed needs to be increased with
increasing volume and depth. Other variants of “deep cultures” developed more recently
are discussed in the following section.

10. Tissue Culture Ware and Devices

Since the pioneering works of Bass and Bass and Johns [17,18], a wide variety of culture
vessels, including glass tubes, Erlenmeyer flasks, flow vials, and others, have been used for
the in vitro cultivation of malaria parasites. Glassware had been commonly used in the past
for cell culture in general. Nowadays, plasticware (usually polystyrene or polycarbonate)
has largely replaced glass for culture. Culture-grade plastics are treated to produce a
wettable, charged surface, a property that is especially important for adherent cells.

For malaria cultivation, any sterile, flat-bottom, tissue-grade plastic (usually polystyrene) may be used. An open vessel (microtiter plate, Petri dish, tissue culture flask) is designed to equilibrate bicarbonate in the medium with gaseous CO₂ in the incubator. An airtight closed vessel is more appropriate if pre-mixed gas is flushed into the vessel and placed in an incubator without high CO₂ content. It is important to fill an appropriate volume of blood-medium mixture in each well, Petri dish, or culture flask to avoid the formation of a meniscus, which would result in suboptimal parasite growth [148].

In the original method developed by Trager and Jensen, both continuous flow vessels and Petri dishes were shown to be suitable [27]. Several semi-automated or manually operated perfusion or continuous flow devices were described and developed to propagate malaria parasites in the past [16,32,240–247]. These special culture devices, which are not commercially available, are too sophisticated for routine use in most laboratories and are not necessary for most research works, with the possible exception of the production of infective gametocytes. Depending on the purpose, *P. falciparum* can be cultivated in 6-, 24-, 48-, or 96-well microtitre plates, Petri dishes of various diameters, and tissue culture flasks of various sizes.

Since the early days of malaria cultivation, investigators have been attempting to
adapt the basic methods of “small-scale” cultivation to produce large quantities of parasites
for various experimental purposes, in particular biochemical studies [53,242,245,247–249].
For large-scale preparation of parasite material, Trager advocated the use of the flow
vessel [250,251]. Some modifications, such as additional glucose in culture medium, sus-
pension culture, and increased frequency of medium change, resulted in higher parasitemia,
but limited volume posed a major technical difficulty.

The multiplication of culture vessels, for example, cultivation in several large culture flasks, would be cumbersome to handle. In the protocol developed by Radfar et al. [122], however, the number of 150 cm² culture flasks (with up to 140 mL of culture medium per flask; RPMI 1640, 25 mM HEPES, 1.77 mM NaHCO₃, 0.5% Albumax, 100 µM hypoxanthine; incubation in 5% CO₂ incubator) at any one time is limited to three. High parasitemias (about 50%; hematocrit 0.8–1.5%) of synchronous cultures of laboratory-adapted reference clones (3D7 and Dd2) can be attained in three 150 cm² flasks every four days, two of which are harvested, and the content of the third flask is divided and subcultured in three new 150 cm² flasks. The culture medium is replaced once a day, and fresh erythrocytes (stored <14 days) are used for subcultures. It was reported that 1.2 mg of parasite protein per mL of harvested culture can be obtained in a week using this protocol.

To overcome the limit imposed by a relatively small volume of culture medium in tra-
ditional culture vessels, a method of deeply stirred cultivation using a large vessel (5–15 L
glass bottles with 3–8 L of culture medium and a system of semi-automatic medium re-
placement) was described and shown to be feasible with a laboratory-adapted *P. falciparum*
strain [239].

Over the past decades, several alternative techniques have been developed to maxi-
mize the yield of cultures of various cells. One of these is the three-dimensional hollow-fiber
capillary bioreactor [252,253], which is also called “artificial capillaries”, originally devel-
oped for hemodialysis. This device consists of a cartridge containing thousands of hollow,
semipermeable, capillary-like plastic tubules or fibers. Cells are inoculated in the space
increasing volume and depth. Other variants of “deep cultures” developed more recently
are discussed in the following section.

10. Tissue Culture Ware and Devices

Since the pioneering works of Bass and Bass and Johns [17,18], a wide variety of culture
vessels, including glass tubes, Erlenmeyer flasks, flow vials, and others, have been used for
the in vitro cultivation of malaria parasites. Glassware had been commonly used in the past
for cell culture in general. Nowadays, plasticware (usually polystyrene or polycarbonate)
has largely replaced glass for culture. Culture-grade plastics are treated to produce a
wettable, charged surface, a property that is especially important for adherent cells.

For malaria cultivation, any sterile, flat-bottom, tissue-grade plastic (usually polystyrene) may be used. An open vessel (microtiter plate, Petri dish, tissue culture flask) is designed to equilibrate bicarbonate in the medium with gaseous CO2 in the incubator. An airtight closed vessel is more appropriate if pre-mixed gas is flushed into the vessel and placed in an incubator without high CO2 content. It is important to fill an appropriate volume of blood-medium mixture in each well, Petri dish, or culture flask to avoid the formation of a meniscus, which would result in suboptimal parasite growth [148].

In the original method developed by Trager and Jensen, both continuous flow vessels and Petri dishes were shown to be suitable [27]. Several semi-automated or manually operated perfusion or continuous flow devices were described and developed to propagate malaria parasites in the past [16,32,240–247]. These special culture devices, which are not commercially available, are too sophisticated for routine use in most laboratories and are not necessary for most research works, with the possible exception of the production of infective gametocytes. Depending on the purpose, *P. falciparum* can be cultivated in 6-, 24-, 48-, or 96-well microtitre plates, Petri dishes of various diameters, and tissue culture flasks of various sizes.

Since the early days of malaria cultivation, investigators have been attempting to
adapt the basic methods of “small-scale” cultivation to produce large quantities of parasites
for various experimental purposes, in particular biochemical studies [53,242,245,247–249].
For large-scale preparation of parasite material, Trager advocated the use of the flow
vessel [250,251]. Some modifications, such as additional glucose in culture medium, sus-
pension culture, and increased frequency of medium change, resulted in higher parasitemia,
but limited volume posed a major technical difficulty.

The multiplication of culture vessels, for example, cultivation in several large culture flasks, would be cumbersome to handle. In the protocol developed by Radfar et al. [122], however, the number of 150 cm² culture flasks (with up to 140 mL of culture medium per flask; RPMI 1640, 25 mM HEPES, 1.77 mM NaHCO₃, 0.5% Albumax, 100 µM hypoxanthine; incubation in 5% CO₂ incubator) at any one time is limited to three. High parasitemias (about 50%; hematocrit 0.8–1.5%) of synchronous cultures of laboratory-adapted reference clones (3D7 and Dd2) can be attained in three 150 cm² flasks every four days, two of which are harvested, and the content of the third flask is divided and subcultured in three new 150 cm² flasks. The culture medium is replaced once a day, and fresh erythrocytes (stored <14 days) are used for subcultures. It was reported that 1.2 mg of parasite protein per mL of harvested culture can be obtained in a week using this protocol.

To overcome the limit imposed by a relatively small volume of culture medium in tra-
ditional culture vessels, a method of deeply stirred cultivation using a large vessel (5–15 L
glass bottles with 3–8 L of culture medium and a system of semi-automatic medium re-
placement) was described and shown to be feasible with a laboratory-adapted *P. falciparum*
strain [239].

Over the past decades, several alternative techniques have been developed to maxi-
mize the yield of cultures of various cells. One of these is the three-dimensional hollow-fiber
capillary bioreactor [252,253], which is also called “artificial capillaries”, originally devel-
oped for hemodialysis. This device consists of a cartridge containing thousands of hollow,
semipermeable, capillary-like plastic tubules or fibers. Cells are inoculated in the space
between the fibers. The culture medium is perfused by a pump over the large surface area per unit volume to provide maximal nutrient and gas exchange. Pressure is maintained and controlled by a microprocessor to ensure a homogenous medium flow and distribution.

In the first study that assessed bioreactor for malaria cultivation, Li et al. reported that this device allowed *P. falciparum* cultures with glucose- and hypoxanthine-supplemented RPMI 1640-HEPES-NaHCO₃ media at high hematocrit (50-100%) [254]. The increase in the yield of infected erythrocytes with the hollow-fiber capillary bioreactor can attain 81-fold over 4-5 days compared with flask cultures. Using this culture system, a laboratory-adapted strain was maintained at the hematocrit of 50% for 16 days, with subcultures every four days. The maximum parasite density that can be attained with this device seems to be limited to 12% (>20% with a Petri dish or continuous flow method, depending on the hematocrit), but with 50-100% hematocrit, this would be desirable. A British-Thai research group reported that when using a laboratory-adapted mefloquine-sensitive Thai strain TM036, the total parasite yield in the bioreactor was equivalent to 19 T25 culture flasks (with a surface area of 25 cm²) on day 9 and 152 T25 flasks on day 35 of continuous culture [255,256]. In their study, 250 mL/day of a culture medium (RPMI 1640 supplemented with 10% human serum or 0.5% Albumax® II + hypoxanthine + D-glucose; 40% hematocrit; change in the culture medium, once daily) was required.

A somewhat less sophisticated, single-use plastic bioreactor bag has been widely used in pharmaceutical and biotechnological sectors over the last two-to-three decades. The plastic bag is typically made of a multilayered high-quality polymer film. The bioreactors are usually designed for a suspension or shaking culture, with either a stirrer integrated into the bag or a support that produces a rocking motion [257,258]. The bioreactor was adapted for the cultivation of laboratory-adapted *P. falciparum* strains [259]. The parasites (3D7 and Dd2 strains) were grown in a bioreactor containing 1 L of medium (RPMI 1640 supplemented with 0.5% Albumax® and 1 mg/L hypoxanthine; 2-4% hematocrit; equivalent of 20 T150 culture flasks) over 2-3 intraerythrocytic cycles. The bioreactor was placed on a rocking temperature-controlled support and was directly supplied with a gas mixture of 3% O₂, 5% CO₂, and 92% N₂. The authors reported that the multiplication rate of the parasites in the control static culture flask and wave bioreactor was similar (starting parasitemia, 0.36%; final parasitemia after 3-5 days, 4-5%) and, as expected, multiply infected erythrocytes were observed more frequently in the static cultures.

