- Email: [email protected]
A method for large-volume cultivation of malaria parasites based on the principle of the Trager-Jensen culture method
334 TRANSACTIONS
OP THE
ROYALSOCIETY
OF TROPICAL
MEDICINE
AND HYGIENE,
VOL.
73, No.
3, 1979
A method for large-volume cultivation of malaria parasites based on the principle of the Trager-Jensen culture method WILLIAM CHIN Vector Biology and Control Division, Bureau of Tropical Diseases, Center for Disease Control, Public Health Service, U.S. Department of Health, Education and Welfare, Atlanta, Georgia 30333
The production of malaria antigen for diagnostic, immunologic and other studies is now a possibility following the development of a continuous in vitro culture of Plasmodium falciparum by TRAGER & JENSEN(1976). We have recently devised a method for large-volume cultivation of malaria parasites, using readily available laboratory equipment and based on the principles of the Trager- Jensen method. This method is capable of culturing a maximum of eight flasks, each with a culture volume of 50 ml. The number of flasks used is limited by the capacity
of our peristaltic pump conveniently to handle no more than eight sets of tubings. The description of the method follows : The flask culture set-up is illustrated in Fig. 1. Sterile, disposable 150 cm2 flat-bottom tissueculture flasks with screw caps are used (Corning Glass Works, Corning, New York 14830*). A flask * Use of commercial sources does not constitute endorsement by the Public Health Service or by the U.S. Department of Health, Education and Welfare.
TTON PLUG
‘TRAY
INCUBATOR - 37” C
Fig. 1. Schematic diagram of a large-volume method for the cultivation of malaria parasites.
335
W, CHIN
adapter (A) is made by drilling three holes in a No. 3 silicone rubber stopper to accommodate the following 4 mm COD) alass tubinas : The &be from‘ the’ medium reservoir is 26 cm long and has an elbow 1 to 2 cm from the tip so that when in place, the tip of the tube is 1 cm above the bottom surface of the flask and 1 to 2 mm from touching the side of the flask. The gas inlet tube is 20 cm long with the distal end bent slightly upwards. The gas outlet tube is 6 cm long and plugged loosely with cotton. The outer end of the tube is capped with a plug made of silicone rubber tubing sealed at one end with a short piece of glass rod. A screw cap, used to secure the adapter in place, is made by cutting out the central portion of the flask cap. A 150 ml bottle is used as a medium reservoir stoppered by a terminal adapter (B) made by boring two holes into a No. 2 silicone rubber stopper to accommodate two tubings. The longer tube, 15 cm, is adjusted on the stopper so that the distal end touches the bottom of a 150 ml bottle. The second tube, 5 cm, is plugged loosely with cotton and serves as a vent. A rubber umbrella, made by cutting a piece of dental dam latex of 6 cm in diameter, is fitted over the stopper and through the two tubes. This guards the narrow edge of the stopper from contamination when the stopper is manipulated by hand. A 150 ml bottle (C), with glass tubes and tubing in place as shown, contains sterile water and is used as a visual indicator of the flow rate of a compressed gas mixture (3% CO,, 10% 0,, balance N,). Gas is filtered through a 0.2~ millipore membrane (D) before entering the water bottle. To sterilize components, all the silicone rubber tubings connecting units A, B and C are disconnected and the ends wrapped with foil and autoclaved. The other units are also wrapped with foil and are similarly sterilized. To assemble the components and initiate a culture, aseptic technique is used in the following procedure : 1. Affix flask adapter in place and screw on perforated cap to secure stopper in position. Place unit incubator (37°C). 2. Connect the gas tubings. 3. Prepare 50 ml of culture with RPM1 1640 (Grand Island Biological Company, Grand Island, New York 14072”) and 10% human serum having a red cell concentration of 8 y0 (v/v) with a parasitaemia of approximately 0.5 %. Place the culture in a 150 ml bottle, position terminal adapter, and loosen latex umbrella. Connect medium tubing to the adapter. 4. Insert the gas and medium tubes into incubator and connect them to the flask adapter. 5. Remove outlet plug on flask adapter. 6. Turn on gas at a rate of four to five bubbles/ second. 7. Place medium tubing in peristaltic pump and
