Yury M. Colton and Dr. James B. Jensen, Microbiology
Continuous cultivation of the erythrocytic stages of Plasmodiumfalciparum has greatly increased the possibilities of malaria research (Trager and Jensen 1976). However, the need for human serum in the culture medium has proved to be limiting in some situations and the necessary serum is becoming very difficult and costly to acquire. This paper describes the development of culture media in which the serum requirement is reduced by ten-fold and Plasmodiumfalciparum can be maintained indefinitely using standard culture techniques.
Cultures of Plasmodiumfalciparum were maintained using the methods of Trager and Jensen(1976). The cultures were kept 50 ml culture plates using human erythrocytes that were infected with P. falcipariim. The erythrocytes were obtained from a volunteer and stored at 4 C for subsequent use. The plates were incubated in gas chambers under 5% oxygen, 5% carbon dioxide, and 90% nitrogen. The medium was changed daily and fresh uninfected erythrocytes were added every 4 days. For observation and parasite count thin blood smears were made and stained with Giemsa stain and examined microscopically. The stock medium consisted of 16. 2 g RPMI 1640 powdered medium (GIBCO) with 25 mM Hepes buffer and L-glutamine. This powder was dissolved in 1 liter of double distilled water and filter sterilized with 0.2 nm Nalgene filters and divided into 100 ml aliquots stored at 4 C. At the time of use the pH of a 100 ml aliquot was adjusted by adding 4.5 ml of 7.5% sodium bicarbonate solution and to limit contamination 2 ml penicillin and streptomycin solution (Sigma) were also added. Erythrocytes were washed twice in plain RPMI 1640 medium. After washing, 5% erythrocyte (v/v) suspensions were prepared in the culture plates using the appropriate RPMI 1640 medium formulated for the experiment.
The control cultures were made from the stock RPMI 1640 medium and supplemented with human serum to a 10% (v/v) serum concentration. For these specific experiments the human serum was replaced by Cosmic Calf serum (Hyclone) which had been heat inactivated and divided into 10 ml aliquots and frozen down for storage until needed. The calf serum supports cultures nearly as well as human serum and is easier to obtain in the needed quantities.
The experimental cultures were based on previous attempts in serum-free culture medium (Ofulla et al. 1993). The following were added to 100 ml of RPMI 1640 stock medium: 0. 2g glucose, 0.006 g hypoxanthine, 0.5g Cohn fraction V bovine albumin, and 1 ml lipidscholesterolrich mixture. After completely dissolving these reagents in the RPMI 1640 the medium was filter sterilized and then Cosmic Calf serum was added to create a 1% (v/v) serum supplemented defined medium. The negative control was the same as above but without the calf serum supplement.
The continuous cultivation of parasites was achieved using the 1% serum supplemented defined medium. The results were repeated using two different strains of P. falciparum with only slightly lower parasite numbers than the control cultures after 8 weeks of growth. The defined medium without the serum supplement showed continued growth of the parasites for 6 days and then the parasites died off. Serum supplements of 0.5% and 2.0% were also explored but the 0.5% serum medium would not support the cultures for more than 7 days and the 2.0% serum medium did not support the culture any better than the 1.0% serum medium.
The malaria parasite is very sensitive to the medium used for in vitro cultivation. Many methods have been proposed for serum-free cultivation using reagents to substitute the serum requirements for parasite growth. From my experiments it seems that there is a nutrient necessary only in very small concentrations and this micro-nutrient is found in the components of blood serum. Further exploration will yield the exact nutrient requirements for the growth of the parasite.
References
- Ayub V. O. Ofulla et al. 1993. Cultivation of Plasniodiunifalciparum parasites in a serum-free medium. Am. J Trop. Med Hyg. 49(3), 1
335-340. - Trager, W., and Jensen, J. B. 1976. Human malaria parasites in continuous culture. Science 193, 673-675.