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Human Malaria Parasites in Continuous Culture

Abstract. Plasmodium falciparum can now be maintained in continuous culture inhuman erythrocytes incubated at 38°C in RPMI 1640 medium with human serum un-

der an atmosphere with 7 percent carbon dioxide and low oxygen (I or 5 percent).The original parasite material, derived from an infected Aotus trivirgatus monkey,was diluted more than 100 million times by the addition ofhuman erythrocytes at 3- or

4-day intervals. The parasites continued to reproduce in their normal asexual cycleof approximately 48 hours but were no longer highly synchronous. They have re-

mained infective to Aotus.

Prerequisite to the ultimate goal of avaccine against malaria, a disease thatstill claims 96 million cases annually witha million deaths in Africa alone, is amethod for in vitro propagation of theparasites (1). This has now beenachieved.

Since the first attempts by Bass andJohns (2) and including all the more re-cent work with the erythrocytic stages ofboth human malaria and malarial para-sites of experimental animals, only oneor at best a few cycles of development invitro have been obtained (1, 3, 4). Suchshort-term experiments have been usefulin chemotherapeutic, biochemical, andimmunological studies (4, 5). It was in ex-periments of this type (6) that the findingwas made that medium RPMI 1640 (7),supplemented with Hepes buffer andmonkey (Macaca mulata) serum, sup-ported much better development of themonkey malaria Plasmodium coatneyithan did a variety of other media.

This medium with human serum wastherefore tried in the continuous flow sys-tem previously found to support 4-daydevelopment of both P. coatneyi and P.falciparum (4). The principle of thismethod, which has been well described,is to have the erythrocytes in a shallowstationary layer covered by a shallow lay-er of medium that flows slowly and con-tinuously over the settled cells. Two cul-tures were begun in the following way.Heparinized blood, from an Aotus tri-virgatus monkey (8) infected with P. fal-ciparum [FVO chloroquine-resistantstrain obtained originally from Dr. W. A.Siddiqui (9)] and showing a parasitemiaof 15 percent, was centrifuged, theplasma and buffy coat were removed,20 AUGUST 1976

and the cells were resuspended in culturemedium consisting of RPMI 1640 pow-dered medium (Grand Island) supple-mented with Hepes buffer (25 mM) and 0.2percent NaHCO3 (hereafter abbreviatedas RP) with 10 percent type AB humanserum and with a volume of heparin solu-tion (30 mg of heparin per 100 ml ofblood in 0.85 percent NaCI) equivalent toone-tenth the volume of the resuspendedcells. A suspension of normal humanerythrocytes [type AB, Rh+ blood col-lected in acid, citrate, and dextrose(ACD) (10)] had meanwhile been pre-

pared. Type AB blood was used becausethis or type B, but not type A, could bemixed with Aotus blood without dangerof agglutination of the Aotus cells (4).The blood was centrifuged for 10 min-utes at 1000g, the supernatant and buffycoat were removed, and the cells weresuspended in an equal volume of RP andcentrifuged again. This washing was re-peated, and a 45 percent suspension ofthe washed cells was then made in RPwith 15 percent type AB human serum(10). Eighteen milliliters of this suspen-sion were mixed with 2 ml of the infectedAotus blood suspension. One milliliter ofthis mixture was placed in each of twoflat-bottomed vials equipped with anoverflow tube that is raised 3 mm abovethe bottom of the vial and delivers in-to an overflow collecting flask (4). Eachvial was connected aseptically to a reser-voir flask holding 120 ml of RP mediumwith 15 percent type AB serum. Both thevial and the reservoir flask received aslow current of a gas mixture consistingof 7 percent C02, 5 percent 02, and 88percent N2. The flow of medium, con-trolled by a peristaltic pump, was startedand adjusted to a rate of 50 ml/day.At intervals of 2 days, or sometimes of

1 day, each vial was transferred to a newoverflow flask and a new reservoir flaskof medium was attached. At the sametime, small samples of the settlederythrocytes were taken with a very finecapillary pipette and used for the prepara-tion of wet mounts and a Giemsa-stainedfilm. At intervals of 3 or 4 days, fresh hu-

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Fig. 1. Plasmodium falciparum after 50 days in continuous culture in human erythrocytes.(A) x 3500; (B-D) x 5400. Note all stages of the asexual cycle. In (B) one erythrocyte con-tains three late schizonts.

