Studies on serum requirements for the cultivation of Plasmodium falciparum

Bulletin of the World Health Organization, 60 (4): 565-569 (1982) Studies on serum requirements for the cultivation of Plasmodium falciparum. 1. Animal sera* A. A. Divo & J. B. JENSEN2 Pooling a number offreshly collected lots of human serum eliminated the variability observed between individual serum samples, and reduced the amount of human serum requiredfor optimum parasite growth in continuous culture. In thepresent experiments the addition of 50 ml of pooled human serum per litre of RPMI (5% serum) resulted in optimum growth. Batches ofRPMI 1640 supplemented with freshly collected and pooled lots ofbovine, porcine, goat, equine, or ovine sera, as well as commercially availablefetal- and young-calf sera, were tested and compared with 5% pooled human serum. Various combinations ofanimal sera with and without Neopeptone were also examined as supple- ments to the basic culture medium. As an alternative to human serum, only bovine serum supplemented with Neopeptone could support continuous parasite growth, but at significantly reduced levels. Continuous parasite growth was obtained by transferring parasites directlyfrom 5% human serum into medium plusfreshly collected, Neopeptone- supplemented, pooled bovine serum, without any needfor an adaptation period. With the advent of the Trager & Jensen method (1), continuous cultures of Plasmodium falciparum are routinely maintained in various laboratories through- out the world. This technique has afforded innumer- able opportunities to study parasite biology, bio- chemistry, and immunology; and these studies should facilitate the development of superior chemothera- peutic agents and possibly an effective vaccine based on antigens derived from in vitro cultures of the parasite. Currently, the parasite cultures require the addition of 100 ml of human serum per litre of RPMI 1640 for optimum parasite growth. This requirement places a number of constraints on the investigator; some laboratories are located in endemic areas where human serum may be inhibitory to the cultures owing to antimalarial antibody or antimalarial drugs present in locally procured sera. Even in some laboratories in developed countries, fresh human serum is difficult to obtain. Furthermore, any widely used vaccine should not be grown in human serum when there is a real possibility of contamination with infectious agents. * Michigan Agricultural Experiment Station journal article No. 10022. From the Department of Microbiology and Public Health, Michigan State University, East Lansing, MI 48824, USA. This investigation received the financial support of the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. ' Graduate Assistant. 2 Assistant Professor. For these reasons, and others, a suitable replacement for human serum would be a beneficial develop- ment. Previous experiments have indicated that commer- cially available animal sera are inadequate for continuous cultures of P.falciparum (2); additional reports support the claim that commercial processing of animal sera detracts from their quality in tissue culture applications (3). At the same time, it was demonstrated that commercially prepared human serum was inferior to that freshly obtained from local blood banks. In addition, freshly collected human serum varies significantly between individuals in its ability to support continuous parasite growth (2). Other investigators (4- 6) have reported the replacement of human serum with a variety of supple- ments. Their data indicate that enriched fetal- or young-calf serum yields parasite growth comparable with that obtained in human serum. In our hands the bovine alternatives have given variable results, but growth has always been inferior to that obtained using human serum. In this report we discuss the minimum amount of freshly collected and pooled human serum required for optimum parasite growth, and compare this with various animal sera prepared in the same manner. Bovine serum from a number of different sources was supplemented with Neopeptone according to the method of Ifediba& Vanderberg (4), and examined as a human serum replacement. 