Two gametocyte-forming clones, HB-3 and 3D7, were used. Concentrates of late stage parasites were mixed with bloods containing different proportions of young erythrocytes, and the parasitemia and proportion of gametocytes determined after 2, 3 or 4 days of culture. Significantly more gametocytes were formed in light cells than in heavy cells separated from the same normal blood samples. Up to seven times more gametocytes were formed in reticulocyte-rich bloods from patients with sickle cell anemia than in normal control blood.

No abstract

No abstract

We have earlier reported conditions that support the axenic development in vitro of a complete asexual erythrocytic cycle of Plasmodium fakciparum. Up to 30%o of merozoites showed initial differentiation into trophic forms (rings) viable at 14 hr. However, only -1% of the merozoites would develop further into trophozoites and early schizonts viable at 36 hr. In efforts to increase the number of late stage parasites, we have now found a significant favorable effect of the addition of erythrocyte ghosts. Doubling the quantity of erythrocyte membrane in the erythrocyte sonicate medium resulted in approximate doubling of the number of trophozoites and early schizonts. These results indicate that components of the erythrocyte membrane are essential for the complete development of the erythrocytic cycle.We have described a method that supports a complete cycle of extracellular development in vitro of one of the many kinds of obligate intracellular parasitic protozoa, the malaria parasite Plasmodium falciparum (1,2). These results open a way to investigate the detailed relationships between the parasite and its host cell and to understanding of the nature of its dependence on the host cell. The importance of extracellular cultivation of intracellular parasites has been well emphasized by Moulder (3). At present, however, our methods do not provide for continuous extracellular culture. Although up to 30% of erythrocytic merozoites of P. falciparum will differentiate into young rings and be viable at 14 hr into their 48-hr cycle of schizogonic development, only -1% develop into trophozoites and early schizonts viable at 36 hr (1). Most of these go on to complete schizogony with formation of infective merozoites. These merozoites can again develop extracellularly (unpublished results) but their numbers are insufficient for continuous maintenance.Previous work (4) had shown that initial development was better in sonicated human erythrocyte extract, containing disrupted erythrocyte membranes, than in frozen-thawed extract from which the stroma had been removed. It therefore seemed reasonable to attempt to improve the medium by the provision of additional erythrocyte membrane. We present here results showing that constituents of the erythrocyte membrane favor the development of rings into trophozoites and schizonts.MATERIALS AND METHODS Parasites. Stock cultures of P. falciparum were maintained in flow vessels (5). We used two clones, HB-3 (6) of Honduras I/CDC isolate and A-2 of FCR-3/Gambia (7). Cultures were synchronized and purified to produce a 2-5% (vol/vol) schizont suspension as described (2). This suspension was incubated at 37°C for 1.25 hr and agitated at 10-min intervals. Incubated suspension was divided in two equal volumes and then centrifuged at 200 X g for 10 min to sediment schizonts. The supernatant containing merozoites was then centrifuged for 10 min at 1500 x g. The supernatant was discarded and the pellets containing merozoites were resuspended in a minimal amount of control or membrane-suppleme...

Continuous culture of the erythrocytic cycle of Plasmodium falciparum within human erythrocytes maintained under appropriate conditions in vitro has facilitated a large body of fundamental investigations with this important pathogen. It has revealed complex relationships between the developing intracellular parasite and its host erythrocyte. In pursuit of further understanding of these relationships, we have now developed,.and here describe, methods that support extracellular development of the erythrocytic stages of P falciparum.Since only 1 to 2% of an inoculum of free merozoites can develop extracellularly through the entire schizogonic cycle to again form merozoites, the method does not support continuous extracellular culture. The work shows that the parasitophorous membrane and vacuole are not essential. Development of the parasites beyond the ring stage does however require constituents of the erythrocyte membrane.Abbreviations: CPD = citrate-phosphate dextrose; KIS = KI medium with 10% human serum; RP-C = RP medium without bicarbonate or serum; RPS = RP medium with bicarbonate and serum