• View in gallery

    (A) Weekly in vitro parasitemia under different atmospheric conditions determined by microscopy. Group 1 (white bars), culture flasks in 5% CO2 (positive control); group 2 (gray bars), culture flasks in normal air; group 3 (black bars), Petaka G3 device under the same conditions as for group 2. After day 15, viable asexual parasites were not observed in group 2. *Number of dilutions made before the evaluation of parasitemia. (B) Daily parasite growth of 3D7 clone in three different atmospheric conditions (group 1, white squares; group 2, black circles; and group 3, black triangles). The hatched vertical bars denote days when cultures were diluted but parasitemia was not determined. Culture media were not changed during weekends and national holidays. Growth was optimal until day 15 in all three groups. Both groups 1 and 3 showed suboptimal growth between days 16 and 29 because of inadequate dilution with fresh uninfected erythrocytes. Group 2 failed to grow beyond day 15. * Dilution of parasitemia with fresh uninfected erythrocytes.

  • View in gallery

    Illustration of the cytometric count of various cell components in the blood–culture medium suspension by CytoFLEX flow cytometer (Beckman Coulter) with CytExpert 2.0 software (Beckman Coulter). Here, the term “erythrocytes” denotes uninfected red blood cells; P2, red blood cells with young trophozoites (rings); P3, cell debris; P4, red blood cells with mature trophozoites and schizonts; and P5, free merozoites.18 Forward scatter area (FSC-A) denotes particle size. Near ultraviolet 450 area (NUV-450-A) corresponds to the Hoescht fluorescence emission. Both X and Y axes are expressed as logarithmic values of fluorescence intensity.

  • View in gallery

    Weekly in vitro parasite growth under different atmospheric conditions (group 1, culture flasks in 5% CO2; group 2, culture flasks in normal air; and group 3, Petaka G3 device in normal air) determined by cytometry, with a starting parasitemia of 0.5% on day 0. Dilution factors were included to adjust parasite growth. The box and whisker plots show the median percentage of parasite growth (horizontal bars), their quartiles (box), and range (whiskers). Measurements were obtained from 12 replicates of each group. *One outlier value was observed in group 1. After day 15, asexual parasites with normal morphology and schizonts were not observed in group 2 by microscopic examination of thin smears.

  • 1.

    Bass CC, 1911. A new conception of immunity. Its application to the cultivation of protozoa and bacteria from the blood and to therapeutic measures. J Am Med Assoc 57: 1534.

    • Search Google Scholar
    • Export Citation
  • 2.

    Bass CC, Johns FM, 1912. The cultivation of malarial plasmodia (Plasmodium vivax and Plasmodium falciparum) in vitro. J Exp Med 16: 567579.

    • Search Google Scholar
    • Export Citation
  • 3.

    Haynes JD, Diggs CL, Hines FA, Desjardins RE, 1976. Culture of human malaria parasites P. falciparum. Nature 263: 767769.

  • 4.

    Trager W, Jensen JB, 1976. Human malaria parasites in continuous culture. Science 193: 673675.

  • 5.

    Grun JL, Weidanz WP, 1987. Cultivation of Plasmodium falciparum in commercially available sera depleted of natural antibodies reactive with human erythrocytes. J Parasitol 73: 384388.

    • Search Google Scholar
    • Export Citation
  • 6.

    Basco LK, 2003. Molecular epidemiology of malaria in Cameroon. XV. Experimental studies on serum substitutes and supplements and alternative culture media for in vitro drug sensitivity assays using fresh isolates of Plasmodium falciparum. Am J Trop Med Hyg 69: 168173.

    • Search Google Scholar
    • Export Citation
  • 7.

    Basco LK, 2006. Molecular epidemiology of malaria in Cameroon. XXIII. Experimental studies on serum substitutes and alternative culture media for in vitro drug sensitivity assays using clinical isolates of Plasmodium falciparum. Am J Trop Med Hyg 75: 777782.

