• 疟原虫的培养方法

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• Subcultivate the parasites when necessary (see comments below).


Comments
• The Albumax II and hypoxanthine we use cannot be considered sterile. We thus make a stock solution of 100 g of Albumax II and 400 mg of hypoxanthine in 2 liters of RPMI 1640 and filter the solution through 0.8-祄 and 0.2-祄 filters. We then use 50 mL of this stock solution per 500-mL bottle of culture medium.
• It is important to keep all media preheated to 37 癈 and to minimize handling time outside the 37 癈 incubator. (No coffee breaks while handling cultures!)
• When gassing the culture flasks, fit the gas hose with a 0.2-祄 filter unit, and use a sterile needle (preferably blunt to avoid accidents) (see the figure).
• If initial patient parasitemia is high (>0.4%), change the medium the next day, otherwise leave it for 48 to 72 h before the first change of culture medium.
• Subcultivate patient isolates to keep parasitemia below 1 to 2% by adding fresh uninfected RBC. Although many laboratory-adapted parasite lines tolerate 5% parasitemia or more, this is not the case for most patient isolates.
• Do not leave parasites in freezing or thawing solution longer than absolutely necessary, as these solutions are harmful to the parasites.
• Washed uninfected RBC for subculture should be kept in the refrigerator for 24 h before first usage (to discourage any remaining leukocytes), and can be kept in the refrigerator for up to 14 days.


References
Cranmer SL Magowan C, Liang J, Coppel RL, Cooke BM. 1997. An alternative to serum for cultivation of Plasmodium falciparum in vitro. Trans R Soc Trop Med Hyg 91(3):363-365.

Jensen JB, Trager W. 1977. Plasmodium falciparum in culture: use of outdated erthrocytes and description of the candle jar method. J Parasitol 63(5):883-886.

Nielsen MA, Staalsoe T, Kurtzhals JA, Goka BQ, Dodoo D, Alifrangis M, Theander TG, Akanmori BD, Hviid L. 2002. Plasmodium falciparum variant surface antigen expression varies between isolates causing severe and non-severe malaria and is modified by acquired immunity. J Immunol 168(7):3444-3450.

Trager W, Jensen JB. 1976. Human malaria parasites in continuous culture. Science 193(4254):673-675.

I:C. Short-term cultivation of Plasmodium falciparum isolates from patient blood
by Malin Haeggström and Martha Schlichtherle
Microbiology and Tumor Biology Center (MTC), Karolinska Institutet and Swedish Institute for Infectious Disease Control, Box 280, SE-171 77 Stockholm, Sweden
e-mail: malin.haeggstrom@smi.ki.se

We give here a protocol for growing P. falciparum from patient blood. The vast majority of cultures will survive only the first cycle (i.e., from the ring stage to trophozoites/schizonts) and will not be able to reinvade new RBCs. In our hands, about 90% of patient isolates grew for at least one cycle. For establishment of long-term cultivation, see PARASITES section I:B.

Equipment
37 °C incubator
centrifuge for 500-3000 g
candle-jar or gas (92% N2, 5% CO2, 3% O2)

Materials and reagents
venous blood from malaria patient
heparinized or EDTA tubes
Polymorphprep (Axis-Shield)
RPMI 1640 (optimal HEPES-buffered)
O+ RBCs (or autologous blood group)
MCM with AB+ serum
culture flasks or plates

Procedure
• Collect 2 to 5 mL of venous blood into heparinized/EDTA tubes and keep at 4 °C. Process the sample within 1 hour.
• Carefully layer the blood over 5 mL of Polymorphprep and centrifuge at 500 x g for 15 min.
• Collect the cell layer of interest. Erythrocytes are in the bottom of the tube.
• Add 10 mL of sterile RPMI 1640 (37 °C) to the cells, resuspend, and centrifuge at 500 x g for 5 min.
• Aspirate the supernatant and repeat the wash twice.
• Estimate the pellet volume and mix with equal amount RPMI 1640.


If you wish to keep the monocytes, transfer the cell layer containing monocytes into a sterile 15-mL tube (see the manufacturer’s protocol for Polymorphprep). Wash once with RPMI 1640 by centrifugation at 3,000 to 5,000 x g for 2 min. Remove the supernatant and add 1 mL of 10% DMSO in fetal calf serum. Freeze the cells immediately at –70 °C and then transfer to liquid nitrogen.

Reference:
Haeggström M, Kironde F, Berzins K, Chen Q, Wahlgren M, Fernandez V. 2004. Common trafficking pathway for variant antigens destined for the surface of the Plasmodium falciparum-infected erythrocyte. Mol Biochem Parasitol 133(1):1-14.

I. Growing Plasmodium falciparum cultures at high parasitemia
by Malin Haeggström and Martha Schlichtherle
Microbiology and Tumor Biology Center (MTC), Karolinska Institutet and Swedish Institut
for Infectious Disease Control, Box 280, SE-171 77 Stockholm, Sweden
e-mail: malin.haeggstrom@mtc.ki.se

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