The availability of research-grade biological samples, such as asexual blood-stage malaria parasites, that are safe and suitable for administration to human volunteers can facilitate the implementation and evaluation of clinical trials on vaccine candidates and novel antimalarial drugs [260]. Such *P. falciparum* cell banks have been produced over 16 days of continuous culture using a wave bioreactor (a final culture volume of 2 L in a 10 L cellbag; 6.3% parasitemia, synchronized to obtain 96% of the parasites at ring stage) [261].

Unlike “home-made” culture vessels used by a number of investigators, bioreactors are commercially available. Cultures are performed in a sterile, closed system. The closed system offers many advantages over the traditional methods of static and suspension cultures in Petri dishes or culture flasks, including higher parasite yield and less labor-intensive manipulation, but it requires a relatively high initial investment to acquire the apparatus (at least for hollow-fiber capillary bioreactors). These devices may be of interest to investigators requiring a large quantity of parasite material. One of the disadvantages of the large-scale cultivation system using bioreactors is that since they were not specifically designed for malaria cultivation, removal and replenishment of the culture medium and uninfected erythrocytes may have to be performed in a laminar flow hood. Some models may also require a setup inside an incubator with an appropriate gas mixture. Moreover, long-term cultivation of *P. falciparum* strains beyond 16 days in a bioreactor does not seem to have been performed, reported, or published.
between the fibers. The culture medium is perfused by a pump over the large surface area per unit volume to provide maximal nutrient and gas exchange. Pressure is maintained and controlled by a microprocessor to ensure a homogenous medium flow and distribution.

In the first study that assessed bioreactor for malaria cultivation, Li et al. reported that this device allowed *P. falciparum* cultures with glucose- and hypoxanthine-supplemented RPMI 1640-HEPES-NaHCO₃ media at high hematocrit (50-100%) [254]. The increase in the yield of infected erythrocytes with the hollow-fiber capillary bioreactor can attain 81-fold over 4-5 days compared with flask cultures. Using this culture system, a laboratory-adapted strain was maintained at the hematocrit of 50% for 16 days, with subcultures every four days. The maximum parasite density that can be attained with this device seems to be limited to 12% (>20% with a Petri dish or continuous flow method, depending on the hematocrit), but with 50-100% hematocrit, this would be desirable. A British-Thai research group reported that when using a laboratory-adapted mefloquine-sensitive Thai strain TM036, the total parasite yield in the bioreactor was equivalent to 19 T25 culture flasks (with a surface area of 25 cm²) on day 9 and 152 T25 flasks on day 35 of continuous culture [255,256]. In their study, 250 mL/day of a culture medium (RPMI 1640 supplemented with 10% human serum or 0.5% Albumax® II + hypoxanthine + D-glucose; 40% hematocrit; change in the culture medium, once daily) was required.

A somewhat less sophisticated, single-use plastic bioreactor bag has been widely used in pharmaceutical and biotechnological sectors over the last two-to-three decades. The plastic bag is typically made of a multilayered high-quality polymer film. The bioreactors are usually designed for a suspension or shaking culture, with either a stirrer integrated into the bag or a support that produces a rocking motion [257,258]. The bioreactor was adapted for the cultivation of laboratory-adapted *P. falciparum* strains [259]. The parasites (3D7 and Dd2 strains) were grown in a bioreactor containing 1 L of medium (RPMI 1640 supplemented with 0.5% Albumax® and 1 mg/L hypoxanthine; 2-4% hematocrit; equivalent of 20 T150 culture flasks) over 2-3 intraerythrocytic cycles. The bioreactor was placed on a rocking temperature-controlled support and was directly supplied with a gas mixture of 3% O₂, 5% CO₂, and 92% N₂. The authors reported that the multiplication rate of the parasites in the control static culture flask and wave bioreactor was similar (starting parasitemia, 0.36%; final parasitemia after 3-5 days, 4-5%) and, as expected, multiply infected erythrocytes were observed more frequently in the static cultures.

The availability of research-grade biological samples, such as asexual blood-stage malaria parasites, that are safe and suitable for administration to human volunteers can facilitate the implementation and evaluation of clinical trials on vaccine candidates and novel antimalarial drugs [260]. Such *P. falciparum* cell banks have been produced over 16 days of continuous culture using a wave bioreactor (a final culture volume of 2 L in a 10 L cellbag; 6.3% parasitemia, synchronized to obtain 96% of the parasites at ring stage) [261].

Unlike “home-made” culture vessels used by a number of investigators, bioreactors are commercially available. Cultures are performed in a sterile, closed system. The closed system offers many advantages over the traditional methods of static and suspension cultures in Petri dishes or culture flasks, including higher parasite yield and less labor-intensive manipulation, but it requires a relatively high initial investment to acquire the apparatus (at least for hollow-fiber capillary bioreactors). These devices may be of interest to investigators requiring a large quantity of parasite material. One of the disadvantages of the large-scale cultivation system using bioreactors is that since they were not specifically designed for malaria cultivation, removal and replenishment of the culture medium and uninfected erythrocytes may have to be performed in a laminar flow hood. Some models may also require a setup inside an incubator with an appropriate gas mixture. Moreover, long-term cultivation of *P. falciparum* strains beyond 16 days in a bioreactor does not seem to have been performed, reported, or published.
# 11. Atmosphere

In the human host, O₂ concentrations under normal physiological conditions vary widely in different parts of the body: 13.2–13.7% (partial pressure, 100–104 mm Hg) in the alveolar air and pulmonary capillaries, 12.5% (95 mm Hg) in the arterial blood, 5.3% (40 mm Hg) in the venous blood and interstitial fluid, and 0.6–5.3% (5–40 mm Hg) within the cells and tissues (for comparison, 20.95% O₂ (159 mm Hg) in the atmospheric dry air). The diffusion of oxygen occurs from the alveolus (oxygen partial pressure, 100–104 mm Hg) into the erythrocytes passing through afferent pulmonary capillaries (partial pressure, 40 mm Hg) due to the difference in oxygen percentage composition. Most oxygen molecules in circulating blood (97%) are reversibly bound to the heme iron of the hemoglobin (up to four heme binding sites per hemoglobin molecule) and transported to the cells. A small proportion of oxygen (3%) is dissolved in the plasma. Oxygen is removed as blood passes through the tissues outside the lung, resulting in a low partial pressure of oxygen in the afferent pulmonary capillaries of 40 mm Hg in the venous end of the capillaries. Oxygen is used as the terminal electron acceptor of aerobic respiration in mitochondria in human tissues and passes back into the blood as CO₂ and H₂O.

The diffusion of carbon dioxide occurs in a direction opposite to oxygen. Carbon dioxide is produced by cells, is diffused into the local capillaries, and is transported by the blood to the lungs in three different forms: (i) bicarbonate ions (70%), (ii) dissolved CO₂ in plasma (7%), and (iii) carbamino compounds, i.e., reversible binding of CO₂ with hemoglobin and plasma proteins (23%). Carbon dioxide enters into the erythrocytes, and bicarbonate ions (HCO₃⁻) are formed by the hydroxylation of CO₂ catalyzed by carbonic anhydrase that is present in the erythrocytes but not plasma. HCO₃⁻ diffuses out of the erythrocytes, and chloride ions enter the erythrocytes to maintain the ionic balance (a phenomenon called the chloride shift). H⁺ ions are buffered by hemoglobin. Unlike O₂, CO₂ concentrations vary only slightly (range, 5.3–6.1% (40–46 mm Hg); for comparison, 0.03–0.04% (0.2–0.3 mm Hg) in the atmospheric dry air) in different organs and tissues of the human body under normal physiological conditions, but the slight difference is enough to ensure CO₂ diffusion and transport from the peripheral tissues to the lungs. The remaining gases in the human body are mostly composed of N₂ (74.9–75.4% (569–573 mm Hg); for comparison, 78–79% (594–601 mm Hg) in atmospheric dry air) and water vapor (6.2% (47 mm Hg)) at sea level at 37 °C.

Malaria parasites are obligate microaerophilic organisms, i.e., the parasites require O₂, but at low concentrations. They do not grow under strictly anaerobic conditions. At the other extreme, the normal O₂ content (20.95%; partial pressure, 159 mm Hg) in the atmosphere has been suggested to be too high for optimal parasite growth. In the human host, erythrocytes infected with ring-stage *P. falciparum* parasites passing through the lung capillaries may be exposed to 13–14% O₂ (partial pressure, approximately 100–104 mm Hg). However, in the venous end of the capillaries of deep organs where erythrocytes harboring *P. falciparum* trophozoites and schizonts are sequestered, O₂ concentration falls to approximately 5% (partial pressure, 38 mm Hg). A low O₂ tension in an incubator mimics the in vivo conditions to which human malaria parasites are naturally subjected. Therefore, it is not surprising that a microaerophilic environment favors parasite growth.

The understanding of what is now basic fact was one of the key experimental factors that eventually led to the development of continuous culture. Trager and Jensen developed the flow vial method in an atmosphere of 7% CO₂, 5% O₂, and 88% N₂ [27]. Haynes et al. developed their culture system in an atmosphere of 3% CO₂–6.6% O₂ [26]. An alternative method developed by Trager and Jensen, called “the candle jar method”, requires a simple, inexpensive air-tight device in which cultures in Petri dishes or culture plates are placed, and a candle is lit to consume O₂ within the device [27]. When the candle goes out, an atmosphere of approximately 3% CO₂, 17–18% O₂, and 79–80% N₂ is created within the candle jar [27,81,107,115,262] (Table 4).
# 11. Atmosphere

In the human host, O₂ concentrations under normal physiological conditions vary widely in different parts of the body: 13.2–13.7% (partial pressure, 100–104 mm Hg) in the alveolar air and pulmonary capillaries, 12.5% (95 mm Hg) in the arterial blood, 5.3% (40 mm Hg) in the venous blood and interstitial fluid, and 0.6–5.3% (5–40 mm Hg) within the cells and tissues (for comparison, 20.95% O₂ (159 mm Hg) in the atmospheric dry air). The diffusion of oxygen occurs from the alveolus (oxygen partial pressure, 100–104 mm Hg) into the erythrocytes passing through afferent pulmonary capillaries (partial pressure, 40 mm Hg) due to the difference in oxygen percentage composition. Most oxygen molecules in circulating blood (97%) are reversibly bound to the heme iron of the hemoglobin (up to four heme binding sites per hemoglobin molecule) and transported to the cells. A small proportion of oxygen (3%) is dissolved in the plasma. Oxygen is removed as blood passes through the tissues outside the lung, resulting in a low partial pressure of oxygen in the afferent pulmonary capillaries of 40 mm Hg in the venous end of the capillaries. Oxygen is used as the terminal electron acceptor of aerobic respiration in mitochondria in human tissues and passes back into the blood as CO₂ and H₂O.