    • Search Google Scholar
    • Export Citation
  • 8.

    Srivastava K, Singh S, Singh P, Puri SK, 2007. In vitro cultivation of Plasmodium falciparum: studies with modified medium supplemented with Albumax II and various animal sera. Exp Parasitol 116: 171174.

    • Search Google Scholar
    • Export Citation
  • 9.

    Zolg JW, Macleod AJ, Dickson IH, Scaife JG, 1982. Plasmodium falciparum: modifications of the in vitro culture conditions improving parasitic yields. J Parasitol 68: 10721080.

    • Search Google Scholar
    • Export Citation
  • 10.

    Ferrer J, Rosal MD, Vidal JM, Prats C, Valls J, Herreros EA, Lopez D, Gargallo D, 2008. Effect of the haematocrit layer geometry on Plasmodium falciparum static thin-layer in vitro cultures. Malar J 7: 203.

    • Search Google Scholar
    • Export Citation
  • 11.

    Allen RJW, Kirk K, 2010. Plasmodium falciparum culture: the benefits of shaking. Mol Biochem Parasitol 169: 6365.

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    Scheibel LW, Ashton SH, Trager W, 1979. Plasmodium falciparum: microaerophilic requirements in human red blood cells. Exp Parasitol 47: 410418.

    • Search Google Scholar
    • Export Citation
  • 13.

    Waki S, Miyagami T, Nakazawa S, Igarashi I, Suzuki M, 1987. Maintenance and propagation of human malaria parasites in culture using an ordinary CO2 incubator. Trans R Soc Trop Med Hyg 78: 418.

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    • Export Citation
  • 14.

    Binh VQ, Luty AJF, Kremsner PG, 1997. Differential effects of human serum and cells on the growth of Plasmodium falciparum adapted to serum-free in vitro culture conditions. Am J Trop Med Hyg 57: 594600.

    • Search Google Scholar
    • Export Citation
  • 15.

    Haruki K, Kobayashi F, Fujino T, Matsui T, Tsuji M, 2001. Evaluation of Anaeropack® (AnP) type as tools of Plasmodium falciparum cultivation and drug sensitivity tests. Jpn J Trop Med Hyg 29: 365370.

    • Search Google Scholar
    • Export Citation
  • 16.

    Pascual A, Basco LK, Baret E, Amalvict R, Travers D, Rogier C, Pradines B, 2011. Use of the atmospheric generators for capnophilic bacteria Genbag-CO2 for the evaluation of in vitro Plasmodium falciparum susceptibility to standard anti-malarial drugs. Malar J 20: 8.

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    Grunwald D, Mayol J-F, Ronot X, 2010. Cycle Cellulaire et Cytométrie en Flux. Cachan, France: Lavoisier, 217219.

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    Janse CJ, Haghparast A, Sperança MA, Ramesar J, Kroeze H, del Portillo HA, Waters AP, 2003. Malaria parasites lacking eEF1A have a normal S/M phase yet grow more slowly due to a longer G1 phase. Mol Microbiol 50: 15391551.

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    Krungkrai J, Burat D, Kudan S, Krungkrai S, Prapunwattana P, 1999. Mitochondrial oxygen consumption in asexual and sexual bloodstages of the human malarial parasite, Plasmodium falciparum. Southeast Asian J Trop Med Public Health 30: 636642.

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Long-Term In vitro Cultivation of Plasmodium falciparum in a Novel Cell Culture Device

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  • 1 Centre François Baclesse, Normandie Université, UNICAEN, UR ABTE EA 4651, Caen, France;
  • 2 Aix Marseille Univ, IRD, AP-HM, SSA, VITROME, Marseille, France;
  • 3 Normandie Université, UNIROUEN, CETAPS EA 3832, Mont Saint Aignan Cedex, France;
  • 4 Normandie Université, UNICAEN, INSERM U 1075 COMETE, Caen, France;
  • 5 Normandie Université, UNICAEN, Plateau de Cytométrie en Flux, ICORE, Caen, France

