Malaria pp 3-15 | Cite as

In Vitro Culturing Plasmodium falciparum Erythrocytic Stages

  • Alexander G. MaierEmail author
  • Melanie Rug
Part of the Methods in Molecular Biology book series (MIMB, volume 923)


The in vitro cultivation of Plasmodium falciparum is absolutely essential for the molecular dissection of parasite biology and still poses several challenges. The dependence on, and interaction with host red blood cells, the tightly regulated stage-specific expression of proteins, and the parasite peculiar demands on nutrients and gaseous environments are only a few aspects that need to be addressed to successfully cultivate P. falciparum in vitro. In this chapter, we present techniques for normal maintenance of the erythrocytic stages of P. falciparum cultures, their synchronization and the generation of clonal cell lines.

Key words

Malaria Plasmodium falciparum In vitro culture Erythrocyte Synchronization 



We would like to acknowledge the efforts of generations of researchers establishing and fine-tuning the various techniques. It is the nature of chapters like this that it is biased towards our own experiences and cannot be comprehensive. Our work is supported by the Australian Research Council (ARC), the National Health and Medical Research Council of Australia and the Hugh D T Williamson Foundation (managed by ANZ Trustees). We thank the Australian Red Cross Blood Service for the provision of human blood and serum. AGM is an ARC Australian Research Fellow.