The diffusion of carbon dioxide occurs in a direction opposite to oxygen. Carbon dioxide is produced by cells, is diffused into the local capillaries, and is transported by the blood to the lungs in three different forms: (i) bicarbonate ions (70%), (ii) dissolved CO₂ in plasma (7%), and (iii) carbamino compounds, i.e., reversible binding of CO₂ with hemoglobin and plasma proteins (23%). Carbon dioxide enters into the erythrocytes, and bicarbonate ions (HCO₃⁻) are formed by the hydroxylation of CO₂ catalyzed by carbonic anhydrase that is present in the erythrocytes but not plasma. HCO₃⁻ diffuses out of the erythrocytes, and chloride ions enter the erythrocytes to maintain the ionic balance (a phenomenon called the chloride shift). H⁺ ions are buffered by hemoglobin. Unlike O₂, CO₂ concentrations vary only slightly (range, 5.3–6.1% (40–46 mm Hg); for comparison, 0.03–0.04% (0.2–0.3 mm Hg) in the atmospheric dry air) in different organs and tissues of the human body under normal physiological conditions, but the slight difference is enough to ensure CO₂ diffusion and transport from the peripheral tissues to the lungs. The remaining gases in the human body are mostly composed of N₂ (74.9–75.4% (569–573 mm Hg); for comparison, 78–79% (594–601 mm Hg) in atmospheric dry air) and water vapor (6.2% (47 mm Hg)) at sea level at 37 °C.

Malaria parasites are obligate microaerophilic organisms, i.e., the parasites require O₂, but at low concentrations. They do not grow under strictly anaerobic conditions. At the other extreme, the normal O₂ content (20.95%; partial pressure, 159 mm Hg) in the atmosphere has been suggested to be too high for optimal parasite growth. In the human host, erythrocytes infected with ring-stage *P. falciparum* parasites passing through the lung capillaries may be exposed to 13–14% O₂ (partial pressure, approximately 100–104 mm Hg). However, in the venous end of the capillaries of deep organs where erythrocytes harboring *P. falciparum* trophozoites and schizonts are sequestered, O₂ concentration falls to approximately 5% (partial pressure, 38 mm Hg). A low O₂ tension in an incubator mimics the in vivo conditions to which human malaria parasites are naturally subjected. Therefore, it is not surprising that a microaerophilic environment favors parasite growth.

The understanding of what is now basic fact was one of the key experimental factors that eventually led to the development of continuous culture. Trager and Jensen developed the flow vial method in an atmosphere of 7% CO₂, 5% O₂, and 88% N₂ [27]. Haynes et al. developed their culture system in an atmosphere of 3% CO₂–6.6% O₂ [26]. An alternative method developed by Trager and Jensen, called “the candle jar method”, requires a simple, inexpensive air-tight device in which cultures in Petri dishes or culture plates are placed, and a candle is lit to consume O₂ within the device [27]. When the candle goes out, an atmosphere of approximately 3% CO₂, 17–18% O₂, and 79–80% N₂ is created within the candle jar [27,81,107,115,262] (Table 4).
<table><caption>**Table 4.** A comparison of partial pressures and/or concentrations of oxygen and carbon dioxide in in vivo and in vitro conditions.</caption><thead><tr><th>Site</th><th>Oxygen (mm Hg)</th><th>Carbon Dioxide (mm Hg)</th></tr></thead><tbody><tr><td>Atmospheric dry air <sup>1</sup></td><td>159 (20.95%)</td><td>0.2–0.3 (0.03–0.04%)</td></tr><tr><td>Human body <sup>2</sup></td><td></td><td></td></tr><tr><td>Pulmonary capillaries</td><td>100–104 (13.2–13.7%)</td><td>40 (5.3%)</td></tr><tr><td>Arterial blood <sup>3</sup></td><td>95 (12.5%)</td><td>40 (5.3%)</td></tr><tr><td>Venous blood</td><td>40 (5.3%)</td><td>45 (5.9%)</td></tr><tr><td>Tissue (intracellular)</td><td>5–40 (0.6–5.3%)</td><td>46 (6.1%)</td></tr><tr><td>Candle jar <sup>4</sup></td><td>17–18% (range, 14.5–17.8%)</td><td>3% (range, 1.1–3.3%)</td></tr><tr><td>CO<sub>2</sub> incubator</td><td>19–20%</td><td>5%</td></tr></tbody></table>

¹ Nitrogen, 594–601 mm Hg (78–79%). ² Values cited in Guyton and Hall [263] and Boron and Boulpaep [264]. The values in alveolar air are the same as pulmonary capillaries. The values in venous blood are the same as interstitial fluid. The partial pressures of N₂ and H₂O in the human body at sea level are 569–573 mm Hg (74.9–75.4%) and 47 mm Hg (6.2%) at 37 °C, respectively. ³ Partial pressure of oxygen decreases to 95 mm Hg in the arterial blood due to the mixture of oxygenated blood that passes through the alveolar capillaries and unoxygenated blood returning from the deep lung tissues away from the gas exchange areas. ⁴ Approximate values and ranges measured by several authors [27,81,107,115,262]. The remaining gas content is mostly nitrogen; there is a small quantity of water vapor in the candle jar.

The first study on the various combinations of O₂–CO₂ gas mixtures showed that the optimal growth of a *P. falciparum* strain occurs in a mixture of 2–3% O₂, 1–2% CO₂, and 95% N₂ over a 96–144 h incubation period [107]. The parasite strain (FCR-3/The Gambia) also grew in a 5% CO₂ incubator (about 19–20% O₂) at a similar multiplication rate as the 3 or 5% O₂–5% CO₂ mixture or in a candle jar (16.1–17.8% O₂ and 1.1–2.1% CO₂) for up to 72 h. The multiplication rate in 5% CO₂ in the air decreased to about 50% of that attained in the 3 or 5% O₂–5% CO₂ mixture or in a candle jar after 96–120 h of incubation. The growth rates at the microaerophilic level of 0.5% O₂ were generally similar to, or slightly less than, those with 1–5% O₂, depending on the CO₂ level and the duration of incubation. The gas mixture of 3% CO₂ in the air and the total anaerobic atmosphere did not support parasite growth. In another study, parasite growth assessed for three days was shown to be similar with two different gas mixtures (5% O₂–5% CO₂ versus 10% O₂–5% CO₂) and in a candle jar (about 17–18% O₂–3% CO₂), but O₂ content <5% was reported to be inhibitory [29].

As reported by Scheibel et al. [107], other authors have also suggested that 5% CO₂ in the air (i.e., approximately 19–20% O₂–5% CO₂) may not support the growth of malaria parasites in vitro. Butcher reported that a laboratory-adapted strain (HG13/The Gambia) grew for one or two intraerythrocytic cycles in 5% CO₂ in the air, but the normal atmospheric content of O₂ inhibited the growth of the parasites beyond two intraerythrocytic cycles [29]. Similar findings were reported by Binh et al. [167] in a single laboratory-adapted isolate (BINH/Kenya) maintained in a serum-free medium and cultivated in 5% CO₂ in the air. On the contrary, other investigators have found that the atmosphere of 5% CO₂ in the air (in the CO₂ incubator) is suitable (but not always optimal compared to cultures in a gas mixture of 5% O₂, 5% CO₂, and 90% N₂), for both the short-term and long-term cultivation of freshly isolated parasites and laboratory-adapted *P. falciparum* strains [103,106,265].

The incubation of laboratory-adapted strains (3D7 and W2) in the 21% O₂, 5% CO₂, and 74% N₂ gas mixtures may have an adverse effect on parasite growth [266,267]. Morphological alteration was not observed under an optical microscope compared with the controls (5% O₂, 5% CO₂, and 90% N₂; 10% O₂, 5% CO₂, and 85% N₂ gas mixtures), but parasitemia decreased in 3D7 during the second intraerythrocytic cycle when exposed to 21% O₂. The authors also noted that the duration of schizogony was prolonged in parasites (mature trophozoites and schizonts) exposed to 21% O₂, leading to a 4 h delay in the schizogonic phrase of 3D7 (45 h cycle in 5% O₂ versus 49 h cycle in 21% O₂). This modification was due to the later initiation of nucleic division, as evidenced by the delayed appearance of schizonts. Short-term cultivation (two intraerythrocytic cycles) of 3D7 in 10% O₂ had no
<table><caption>**Table 4.** A comparison of partial pressures and/or concentrations of oxygen and carbon dioxide in in vivo and in vitro conditions.</caption><thead><tr><th>Site</th><th>Oxygen (mm Hg)</th><th>Carbon Dioxide (mm Hg)</th></tr></thead><tbody><tr><td>Atmospheric dry air <sup>1</sup></td><td>159 (20.95%)</td><td>0.2–0.3 (0.03–0.04%)</td></tr><tr><td>Human body <sup>2</sup></td><td></td><td></td></tr><tr><td>Pulmonary capillaries</td><td>100–104 (13.2–13.7%)</td><td>40 (5.3%)</td></tr><tr><td>Arterial blood <sup>3</sup></td><td>95 (12.5%)</td><td>40 (5.3%)</td></tr><tr><td>Venous blood</td><td>40 (5.3%)</td><td>45 (5.9%)</td></tr><tr><td>Tissue (intracellular)</td><td>5–40 (0.6–5.3%)</td><td>46 (6.1%)</td></tr><tr><td>Candle jar <sup>4</sup></td><td>17–18% (range, 14.5–17.8%)</td><td>3% (range, 1.1–3.3%)</td></tr><tr><td>CO<sub>2</sub> incubator</td><td>19–20%</td><td>5%</td></tr></tbody></table>

¹ Nitrogen, 594–601 mm Hg (78–79%). ² Values cited in Guyton and Hall [263] and Boron and Boulpaep [264]. The values in alveolar air are the same as pulmonary capillaries. The values in venous blood are the same as interstitial fluid. The partial pressures of N₂ and H₂O in the human body at sea level are 569–573 mm Hg (74.9–75.4%) and 47 mm Hg (6.2%) at 37 °C, respectively. ³ Partial pressure of oxygen decreases to 95 mm Hg in the arterial blood due to the mixture of oxygenated blood that passes through the alveolar capillaries and unoxygenated blood returning from the deep lung tissues away from the gas exchange areas. ⁴ Approximate values and ranges measured by several authors [27,81,107,115,262]. The remaining gas content is mostly nitrogen; there is a small quantity of water vapor in the candle jar.