The standard in vitro cultivation procedure for Plasmodium falciparum requires gas exchange and a microaerophilic atmosphere. A novel system using a commercially available cell culture device (Petaka G3; Celartia Ltd., Powell, OH) was assessed for long-term cultivation of a P. falciparum reference laboratory clone in normal air. Parasite growth during 30 days was similar, or better, in Petaka G3 than that in the standard cultivation method with gas exchange in a CO2 incubator. The successful cultivation of P. falciparum in the Petaka G3 device suggests that low O2 content available in hemoglobin and dissolved gas in the blood is sufficient for long-term cultivation. This finding may open the way to novel methods to cultivate and adapt P. falciparum field isolates to in vitro conditions with more ease.

INTRODUCTION

Long-term cultivation of Plasmodium falciparum malaria parasites is one of the research tools that have played a crucial role in drug development and in the studies on parasite biology and immunology. The understanding of in vitro requirements and development of long-term cultivation techniques took more than 60 years of research since the first attempt by Bass to maintain a successful short-term cultivation during one or two parasite life cycles.1,2 In 1976, two independent American research teams had finally discovered the set of requirements for successful continuous in vitro cultivation.3,4 These include culture media supplemented with buffers and serum, culture vessels that allow shallow static culture favoring gas exchange (flask, microtiter plate, Petri dish, or flow vial), and microaerophilic atmosphere. Other basic requirements include a constant temperature (37–38°C), humidity, and human (or simian) erythrocytes. The original methods used either medium 199 with Earle’s modified salts (enriched with 2 mg/mL glucose, 2 mM glutamine, 3 × 10−5 M 2-mercaptoethanol, and 30 µg/mL α-tocopherol) or RPMI 1640 (Dominique Dutscher SAS, Brumath, France) buffered with either 10 mM TES (for medium 199) or 25 mM HEPES-25 mM NaHCO3 (for RPMI 1640) and supplemented with 10–15% fetal bovine serum or human serum. Subsequent experiments have shown that several standard media for eukaryotic cell culture, animal sera, and serum substitutes (e.g., lipid-enriched bovine albumin [Albumax®; Gibco, Grand Island, NY]) also support P. falciparum growth in vitro for short-term and long-term cultivation.58 Static culture, as opposed to suspension culture, was also found to be unnecessary.911 The method developed by Trager and Jensen,4 rather than the similar technique of Hayes et al.,3 has become a standard laboratory procedure and is currently used with or without minor modifications in malaria research laboratories around the world.

Plasmodium falciparum cultivation has been performed under microaerophilic conditions since 1976. In the original methods developed by Haynes et al.3 and Trager and Jensen,4 cultures were placed in a controlled gas mixture of 3–7% CO2 and 1–6.6% O2 in CO2 incubators with variable oxygen control. Subsequent experiments have shown that sophisticated incubators with oxygen control are not necessary. A premixed CO2–O2–N2 gas can be flushed into culture flasks and their caps closed airtight before placing them in a room-air incubator. A suitable atmosphere can also be obtained by various alternative ways, including 5% CO2 incubators, which lower the O2 content to about 17%; candle jar (approximately 5% CO2 and 17% O2 when the candle burns out); or generator sachet placed in an airtight box or plastic bag.1216

The requirement for a microaerophilic atmosphere has been one of the limiting factors that have hampered the more widespread application of P. falciparum cultivation in field laboratories. A novel system designed to perform eukaryotic cell cultivation independently of CO2 and humidity has been developed in recent years. Moreover, the contents of the culture vessel are isolated from the external environment, reducing the risk of microbial contamination. The aim of the present study was to assess the performance of long-term P. falciparum cultivation using the Petaka G3 device (Celartia Ltd.).

MATERIALS AND METHODS

Cell culture.

