Isolation of Plasmodium berghei ookinetes in culture using Nycodenz density gradient columns and magnetic isolation
© Carter et al; licensee BioMed Central Ltd. 2003
Received: 09 September 2003
Accepted: 03 November 2003
Published: 03 November 2003
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.
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.
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.
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.
Studies on ookinete stages of the malaria parasite were greatly aided by the development, over 30 years ago, of a method for the production of ookinetes in vitro following 24 h culture of gametocytaemic blood. The basic protocol for isolation of gametocytes from infected blood and subsequent ookinete development was described by Weiss & Vanderberg  and has since been modified and improved . Experimenters have different opinions concerning the need to remove white blood cells prior to culture. This is advisable as we have shown a significant decrease in the number of ookinetes undergoing apoptosis during development, if this is performed . Following culture for 20–24 h, the usual method for ookinete separation and enrichment is on density gradients. Silica gradients (Percoll) have been used since the 1980's for gametocyte concentration (e.g. [4, 5]); however, Nycodenz cushions have largely replaced Percoll due to its ease of use and reduced toxicity to Plasmodium . Differing cushion concentrations allow enrichment of either zygotes or ookinetes . Inhibitors of DNA synthesis (such as mitomycin C, aphidicolin and Berenil [8, 9]) have been successfully employed as tools to further enrich ookinete preparations by eliminating asexual stages from gametocytaemic blood. This objective has also been achieved using pyrimethamine . In addition, magnetized beads coated in an antibody raised against a major ookinete surface protein, P28, have been used to isolate ookinetes from erythrocytes .
Ookinete enrichment is labour intensive and time consuming and is likely to dramatically reduce ookinete yield. Furthermore, complete separation to the exclusion of all other blood cells is difficult. Here we describe a novel method for purification based on the magnetic properties of the parasite itself and compare this with enrichment on a Nycodenz cushion.
Infections and blood processing
CD mice were treated with phenylhydrazine two days prior to infection by intra peritoneal inoculation with P. berghei ANKA strain, obtained from a donor mouse between the second and sixth passage from cryopreserved stock . Parasitaemia was checked on day three post-infection by microscopic examination of thin blood films and the presence of exflagellating gametocytes was determined in thick blood films. Gametocytaemic blood was collected into a heparinized syringe via cardiac puncture and retained on ice prior to removal of white blood cells. This was achieved by passing the blood through a pre-equilibrated 5 ml sterile column containing 1 ml glass wool and 3 ml of Whatmann CF11 cellulose powder (Beckton & Dickenson). The blood was washed through the column with 10 mls of modified RPMI 1640 medium (9 ml RPMI [per litre: 16.4 g RPMI medium (Sigma); 2 g sodium bicarbonate; 10 ml penicillin (1000 U/ml) / streptomycin (1 mg/ml); 50 mg hypoxanthine] with 1 ml heat inactivated foetal bovine serum (Invitrogen)). The blood was collected into T25 culture flasks, leaving a final culture volume of approximately 11 ml. The majority of ookinetes had developed during 20 hours incubation at 19°C. Cultures from different mice were pooled, ookinetes counted and the culture divided equally between methods of purification.
Ookinete isolation methods
Two methods were employed for comparison of yield and efficiency of ookinete isolation from uninfected erythrocytes, namely: suspension on a Nycodenz cushion and a novel technique using a magnet to retain ookinetes.
Enrichment on a Nycodenz cushion was performed as previously described . Briefly, following incubation erythrocytes were lysed with 0.34 M NH4Cl and the suspension was then layered onto a cushion of 17% Nycodenz at 20°C and spun for 30 min at 1660 g. Ookinetes were removed from the interface, washed and counted.
To compare the viability of ookinetes isolated using the different methods, parasites were maintained in vitro as described previously . Ookinetes were seeded into 8-well slides at a density of 1 × 104 and the number of oocyst per well were counted after nine days of culture in oocyst medium in the presence of Drosophila S2 cells at 1 × 105 per well.
In an attempt to increase the yield of ookinetes from magnet isolation, the speed of flow of blood through the column was reduced by decreasing the gauge of needle used. Assessments were carried out with needle sizes of 21G (0.8 × 40 mm) (Terumo, Europe), 23G (0.6 × 25 mm) and 25G (0.5 × 16 mm) (Sherwood, UK). Yield from the magnetized columns was also compared using the syringe plunger supplied with the column, or allowing the elution to occur under gravity.