4206 565- 566 A. A. DIVO & J. B. JENSEN MATERIALS AND METHODS All sera were tested in cultures of P.falciparum maintained using the Petri dish-candle jar method (7). Experimental groups of 4 Petri dishes each were subcultured from a common pool of infected blood that had been cultured in RPMI 1640a supplemented with human type A Rh' serum; initial parasitaemias of 0. I% were used. All experiments were conducted using P.falciparum strain FCR3 (8) and parasite development was monitored over a 96-h period. Para- sitaemias were determined by counting parasites per 10 000 erythrocytes. Freshly collected sera Human serum was obtained from freshly clotted whole blood purchased from the local American Red Cross Blood Bank. A pooled human serum lot (PHS) was prepared by combining equal portions of 18 serum samples. Since initial experiments suggested that 50 ml of PHS was as good as 100 ml of most individual samples per litre, the former level became the standard for comparison with animal sera. Fresh bovine and porcine blood samples were collected from a local abattoir. A pooled lot of bovine serum (PBS) included samples from 15 adult Holstein cows and 1 black angus steer. The porcine pool (PPS) contained serum from 8 different animals, all sows. The equine (PES), goat (PGS), and ovine (POS) sera were kindly provided by Dr J. F. Williamsb from farm animals in his care. These sera were tested in pools of 2-6 samples each. The animal sera were tested as supplements to the RPMI medium at concentrations of 100 ml per litre. Combinations of these sera were tested using equal portions of each serum, to a total of 100 ml per litre. All animal sera were sterilized by filtration. Commercially prepared sera Some commercial sources of fetal- or young-calf sera provided a "high quality" specially processed product, which is reputed to be a superior material for culture purposes. We tested sera of this type from Bio- cellc (newborn calf serum) and AMFd (ZetaSera-D) and compared these with our freshly collected pooled adult bovine serum (PBS). A sample from Gibco Laboratories (calf serum) was also tested as a rep- resentative of a "standard commercial" bovine serum. Three types of processed human serum were also tested, these include human serum defibrinated (HSD),d clarified human serum defibrinated 0 Gibco Laboratories, Grand Island, NY, USA. b Michigan State University Veterinary Clinic. cBiocell, Carson, CA, USA. d AMF Immuno-Reagents, Sequin, TX, USA. (CHSD),d and T3/T4 free human serum (TFHS).d These human sera were obtained from plasma sep- arated from outdated blood. Neopeptonee solution was prepared at a concen- tration of 150 g per litre and used at a concentration of 12 ml per litre of complete medium according to Ifediba & Vanderberg (4). All animal sera were heat inactivated at 56 °C for 30 min and stored at - 20 °C until tested. To prevent RBC agglutination, the swine serum required adsorption against human erythro- cytes before use. RESULTS AND DISCUSSION The data illustrated in Fig. 1 indicate that 5% pooled human serum (PHS) (50 ml per litre of medium) supports optimum parasite growth. Our 50r ._______________T TT 4 0- T/ 320 /; 50 10.0 150 °b POOLED HUMAN SERUM Fig. 1. Parasitaemia obtained with different concen- trations of pooled human serum using P. falciparum strain FCR3 (mean ± SD for 4 observations). laboratory now routinely uses 5%o PHS to supplement RPMI 1640 with all strains of P.falciparum. We have no reason to believe that 5%o PHS would not support newly isolated parasite cultures, but this has yet to be demonstrated. The observed reduction in the concen- tration of pooled serum required for optimum para- site growth was expected, considering that Jensen (2) found that many individual serum samples would support growth at much less than the 10% level, with only the poorest samples requiring 10%o. Work in our laboratory indicates that 15-20 individual samples should be pooled in order to ensure an adequate pool of serum. At present we divide each serum lot into ' Difco Laboratories, Detroit, MI, USA. CULTIVATION OF P. FALCIPARUM. 1. 50-ml aliquots and add one aliquot to each 1-litre bottle; the bottles are then kept frozen until 20 such aliquots have been combined in each bottle. Each pool of serum is then melted and aseptically distri- buted into 5-ml portions and refrozen until used. Our work has shown that high parasitaemias can be maintained with 5% PHS using the same methods as for cultures with 10% human serum. When the culture medium is continuously renewed or changed daily, 5% PHS performs as well as 10/o serum in all applications so far tested. Furthermore, 2.5% PHS will support continuous parasite growth consistently at the rate indicated in Fig. 1, but lower concen- trations give variable results after repeated sub- cultures. The data shown in Table 1 indicate that 5% PHS is superior to any of the freshly collected and pooled animal sera tested. Initially the PPS, PGS, and combinations of PBS, PPS, and PGS were encourag- ing, but after the second subculture