The drug-sensitive P. falciparum reference clone 3D7 (of unknown origin derived from NF54 isolate from a case of airport malaria in Amsterdam, the Netherlands) was cultivated for 30 days under three different conditions using culture flasks or the Petaka G3 device. This latter device, a rectangular chamber (size 85.5 × 127.5 × 5.0 mm and 25 mL of internal volume), was specifically designed for cell cultivation on a large surface area without any internal air space to avoid contamination and allows minimal gas exchange with normal air (internal oxygen tension: 15–45 mmHg) through a 0.2-µm filter for an automatic gas diffusion control system. Its injection port and 0.2-µm air exhaust filter allow introduction of fresh culture medium and removal of spent medium, with or without cell retrieval, in sterile conditions. Many of the cultivation steps can be performed on a laboratory bench without a laminar flow system.

The culture medium–blood mixture was slightly modified from the original methods developed by Haynes et al.3 and Trager and Jensen.4 For each group, it consisted of type A+ erythrocytes at 5% hematocrit suspended in RPMI 1640 medium supplemented with 25 mM HEPES, 25 mM NaHCO3, 1 mg/L hypoxanthine, 2 mM l-glutamine (GE Healthcare Europe, Vélizy-Villacoublay, France), and 0.5% Albumax II®. The initial parasitemia was set at 0.5%.

Group 1 was the control culture performed in 25-cm2 culture flasks containing 5 mL of culture medium–blood mixture closed with a vented cap containing a hydrophobic filter for gas exchange (total volume capacity, 50 mL; Falcon®, Corning, NY) and placed in a CO2 incubator (5% CO2 controlled atmosphere with 95% humidity; INCO 108, Memmert GmbH, Schwabach, Germany). Negative control group 2 was identical to group 1 but was placed in regular room-air microbiological incubator type B15 (Thermo-Scientific, Langenselbold, Germany). Group 3 (experimental group) consisted of a 75-cm2 Petaka G3 nonadherent cell device containing 25 mL of culture medium–blood mixture to fill the internal chamber completely without leaving any air space inside, as recommended by the manufacturer, which was placed in the room-air microbiological incubator, as in group 2. All culture flasks and devices were incubated at 37°C. Cultures were maintained in 12 flasks or Petaka G3 devices for each group, that is, a total of 36 flasks or culture devices, for 30 days. The culture medium was changed on a daily basis in groups 1 and 2 and every 4 days in group 3 using sterile syringes and needles to remove the spent supernatant, except during holidays and weekends. In all groups, fresh uninfected type A+ erythrocytes were added to reduce the parasitemia by half when parasitemia attained 10%. The red cell pellet (2 mL) was divided into two equal fractions. One fraction was cryopreserved, whereas the other fraction was returned to its original flask or device, to which 1 mL of uninfected erythrocytes was added. The dilution factor was taken into account to calculate parasitemia and multiplication rate.

Parasite growth.

Parasitemia was evaluated daily in one of the randomly selected replicates from each group by microscopic examination of Giemsa-stained thin blood films. The number of infected erythrocytes was counted against 2,000 erythrocytes and expressed as the percentage of infected erythrocytes.

For cytometric parasite count, parasite growth was measured weekly, that is, on days 8, 15, 22, and 29. The culture medium–blood mixture (25 µL) was transferred from 36 flasks or culture devices to a 96-well plate and fixed with 200 µL of paraformaldehyde and stained with 20 µL of Hoechst 33342 solution (44 µM) (H3570; Life Technologies Corporation, Eugene, OR) in each well.17 The plate was incubated for 45 minutes at 4°C in the dark, and 50,000 occurrences were analyzed for each sample or measured for 3 minutes when there was a high degree of hemolysis (especially in the negative control group 2), using a CytoFLEX flow cytometer (Beckman Coulter, Villepinte, France).

Statistical analysis.