The first study on the various combinations of O₂–CO₂ gas mixtures showed that the optimal growth of a *P. falciparum* strain occurs in a mixture of 2–3% O₂, 1–2% CO₂, and 95% N₂ over a 96–144 h incubation period [107]. The parasite strain (FCR-3/The Gambia) also grew in a 5% CO₂ incubator (about 19–20% O₂) at a similar multiplication rate as the 3 or 5% O₂–5% CO₂ mixture or in a candle jar (16.1–17.8% O₂ and 1.1–2.1% CO₂) for up to 72 h. The multiplication rate in 5% CO₂ in the air decreased to about 50% of that attained in the 3 or 5% O₂–5% CO₂ mixture or in a candle jar after 96–120 h of incubation. The growth rates at the microaerophilic level of 0.5% O₂ were generally similar to, or slightly less than, those with 1–5% O₂, depending on the CO₂ level and the duration of incubation. The gas mixture of 3% CO₂ in the air and the total anaerobic atmosphere did not support parasite growth. In another study, parasite growth assessed for three days was shown to be similar with two different gas mixtures (5% O₂–5% CO₂ versus 10% O₂–5% CO₂) and in a candle jar (about 17–18% O₂–3% CO₂), but O₂ content <5% was reported to be inhibitory [29].

As reported by Scheibel et al. [107], other authors have also suggested that 5% CO₂ in the air (i.e., approximately 19–20% O₂–5% CO₂) may not support the growth of malaria parasites in vitro. Butcher reported that a laboratory-adapted strain (HG13/The Gambia) grew for one or two intraerythrocytic cycles in 5% CO₂ in the air, but the normal atmospheric content of O₂ inhibited the growth of the parasites beyond two intraerythrocytic cycles [29]. Similar findings were reported by Binh et al. [167] in a single laboratory-adapted isolate (BINH/Kenya) maintained in a serum-free medium and cultivated in 5% CO₂ in the air. On the contrary, other investigators have found that the atmosphere of 5% CO₂ in the air (in the CO₂ incubator) is suitable (but not always optimal compared to cultures in a gas mixture of 5% O₂, 5% CO₂, and 90% N₂), for both the short-term and long-term cultivation of freshly isolated parasites and laboratory-adapted *P. falciparum* strains [103,106,265].

The incubation of laboratory-adapted strains (3D7 and W2) in the 21% O₂, 5% CO₂, and 74% N₂ gas mixtures may have an adverse effect on parasite growth [266,267]. Morphological alteration was not observed under an optical microscope compared with the controls (5% O₂, 5% CO₂, and 90% N₂; 10% O₂, 5% CO₂, and 85% N₂ gas mixtures), but parasitemia decreased in 3D7 during the second intraerythrocytic cycle when exposed to 21% O₂. The authors also noted that the duration of schizogony was prolonged in parasites (mature trophozoites and schizonts) exposed to 21% O₂, leading to a 4 h delay in the schizogonic phrase of 3D7 (45 h cycle in 5% O₂ versus 49 h cycle in 21% O₂). This modification was due to the later initiation of nucleic division, as evidenced by the delayed appearance of schizonts. Short-term cultivation (two intraerythrocytic cycles) of 3D7 in 10% O₂ had no
significant effect on the duration of the intraerythrocytic cycle, parasitemia, or parasite stages compared to the control culture in 5% O₂.

In another study, the growth of parasites (3D7 clone) cultivated in 20% O₂, 5% CO₂, and 75% N₂ for 48 h was reported to be similar to the control parasites cultivated in 5% O₂, 5% CO₂, and 90% N₂, but in the former condition (20% O₂), vitamin E biosynthesis of the parasites increased significantly and the level of lipid peroxidation decreased, protecting plasma membranes from damage due to reactive oxygen species (ROS) (α-tocopherol reduces the amount of ROS produced during oxidative stress) [268]. The study suggested that a relatively high O₂ content may not necessarily lead to deleterious biochemical effects, but the effects of 20% O₂ were not assessed beyond 48 h.

In the experiments conducted by another research group, both the oxygen concentration (5% O₂, 5% CO₂, and 90% N₂ versus 21% O₂, 5% CO₂, and 74% N₂) and the type of serum supplementation (pooled human serum (5% v/v) with Albumax® II (2.5 mg/mL) versus Albumax® II alone (5 mg/mL)) used to cultivate 3D7 clones for several months influenced the duration of intraerythrocytic cycle in vitro. The duration ranged from a 40 h cycle for 3D7 cultivated with the serum Albumax® II mixture supplemented to the standard RPMI 1640 medium in 5% O₂ to a 44 h (3D7 cultivated with Albumax® II, without human serum in 21% O₂) or a 45 h cycle (3D7 cultivated with Albumax® II, without human serum, in 5% O₂) [215].

Contrary to the general consensus among malaria researchers that an optimal long-term in vitro cultivation of *P. falciparum* requires low O₂ concentration (i.e., any value slightly above anaerobic condition and that obtained in a candle jar or 5% CO₂ incubator), a research group reported that several *P. falciparum* reference strains have been maintained in a normal atmospheric gas mixture (20.95% O₂, 0.04% CO₂, 78–79% N₂) in their laboratory for several months to one year in a standard RPMI 1640 medium with 0.5% Albumax® supplemented with hypoxanthine and 2 g/L glucose [269]. The suspension cultures (starting parasitemia > 0.5%) were maintained in culture flasks (with the cap screwed tightly) or hermetic glass bottles (0.1–2 L bottles; 20% of the volume was filled with culture medium and 80% of the volume was occupied by atmospheric gas or the standard gas mixtures of 5% O₂, 5% CO₂, and 90% N₂; the latter was flushed into the culture vessel before screwing the cap) placed on an orbital shaker. When suspension cultures in atmospheric gas and the standard gas mixture were compared, parasite growth during six days was similar. The parasites in static cultures in glass bottles did not grow. The free oxygen contents in uninfected erythrocytes measured after 24 h in the flasks with atmospheric air or the standard gas mixture were 180 µM and 147 µM, respectively (arterial oxygen concentration, 130 µM).

In an earlier experiment, the same research group reported that a reference clone (3D7) was cultivated in their laboratory in an anaerobic condition (i.e., 5% CO₂ and 95% N₂) for 10 days, and the multiplication rate remained similar to the control parasites cultivated in the standard gas mixture (5% O₂, 5% CO₂, and 90% N₂), leading the authors to conclude that the parasite adapted to anaerobic conditions [270]. Details of parasite growth were not described in that study.

Another effect that O₂ (or CO₂) concentration may have in short-term cultivation of malaria parasites is possible changes in the level of susceptibility in vitro to some antimalarial drugs and antibiotics [215,262,266,269,271,272]. Transcriptomic data suggest various metabolic changes in response to hyperoxia [267], but the precise underlying reason for the influence of O₂ concentration on the level of in vitro drug susceptibility is not yet well-known. In vitro response to chloroquine is also influenced by CO₂ concentration; chloroquine IC₅₀ decreases significantly in a chloroquine-resistant laboratory-adapted strain (K1/Thailand) when CO₂ concentration varied from 7% to 5% and 2.7% with a constant O₂ concentration set at 5% [262]. Growth rates in the drug-free control wells were not affected by CO₂ concentration during the 42 h incubation period. The underlying cause of altered IC₅₀ values is due to pH modification (a higher CO₂ concentration leads to lower
significant effect on the duration of the intraerythrocytic cycle, parasitemia, or parasite
stages compared to the control culture in 5% O₂.

In another study, the growth of parasites (3D7 clone) cultivated in 20% O₂, 5% CO₂, and 75% N₂ for 48 h was reported to be similar to the control parasites cultivated in 5% O₂, 5% CO₂, and 90% N₂, but in the former condition (20% O₂), vitamin E biosynthesis of the parasites increased significantly and the level of lipid peroxidation decreased, protecting plasma membranes from damage due to reactive oxygen species (ROS) (α-tocopherol reduces the amount of ROS produced during oxidative stress) [268]. The study suggested that a relatively high O₂ content may not necessarily lead to deleterious biochemical effects, but the effects of 20% O₂ were not assessed beyond 48 h.

In the experiments conducted by another research group, both the oxygen concentra-
tion (5% O₂, 5% CO₂, and 90% N₂ versus 21% O₂, 5% CO₂, and 74% N₂) and the type of
serum supplementation (pooled human serum (5% v/v) with Albumax® II (2.5 mg/mL)
versus Albumax® II alone (5 mg/mL)) used to cultivate 3D7 clones for several months
influenced the duration of intraerythrocytic cycle in vitro. The duration ranged from a
40 h cycle for 3D7 cultivated with the serum Albumax® II mixture supplemented to the
standard RPMI 1640 medium in 5% O₂ to a 44 h (3D7 cultivated with Albumax® II, without
human serum in 21% O₂) or a 45 h cycle (3D7 cultivated with Albumax® II, without human
serum, in 5% O₂) [215].

Contrary to the general consensus among malaria researchers that an optimal long-
term in vitro cultivation of *P. falciparum* requires low O₂ concentration (i.e., any value
slightly above anaerobic condition and that obtained in a candle jar or 5% CO₂ incubator), a
research group reported that several *P. falciparum* reference strains have been maintained in
a normal atmospheric gas mixture (20.95% O₂, 0.04% CO₂, 78–79% N₂) in their laboratory
for several months to one year in a standard RPMI 1640 medium with 0.5% Albumax®
supplemented with hypoxanthine and 2 g/L glucose [269]. The suspension cultures
(starting parasitemia > 0.5%) were maintained in culture flasks (with the cap screwed
tightly) or hermetic glass bottles (0.1–2 L bottles; 20% of the volume was filled with culture
medium and 80% of the volume was occupied by atmospheric gas or the standard gas
mixtures of 5% O₂, 5% CO₂, and 90% N₂; the latter was flushed into the culture vessel before
screwing the cap) placed on an orbital shaker. When suspension cultures in atmospheric
gas and the standard gas mixture were compared, parasite growth during six days was
similar. The parasites in static cultures in glass bottles did not grow. The free oxygen
contents in uninfected erythrocytes measured after 24 h in the flasks with atmospheric
air or the standard gas mixture were 180 µM and 147 µM, respectively (arterial oxygen
concentration, 130 µM).

