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Plasmodium falciparum: modified medium composition supports continuous cultivation with foetal bovine serum
Experimental Parasitology 108 (2004) 74–75 www.elsevier.com/locate/yexpr
Research Brief
Plasmodium falciparum: modified medium composition supports continuous cultivation with foetal bovine serum Kumkum Srivastava, S.K.Puri* Parasitology Division, Central Drug Research Institute, Lucknow 226001, India Received 4 March 2004; accepted 19 July 2004 Available online 26 August 2004
The basic components of the suitable ingredients for continuous cultivation of Plasmodium falciparum in vitro have not undergone much change since the original report by Trager and Jensen (1976). Hence medium RPMI-1640 buffered with 25 mM Hepes, 25 mM NaHCO3, and supplemented with 10% human serum continues to be the medium of choice in laboratories around the world. The intervening years have seen occasional reports on attempts to substitute animal serum or serum components for long term cultivation of P. falciparum although not many of these have found successful applications. The role played by serum in promoting parasite growth is still undefined and the lack of availability of good quality human serum, particularly in malaria endemic zones, continues to limit wider applications of in vitro culture resource. Ofulla et al. (1993) observed that albumin and lipids are essential serum components needed to sustain growth and a commercially available lipid enriched bovine serum albumin product Albumax II is nowadays being used for continuous cultivation in a few laboratories (Basco, 2003; Cranmer et al., 1997; Flores et al., 1997). Compared with attempts to find substitutes for human serum, there are not many reports on continuous cultivation of P. falciparum using medium other than RPMI 1640. Studies by Haynes et al. (1976) and Chen et al. (1980) employed use of medium 199 and HamÕs-12 nutrient mixture respectively for parasite growth. Divo and Jensen (1982) compared M199 in both EagleÕs and HanksÕ salts and HamÕs F-12 with RPMI-1640 and found that the latter was superior to both formulations of M199 and equal to HamÕs F-12 for cultivation of P. falciparum. Commercially available growth media like NCTC 135 and IMDM apart from their applications for maintaining cell lines have been successfully used for growth and development of metazoan filariid parasites which obviously need higher nutrient requirements (Franke et al., 1987). Our studies showed that a combination of medium RPMI 1640, medium NCTC 135, and medium IMDM constituted in the ratio of 2:1:1 (designated modified medium RPNI) consistently yielded better growth of P. falciparum as compared to that with conventionally used medium RPMI 1640, when 10% human serum was used as the routine supplement. Plasmodium falciparum (NF 54 strain) is routinely maintained in culture medium consisting of RPMI-1640 (Gibco) supplemented with Hepes buffer (25 mM), 0.2% NaHCO3, antibiotic (Gentamycin 40 lg/
*
Corresponding author. Fax: +91 0522 2223405. E-mail address: [email protected] (S.K. Puri).
0014-4894/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2004.07.008
ml; Sigma), antimycotic (Fungizone 0.25 lg/ml; Gibco) and 10% compatible normal human serum using the candle jar method (Trager and Jensen, 1976). The spent culture medium is replaced once every 24 h. The modified medium was constituted by combination of RPMI 1640, NCTC 135 (Sigma), and IMDM (Sigma) in appropriate ratios as indicated. A representative growth profile of P. falciparum using different combinations of the three media is shown in Fig. 1. Parasite cultures initiated at 0.5% parasitaemia attained above 10% level on day 8 with modified medium RPNI. On the other hand, cultures incubated with
Fig. 1. Growth kinetics of P. falciparum (NF-54) in different media combinations supplemented with 10% human serum.
K. Srivastava, S.K.Puri / Experimental Parasitology 108 (2004) 74–75
75
also eliminates the requirement of blood group compatibility between erythrocytes and serum as well as variations accounted to collection of serum from different donors. Moreover, it also obviates bio-hazardous risks associated with handling of a blood component by researchers.
Acknowledgment The authors are grateful to the Director CDRI for continued encouragement and support (Contribution number 6614 from CDRI).
References
Fig. 2. Growth of P. falciparum (NF-54) in modified medium RPNI supplemented with 10% Human (¤) or FBS (n).