Weekly parasite growth determined individually from 12 replicates in each group using flow cytometry was expressed as the median percentage of parasite growth, quartiles, and range. The Kruskal–Wallis test was used to compare the median of the 3 groups, followed by a 2 × 2 Mann–Whitney test. The significant level was fixed at P < 0.05. All analyses were conducted using SAS version 9.4 (SAS Institute, Cary, NC).

RESULTS

The daily parasite growth evaluated using microscopy from day 1 (the first day of cultivation is denoted Day 0) is illustrated in Figure 1A and B. Flow cytometry clearly distinguished between red blood cells infected with different stages of P. falciparum and uninfected red blood cells (Figure 2). However, the results obtained with cytometry overestimated the actual parasitemia since all nucleated cells, including multiple trophozoites in a single erythrocyte, or cellular elements, such as merozoites in schizonts, were counted as individual parasites (whereas in microscopy, multiple parasites or merozoites in schizonts in an erythrocyte are counted as a single infected erythrocyte).

Figure 1.
Figure 1.

(A) Weekly in vitro parasitemia under different atmospheric conditions determined by microscopy. Group 1 (white bars), culture flasks in 5% CO2 (positive control); group 2 (gray bars), culture flasks in normal air; group 3 (black bars), Petaka G3 device under the same conditions as for group 2. After day 15, viable asexual parasites were not observed in group 2. *Number of dilutions made before the evaluation of parasitemia. (B) Daily parasite growth of 3D7 clone in three different atmospheric conditions (group 1, white squares; group 2, black circles; and group 3, black triangles). The hatched vertical bars denote days when cultures were diluted but parasitemia was not determined. Culture media were not changed during weekends and national holidays. Growth was optimal until day 15 in all three groups. Both groups 1 and 3 showed suboptimal growth between days 16 and 29 because of inadequate dilution with fresh uninfected erythrocytes. Group 2 failed to grow beyond day 15. * Dilution of parasitemia with fresh uninfected erythrocytes.

Citation: The American Journal of Tropical Medicine and Hygiene 100, 4; 10.4269/ajtmh.18-0527

Figure 2.
Figure 2.

Illustration of the cytometric count of various cell components in the blood–culture medium suspension by CytoFLEX flow cytometer (Beckman Coulter) with CytExpert 2.0 software (Beckman Coulter). Here, the term “erythrocytes” denotes uninfected red blood cells; P2, red blood cells with young trophozoites (rings); P3, cell debris; P4, red blood cells with mature trophozoites and schizonts; and P5, free merozoites.18 Forward scatter area (FSC-A) denotes particle size. Near ultraviolet 450 area (NUV-450-A) corresponds to the Hoescht fluorescence emission. Both X and Y axes are expressed as logarithmic values of fluorescence intensity.

Citation: The American Journal of Tropical Medicine and Hygiene 100, 4; 10.4269/ajtmh.18-0527

Parasites grown in the standard 5% CO2 atmosphere (positive control group 1) showed a regular increase in parasitemia, requiring a regular dilution with fresh uninfected erythrocytes during the first 14 days. By contrast, parasites in the negative control group 2, cultivated in normal air, failed to grow after 14–15 days, as expected. Before day 7, pyknotic forms characteristic of dying parasites were observed in group 2, and at least some of the asexual parasites included in parasite counting between day 7 and day 15 were probably nonviable. The growth curve of parasites cultivated in the Petaka G3 device (group 3) generally followed a similar tendency of parasite growth as that of group 1. The growth curves of groups 1 and 3 indicated parasite multiplication after the addition of fresh uninfected erythrocytes. At the end of the first week (day 8), groups 1 and 3 resulted in comparable percentage of parasite growth (P > 0.05) (Figure 3). Likewise, on day 15 (before dilution with fresh erythrocytes), the growth curves of groups 1 and 3 exhibited similar increasing tendencies. Although parasite growth was not optimal between days 15 and 29 in both groups 1 and 3, probably because of inadequate dilution with fresh uninfected erythrocytes, on day 29, parasites exhibited better growth in group 3 than in group 1 (P < 0.05).