activate infusion control to pump culture material into flask. 8. Allow gas to flush air from the flask for at least five minutes after pumping culture into flask. 9. Turn off gas and replace outlet plug. The medium is changed at least once daily as follows: unplug gas outlet and turn on gas. Very gently and slowly, tilt flask to 10-20” angle by raising adapter end. Proper position is maintained by a suitably sized support. When properly done, the red cells will remain on the bottom and the tip of the medium tube will be immersed in the pooled medium. Activate pump to withdraw position. To obtain red cells for a smear, gently resuspend the red cells towards the end of medium withdrawal to allow the escape of a small sample of red cells. An aliquot of this evacuated sample may then be centrifuged to sediment the red cells for a thin blood smear. To replace the medium, prepare 50 ml of medium in a 150 ml bottle. Grasp the terminal adapter through the rubber umbrella and carefully change the bottles. Reverse the pump to infusion position and pump fresh medium back into flask. Resuspend the red cells gently, turn off gas after complete flushing and replace outlet plug. To subculture, unplug gas outlet, turn on gas and pump out entire flask content by standing flask on end. Depending on parasitaemia, use appropriate volume of evacuated culture material for subculturing with a fresh red cell suspension. This method is currently used in our laboratory to produce malaria parasites from culture-adapted strains for two types of studies: providing parasites of all stages for use as antigen for the ELISA test and merozoites for use in immunization studies. In our experience, using a red blood cell concentration of 8 %, an initial P. falciparum parasitaemia of 0.5 “,$ will increase at least lo-fold every four days when the medium is changed once daily. More frequent changes of medium and/or decreasing the RBC concentration to 5% or less will increase the percentage of red cells infected. To harvest the parasites, the culture is centrifuged initially at 650 g for 10 minutes. This will sediment the red cells which contain parasites of all stages for use as antigen in the ELISA test. The supernatant is centrifuged again, this time at 8 000 g for 20 minutes. The second centrifugation will pack the suspended merozoites. In a typical four-day culture of P. falciparum using four flasks approximately 2 X log merozoites may be recovered from the liquid portion of the culture. Reference Trager, W. & Jensen, J. B. (1976). Human malaria parasites in continuous culture. Science, 193, 673-675.
Accepted for publication 21st December, 1978.
OP THE
ROYALSOCIETY
OF TROPICAL
MEDICINE
AND HYGIENE,
VOL.
73, No.
3, 1979
A method for large-volume cultivation of malaria parasites based on the principle of the Trager-Jensen culture method WILLIAM CHIN Vector Biology and Control Division, Bureau of Tropical Diseases, Center for Disease Control, Public Health Service, U.S. Department of Health, Education and Welfare, Atlanta, Georgia 30333
The production of malaria antigen for diagnostic, immunologic and other studies is now a possibility following the development of a continuous in vitro culture of Plasmodium falciparum by TRAGER & JENSEN(1976). We have recently devised a method for large-volume cultivation of malaria parasites, using readily available laboratory equipment and based on the principles of the Trager- Jensen method. This method is capable of culturing a maximum of eight flasks, each with a culture volume of 50 ml. The number of flasks used is limited by the capacity
of our peristaltic pump conveniently to handle no more than eight sets of tubings. The description of the method follows : The flask culture set-up is illustrated in Fig. 1. Sterile, disposable 150 cm2 flat-bottom tissueculture flasks with screw caps are used (Corning Glass Works, Corning, New York 14830*). A flask * Use of commercial sources does not constitute endorsement by the Public Health Service or by the U.S. Department of Health, Education and Welfare.
TTON PLUG
‘TRAY
INCUBATOR - 37” C
Fig. 1. Schematic diagram of a large-volume method for the cultivation of malaria parasites.