these sera failed to support significant parasite growth. Of the animal sera tested, only PPS supported significant growth beyond 96 h, and of the various combinations, only the PBS/PGS and PBS/PPS/PGS supported signifi- cant parasite growth. It was expected that all combi- nations that included PPS would promote parasite growth for at least two subcultures. Thus it was sur- prising that combinations of these sera would not support continuous cultures. A review of other reports on human serum replace- ment indicates that the work reported by Ifediba & Vanderberg (4) most closely parallels our objectives, which are to replace the human serum required for continuous cultures of P.falciparum without adding complicating factors to the system, or reducing para- site growth. They have reported that RPMI 1640 supplemented with calf serum plus Neopeptone or Proteose peptone No. 3 will support parasite growth as well as RPMI plus 10% human serum. The draw- back of their system, as reported, is that the parasites apparently require an extensive period of adaptation to the bovine serum before continuous growth can be maintained. In our experience, even after 3 months of laborious adaptation, cultures in Neopeptone- bovine serum never grew as well as those in RPMI with 5% PHS. It was of interest to determine whether the source of bovine serum used affected both the rate of growth of the parasite and the length of time required for the parasite to become successfully adapted. Table 2 illustrates data from experiments using bovine serum from a number of different sources. Three types of specially processed human serum were also tested. Some of our experiments with Neopeptone were run using goat, porcine, and bovine Table 1. Comparison of 5% pooled human serum with 10% freshly collected, pooled, but unsupplemented animal sera Sera' % of growth obtained % Growthin 5% PHSb upon subculture Comments PHS 100 ± 6.25 100 ± 10.6 Represents a 55-fold increase in parasitaemia over a 96-h period PPS 76.6 ± 4.9 65.2 ± 4.4 Subsequent subcultures failed PGS 49.7 ± 4.4 7.3 ± 1.0 Subsequent subcultures failed PES 19.3 ± 4.8 NAGd Difficult to sterilize by filtration PBS 6.3 ± 1.2 NAG POS NAG PBS/PPS 54.4 ± 1.2 NAG PBS/PGS 41.6 ± 3.7 28.9 ± 5.0 Subsequent subcultures failed PGS/PPS 65.0 ± 1.7 NAG PBS/PSS/PGS 50.3 ± 3.6 30.2 ± 2.8 Subsequent subcultures failed PHS (pooled human serum), PPS (pooled porcine serum), PGS (pooled goat serum), PES (pooled equine serum), POS (pooled ovine serum), PBS (pooled bovine serum). When combinations were used, equal portions of animal sera were combined to a total of 10%. b Growth for the first 96 h in vitro. Mean ± SD for 4 observations. ' Mean ± SD for 4 observations. d NAG = no appreciable growth, less than 5% of the control. 567 A. A. DIVO & J. B. JENSEN Table 2. Parasite growth in 10% freshly collected, pooled adult bovine serum (PBS), "high quality" commercially prepared bovine sera, and three types of commercially processed human serum, with and without Neopeptone Sera' % of growth obtained Commentsin 5% PHSb PHS 100 ± 2.1 Represents a 51-fold increase in parasitaemia over a 96-h period PHS + Neopeptone 99 ± 6.1 PBS + Neopeptone 65.1 ± 4.4 PBS + Neopeptone (after adaptation period) 68.2 ± 4.5 Growth continues at initial rate for subsequent subcultures Biocell + Neopeptone 63.7 ± 2.1 AMF + Neopeptone 64.5 ± 3.3 Gibco + Neopeptone NAG Processed human serad CHSD NAG CHSD + Neopeptone NAG TFHS 16.5 ± 1.4 TFHS + Neopeptone 31.4 ± 2.7 Causes slight haemolysis over 96-h period HSD + Neopeptone 31.2 ± 2.8 a PHS (pooled human serum), PBS (pooled bovine serum), Biocell (newborn calf serum), AMF (ZetaSera-D), Ventrex (fetal calf replacement serum), Gibco (calf serum), CHSD (clarified human serum defibrinated), TFHS (T3/T4 free human serum), HSD (human serum defibrinated). b Mean ± SD for 4 observations. c NAG = no appreciable growth, less than 5% of the control (PHS). d Sera derived from outdated CPD-preserved blood. sera and it was found to have a positive effect only when used in bovine serum. The data presented in Table 2 indicate that PBS (freshly collected and pooled adult bovine serum) and the "high quality" commercially processed bovine sera, when supplemented with Neopeptone, will sup- port parasite growth at about 60-65%To of the rate obtained with 5%o PHS. Standard sources of bovine serum generally supplied a product poorer in quality, which gave inconsistent results in parasite cultures. We have found that freshly collected adult bovine serum will maintain continuous parasite cultures without an adaptation period. Furthermore, after an