Figure 3.
Figure 3.

Weekly in vitro parasite growth under different atmospheric conditions (group 1, culture flasks in 5% CO2; group 2, culture flasks in normal air; and group 3, Petaka G3 device in normal air) determined by cytometry, with a starting parasitemia of 0.5% on day 0. Dilution factors were included to adjust parasite growth. The box and whisker plots show the median percentage of parasite growth (horizontal bars), their quartiles (box), and range (whiskers). Measurements were obtained from 12 replicates of each group. *One outlier value was observed in group 1. After day 15, asexual parasites with normal morphology and schizonts were not observed in group 2 by microscopic examination of thin smears.

Citation: The American Journal of Tropical Medicine and Hygiene 100, 4; 10.4269/ajtmh.18-0527

DISCUSSION

Several attempts have been made to improve the yield of P. falciparum cultivation based on methods modified from the original technical procedures developed by Haynes et al.3 and Trager and Jensen.4 For this purpose, modified culture vessels have been developed and applied to P. falciparum cultivation, mostly for large-scale production of parasites. In the first attempt, deep stirred cultivation was developed to produce high parasite yields in a single, large vessel (15 L) with semi-automatic medium replacement.19 In this technique, one-third of the deep culture vessel volume is filled with a gas mixture (5% O2, 5% CO2, and 90% N2) for optimal parasite growth. This deep cultivation system proved to be suitable for optimal parasite growth and yield. More recent experiments have used bioreactors. Cultivation of malaria parasites in a hollow-fiber capillary bioreactor has been shown to increase parasite yield and can be performed even at 100% hematocrit.20 The entire system consisting of the bioreactor (cartridge), pump, and bottle with culture medium is kept either inside an airtight box filled with 5% CO2, 5% O2, and 90% N2 in a room-air incubator or in a 5% CO2 incubator.20,21 A wave bioreactor, in which a controlled wave motion is produced within the device to create and enhance hydrodynamic conditions favorable for cell cultivation and gas exchange (1–2 hours of 3% O2, 5% CO2, and 92% N2 gas flow into the Cellbag), has also been used successfully to maintain the same rate of P. falciparum growth as in the static control flask.22 However, cultivation in deep culture vessel or bioreactors still requires humidity and gas exchange to obtain microaerophilic conditions.

The development of asexual intraerythrocytic P. falciparum parasites is characterized by a 48-hour cycle. During the first 24 hours, young trophozoites maintain a low metabolic rate and do not display any major morphological change other than thickening of the cytoplasm. During the second 24-hour period, intense metabolism, accompanied by a large consumption of glucose, increased production of lactic acid, increased cytoplasmic volume occupying most of the available space in the erythrocyte, DNA replication, production of hemozoin, and formation of merozoites, occurs in mature trophozoites and schizonts.23 Oxygen is the terminal electron acceptor of mitochondrial respiration in humans and malaria parasites. Plasmodium falciparum possesses a single mitochondrion with few cristae,24,25 which may partly explain why the parasite requires O2 at a much lower extent, as compared with the human host cells. In vivo, only young trophozoites come in contact with relatively high O2 levels (15–21% O2 in expired and inhaled air, respectively) during their short transit in the pulmonary alveolar capillaries. Most O2 molecules (97%) are reversibly bound to the heme iron of the hemoglobin for transport to tissues and cells in humans. A small proportion of O2 (3%) is dissolved in the plasma. In vivo, mature trophozoites and schizonts obtain most of the required O2 from gas dissolved in the blood in small vessels of deep organs to which they adhere, where the O2 content at tissue and cellular levels ranges from 0.6 to 5.3%.26,27 Mature intraerythrocytic stage parasites may also possibly obtain O2 molecules carried by hemoglobin during hemoglobin digestion. O2 as seen in the microaerophilic environment has been shown to be essential to the parasite’s growth.28,29

