Isolation of Plasmodium berghei ookinetes in culture using Nycodenz density gradient columns and magnetic isolation

Abstract

BACKGROUND: Large scale in vitro production of the mosquito stages of malaria parasites remains elusive, with only limited success for complete sporogonic development and only one report of development through to infective sporozoites. The initial step in this process is the production, in vitro, of ookinetes from gametocytaemic blood. Methods for isolation of these ookinetes from blood cells have been described; however, in addition to yield often being low, processing time and potential for contamination by erythrocytes remain high. METHODS: This study compares two procedures for retaining mature ookinetes from blood stage cultures, whilst removing red blood cells and other contaminants prior to further culture of the parasite. The well established method of isolation on Nycodenz cushions is compared with a novel method utilizing the innate magnetic properties of the haem pigment crystals found in the cytoplasm of ookinetes. RESULTS: Yield and viability of ookinetes were similar with both isolation methods. However, in our hands magnetic isolation produced a cleaner ookinete preparation much more quickly. Moreover, decreasing the flow rate through the magnetic column could further enhance the yield. CONCLUSION: We recommend the enrichment of an ookinete preparation prior to further culture being performed using the magnetic properties of Plasmodium berghei ookinetes as an alternative to their density. The former technique is faster, removes more erythrocytes, but day-to-day costs are greater.

Figure 1

Equipment for isolation using the magnetic properties of ookinetes. Gametocytaemic mouse blood that has been cultured for 20 h is added to the LS column via a pre-separation filter. The speed of flow through the column is reduced by adding a needle to the base, allowing time for ookinetes to be captured in the magnetized column. Parasites are eluted with oocyst culture medium when the column is released from the magnet.

Figure 2

Comparison of the degree of red blood cell contamination using two different purification methods. Enriched ookinete preparations were resuspended in an equal volume of oocyst medium and the degree of contamination by red blood cells and debris was assessed using an equal volume of parasite material. Preparations were viewed at a magnification of × 400. Ookinetes are indicated by an arrow. Scale bar = 10 μm. Note heavy contamination with other parasite stages/cellular debris following purification on a Nycodenz cushion.