adaptation period of 3 months, the rate of parasite growth is not significantly different from that observed when the parasites were initially introduced into the Neopeptone-supplemented bovine serum (65% of the rate obtained with 5%0 PHS for non- adapted parasites as compared with 68%o after adaptation). An adaptation period appears to be required only for bovine serum of inferior quality, such as the Gibco Laboratories calf serum tested. Elimination of an adaptation period has the advan- tage of not selecting for any particular subpopulation of P.falciparum and also allows the parasite to be freely transferred from RPMI plus bovine serum into RPMI plus human serum without any adverse effects. Our laboratory has also found that high parasitaemia can be maintained using 10% PBS plus Neopeptone. The practical aspects of using bovine serum for con- tinuous parasite cultures must still be rigorously evaluated. We do not know whether growth in bovine serum alters the antigenic characteristics of P.falci- parum, or whether it has an effect on in vitro testing of drug sensitivity. The three commercial types of processed human serum were inadequate for culture purposes (Table 2). It is interesting that Neopeptone has a positive effect on parasite growth when used with these sera, whereas it does not with 5% PHS. Commercial pro- cessing must remove or degrade essential components required for parasite growth. Currently, our laboratory is investigating the basis of the improvement in parasite growth in bovine serum seen with Neopeptone. Obviously, the Neopep- tone must enrich the culture medium with a required nutrient that is not present in bovine serum. In experiments to be reported elsewhere, hypoxanthine appears to replace Neopeptone for continuous parasite cultivation in bovine serum. 568 CULTIVATION OF P. FALCIPARUM. 1. 569 RtSUMt ETUDES SUR LES BESOINS EN SERUMS POUR LA CULTURE DE PLASMODIUMFALCIPARUM. 1. StRUMS ANIMAUX La constitution d'une collection de 18 lots de serum fraichement preleve sur des sujets humains a elimine les variations souvent observees entre des echantillons indivi- duels de serum et a reduit la quantite de serum humain necessaire pour la croissance optimale du parasite. Dans les experiences en cours, l'adjonction de 50 ml du serum humain stocke a 1 litre du milieu de culture RPMI (5% de serum) a permis d'obtenir une croissance optimale. Pour l'adaptation de souches recemment isol6es, il sera peut-etre necessaire d'utiliser des concentrations superieures de serum. Des lots de serums bovins, porcins, caprins, equins et ovins fraichement recueillis et stockes, ainsi que des serums provenant de jeunes veaux ou de foetus de veau, qui sont disponibles dans le commerce, ont e soumis a des epreuves et compares avec le milieu de culture a 5%o de serum humain. Les resultats ont montre que les serums porcins et caprins, et des melanges de serums porcins, caprins et bovins, favori- seraient la croissance du parasite pendant un nombre limite de cycles mais qu'ils ne maintiendraient pas une croissance continue du parasite. Le Neopeptone et des melanges de serums animaux ont et etudies en qualite de supplements possibles au milieu de culture de base, RPMI 1640. Apres le serum humain, seul le serum bovin additionne de Neopeptone pourrait obtenir une croissance continue du parasite mais a des niveaux forte- ment reduits. Lors de l'utilisation de serum bovin de haute qualite, une croissance continue du parasite a pu etre obtenue par le transfert direct de parasites du serum humain au serum bovin sans qu'une periode d'adaptation soit neces- saire. Le serum de bovins adultes fraichement recueilli et stocke s'est revel aussi bon, ou superieur, par rapport aux serums que l'on trouve dans le commerce qui ont e preleves sur de jeunes veaux ou des foetus de veau et soumis a des tests. REFERENCES 1. TRAGER, W. & JENSEN, J. B. Science, 193: 673-675 (1976). 2. JENSEN, J. B. Bulletin of the World Health Organiz- ation, 57 (Suppl. 1): 27 - 31 (1979). 3. BOONE, C. W. ET AL. In vitro, 7: 174- 189 (1972). 4. IFEDIBA, T. & VANDERBERG, J. P. Journal of parasit- ology, 66: 236- 239 (1980). 5. ZHENGREN, C. ET AL. Chinese medical journal, 93: 31-35 (1980). 6. SIDDIQUI, W. A. Continuous in vitro cultivation of Plasmodium falciparum in human erythrocytes: Description of a simple technique to obtain high yields of parasites. In: Practical tissue culture applications. New York, Academic Press, 1979, pp. 267 - 277. 7. JENSEN, J. B. & TRAGER, W. Journal ofparasitology, 63: 883 - 886 (1977). 8. JENSEN, J. B. & TRAGER, W. 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