In an earlier experiment, the same research group reported that a reference clone (3D7)
was cultivated in their laboratory in an anaerobic condition (i.e., 5% CO₂ and 95% N₂) for
10 days, and the multiplication rate remained similar to the control parasites cultivated in
the standard gas mixture (5% O₂, 5% CO₂, and 90% N₂), leading the authors to conclude
that the parasite adapted to anaerobic conditions [270]. Details of parasite growth were not
described in that study.

Another effect that O₂ (or CO₂) concentration may have in short-term cultivation
of malaria parasites is possible changes in the level of susceptibility in vitro to some
antimalarial drugs and antibiotics [215,262,266,269,271,272]. Transcriptomic data suggest
various metabolic changes in response to hyperoxia [267], but the precise underlying reason
for the influence of O₂ concentration on the level of in vitro drug susceptibility is not yet
well-known. In vitro response to chloroquine is also influenced by CO₂ concentration;
chloroquine IC₅₀ decreases significantly in a chloroquine-resistant laboratory-adapted
strain (K1/Thailand) when CO₂ concentration varied from 7% to 5% and 2.7% with a
constant O₂ concentration set at 5% [262]. Growth rates in the drug-free control wells were
not affected by CO₂ concentration during the 42 h incubation period. The underlying cause
of altered IC₅₀ values is due to pH modification (a higher CO₂ concentration leads to lower
pH of culture medium) resulting from an imbalanced CO₂-NaHCO₃ buffer system. These observations have important implications for data interpretation of in vitro drug response.

The candle jar method is practical and affordable in most laboratories, including those in the field. The candle jar still needs to be placed in a simple incubator to maintain it at 37 °C. The possibility of using an ordinary CO₂ incubator, rather than using the more expensive, sophisticated incubator with O₂ regulation, for malaria cultures, including short-term cultures (e.g., for in vitro drug susceptibility assays requiring 24–72 h of incubation), is an important option for moderately equipped field laboratories. Normal air, however, is generally not suitable for malaria cultivation, even for laboratory-adapted *P. falciparum* strains [273]. For fresh clinical isolates, some investigators noted that the use of a commercial gas mixture of 5% CO₂–5% O₂–90% N₂, instead of a candle jar, allowed a higher proportion of isolates to be adapted to long-term in vitro culture conditions [274].

In addition to the use of a candle jar, the normal gas composition in the atmosphere can be modified by (i) using an incubator that can be programmed to regulate the CO₂ and/or O₂ contents and placing Petri dishes or tissue culture flasks with vented caps (or, if they do not have vented caps, place the cap loosely on the flasks) or (ii) flushing pre-mixed gas into culture flasks before replacing the cap tightly. With both of these methods, cylinders containing pure CO₂, pure N₂ (to lower O₂ content), or various mixtures of CO₂, O₂, and N₂ (pre-mixed gas) are required. For maintenance, a 5% CO₂ incubator requires a single source of gas (CO₂), and it is less expensive than the more sophisticated incubator with adjustable levels of O₂ and CO₂, for which two sources of gas, CO₂ and N₂, are required. If a CO₂ incubator, with or without variable O₂ control, is not available, pre-mixed gas can be used, but it is often unavailable in many malaria-endemic countries. Ordering pre-mixed gas from an industrialized country would be costly and would take time. One possible solution to this problem would be to use an adjustable gas mixing device [275]. Such a device still requires a relatively high initial investment to acquire and install the gas supply system. Gas cylinders with pure CO₂, pure N₂, and pure O₂, which may be available locally, are also necessary to operate the system.

Several less sophisticated methods to modify the gas contents for malaria cultivation have been developed. One simple method that does not require any additional equipment is to blow end-tidal-exhaled air into the culture flask [276]. Tidal volume refers to the amount of air inspired and expired with each breath during quiet respirations, i.e., without any added effort during inspiration or expiration. At the end of expiration, the alveolar partial pressure of O₂ increases and lowers the partial pressure of CO₂ to a minimal level. To obtain a suitable gas composition in exhaled air, a person inhales air normally without effort, holds the breath for 15 s, and exhales into the culture flask through a sterile cotton-plugged Pasteur pipette. Immediately after expiration, the flask is tightly sealed. The gas composition of end-tidal-exhaled air was found to contain 5.1% CO₂, 15.5% O₂, and 78% N₂, which are close to the gas mixture in a candle jar. Using end-tidal-exhaled air as the source of gas, a laboratory-adapted *P. falciparum* strain was successfully maintained for 29 days. The growth rate was similar or slightly lower with end-tidal-exhaled air than pre-mixed gas (5% CO₂–5% O₂–90% N₂) for the first 14 days and tended to be about three-to-four-fold lower than pre-mixed gas between day 14 and day 29 of cultures.

Another method involves the introduction of a single-use chemical pack containing ascorbic acid within an airtight culture vessel. Once opened, these packs absorb O₂ from the enclosed air and release CO₂ in a chemical reaction and modify the atmosphere to one of the following pre-determined gas composition, depending on the type of gas sachets (Sugiyama-gen, Tokyo, Japan): (i) 5% CO₂ and 15% O₂, (more adapted for bacterial culture; similar to a candle jar), (ii) 10% CO₂ and 10% O₂, or (iii) 5% CO₂ and 5% O₂ (specifically manufactured for malaria culture). The specified atmosphere is reported to be attained within 2 h in a hermetically sealed container and can be maintained for over 24 h.

Preliminary studies have demonstrated that both fresh clinical isolates and laboratory-adapted *P. falciparum* strains can be cultivated with this novel system [277–280]. The growth rate of laboratory-adapted parasites was similar in three different gas mixtures produced
pH of culture medium) resulting from an imbalanced CO₂-NaHCO₃ buffer system. These observations have important implications for data interpretation of in vitro drug response.

The candle jar method is practical and affordable in most laboratories, including those in the field. The candle jar still needs to be placed in a simple incubator to maintain it at 37 °C. The possibility of using an ordinary CO₂ incubator, rather than using the more expensive, sophisticated incubator with O₂ regulation, for malaria cultures, including short-term cultures (e.g., for in vitro drug susceptibility assays requiring 24–72 h of incubation), is an important option for moderately equipped field laboratories. Normal air, however, is generally not suitable for malaria cultivation, even for laboratory-adapted *P. falciparum* strains [273]. For fresh clinical isolates, some investigators noted that the use of a commercial gas mixture of 5% CO₂–5% O₂–90% N₂, instead of a candle jar, allowed a higher proportion of isolates to be adapted to long-term in vitro culture conditions [274].

In addition to the use of a candle jar, the normal gas composition in the atmosphere can be modified by (i) using an incubator that can be programmed to regulate the CO₂ and/or O₂ contents and placing Petri dishes or tissue culture flasks with vented caps (or, if they do not have vented caps, place the cap loosely on the flasks) or (ii) flushing pre-mixed gas into culture flasks before replacing the cap tightly. With both of these methods, cylinders containing pure CO₂, pure N₂ (to lower O₂ content), or various mixtures of CO₂, O₂, and N₂ (pre-mixed gas) are required. For maintenance, a 5% CO₂ incubator requires a single source of gas (CO₂), and it is less expensive than the more sophisticated incubator with adjustable levels of O₂ and CO₂, for which two sources of gas, CO₂ and N₂, are required. If a CO₂ incubator, with or without variable O₂ control, is not available, pre-mixed gas can be used, but it is often unavailable in many malaria-endemic countries. Ordering pre-mixed gas from an industrialized country would be costly and would take time. One possible solution to this problem would be to use an adjustable gas mixing device [275]. Such a device still requires a relatively high initial investment to acquire and install the gas supply system. Gas cylinders with pure CO₂, pure N₂, and pure O₂, which may be available locally, are also necessary to operate the system.

Several less sophisticated methods to modify the gas contents for malaria cultivation have been developed. One simple method that does not require any additional equipment is to blow end-tidal-exhaled air into the culture flask [276]. Tidal volume refers to the amount of air inspired and expired with each breath during quiet respirations, i.e., without any added effort during inspiration or expiration. At the end of expiration, the alveolar partial pressure of O₂ increases and lowers the partial pressure of CO₂ to a minimal level. To obtain a suitable gas composition in exhaled air, a person inhales air normally without effort, holds the breath for 15 s, and exhales into the culture flask through a sterile cotton-plugged Pasteur pipette. Immediately after expiration, the flask is tightly sealed. The gas composition of end-tidal-exhaled air was found to contain 5.1% CO₂, 15.5% O₂, and 78% N₂, which are close to the gas mixture in a candle jar. Using end-tidal-exhaled air as the source of gas, a laboratory-adapted *P. falciparum* strain was successfully maintained for 29 days. The growth rate was similar or slightly lower with end-tidal-exhaled air than pre-mixed gas (5% CO₂–5% O₂–90% N₂) for the first 14 days and tended to be about three-to-four-fold lower than pre-mixed gas between day 14 and day 29 of cultures.

Another method involves the introduction of a single-use chemical pack containing ascorbic acid within an airtight culture vessel. Once opened, these packs absorb O₂ from the enclosed air and release CO₂ in a chemical reaction and modify the atmosphere to one of the following pre-determined gas composition, depending on the type of gas sachets (Sugiyama-gen, Tokyo, Japan): (i) 5% CO₂ and 15% O₂, (more adapted for bacterial culture; similar to a candle jar), (ii) 10% CO₂ and 10% O₂, or (iii) 5% CO₂ and 5% O₂ (specifically manufactured for malaria culture). The specified atmosphere is reported to be attained within 2 h in a hermetically sealed container and can be maintained for over 24 h.

Preliminary studies have demonstrated that both fresh clinical isolates and laboratory-adapted *P. falciparum* strains can be cultivated with this novel system [277–280]. The growth rate of laboratory-adapted parasites was similar in three different gas mixtures produced
by the sachets, i.e., the 1–3% O₂/3–4% CO₂/93–96% N₂ gas mixture, and a candle jar over a 48 h period. Moreover, the long-term cultivation of laboratory strains was successful for 26 days at similar growth rates in different gas mixtures. In vitro drug susceptibility assays can also be performed in the field with these gas-generating sachets [281–285].