  1. 1.
    Cowman AF, Crabb BS (2006) Invasion of red blood cells by malaria parasites. Cell 124:755–766PubMedCrossRefGoogle Scholar
  2. 2.
    Maier AG et al (2009) Malaria parasite proteins that remodel the host erythrocyte. Nat Rev Microbiol 7:341–354PubMedCrossRefGoogle Scholar
  3. 3.
    Trager W, Jensen JB (1976) Human malaria parasites in continuous culture. Science 193:673–675PubMedCrossRefGoogle Scholar
  4. 4.
    Fang J et al (2004) The effects of glucose concentration on the reciprocal regulation of rRNA promoters in Plasmodium falciparum. J Biol Chem 279:720–725PubMedCrossRefGoogle Scholar
  5. 5.
    LeRoux M et al (2009) Plasmodium falciparum biology: analysis of in vitro versus in vivo growth conditions. Trends Parasitol 25:474–481PubMedCrossRefGoogle Scholar
  6. 6.
    Scheibel LW et al (1979) Plasmodium falciparum: microaerophilic requirements in human red blood cells. Exp Parasitol 47:410–418PubMedCrossRefGoogle Scholar
  7. 7.
    Butcher GA (1981) A comparison of static thin layer and suspension cultures for the maintenance in vitro of Plasmodium falciparum. Ann Trop Med Parasitol 75:7–17PubMedGoogle Scholar
  8. 8.
    Moloney MB et al (1990) Plasmodium falciparum growth in deep culture. Trans R Soc Trop Med Hyg 84:516–518PubMedCrossRefGoogle Scholar
  9. 9.
    Puthia MK, Tan KS (2005) Plasmodium falciparum: a simplified technique for obtaining singly infected erythrocytes. Parasitol Res 95:176–178PubMedCrossRefGoogle Scholar
  10. 10.
    Allen RJ, Kirk K (2010) Plasmodium falciparum culture: the benefits of shaking. Mol Biochem Parasitol 169:63–65PubMedCrossRefGoogle Scholar
  11. 11.
    Trager W (1971) A new method for intraerythrocytic cultivation of malaria parasites (Plasmodium coatneyi and P. falciparum). J Protozool 18:239–242PubMedGoogle Scholar
  12. 12.
    Ponnudurai T et al (1983) An automated large-scale culture system of Plasmodium falciparum using tangential flow filtration for medium change. Parasitology 87(3):439–445PubMedCrossRefGoogle Scholar
  13. 13.
    Li T et al (2003) A new method for culturing Plasmodium falciparum shows replication at the highest erythrocyte densities. J Infect Dis 187:159–162PubMedCrossRefGoogle Scholar
  14. 14.
    O’donnell AJ et al (2011) Fitness costs of disrupting circadian rhythms in malaria parasites. ProcBiol Sci 278:2429–2436CrossRefGoogle Scholar
  15. 15.
    Kwiatkowski D, Nowak M (1991) Periodic and chaotic host-parasite interactions in human malaria. Proc Natl Acad Sci USA 88:5111–5113PubMedCrossRefGoogle Scholar
  16. 16.
    Reece SE et al (2009) Plastic parasites: sophisticated strategies for survival and reproduction? Evol Appl 2:11–23PubMedCrossRefGoogle Scholar
  17. 17.
    Lambros C, Vanderberg JP (1979) Synchronization of Plasmodium falciparum erythrocytic stages in culture. J Parasitol 65:418–420PubMedCrossRefGoogle Scholar
  18. 18.
    Ginsburg H et al (1989) Alkalinization of the food vacuole of malaria parasites by quinoline drugs and alkylamines is not correlated with their antimalarial activity. Biochem Pharmacol 38:2645–2654PubMedCrossRefGoogle Scholar
  19. 19.
    Haynes JD, Moch JK (2002) Automated synchronization of Plasmodium falciparum parasites by culture in a temperature-cycling incubator. Methods Mol Med 72:489–497PubMedGoogle Scholar
  20. 20.
    Blair PL et al (2002) Transcripts of developmentally regulated Plasmodium falciparum genes quantified by real-time RT-PCR. Nucleic Acids Res 30:2224–2231PubMedCrossRefGoogle Scholar
  21. 21.
    Paul F et al (1981) Separation of malaria-infected erythrocytes from whole blood: use of a selective high-gradient magnetic separation technique. Lancet 2:70–71PubMedCrossRefGoogle Scholar
  22. 22.
    Dluzewski AR et al (1984) A simple method for isolating viable mature parasites of Plasmodium falciparum from cultures. Trans R Soc Trop Med Hyg 78:622–624PubMedCrossRefGoogle Scholar
  23. 23.
    Jensen JB (1978) Concentration from continuous culture of erythrocytes infected with trophozoites and schizonts of Plasmodium falciparum. Am J Trop Med Hyg 27:1274–1276PubMedGoogle Scholar
  24. 24.
    Pasvol G et al (1978) Separation of viable schizont-infected red cells of Plasmodium falciparum from human blood. Ann Trop Med Parasitol 72:87–88PubMedGoogle Scholar
  25. 25.
    Goodyer ID et al (1994) Purification of mature-stage Plasmodium falciparum by gelatine flotation. Ann Trop Med Parasitol 88:209–211PubMedGoogle Scholar
  26. 26.
    Boyle MJ et al (2010) Isolation of viable Plasmodium falciparum merozoites to define erythrocyte invasion events and advance vaccine and drug development. Proc Natl Acad Sci USA 107:14378–14383PubMedCrossRefGoogle Scholar
  27. 27.
    Ranford-Cartwright LC et al (2010) New synchronization method for Plasmodium falciparum. Malar J 9:170PubMedCrossRefGoogle Scholar
  28. 28.
    Hotta CT et al (2000) Calcium-dependent modulation by melatonin of the circadian rhythm in malarial parasites. Nat Cell Biol 2:466–468PubMedCrossRefGoogle Scholar