335
W, CHIN
adapter (A) is made by drilling three holes in a No. 3 silicone rubber stopper to accommodate the following 4 mm COD) alass tubinas : The &be from‘ the’ medium reservoir is 26 cm long and has an elbow 1 to 2 cm from the tip so that when in place, the tip of the tube is 1 cm above the bottom surface of the flask and 1 to 2 mm from touching the side of the flask. The gas inlet tube is 20 cm long with the distal end bent slightly upwards. The gas outlet tube is 6 cm long and plugged loosely with cotton. The outer end of the tube is capped with a plug made of silicone rubber tubing sealed at one end with a short piece of glass rod. A screw cap, used to secure the adapter in place, is made by cutting out the central portion of the flask cap. A 150 ml bottle is used as a medium reservoir stoppered by a terminal adapter (B) made by boring two holes into a No. 2 silicone rubber stopper to accommodate two tubings. The longer tube, 15 cm, is adjusted on the stopper so that the distal end touches the bottom of a 150 ml bottle. The second tube, 5 cm, is plugged loosely with cotton and serves as a vent. A rubber umbrella, made by cutting a piece of dental dam latex of 6 cm in diameter, is fitted over the stopper and through the two tubes. This guards the narrow edge of the stopper from contamination when the stopper is manipulated by hand. A 150 ml bottle (C), with glass tubes and tubing in place as shown, contains sterile water and is used as a visual indicator of the flow rate of a compressed gas mixture (3% CO,, 10% 0,, balance N,). Gas is filtered through a 0.2~ millipore membrane (D) before entering the water bottle. To sterilize components, all the silicone rubber tubings connecting units A, B and C are disconnected and the ends wrapped with foil and autoclaved. The other units are also wrapped with foil and are similarly sterilized. To assemble the components and initiate a culture, aseptic technique is used in the following procedure : 1. Affix flask adapter in place and screw on perforated cap to secure stopper in position. Place unit incubator (37°C). 2. Connect the gas tubings. 3. Prepare 50 ml of culture with RPM1 1640 (Grand Island Biological Company, Grand Island, New York 14072”) and 10% human serum having a red cell concentration of 8 y0 (v/v) with a parasitaemia of approximately 0.5 %. Place the culture in a 150 ml bottle, position terminal adapter, and loosen latex umbrella. Connect medium tubing to the adapter. 4. Insert the gas and medium tubes into incubator and connect them to the flask adapter. 5. Remove outlet plug on flask adapter. 6. Turn on gas at a rate of four to five bubbles/ second. 7. Place medium tubing in peristaltic pump and
activate infusion control to pump culture material into flask. 8. Allow gas to flush air from the flask for at least five minutes after pumping culture into flask. 9. Turn off gas and replace outlet plug. The medium is changed at least once daily as follows: unplug gas outlet and turn on gas. Very gently and slowly, tilt flask to 10-20” angle by raising adapter end. Proper position is maintained by a suitably sized support. When properly done, the red cells will remain on the bottom and the tip of the medium tube will be immersed in the pooled medium. Activate pump to withdraw position. To obtain red cells for a smear, gently resuspend the red cells towards the end of medium withdrawal to allow the escape of a small sample of red cells. An aliquot of this evacuated sample may then be centrifuged to sediment the red cells for a thin blood smear. To replace the medium, prepare 50 ml of medium in a 150 ml bottle. Grasp the terminal adapter through the rubber umbrella and carefully change the bottles. Reverse the pump to infusion position and pump fresh medium back into flask. Resuspend the red cells gently, turn off gas after complete flushing and replace outlet plug. To subculture, unplug gas outlet, turn on gas and pump out entire flask content by standing flask on end. Depending on parasitaemia, use appropriate volume of evacuated culture material for subculturing with a fresh red cell suspension. This method is currently used in our laboratory to produce malaria parasites from culture-adapted strains for two types of studies: providing parasites of all stages for use as antigen for the ELISA test and merozoites for use in immunization studies. In our experience, using a red blood cell concentration of 8 %, an initial P. falciparum parasitaemia of 0.5 “,$ will increase at least lo-fold every four days when the medium is changed once daily. More frequent changes of medium and/or decreasing the RBC concentration to 5% or less will increase the percentage of red cells infected. To harvest the parasites, the culture is centrifuged initially at 650 g for 10 minutes. This will sediment the red cells which contain parasites of all stages for use as antigen in the ELISA test. The supernatant is centrifuged again, this time at 8 000 g for 20 minutes. The second centrifugation will pack the suspended merozoites. In a typical four-day culture of P. falciparum using four flasks approximately 2 X log merozoites may be recovered from the liquid portion of the culture. Reference Trager, W. & Jensen, J. B. (1976). Human malaria parasites in continuous culture. Science, 193, 673-675.
Accepted for publication 21st December, 1978.