RPMI 1640 and NCTC 135 (1:1 ratio), NCTC 135 and IMDM (1:1 ratio), RPMI 1640 and IMDM (1:1 ratio) or with RPMI 1640 alone attained parasitaemia levels between 6.5 and 7.6% under identical conditions. As a sequel to these observations, we monitored the growth of P. falciparum in modified medium RPNI supplemented with 10% foetal bovine serum (FBS) and 10% human serum and results have shown comparable growth in the two groups over a 7-day period (Fig. 2). The parasite cultures with FBS supplemented medium RPNI have since been continuously maintained for over 6 months. Furthermore, we have also observed that parasites growing in human serum supplemented RPMI 1640 can be transferred to FBS supplemented RPNI and do not require sequential adaptation in modified medium for improved growth kinetics. The parasites maintained in FBS supplemented RPNI have also been cryopreserved and successfully revived in the same media. Substitution of human serum with foetal bovine serum is a readily available alternative for continuous cultivation of P. falciparum which
Basco, L.K., 2003. Molecular epidemiology of malaria in cameroon.xv. Experimental studies on serum substitute and supplements and alternative culture media for in vitro drug sensitivity assays using fresh isolate of Plasmodium falciparum. American Journal of Tropical Medicine and Hygiene 69, 168–173. Chen, Z., Gao, M., Li, Y., Han, S., Zhang, N., 1980. Studies on the cultivation of erythrocytic stage of Plasmodium in vitro. Chinese Medical Journal 93, 31–35. Cranmer, S.L., Magowan, C., Liang, J., Coppel, R.L., Cooke, B.M., 1997. An alternative to serum for cultivation of Plasmodium falciparum in vitro. Transaction of Royal Society for Tropical Medicine and Hygiene 91, 363–365. Divo, A.A., Jensen, J.B., 1982. Studies on serum requirements for the cultivation of Plasmodium falciparum. I. Animal sera. Bulletin WHO 60, 565–569. Flores, M.V.C., Berger-Eiszele, S.M., Stewart, T.S., 1997. Long term cultivation of Plasmodium falciparum parasites in media with commercial non-serum supplements. Parasitology Research 83, 734–736. Franke, E.D., Riberu, W., Wiady, I., 1987. In vitro cultivation of third stage larvae of Wuchereria bancrofti to the fourth stage. American Journal of Tropical Medicine and Hygiene 37, 370–375. Haynes, J.D., Diggs, C.L., Hines, F.A., Desjardins, R.E., 1976. Culture of human malaria parasites Plasmodium falciparum. Nature 263, 767–769. Ofulla, A.O., Orago, A.S., Githure, J.I., Burans, J.P., Aleman, G.M., Johnson, A., Martin, S.K., 1993. Cultivation of Plasmodium falciparum parasites in a serum-free medium. American Journal of Tropical Medicine and Hygiene 49, 335–340. Trager, W., Jensen, J.B., 1976. Human malaria parasites in continuous culture. Science 193, 673–675.
Research Brief
Plasmodium falciparum: modified medium composition supports continuous cultivation with foetal bovine serum Kumkum Srivastava, S.K.Puri* Parasitology Division, Central Drug Research Institute, Lucknow 226001, India Received 4 March 2004; accepted 19 July 2004 Available online 26 August 2004
The basic components of the suitable ingredients for continuous cultivation of Plasmodium falciparum in vitro have not undergone much change since the original report by Trager and Jensen (1976). Hence medium RPMI-1640 buffered with 25 mM Hepes, 25 mM NaHCO3, and supplemented with 10% human serum continues to be the medium of choice in laboratories around the world. The intervening years have seen occasional reports on attempts to substitute animal serum or serum components for long term cultivation of P. falciparum although not many of these have found successful applications. The role played by serum in promoting parasite growth is still undefined and the lack of availability of good quality human serum, particularly in malaria endemic zones, continues to limit wider applications of in vitro culture resource. Ofulla et al. (1993) observed that albumin and lipids are essential serum components needed to sustain growth and a commercially available lipid enriched bovine serum albumin product Albumax II is nowadays being used for continuous cultivation in a few laboratories (Basco, 2003; Cranmer et al., 1997; Flores et al., 1997). Compared with attempts to find substitutes for human serum, there are not many reports on continuous cultivation of P. falciparum using medium other than RPMI 1640. Studies by Haynes et al. (1976) and Chen et al. (1980) employed use of medium 199 and HamÕs-12 nutrient mixture respectively for parasite growth. Divo and Jensen (1982) compared M199 in both EagleÕs and HanksÕ salts and HamÕs F-12 with RPMI-1640 and found that the latter was superior to both formulations of M199 and equal to HamÕs F-12 for cultivation of P. falciparum. Commercially available growth media like NCTC 135 and IMDM apart from their applications for maintaining cell lines have been successfully used for growth and development of metazoan filariid parasites which obviously need higher nutrient requirements (Franke et al., 1987). Our studies showed that a combination of medium RPMI 1640, medium NCTC 135, and medium IMDM constituted in the ratio of 2:1:1 (designated modified medium RPNI) consistently yielded better growth of P. falciparum as compared to that with conventionally used medium RPMI 1640, when 10% human serum was used as the routine supplement. Plasmodium falciparum (NF 54 strain) is routinely maintained in culture medium consisting of RPMI-1640 (Gibco) supplemented with Hepes buffer (25 mM), 0.2% NaHCO3, antibiotic (Gentamycin 40 lg/
*
Corresponding author. Fax: +91 0522 2223405. E-mail address: [email protected] (S.K. Puri).