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man AB erythrocytes, prepared in the fol-lowing way, were added. An appropriatevolume of blood in ACD (used after stor-age at 4°C for up to 2 weeks) was centri-fuged, the supernatant and buffy coatwere removed, and the cells were resus-pended in an equal volume of RP andcentrifuged again. This washing was re-peated and the cells were finally resus-pended at about 45 percent in RP con-taining 15 percent AB serum. Portions ofthis suspension-2, 2.5, or 3 ml, depend-ing on the dilution desired-were thenadded to the culture vial and mixed thor-oughly with the l-ml content (see above)of the vial. The same volume that hadbeen added was then drawn off, leavingin the vial I ml of blood now diluted 1: 3or 1: 4 with fresh blood cell suspension.This procedure was tantamount to sub-culture, and the 2 to 3 ml of mixture thatwas removed could be used to start newcultures or for other experimental pur-poses. A droplet of the mixture was usedto make a stained slide from which aparasite count was made. The number ofparasites per 10,000 erythrocytes on this

slide thus provided a base for estimatingthe extent of multiplication as seen inslides made on later days.

Representative counts were made(Table 1) for two lines (A, carried to 48days, and B, still being carried as foursublines now more than 2 months in vi-tro). In line A the ipitial inoculum ofyoung, ring forms grew into trophozoitesand dividing forms in 2 days. At 4 daysreinvasion had occurred but there wasno increase in number. Nevertheless, thepreparation was diluted threefold witherythrocyte suspension, reducing theparasite count to only six per 10,000erythrocytes. Two days later it had risento 38, most of which were rings. Such six-fold or better multiplication in a singlecycle has continued (Table 1). In 50 daysthere would be 25 cycles. If we assumean average increase of only fourfold ateach cycle, the overall increase in 50days may be estimated as 425.The gas mixture for line A was

changed on day 36 to 7 percent C02, 1percent 02, and 92 percent N2; this mix-ture was used throughout for line B. It

Table 1. Plasmodium falciparum in continuous culture in human erythrocytes. Representativecounts during the history of two lines. Line A was kept in flow vials continuously. Line B wasderived from line A on day 28 in vitro and was passed through three successive petri dish cul-tores (with a four- and an eightfold dilution at subculture) before being returned to flow vials onday 38 after further fourfold dilution. Dilution refers to the dilution by volume of the culturewith a 45 percent suspension of freshly washed human erythrocytes taken from cold storage inACD (see text). At zero time a suspension of erythrocytes from an infected Aotus trivirgatusmonkey was diluted tenfold with human erythrocyte suspension and this was considered theoriginal undiluted concentration of parasites (32 per 10,000). Subcultures (that is, dilutions withfresh erythrocytes) were done at 3- or 4-day intervals and usually a fourfold dilution of the cul-ture was effected. Abbreviations: R, rings without pigment; T, uninucleate forms with pigment;2N, forms with two nuclei; and >2N, forms with more than two nuclei.

Days Parasites per 10,000 erythrocytesin vitro Dilution R T 2N >2N Total

Line A0 1 32 0 0 0 322 6 23 7 17 534 18 5 0 7 304 3 1 1 0 4 66 27 7 2 2 38

11 36 5 2 1 1 913 19 13 3 3 3814 40 9 0 1 5022 1,728 9 2 0 1 1224 30 20 3 6 5925 29 32 6 15 8228 18,000 11 3 0 0 1431 34 25 3 4 6646 10.8 x 106 3 1 0 0 448 22 5 2 2 31

Line B28 18,000 11 3 0 0 1431 54 14 2 7 7735 580,000 6 3 0 0 938 56 9 3 1 6938 2x106 5 1 0 0 641 29 9 3 1 4246 32x106 8 1 2 0 1150 170 48 28 46 29250 128 x 106 7 5 3 2 1753 144 76 8 14 242

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appears to give better development inthe flow vials than the mixture with 5 per-cent 02. This low 02 concentration wasnot strictly maintained because the cellswere periodically exposed to air whenbeing sampled and when being dilutedwith fresh erythrocytes.

Propagation of the parasites has alsobeet} obtained in simple plastic petri dish-es held in a candle jar, with the mediumbeing changed manually once a day. Acandle jar is a long-known micro-biological method (11) and has been usedfor the culture of parasites of the genusEimeria in tissue culture (12). A whitecandle is centrally placed in a desiccatorequipped with a stopcock. The candleis lit, and the cover is put on withthe stopcock open. When the candlegoes out the stopcock is closed. Thisis a simple but effective way to pro-duce an atmosphere with low 02 andhigh CO2 content. On day 28 in vitro ma-terial (1.5 ml) from a flow vial was placedin 35-mm petri dishes (Falcon) to give adepth of about 2 mm. The dishes wereplaced in a candle jar and incubated at38°C. Fresh medium was provided dailyby gently tipping the dishes and with-drawing the supernatant fluid and replac-ing it with a portion of fresh RP containing15 percent human serum. Fresh erythro-cytes were added every third or fourthday as in the flow vial method. Ex-cellent multiplication ofparasites was ob-served. This method has the advantageof simplicity and the disadvantage of re-

quiring daily attention for the change ofmedium. Both methods provide whatmay be the important requirements: asettled layer of erythrocytes in an appro-priate culture medium, an atmosphere ofrelatively high CO2 and low 02, and a

method for change of medium with mini-mal disturbance of the cells. The dishmethod has also been used to initiate anew culture line.