  29. 29.
    Beraldo FH, Garcia CR (2005) Products of tryptophan catabolism induce Ca2+ release and modulate the cell cycle of Plasmodium falciparum malaria parasites. J Pineal Res 39:224–230PubMedCrossRefGoogle Scholar
  30. 30.
    Divo AA, Jensen JB (1982) Studies on serum requirements for the cultivation of Plasmodium falciparum. 2. Medium enrichment. Bull World Health Organ 60:571–575PubMedGoogle Scholar
  31. 31.
    Divo AA et al (1985) Isolation and cultivation of Plasmodium falciparum using adult bovine serum. J Parasitol 71:504–509PubMedCrossRefGoogle Scholar
  32. 32.
    Geary TG et al (1985) Nutritional requirements of Plasmodium falciparum in culture. III. Further observations on essential nutrients and antimetabolites. J Protozool 32:608–613PubMedGoogle Scholar
  33. 33.
    Trager W, Jensen JB (1997) Continuous culture of Plasmodium falciparum: its impact on malaria research. Int J Parasitol 27:989–1006PubMedCrossRefGoogle Scholar
  34. 34.
    Martin RE, Kirk K (2007) Transport of the essential nutrient isoleucine in human erythrocytes infected with the malaria parasite Plasmodium falciparum. Blood 109:2217–2224PubMedCrossRefGoogle Scholar
  35. 35.
    Roth EF Jr (1987) Malarial parasite hexokinase and hexokinase-dependent glutathione reduction in the Plasmodium falciparum-infected human erythrocyte. J Biol Chem 262:15678–15682PubMedGoogle Scholar
  36. 36.
    Zolg JW et al (1984) The accumulation of lactic acid and its influence on the growth of Plasmodium falciparum in synchronized cultures. In Vitro 20:205–215PubMedCrossRefGoogle Scholar
  37. 37.
    Lian LY et al (2009) Glycerol: an unexpected major metabolite of energy metabolism by the human malaria parasite. Malar J 8:38PubMedCrossRefGoogle Scholar
  38. 38.
    Olszewski KL et al (2010) Branched tricarboxylic acid metabolism in Plasmodium falciparum. Nature 466:774–778PubMedCrossRefGoogle Scholar
  39. 39.
    Grellier P et al (1991) Lipid traffic between high density lipoproteins and Plasmodium falciparum-infected red blood cells. J Cell Biol 112:267–277PubMedCrossRefGoogle Scholar
  40. 40.
    Mi-Ichi F et al (2006) Intraerythrocytic Plasmodium falciparum utilize a broad range of serum-derived fatty acids with limited modification for their growth. Parasitology 133:399–410PubMedCrossRefGoogle Scholar
  41. 41.
    Guillaume C et al (2004) Anti-Plasmodium properties of group IA, IB, IIA and III secreted phospholipases A2 are serum-dependent. Toxicon 43:311–318PubMedCrossRefGoogle Scholar
  42. 42.
    Frankland S et al (2006) Delivery of the malaria virulence protein PfEMP1 to the erythrocyte surface requires cholesterol-rich domains. Eukaryot Cell 5:849–860PubMedCrossRefGoogle Scholar
  43. 43.
    Frankland S et al (2007) Serum lipoproteins promote efficient presentation of the malaria virulence protein PfEMP1 at the erythrocyte surface. Eukaryot Cell 6:1584–1594PubMedCrossRefGoogle Scholar
  44. 44.
    Cranmer SL et al (1997) An alternative to serum for cultivation of Plasmodium falciparum in vitro. Trans R Soc Trop Med Hyg 91:363–365PubMedCrossRefGoogle Scholar
  45. 45.
    Gerold P et al (1996) Structural analysis of the glycosyl-phosphatidylinositol membrane anchor of the merozoite surface proteins-1 and -2 of Plasmodium falciparum. Mol Biochem Parasitol 75:131–143PubMedCrossRefGoogle Scholar
  46. 46.
    Singh K et al (2007) Growth, drug susceptibility, and gene expression profiling of Plasmodium falciparum cultured in medium supplemented with human serum. J Biomol Screen 12:1109–1114PubMedCrossRefGoogle Scholar
  47. 47.
    Ferrer J et al (2008) Effect of the haematocrit layer geometry on Plasmodium falciparum static thin-layer in vitro cultures. Malar J 7:203PubMedCrossRefGoogle Scholar
  48. 48.
    Weatherall DJ, Clegg JB (2001) Inherited haemoglobin disorders: an increasing global health problem. Bull World Health Organ 79:704–712PubMedGoogle Scholar
  49. 49.
    Zolg JW et al (1982) Plasmodium falciparum: modifications of the in vitro culture conditions improving parasitic yields. J Parasitol 68:1072–1080PubMedCrossRefGoogle Scholar
  50. 50.
    Mutai BK, Waitumbi JN (2010) Apoptosis stalks Plasmodium falciparum maintained in continuous culture condition. Malar J 9(Suppl 3):S6PubMedCrossRefGoogle Scholar
  51. 51.
    Biggs BA et al (1989) Subtelomeric chromosome deletions in field isolates of Plasmodium falciparum and their relationship to loss of cytoadherence in vitro. Proc Natl Acad Sci USA 86:2428–2432PubMedCrossRefGoogle Scholar
  52. 52.
    Bourke PF et al (1996) Disruption of a novel open reading frame of Plasmodium falciparum chromosome 9 by subtelomeric and internal deletions can lead to loss or maintenance of cytoadherence. Mol Biochem Parasitol 82:25–36PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Research School of BiologyThe Australian National UniversityCanberraAustralia

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