The respiratory physiology of the malaria parasites suggests that P. falciparum does not require much gas exchange between the atmosphere and liquid medium during cultivation. The successful cultivation of P. falciparum in the Petaka G3 device suggests that low O2 content available in hemoglobin and dissolved gas is sufficient for long-term cultivation without additional CO2. Further experiments will be required to extend our observation to a panel of reference laboratory strains of P. falciparum and freshly collected isolates. If P. falciparum from various sources can be adapted to this cultivation system, malaria parasites can be collected and cultivated in moderately well-equipped field laboratories with more ease in normal air microbiological incubators and less risk of external contamination. Although the unit price of the Petaka G3 device is about 5–6 times more expensive than classical culture flasks (10.00 versus 1.82 US dollars), it is largely offset by the lower cost of a microbiology incubator (with the ambient air), the cost of which is about one-fifth of the 5% CO2 incubator (including the cost of CO2 gas supply). The lighter weight and greater mechanical simplicity of the microbiology incubator are also more amenable to use in field laboratories.

This study showed that it is possible to use the Petaka G3 device to cultivate P. falciparum under simple working conditions, without high CO2 concentration to create a low O2 atmosphere. Before testing the Petaka G3 device in the field, it is important to further develop the standard operating conditions that are suitable for different P. falciparum strains.

Acknowledgments:

We would like to express our thanks to Dr. Karima Brahimi (Vac4All Company, Paris, France) for kindly providing the 3D7 Plasmodium falciparum clone.

REFERENCES

  • 1.

    Bass CC, 1911. A new conception of immunity. Its application to the cultivation of protozoa and bacteria from the blood and to therapeutic measures. J Am Med Assoc 57: 1534.

    • Search Google Scholar
    • Export Citation
  • 2.

    Bass CC, Johns FM, 1912. The cultivation of malarial plasmodia (Plasmodium vivax and Plasmodium falciparum) in vitro. J Exp Med 16: 567579.

    • Search Google Scholar
    • Export Citation
  • 3.

    Haynes JD, Diggs CL, Hines FA, Desjardins RE, 1976. Culture of human malaria parasites P. falciparum. Nature 263: 767769.

  • 4.

    Trager W, Jensen JB, 1976. Human malaria parasites in continuous culture. Science 193: 673675.

  • 5.

    Grun JL, Weidanz WP, 1987. Cultivation of Plasmodium falciparum in commercially available sera depleted of natural antibodies reactive with human erythrocytes. J Parasitol 73: 384388.

    • Search Google Scholar
    • Export Citation
  • 6.

    Basco LK, 2003. Molecular epidemiology of malaria in Cameroon. XV. Experimental studies on serum substitutes and supplements and alternative culture media for in vitro drug sensitivity assays using fresh isolates of Plasmodium falciparum. Am J Trop Med Hyg 69: 168173.

    • Search Google Scholar
    • Export Citation
  • 7.

    Basco LK, 2006. Molecular epidemiology of malaria in Cameroon. XXIII. Experimental studies on serum substitutes and alternative culture media for in vitro drug sensitivity assays using clinical isolates of Plasmodium falciparum. Am J Trop Med Hyg 75: 777782.

    • Search Google Scholar
    • Export Citation
  • 8.

    Srivastava K, Singh S, Singh P, Puri SK, 2007. In vitro cultivation of Plasmodium falciparum: studies with modified medium supplemented with Albumax II and various animal sera. Exp Parasitol 116: 171174.

    • Search Google Scholar
    • Export Citation
  • 9.

    Zolg JW, Macleod AJ, Dickson IH, Scaife JG, 1982. Plasmodium falciparum: modifications of the in vitro culture conditions improving parasitic yields. J Parasitol 68: 10721080.

    • Search Google Scholar
    • Export Citation
  • 10.

    Ferrer J, Rosal MD, Vidal JM, Prats C, Valls J, Herreros EA, Lopez D, Gargallo D, 2008. Effect of the haematocrit layer geometry on Plasmodium falciparum static thin-layer in vitro cultures. Malar J 7: 203.