The general consensus among malaria researchers is that, for the long-term growth of parasites under optimal conditions, O₂ level should be lower than 21% found in the air. Until today, however, there is no consensus as to what should be the actual O₂ concentration for malaria cultivation [286,287]. According to Jensen and Trager, the actual O₂ level is not critical for malaria cultivation, and it may range from 5 to 17%, with 10–15% giving the best results [81]. More recently, other authors have argued that O₂ levels between 1 and 5% should be considered as the physiological normoxic O₂ range for asexual stages of malaria parasites in the human host [287]. During the ring stage in vivo (for about 24 h), *P. falciparum* parasites come in contact with relatively high O₂ (>13%) levels for a brief moment while in transit in the pulmonary circulation and circulate during the rest of the time in arterial (12.5% O₂), venous (5.3% O₂), and capillary blood (see Table 4). The mature *P. falciparum* trophozoites and schizonts spend the next 24 h sequestered in deep organs, where oxygen concentration falls to 0.6–5.3%. Based on human physiology and the fact that *P. falciparum* has adapted to fluctuating O₂ levels in the host, mostly to oxygen tension below that found in pulmonary capillaries, it can reasonably be argued that the levels of O₂ ranging from 0.6% to 13% in in vitro cultivation probably best mimic the in vivo condition.

For CO₂, even though its concentration can be as high as 5.9–6.1% in the deep venous circulation and at the tissue level (see Table 4), levels >5% CO₂ are too high for the CO₂ bicarbonate buffer system to maintain optimal pH. In vivo, the range of CO₂ concentration is narrow. The standard medium containing RPMI 1640 and the double buffer system, 25 mM HEPES and 25 mM NaHCO₃, was conceived to maintain the pH within the physiological range in an atmosphere with 5% CO₂. The percentage of CO₂ in the atmosphere influences the pH of the culture medium depending on the buffer system. A relatively high (5%) CO₂ is required to maintain the pH of the RPMI 1640–HEPES–NaHCO₃ medium to approximately 7.2. At lower (1–2%) CO₂, the pH of the medium rises to 7.5–7.7. For a given CO₂ concentration, a wide range of O₂ content between 0.5% and 21% does not modify the pH. The optimal CO₂ level for in vitro cultivation of *P. falciparum* is probably 3–5%.

Most strains of *P. falciparum* grow within a wide range of O₂ levels, but at higher levels (17–21%), the growth rate tends to decrease unless the CO₂ level is raised to 5%. The requirement for low O₂ may be strain-dependent. Some strains, in particular chloroquine-susceptible parasites, may require a lower O₂ level (3–5%) and/or human serum (instead of serum-free substitutes, such as Albumax®) for optimal long-term cultivation. Researchers may have to check if *P. falciparum* strains in their laboratory are well-adapted to certain O₂ and CO₂ concentrations.

In a recent experimental work conducted by Géry et al. [288], the authors reported successful long-term cultivation of a laboratory-adapted *P. falciparum* clone (3D7) using a Petaka G4™ device (Celartia, Columbus, OH). This relatively simple, compact, and light cell culture vessel with an auto-controlled gas exchange system was designed to cultivate various cells without any internal air space. The vessel is filled with complete culture media and parasite-infected erythrocytes, leaving no air space within the vessel. Minimal gas exchange with external air occurs through its 0.2 µm filter. The removal of the spent medium and the introduction of fresh media and uninfected erythrocytes are performed through its injection port. Due to its completely closed system, many of the routine steps involved in malaria cultivation can be performed outside a laminar flow hood. It could be hypothesized that a small proportion of O₂ that is dissolved in the plasma and available in hemoglobin molecules may be sufficient for *P. falciparum* parasites to propagate in this closed system. Although further studies are necessary, this novel system to cultivate malaria parasites provides an avenue worth exploring to understand the O₂ and CO₂ requirements of malaria parasites.
by the sachets, i.e., the 1–3% O₂/3–4% CO₂/93–96% N₂ gas mixture, and a candle jar over a 48 h period. Moreover, the long-term cultivation of laboratory strains was successful for 26 days at similar growth rates in different gas mixtures. In vitro drug susceptibility assays can also be performed in the field with these gas-generating sachets [281–285].

The general consensus among malaria researchers is that, for the long-term growth of parasites under optimal conditions, O₂ level should be lower than 21% found in the air. Until today, however, there is no consensus as to what should be the actual O₂ concentration for malaria cultivation [286,287]. According to Jensen and Trager, the actual O₂ level is not critical for malaria cultivation, and it may range from 5 to 17%, with 10–15% giving the best results [81]. More recently, other authors have argued that O₂ levels between 1 and 5% should be considered as the physiological normoxic O₂ range for asexual stages of malaria parasites in the human host [287]. During the ring stage in vivo (for about 24 h), *P. falciparum* parasites come in contact with relatively high O₂ (>13%) levels for a brief moment while in transit in the pulmonary circulation and circulate during the rest of the time in arterial (12.5% O₂), venous (5.3% O₂), and capillary blood (see Table 4). The mature *P. falciparum* trophozoites and schizonts spend the next 24 h sequestered in deep organs, where oxygen concentration falls to 0.6–5.3%. Based on human physiology and the fact that *P. falciparum* has adapted to fluctuating O₂ levels in the host, mostly to oxygen tension below that found in pulmonary capillaries, it can reasonably be argued that the levels of O₂ ranging from 0.6% to 13% in in vitro cultivation probably best mimic the in vivo condition.

For CO₂, even though its concentration can be as high as 5.9–6.1% in the deep venous circulation and at the tissue level (see Table 4), levels >5% CO₂ are too high for the CO₂ bicarbonate buffer system to maintain optimal pH. In vivo, the range of CO₂ concentration is narrow. The standard medium containing RPMI 1640 and the double buffer system, 25 mM HEPES and 25 mM NaHCO₃, was conceived to maintain the pH within the physiological range in an atmosphere with 5% CO₂. The percentage of CO₂ in the atmosphere influences the pH of the culture medium depending on the buffer system. A relatively high (5%) CO₂ is required to maintain the pH of the RPMI 1640-HEPES-NaHCO₃ medium to approximately 7.2. At lower (1–2%) CO₂, the pH of the medium rises to 7.5–7.7. For a given CO₂ concentration, a wide range of O₂ content between 0.5% and 21% does not modify the pH. The optimal CO₂ level for in vitro cultivation of *P. falciparum* is probably 3–5%.

Most strains of *P. falciparum* grow within a wide range of O₂ levels, but at higher levels (17–21%), the growth rate tends to decrease unless the CO₂ level is raised to 5%. The requirement for low O₂ may be strain-dependent. Some strains, in particular chloroquine-susceptible parasites, may require a lower O₂ level (3–5%) and/or human serum (instead of serum-free substitutes, such as Albumax®) for optimal long-term cultivation. Researchers may have to check if *P. falciparum* strains in their laboratory are well-adapted to certain O₂ and CO₂ concentrations.

In a recent experimental work conducted by Géry et al. [288], the authors reported successful long-term cultivation of a laboratory-adapted *P. falciparum* clone (3D7) using a Petaka G4™ device (Celartia, Columbus, OH). This relatively simple, compact, and light cell culture vessel with an auto-controlled gas exchange system was designed to cultivate various cells without any internal air space. The vessel is filled with complete culture media and parasite-infected erythrocytes, leaving no air space within the vessel. Minimal gas exchange with external air occurs through its 0.2 µm filter. The removal of the spent medium and the introduction of fresh media and uninfected erythrocytes are performed through its injection port. Due to its completely closed system, many of the routine steps involved in malaria cultivation can be performed outside a laminar flow hood. It could be hypothesized that a small proportion of O₂ that is dissolved in the plasma and available in hemoglobin molecules may be sufficient for *P. falciparum* parasites to propagate in this closed system. Although further studies are necessary, this novel system to cultivate malaria parasites provides an avenue worth exploring to understand the O₂ and CO₂ requirements of malaria parasites.
**12. Incubator**

The requirement for an incubator or another piece of equipment, such as a water bath or temperature-controlled orbital shaker, that maintains a constant temperature of 37 °C for at least 24–72 h (to perform in vitro drug susceptibility assays or initiate in vitro cultivation) is one of the limiting factors for a wide application of malaria cultures in the field. In developed countries, almost every microbiology laboratory possesses an incubator. It may be a simple incubator with normal atmospheric air or a more sophisticated incubator with 5% CO₂ in the air, or it may have options to adjust both CO₂ and O₂ concentrations. In developing countries, even if an incubator were available, the power supply may not be reliable in some endemic areas due to frequent power cuts and voltage fluctuations.

Few attempts have been made to develop portable incubators designed for field use. A “home-made” portable incubator heated by a car taillight bulb and powered by a car battery was designed and tested by Eastham and Rieckmann [289]. An oven thermostat is connected to the circuit to switch the electric bulb on and off to maintain the temperature at 37 °C. A candle jar desiccator made from large glass jars that can accommodate microtiter plates is placed in the incubator. Such a device may be useful in a remote field site where there is no source of electricity.

Another option is offered by a Japanese manufacturer that has developed a portable incubator in which culture plates or Petri dishes and gas-generating ascorbic acid sachets (5% CO₂–5% O₂ or 5% CO₂ in air) are placed within an airtight box (AnaeroPack® malaria culture system; Sugiyama-gen Co., Ltd., Tokyo, Japan) [279,283,284]. The incubator requires a constant electrical supply source, which may be from a car battery. The available model functions on 110 V (no built-in transformer for countries with 220 V) and is relatively expensive.

Where there is at least a power line, other devices may be used. Water baths, which can be found in most laboratories, may serve as an incubator [290]. If a field-compatible incubator, water bath, or temperature-controlled orbital shaker is not available for on-site in vitro assays, blood samples would have to be transported to the nearest research laboratory.