Assessment of methods
Several criteria were used to compare ookinete enrichment methods: 1) yield was calculated as the percentage of parasites recovered from each method of purification compared to the initial density of parasites, 2) observations were made of the degree of contamination with erythrocyte material after re-suspension of equal volumes of purified parasites, 3) potential to transform into oocysts and develop in culture was also compared, 4) assessments of preparation time and costs of each method were made. Finally, percentage yield when using needles of different gauge was calculated. Statistical analysis of the comparison of yield with varying needle size was performed by one-way analysis of variance (ANOVA). All other analyses were performed using the Student T test where a P value <0.05 was considered significant.
Results and Discussion
Comparison of ookinete yield using different separation techniques
Comparison of ookinete yield from two purification methods. For each purification method (Nycodenz cushion v magnet), the initial density of ookinetes is expressed per ml of blood. Final yield is expressed as a percentage ± SEM of the pre-separation density, n = 4 experiments, each using the pooled blood from two infected mice. No significant difference was observed in yield between the two methods (Student T-test, p > 0.05).
Nycodenz density gradient
3.75 ± 0.48 × 107/ml
3.8 ± 0.56 × 105/ml
4.1 ± 0.48 × 105/ml
1.04 ± 0.17 %
1.11 ± 0.12 %
Comparison of ookinete purity and viability using different separation techniques
Oocyst development following different separation methods. Ookinetes from Nycodenz cushion and magnet separations were cultured for nine days. The total number of oocysts per well was then counted for three wells. The experiment was replicated four times, using blood from different mice. No significant difference in oocyst numbers was observed between or within experiments (Student's T test, p = >0.05).
Magnet Mean no. oocysts per well ± SEM
Nycodenz Mean no. oocysts per well ± SEM
788 ± 67.7
936 ± 52.6
872 ± 56.7
810 ± 64.7
773 ± 14.0
793 ± 25.1
843 ± 34.1
755 ± 54.2
Comparison of ookinete yield with varying needle size
Ookinete yield from the magnetised column using different needle sizes. Ookinetes were separated from the remaining blood culture using the magnetized column with varying needle sizes to reduce the speed of blood flow. Ookinete yield is expressed as a percentage of the pre-separation density, calculated from pooled blood from four mice, n = 4 experiments. All three needles were used in conjunction with the accompanying column plunger, and the most effective needle in terms of yield and lack of contaminants, 23G, was also assessed without using the plunger. * = mean values with different letters are significantly different (ANOVA, p < 0.05)
% yield with 21G needle
% yield with 23G needle
% yield with 23G needle, no plunger
% yield with 25G needle
Mean ± SEM
4.07 ± 0.51
7.75 ± 0.57
7.03 ± 0.76
14.83 ± 2.46
Comparison of time/cost for ookinete separation methods
One of the major advantages of the magnet separation is the speed with which ookinetes can be purified ready for further culture to oocysts. From the initial removal of the 20–24 h parasite culture from the flask, purification and counting can be completed in less than 45 min whereas this process takes 2 h 30 min if red blood cells are lysed and separated on a Nycodenz cushion.
Each magnet separation is, however, more costly than purification on Nycodenz cushions. Current costs in the UK amount to approximately £14 for purification of ookinetes obtained from two infected mice (approximately 7.5 × 105 ookinetes) compared with just over £2 for the Nycodenz cushion (Nycodenz, £1.60, ammonium chloride £0.35 and PBS £0.06). The MidiMACs column and pre-separation filter account for most of this cost (£10 and £50 respectively).
The main advantage of magnetic separation for routine isolation of ookinetes is its speed and simplicity. Apart from the magnet, no additional equipment is required and no skill, other than observance of routine aseptic technique, is necessary. Thus this method provides an alternative approach that other laboratories may wish to adopt. This technique has recently been used successfully to purify P. falciparum ookinetes grown in culture (JW unpublished observations).
We are grateful for financial support from the U.S. Army Medical Research and Material Command (JW, HC), the Wellcome Trust (VC) and the generous support of the Naval Medical Research Centre.
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