    • Search Google Scholar
    • Export Citation
  • 11.

    Allen RJW, Kirk K, 2010. Plasmodium falciparum culture: the benefits of shaking. Mol Biochem Parasitol 169: 6365.

  • 12.

    Scheibel LW, Ashton SH, Trager W, 1979. Plasmodium falciparum: microaerophilic requirements in human red blood cells. Exp Parasitol 47: 410418.

    • Search Google Scholar
    • Export Citation
  • 13.

    Waki S, Miyagami T, Nakazawa S, Igarashi I, Suzuki M, 1987. Maintenance and propagation of human malaria parasites in culture using an ordinary CO2 incubator. Trans R Soc Trop Med Hyg 78: 418.

    • Search Google Scholar
    • Export Citation
  • 14.

    Binh VQ, Luty AJF, Kremsner PG, 1997. Differential effects of human serum and cells on the growth of Plasmodium falciparum adapted to serum-free in vitro culture conditions. Am J Trop Med Hyg 57: 594600.

    • Search Google Scholar
    • Export Citation
  • 15.

    Haruki K, Kobayashi F, Fujino T, Matsui T, Tsuji M, 2001. Evaluation of Anaeropack® (AnP) type as tools of Plasmodium falciparum cultivation and drug sensitivity tests. Jpn J Trop Med Hyg 29: 365370.

    • Search Google Scholar
    • Export Citation
  • 16.

    Pascual A, Basco LK, Baret E, Amalvict R, Travers D, Rogier C, Pradines B, 2011. Use of the atmospheric generators for capnophilic bacteria Genbag-CO2 for the evaluation of in vitro Plasmodium falciparum susceptibility to standard anti-malarial drugs. Malar J 20: 8.

    • Search Google Scholar
    • Export Citation
  • 17.

    Grunwald D, Mayol J-F, Ronot X, 2010. Cycle Cellulaire et Cytométrie en Flux. Cachan, France: Lavoisier, 217219.

  • 18.

    Janse CJ, Haghparast A, Sperança MA, Ramesar J, Kroeze H, del Portillo HA, Waters AP, 2003. Malaria parasites lacking eEF1A have a normal S/M phase yet grow more slowly due to a longer G1 phase. Mol Microbiol 50: 15391551.

    • Search Google Scholar
    • Export Citation
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Author Notes

Address correspondence to Philippe Eldin de Pécoulas, Centre François Baclesse, Normandie Université, UNICAEN, UR ABTE EA 4651, Caen 14000, France. E-mail: philippe.eldin-depecoulas@unicaen.fr

Financial support: This work was supported by a grant from the Royal Institute of Cambodian Traditional Medicine (IRMTC_ABTE-ToxEMAC_ValMyc-2015_Ext. 2017).

Authors’ addresses: Antoine Géry, Ho-Mai-Thy N’Guyen, David Garon, and Philippe Eldin de Pécoulas, Centre François Baclesse, Normandie Université, UNICAEN, UR ABTE EA 4651, Caen, France, E-mails: antoine.gery@gmail.com, maithy2511@gmail.com, estelle.richard@unicaen.fr, david.garon@unicaen.fr, philippe.eldin-depecoulas@unicaen.fr. Leonardo K. Basco, IRD, AP-HM, SSA, VITROME, IHU-Méditerranée Infection, Aix Marseille University, Marseille, France, E-mail: lkbasco@yahoo.fr. Natacha Heutte, Normandie Université, UNIROUEN, CETAPS EA 3832, Mont Saint Aignan Cedex, France, E-mail: natacha.heutte@univ-rouen.fr. Marilyne Guillamin, Normandie Université, UNICAEN, INSERM U 1075 COMETE, Caen, France, and Normandie Université, UNICAEN, Plateau de Cytométrie en Flux, ICORE, Caen, France, E-mail: marilyne.duval@unicaen.fr.

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