0014-4894/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2004.07.008
ml; Sigma), antimycotic (Fungizone 0.25 lg/ml; Gibco) and 10% compatible normal human serum using the candle jar method (Trager and Jensen, 1976). The spent culture medium is replaced once every 24 h. The modified medium was constituted by combination of RPMI 1640, NCTC 135 (Sigma), and IMDM (Sigma) in appropriate ratios as indicated. A representative growth profile of P. falciparum using different combinations of the three media is shown in Fig. 1. Parasite cultures initiated at 0.5% parasitaemia attained above 10% level on day 8 with modified medium RPNI. On the other hand, cultures incubated with
Fig. 1. Growth kinetics of P. falciparum (NF-54) in different media combinations supplemented with 10% human serum.
K. Srivastava, S.K.Puri / Experimental Parasitology 108 (2004) 74–75
75
also eliminates the requirement of blood group compatibility between erythrocytes and serum as well as variations accounted to collection of serum from different donors. Moreover, it also obviates bio-hazardous risks associated with handling of a blood component by researchers.
Acknowledgment The authors are grateful to the Director CDRI for continued encouragement and support (Contribution number 6614 from CDRI).
References
Fig. 2. Growth of P. falciparum (NF-54) in modified medium RPNI supplemented with 10% Human (¤) or FBS (n).
RPMI 1640 and NCTC 135 (1:1 ratio), NCTC 135 and IMDM (1:1 ratio), RPMI 1640 and IMDM (1:1 ratio) or with RPMI 1640 alone attained parasitaemia levels between 6.5 and 7.6% under identical conditions. As a sequel to these observations, we monitored the growth of P. falciparum in modified medium RPNI supplemented with 10% foetal bovine serum (FBS) and 10% human serum and results have shown comparable growth in the two groups over a 7-day period (Fig. 2). The parasite cultures with FBS supplemented medium RPNI have since been continuously maintained for over 6 months. Furthermore, we have also observed that parasites growing in human serum supplemented RPMI 1640 can be transferred to FBS supplemented RPNI and do not require sequential adaptation in modified medium for improved growth kinetics. The parasites maintained in FBS supplemented RPNI have also been cryopreserved and successfully revived in the same media. Substitution of human serum with foetal bovine serum is a readily available alternative for continuous cultivation of P. falciparum which
Basco, L.K., 2003. Molecular epidemiology of malaria in cameroon.xv. Experimental studies on serum substitute and supplements and alternative culture media for in vitro drug sensitivity assays using fresh isolate of Plasmodium falciparum. American Journal of Tropical Medicine and Hygiene 69, 168–173. Chen, Z., Gao, M., Li, Y., Han, S., Zhang, N., 1980. Studies on the cultivation of erythrocytic stage of Plasmodium in vitro. Chinese Medical Journal 93, 31–35. Cranmer, S.L., Magowan, C., Liang, J., Coppel, R.L., Cooke, B.M., 1997. An alternative to serum for cultivation of Plasmodium falciparum in vitro. Transaction of Royal Society for Tropical Medicine and Hygiene 91, 363–365. Divo, A.A., Jensen, J.B., 1982. Studies on serum requirements for the cultivation of Plasmodium falciparum. I. Animal sera. Bulletin WHO 60, 565–569. Flores, M.V.C., Berger-Eiszele, S.M., Stewart, T.S., 1997. Long term cultivation of Plasmodium falciparum parasites in media with commercial non-serum supplements. Parasitology Research 83, 734–736. Franke, E.D., Riberu, W., Wiady, I., 1987. In vitro cultivation of third stage larvae of Wuchereria bancrofti to the fourth stage. American Journal of Tropical Medicine and Hygiene 37, 370–375. Haynes, J.D., Diggs, C.L., Hines, F.A., Desjardins, R.E., 1976. Culture of human malaria parasites Plasmodium falciparum. Nature 263, 767–769. Ofulla, A.O., Orago, A.S., Githure, J.I., Burans, J.P., Aleman, G.M., Johnson, A., Martin, S.K., 1993. Cultivation of Plasmodium falciparum parasites in a serum-free medium. American Journal of Tropical Medicine and Hygiene 49, 335–340. Trager, W., Jensen, J.B., 1976. Human malaria parasites in continuous culture. Science 193, 673–675.