Serum- free culture media for in vitro studies of plasmodium falciparum.
Offulla, Ayub V. O.
MetadataShow full item record
Plasmodium falciparum has routinely been cultured in in vitro in growth media supplemented with compatible human serum. However, serum is undefined and could introduce unknown variables to experiments. Procurement of adequate amounts of suitable serum for research and fieldwork is costly and erratic and serum needs to be stored frozen until just before use. Serum - free bovine albumin culture media (BAM) containing RPMI 1640, Hepes buffer, sodium bicarbonate and bovine albumin had previously been tried for in vitro growth of P. falciparum but growth rates were significantly lower than those observed in serum supplemented medium (SSM). In this subject, studies were designed to improve P. falciparum growth rates in BAM. Also, suitable serum free media for a) short term storage/ transport of P. falciparum parasites and b) determination of P. falciparum antimalarial drug sensitivity was formulated and tested. A culture medium (BAM-GLH) containing 5g/l bovine albumin (Cohn fraction V), 5.96g/l Hepes buffer, 2.65g/l sodium bicarbonate, 2g/l additional glucose, 50-60mg/l hypoxanthine, and 10ml/l lipids-cholesterol rich mixture was found to significantly improve in vitro growth of P. falciparum. BAM-GLH could be used for continuous cultivation of P. falciparum without compromising parasite growth rates. There was no statistically significant difference in continuous growth of two P. falciparum isolates in serum free BAM-GLH compared to the routine serum-supplemented medium (SSM) by paired Students t-test (p0.1, n=10). The BAM-GLH without hypoxanthine (BAM-GL) was useful in the measurement of P. falciparum chemosensitivity to various antimalarial drugs using the tritiated hypoxanthine uptake method. Antimalarial drug sensitivity data (IC 50 s) could also be obtained using BAM without the additional glucose and the lipids-cholesterol rich supplement. There was a significant correlation between IC50 values obtained with the serum-free media (BAM-GL or BAM) and the serum supplemented medium (r=0.6, n=60, p<0.01; linear regression analysis). Suitable conditions for short-term storage of P. falciparum in BAM (compared with SSM) for 0-10 days without liquid nitrogen cryopreservation was also determined. Laboratory adapted and freshly isolated P. falciparum stored/transported at 4oC (or on wet ice) for up to six days could easily adapt and continuously grow in culture. They could also be used to directly set up drug sensitivity assays using the 3H-hypoxanthine uptake technique or the more rapid, newly developed parasite lactate dehydrogenase (pLDH) enzyme assay.