**13. Temperature**

Despite the fact that parasites may be subjected to a relatively high temperature (>40 °C) in the human host, at least for a few hours during febrile episodes, in vitro experiments have suggested that prolonged hyperthermia (i.e., continuous exposure to ≥40 °C for 24–72 h) may actually be deleterious for parasite growth [291–294]. In the experiments conducted by Kwiatkowski [291], it was observed that laboratory-adapted parasite strains (ring stage) exposed to 40 °C during the first half of the intraerythrocytic cycle (i.e., 0–24 h), followed by cultivation at 37 °C for the remaining half of the intraerythrocytic cycle (i.e., 24–48 h), grew almost as well as the control parasites cultivated at 37 °C for 48 h. By contrast, when the parasites were cultivated first at 37 °C for 24 h, followed by incubation at 40 °C for the next 24 h, the 48 h parasitemia decreased considerably compared to the control parasites cultivated at 37 °C for 48 h, and pyknotic schizonts were observed. This earlier finding was not confirmed in a more recent study. In the experiment conducted by Singhaboot et al. [294], even a short 2 h exposure of a laboratory-adapted strain in the ring stage to 40 °C, followed by culture at 37 °C for the next 46 h, led to not only a lower multiplication rate but also morphological alteration, suggesting parasite death. Those authors have reported that cultivation at 38 °C and 39 °C continuously for 48 h also decreases the multiplication rate (roughly 50% of the multiplication rate of the control parasites incubated at 37 °C), but there was no significant difference in the multiplication rates between 38 °C and 39 °C [294]. Exposing the parasites to 38 °C or 39 °C during the first 2 h of the intraerythrocytic cycle followed by incubation at 37 °C for the rest of the first intraerythrocytic cycle did not affect the multiplication rate measured at the end of the first intraerythrocytic cycle. In the experiments conducted by Long et al., it was shown that if the parasites are incubated continuously at a high temperature (38–39 °C), parasite growth decreases significantly at 38.5 °C and 39 °C but is not affected to a significant extent
## 12. Incubator

The requirement for an incubator or another piece of equipment, such as a water bath or temperature-controlled orbital shaker, that maintains a constant temperature of 37 °C for at least 24–72 h (to perform in vitro drug susceptibility assays or initiate in vitro cultivation) is one of the limiting factors for a wide application of malaria cultures in the field. In developed countries, almost every microbiology laboratory possesses an incubator. It may be a simple incubator with normal atmospheric air or a more sophisticated incubator with 5% CO₂ in the air, or it may have options to adjust both CO₂ and O₂ concentrations. In developing countries, even if an incubator were available, the power supply may not be reliable in some endemic areas due to frequent power cuts and voltage fluctuations.

Few attempts have been made to develop portable incubators designed for field use. A “home-made” portable incubator heated by a car taillight bulb and powered by a car battery was designed and tested by Eastham and Rieckmann [289]. An oven thermostat is connected to the circuit to switch the electric bulb on and off to maintain the temperature at 37 °C. A candle jar desiccator made from large glass jars that can accommodate microtiter plates is placed in the incubator. Such a device may be useful in a remote field site where there is no source of electricity.

Another option is offered by a Japanese manufacturer that has developed a portable incubator in which culture plates or Petri dishes and gas-generating ascorbic acid sachets (5% CO₂–5% O₂ or 5% CO₂ in air) are placed within an airtight box (AnaeroPack® malaria culture system; Sugiyama-gen Co., Ltd., Tokyo, Japan) [279,283,284]. The incubator requires a constant electrical supply source, which may be from a car battery. The available model functions on 110 V (no built-in transformer for countries with 220 V) and is relatively expensive.

Where there is at least a power line, other devices may be used. Water baths, which can be found in most laboratories, may serve as an incubator [290]. If a field-compatible incubator, water bath, or temperature-controlled orbital shaker is not available for on-site in vitro assays, blood samples would have to be transported to the nearest research laboratory.

## 13. Temperature

Despite the fact that parasites may be subjected to a relatively high temperature (>40 °C) in the human host, at least for a few hours during febrile episodes, in vitro experiments have suggested that prolonged hyperthermia (i.e., continuous exposure to ≥40 °C for 24–72 h) may actually be deleterious for parasite growth [291–294]. In the experiments conducted by Kwiatkowski [291], it was observed that laboratory-adapted parasite strains (ring stage) exposed to 40 °C during the first half of the intraerythrocytic cycle (i.e., 0–24 h), followed by cultivation at 37 °C for the remaining half of the intraerythrocytic cycle (i.e., 24–48 h), grew almost as well as the control parasites cultivated at 37 °C for 48 h. By contrast, when the parasites were cultivated first at 37 °C for 24 h, followed by incubation at 40 °C for the next 24 h, the 48 h parasitemia decreased considerably compared to the control parasites cultivated at 37 °C for 48 h, and pyknotic schizonts were observed. This earlier finding was not confirmed in a more recent study. In the experiment conducted by Singhaboot et al. [294], even a short 2 h exposure of a laboratory-adapted strain in the ring stage to 40 °C, followed by culture at 37 °C for the next 46 h, led to not only a lower multiplication rate but also morphological alteration, suggesting parasite death. Those authors have reported that cultivation at 38 °C and 39 °C continuously for 48 h also decreases the multiplication rate (roughly 50% of the multiplication rate of the control parasites incubated at 37 °C), but there was no significant difference in the multiplication rates between 38 °C and 39 °C [294]. Exposing the parasites to 38 °C or 39 °C during the first 2 h of the intraerythrocytic cycle followed by incubation at 37 °C for the rest of the first intraerythrocytic cycle did not affect the multiplication rate measured at the end of the first intraerythrocytic cycle. In the experiments conducted by Long et al., it was shown that if the parasites are incubated continuously at a high temperature (38–39 °C), parasite growth decreases significantly at 38.5 °C and 39 °C but is not affected to a significant extent
at 38.0 °C [292]. Some of these experimental studies have shown that ring-stage parasites are relatively more thermoresistant than mature trophozoites and schizonts [291,292].

A more elaborate experimental design involving the exposure of *P. falciparum* ring-stage cultures to 40 °C for 2 h (first heat shock) and the recovery phase at 37 °C for 10 h followed by a second exposure to 40 °C for 24 h (second heat shock; then incubation at 37 °C during the second intraerythrocytic cycle) showed that the growth of the parasites exposed to these two phases of heat shock was not affected during the first intraerythrocytic cycle and increased significantly during the second intraerythrocytic cycle [295]. The control cultures maintained at 37 °C and exposed to the second heat shock, without being exposed to the first heat shock, resulted in a marked diminution in parasite growth during the first intraerythrocytic cycle. The authors concluded that exposure to a short period of high temperature protects the parasites from subsequent heat shock and results in a higher multiplication rate. The clinical implications of the in vitro experiments described above are not clear.

When laboratory-adapted *P. falciparum* parasites are cultivated continuously at low temperatures (28–34 °C) for 48–72 h, maturation is considerably delayed, and/or the multiplication rate is considerably reduced [293,294,296]. A short 2 h exposure of ring-stage parasites to 32–35 °C followed by incubation at 37 °C for 48 h does not significantly affect the parasitemia level or morphology of the parasites [294]. At a much lower temperature (0–4 °C), the growth of laboratory-adapted *P. falciparum* strains is completely arrested. Mature developmental stages (i.e., mature trophozoites and schizonts) are killed when kept at 0–4 °C for 24–48 h [297,298]. Ring-stage parasites may remain viable for up to 14 days at 4 °C; when replaced in suitable culture conditions at 37 °C, in vitro cultivation can be pursued [299]. For fresh blood samples, many field investigators have been transporting samples obtained from malaria-infected patients in remote areas to research laboratories in a cooler or insulated box with cold packs (gel packs or ice packs) or wet ice, which usually maintains the inside temperature within the range of 2–8 °C, depending on the ambient temperature, container, and the number of cold packs or the amount of wet ice in the box. Many, but not all, fresh clinical isolates of *P. falciparum* can be preserved in wet ice for up to 10 days before initiating in vitro cultivation or ex vivo drug susceptibility assays [300].

The first successful continuous cultivation of asexual erythrocytic stages of *P. falciparum* was attained at an incubation temperature of 37–38 °C [26,27]. It has been, and still is, recommended to maintain in vitro cultures at 37 °C for routine maintenance of asexual *P. falciparum* parasites.

## 14. Axenic Culture

Axenic culture is a pure culture without any "contaminating" cells. It may be considered as an ultimate goal to be attained in most, if not all, cultivation procedures. In the case of asexual intraerythrocytic stages of malaria parasites, axenic culture means the propagation of the parasites extracellularly, without the presence of intact erythrocytes. In some early works on non-human malaria parasites, investigators have attempted to remove the asexual parasites from their host erythrocytes for cellular and biochemical studies [16,301–303]. The parasites survived and developed for up to four days, but there was no propagation.

Axenic culture of the erythrocytic stages of *P. falciparum* has been attempted by William Trager with limited success since the 1970s [16]. In the initial studies, schizonts incubated for 20–24 h in a modified, high-potassium RPMI 1640 medium supplemented with human red cell extract, human serum, ATP dipotassium salt, and sodium pyruvate (without intact erythrocytes) developed into extracellular young trophozoites [304]. In later studies, axenic culture initiated from both purified merozoites and young rings freed by lysis from their host erythrocytes resulted in developing extracellular late rings and trophozoites after 14–18 h using a similar culture medium to their earlier studies [305,306]. These extracellular trophozoites had essentially the same ultrastructure as the intracellular trophozoites.
at 38.0 °C [292]. Some of these experimental studies have shown that ring-stage parasites are relatively more thermoresistant than mature trophozoites and schizonts [291,292].

A more elaborate experimental design involving the exposure of *P. falciparum* ring-stage cultures to 40 °C for 2 h (first heat shock) and the recovery phase at 37 °C for 10 h followed by a second exposure to 40 °C for 24 h (second heat shock; then incubation at 37 °C during the second intraerythrocytic cycle) showed that the growth of the parasites exposed to these two phases of heat shock was not affected during the first intraerythrocytic cycle and increased significantly during the second intraerythrocytic cycle [295]. The control cultures maintained at 37 °C and exposed to the second heat shock, without being exposed to the first heat shock, resulted in a marked diminution in parasite growth during the first intraerythrocytic cycle. The authors concluded that exposure to a short period of high temperature protects the parasites from subsequent heat shock and results in a higher multiplication rate. The clinical implications of the in vitro experiments described above are not clear.

