| 疟原虫的培养方法 | | 点击： 作者： 来源：dxy 时间： 2007-12-20 本站论坛 |
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• Replace the MCM every day (not necessary the day after subcultivation).
• Subculture the cultures 2 times/week.
Subcultivation
• Stain a drop of the culture with acridine orange (10 µg /mL) on a glass slide and put on a coverslip or by Giemsa staining of a thin smear (see PARASITES, section III:A or
• Count the parasitemia (i.e., the percentage of infected cells, see PARASITES, section III:C).
• Prepare freshly washed O+ blood in cMCM (5% hematocrit) and add it to the culture to obtain a parasitemia of not more than 1%, preferably 0.1 to 0.5% if two cycles until next subculturing, 0.5 to 1% if one cycle. Parasitemia should never exceed 15%.
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 Trop Med Hyg 91(3):363-365.
Trager W, Jensen JB. 1976. Human malaria parasites in continuous culture. Science 193(4254):673-675.
I:B. Establishment of long-term in vitro cultures of Plasmodium falciparum from patient blood
by Morten A. Nielsen and Trine Staalsoe
Centre for Medical Parasitology, Department of Infectious Diseases M7641, Rigshospitalet, Copenhagen, Denmark
e-mail: mncmp@rh.dk
Careful adherence to the procedures described below should allow a very high success rate with immediate culture (provided parasites are not already damaged by antimalarial drugs), and even when using cryopreserved parasite stabilates, a success rate of >70% should be within reach.
Equipment
centrifuge
incubator
Materials and reagents
culture medium:
500 mL RPMI 1640 (Gibco)
25 mg gentamycin (Gibco)
91.6 mg L-glutamine (Sigma)
2.5 g Albumax II (Invitrogen) (See comments below.)
10 mL normal human serum
10 mg hypoxanthine (Sigma) (See comments below.)
washing medium:
500 mL RPMI 1640
25 mg gentamycin (Gibco)
freezing solution:
sterile, distilled water
3.0% sorbitol (Sigma)
28.0% glycerol (Sigma)
0.65% NaCl
thawing solution:
sterile, distilled water
3.5% NaCl
Giemsa stain:
10% Giemsa in phosphate buffer
gas mixture:
2% O2, 5.5% CO2, 92.5% N2 (Special gas mixtures such as this one can be bought from suppliers of compressed gases.)
red blood cells (RBC) for subcultivation:
Blood type O Rh− or O Rh+ RBC in CPD buffer, washed 3 times in washing medium to remove the Buffy coat (See comments below.)
Step-by-step manipulations
• Collect heparin or CPD anticoagulated venous blood from patient. Do not use EDTA blood, which does not support parasite survival. The collected blood can be used for cultivation immediately (“straight out of the arm”) or after cryopreservation.
Preparation for immediate cultivation (“straight out of the arm”) (optional)
• Wash the anticoagulated blood sample 3 times in prewarmed (37 °C) washing medium (centrifuged for 8 min at 600 g). Remove buffy coat after each centrifugation.
• Continue as described under Cultivation of fresh patient isolates below.
Cryopreservation (optional—Do only if immediate culture is not possible or desirable.)
• Spin the anticoagulated blood sample (centrifuged for 8 min at 600 g) and remove plasma.
• Mix equal amounts of ring-stage parasitized packed RBC and freezing solution (see comments below).
• Distribute the mixture immediately into 1.8-mL screw-cap tubes and snap-freeze in liquid nitrogen.
• Store the tubes in liquid nitrogen until use.
• On day of use, thaw the RBC suspension in a 37 癈-water bath.
• Spin the tube (8 min at 600 g) immediately after thawing (see comments below).
• Discard the supernatant, add an equal amount of thawing solution, mix well, and incubate the cells for 1 min (see comments below).
• Fill the tube with prewarmed (37 癈) washing medium and repeat centrifugation as above.
• Wash the RBC 2 times in prewarmed (37 °C) washing medium (centrifuge for 8 min at 600 g).
• Continue as described under Cultivation of fresh patient isolates below.
Cultivation of fresh patient isolates
• Suspend RBC at ~4% hematocrit in prewarmed (37 癈) culture medium.
• Add 5 mL of the RBC suspension to a 50-mL cell culture flask (T flask) or 25 mL to a 250-mL cell culture flask.
• Flush 50-mL flasks for ~30 s with gas mixture (250-mL flasks for ~90 s) at 1.5- to 2-bar pressure (see figure and comments below).
• Incubate the flasks at 37 °C for 24 h.
• Remove the culture flask gently from the incubator and remove spent supernatant.
• Remove a tiny amount of RBC with a sterile Pasteur pipette and use these RBC to make a Giemsa-stained thin smear.
• Add new (prewarmed) culture medium to the flask, gas it as above, and return it to incubation at 37 °C.
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