When laboratory-adapted *P. falciparum* parasites are cultivated continuously at low temperatures (28–34 °C) for 48–72 h, maturation is considerably delayed, and/or the multiplication rate is considerably reduced [293,294,296]. A short 2 h exposure of ring-stage parasites to 32–35 °C followed by incubation at 37 °C for 48 h does not significantly affect the parasitemia level or morphology of the parasites [294]. At a much lower temperature (0–4 °C), the growth of laboratory-adapted *P. falciparum* strains is completely arrested. Mature developmental stages (i.e., mature trophozoites and schizonts) are killed when kept at 0–4 °C for 24–48 h [297,298]. Ring-stage parasites may remain viable for up to 14 days at 4 °C; when replaced in suitable culture conditions at 37 °C, in vitro cultivation can be pursued [299]. For fresh blood samples, many field investigators have been transporting samples obtained from malaria-infected patients in remote areas to research laboratories in a cooler or insulated box with cold packs (gel packs or ice packs) or wet ice, which usually maintains the inside temperature within the range of 2–8 °C, depending on the ambient temperature, container, and the number of cold packs or the amount of wet ice in the box. Many, but not all, fresh clinical isolates of *P. falciparum* can be preserved in wet ice for up to 10 days before initiating in vitro cultivation or ex vivo drug susceptibility assays [300].

The first successful continuous cultivation of asexual erythrocytic stages of *P. falciparum* was attained at an incubation temperature of 37–38 °C [26,27]. It has been, and still is, recommended to maintain in vitro cultures at 37 °C for routine maintenance of asexual *P. falciparum* parasites.

## 14. Axenic Culture

Axenic culture is a pure culture without any "contaminating" cells. It may be considered as an ultimate goal to be attained in most, if not all, cultivation procedures. In the case of asexual intraerythrocytic stages of malaria parasites, axenic culture means the propagation of the parasites extracellularly, without the presence of intact erythrocytes. In some early works on non-human malaria parasites, investigators have attempted to remove the asexual parasites from their host erythrocytes for cellular and biochemical studies [16,301–303]. The parasites survived and developed for up to four days, but there was no propagation.

Axenic culture of the erythrocytic stages of *P. falciparum* has been attempted by William Trager with limited success since the 1970s [16]. In the initial studies, schizonts incubated for 20–24 h in a modified, high-potassium RPMI 1640 medium supplemented with human red cell extract, human serum, ATP dipotassium salt, and sodium pyruvate (without intact erythrocytes) developed into extracellular young trophozoites [304]. In later studies, axenic culture initiated from both purified merozoites and young rings freed by lysis from their host erythrocytes resulted in developing extracellular late rings and trophozoites after 14–18 h using a similar culture medium to their earlier studies [305,306]. These extracellular trophozoites had essentially the same ultrastructure as the intracellular trophozoites.
Further improvement in the axenic development of one complete asexual erythrocytic cycle (and the early development of extracellular gametocytes) was attained by embedding purified merozoites in a gelatinous matrix overlaid with high-potassium RPMI 1640 supplemented with human red cell extract, serum, ATP, and sodium pyruvate [307–309]. As many as 30% of embedded merozoites differentiated into extracellular trophozoites, but only 1% reached the schizont stage after 36 h. The addition of human erythrocyte membranes (i.e., erythrocyte ghosts prepared by hypotonic lysis) increased the yield of viable extracellular trophozoites and early schizonts at 36 h. Early stages of gametocytes (stages II and III) were also observed. Merozoites produced by the extracellular schizonts during the first intraerythrocytic cycle of axenic culture were viable.

In the final study on axenic culture conducted in William Trager’s laboratory before its closure in 1998, further improvement was obtained with the addition of carnitine (100–200 µM), magnesium sulfate (10 mM), calcium nitrate (0.4 mM), GF21 serum substitute (10–20%), and higher erythrocyte membrane content (in addition to supplements such as 2.1 mM ATP, 5.4 mM sodium pyruvate, and 100 µM coenzyme A sodium salt used in earlier experiments [307–309]) and incubation in 0.5% O₂, 3% CO₂, and 96.5% N₂. These modifications allowed three cycles of schizogony over a six-day period, with a five-fold increase in the number of parasites after each complete cycle [310]. When the extracellular schizonts obtained at the end of the experiment on day 6 were cultivated with intact uninfected erythrocytes, intracellular rings developed on day 7, demonstrating the viability and infectivity of parasites that developed extracellularly for three erythrocytic cycles. The experimental conditions need further improvements to ensure a consistently high yield of viable, extracellular parasites after each successive erythrocytic cycle in the continuous culture. Some clues for such improvements have been suggested by William Trager for future researchers [311]. At present, the continuous axenic culture of the erythrocytic stages of malaria parasites is still in its infancy.

**15. Adaptation, Selection, and Phenotypic/Genotypic Modifications during Cultivation**

The in vitro cultivation of *P. falciparum* has become a major tool for malaria research. Many concrete examples can be given to highlight the important roles that in vitro cultivation has played since 1976, including drug discovery, validation of candidate molecular markers of drug resistance, a better understanding of malaria biology, and vaccine development, to mention a few [13]. Yet, in our present state of knowledge, many unanswered questions about its relevance persist. Several “epistemological” questions (i.e., what and how can we know using *P. falciparum* cultivated in vitro?) have been raised by some authors, the writings (reviews and opinion papers) of whom readers are encouraged to peruse for reflection and debate [57,287,312–314]. Some of these questions are discussed below.

First and foremost, it should be borne in mind that *P. falciparum* parasites maintained in continuous in vitro cultivation do not faithfully represent naturally occurring parasites in the human host. The latter, referred to as an “isolate” when it is collected, is defined as “a sample of parasites, not necessarily genetically homogeneous, collected on a single occasion from a wild host and preserved in the laboratory either by passaging or by deep freezing” [315]. Early studies have shown that *P. falciparum* isolates or uncloned laboratory-adapted strains are often composed of several distinct parasite populations, each of which is characterized by an enzyme variant (or electrophoretic form) or genetic profile [316–324]. Molecular studies conducted during the 1990s on field isolates fully support these findings and have quantified the number of distinct parasite populations in an isolate obtained from individual human hosts (asymptomatic carriers or symptomatic patients), referred to as the “multiplicity of infection” (MOI) or the complexity of infection (COI) [325–330]. The average MOI in a patient population or asymptomatic carriers usually reflects the level of intensity of malaria transmission; it is high in areas characterized by perennial and intense transmission, as in many sub-Saharan African countries [331–334].

The first “barrier” to in vitro cultivation encountered by the parasite populations constituting an isolate is adaptation. Many, but not all, clinical isolates collected from
Further improvement in the axenic development of one complete asexual erythrocytic cycle (and the early development of extracellular gametocytes) was attained by embedding purified merozoites in a gelatinous matrix overlaid with high-potassium RPMI 1640 supplemented with human red cell extract, serum, ATP, and sodium pyruvate [307–309]. As many as 30% of embedded merozoites differentiated into extracellular trophozoites, but only 1% reached the schizont stage after 36 h. The addition of human erythrocyte membranes (i.e., erythrocyte ghosts prepared by hypotonic lysis) increased the yield of viable extracellular trophozoites and early schizonts at 36 h. Early stages of gametocytes (stages II and III) were also observed. Merozoites produced by the extracellular schizonts during the first intraerythrocytic cycle of axenic culture were viable.

In the final study on axenic culture conducted in William Trager’s laboratory before its closure in 1998, further improvement was obtained with the addition of carnitine (100–200 µM), magnesium sulfate (10 mM), calcium nitrate (0.4 mM), GF21 serum substitute (10–20%), and higher erythrocyte membrane content (in addition to supplements such as 2.1 mM ATP, 5.4 mM sodium pyruvate, and 100 µM coenzyme A sodium salt used in earlier experiments [307–309]) and incubation in 0.5% O₂, 3% CO₂, and 96.5% N₂. These modifications allowed three cycles of schizogony over a six-day period, with a five-fold increase in the number of parasites after each complete cycle [310]. When the extracellular schizonts obtained at the end of the experiment on day 6 were cultivated with intact uninfected erythrocytes, intracellular rings developed on day 7, demonstrating the viability and infectivity of parasites that developed extracellularly for three erythrocytic cycles. The experimental conditions need further improvements to ensure a consistently high yield of viable, extracellular parasites after each successive erythrocytic cycle in the continuous culture. Some clues for such improvements have been suggested by William Trager for future researchers [311]. At present, the continuous axenic culture of the erythrocytic stages of malaria parasites is still in its infancy.

**15. Adaptation, Selection, and Phenotypic/Genotypic Modifications during Cultivation**

The in vitro cultivation of *P. falciparum* has become a major tool for malaria research. Many concrete examples can be given to highlight the important roles that in vitro cultivation has played since 1976, including drug discovery, validation of candidate molecular markers of drug resistance, a better understanding of malaria biology, and vaccine development, to mention a few [13]. Yet, in our present state of knowledge, many unanswered questions about its relevance persist. Several “epistemological” questions (i.e., what and how can we know using *P. falciparum* cultivated in vitro?) have been raised by some authors, the writings (reviews and opinion papers) of whom readers are encouraged to peruse for reflection and debate [57,287,312–314]. Some of these questions are discussed below.

First and foremost, it should be borne in mind that *P. falciparum* parasites maintained in continuous in vitro cultivation do not faithfully represent naturally occurring parasites in the human host. The latter, referred to as an “isolate” when it is collected, is defined as “a sample of parasites, not necessarily genetically homogeneous, collected on a single occasion from a wild host and preserved in the laboratory either by passaging or by deep freezing” [315]. Early studies have shown that *P. falciparum* isolates or uncloned laboratory-adapted strains are often composed of several distinct parasite populations, each of which is characterized by an enzyme variant (or electrophoretic form) or genetic profile [316–324]. Molecular studies conducted during the 1990s on field isolates fully support these findings and have quantified the number of distinct parasite populations in an isolate obtained from individual human hosts (asymptomatic carriers or symptomatic patients), referred to as the “multiplicity of infection” (MOI) or the complexity of infection (COI) [325–330]. The average MOI in a patient population or asymptomatic carriers usually reflects the level of intensity of malaria transmission; it is high in areas characterized by perennial and intense transmission, as in many sub-Saharan African countries [331–334].

The first “barrier” to in vitro cultivation encountered by the parasite populations constituting an isolate is adaptation. Many, but not all, clinical isolates collected from