In the cultures all stages of the asexualcycle are readily seen (Fig. 1) at anyone time, since the cultures have lostsome of the synchronicity characteristicof P. falciparum in man (13) and also inAotus. The parasites have retained alto-gether normal morphology. Delicaterings with two chromatin dots are com-mon. Segmenters show 15 to 24 or moremerozoites, the same numbers seen inmaterial from human infections (13). In-fectivity was demonstrated after 35 daysin vitro by the inoculation of culture ma-terial intravenously into a splenecto-mized Aotus monkey. Parasites ap-peared in the monkey 6 days later, andthe infection rose rapidly to a peak of 20percent a week later when the monkeywas killed. Only a few imperfect gameto-

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cytes have so far been noted in prepara-tions after 2 months in vitro, but suffi-ciently thorough examination of theslides for this stage has not yet beendone. Whether infective gametocytescan be produced in vitro and what arethe conditions for their formation areamong the many problems now open toexperimental attack.Of more immediate importance is the

use of the cultures for the preparation ofmerozoites, which may be particularlyimmunogenic (14), and for the study ofmaterials produced by merozoites thatmay function in invasion of erythrocytes(15) and may have a role in induction ofprotective immunity. Of particular inter-est would be a study of the physiologicalcondition of the erythrocyte, especiallywith regard to adenosine triphosphatecontent, in relation to its suitability fordevelopment of the parasites (16), andfor characterization of the requirementsof malaria parasites for extracellular de-velopment in vitro (16).

WILLIAM TRAGERJAMES B. JENSEN

Department ofParasitology,Rockefeller University, New York 10021

References and Notes

1. "Developments in malaria immunology," WHOTech. Rep. Ser. 579 (1975).

2. C. C. Bass and F. M. Johns, J. Exp. Med. 16,567(1912).

3. G. A. Butcher and S. Cohen, Parasitology 62,309 (1971); C. Diggs, K. Pavanand, B. Permpa-nich, V. Numsuwankijkul, R. Haupt, N. Chua-nak, J. Parasitol. 57, 187 (1971); R. S. Phillips,P. I. Trigg, T. J. Scott-Finnegan, R. K. Barthole-mew, Parasitology 65, 525 (1972); W. A. Sid-diqui, J. V. Schnell, S. Richmond-Crum, Am. J.Trop. Med. Hyg. 23, 1015 (1974); J. Parasitol.61, 189 (1975); P. I. Trigg, Parasitology 71, 433(1975).

4. W. Trager,J. Protozool. 18, 232, 239 (1971).5. G. A. Butcher andS. Cohen, Immunology 23,

503 (1972); W. it G. Richards and S. G. Wil-liams, Ann. Trop. Med. Parasitol. 69, 135(1975); W. A. Slddicli, J., V. Schnell, Q. M.Geiman, J. Parasitol. 56, 188 (I1q70).

6. W. Trager, in Biochemistry qf Parasites andHost-Parasite Relqationships, H. van der Bos-sche, Ed. (North-Holland, Amsterdam, inpress).

7. G. E. Moore, R. E. Gemiie", H. A. Franklin, J.Am. Med. Assoc. 199, 519(067).

8. Q. M. Geiman, W. A. $i-4ij, J. v. Schnell,Mil. Med. 134 (Sept. Special Iss ue) 780 (1969).

9. Isolated from a patient retyrned from Vietnamto California [see Q. M. Gei'an and M. J.Meagher, Nature (London) 215, 437 (1967)].

10. T. J. Greenwalt and G. A. Jamieson, The Hu-man Red Cell in Vitro (Grune & Stratton, NewYork, 1974).

11. R. Emerson and A. A. Held, Am. J. Bot. 56,1103 (1969).

12. J. B. Jensen, unpublished work.13. P. C. C. Garnham, Malaria Parasites (Black-

well, Oxford, 1966).14. G. H. Mitchell, G. A. Butcher, S. Cohen, Immu-

nology 29, 397 (1975).15. L. H. Bannister, G. A. Butcher, E. D. Dennis,

G. H. Mitchell, Parasitology 71, 483 (1975); A.Kilejian, T.-H. Liao, W. Trager, Proc. Natl.Acad. Sci. U.S.A. 72, 3057 (1975): L. H. Milleretal., Science 189, 561(1975).

16. W. Trager, J. Protozool. 18, 392 (1971); W.Trager and F. Brohn, Proc. Natl. Acad. Sci.U.S.A. 72, 1834 (1975); F. Brohn and W. Trager,ibid., p. 2456.

17. Supported in part by NIH grant Al 10640 andmainly by AID contract ta-C-1200. We thankR. Klatt for technical assistance.

19 April 1976

20 AUGUST 1976

Neurons Selective for Orientation and Binocular Disparityin the Visual Wulst of the Barn Owl (Tyto alba)

Abstract. The visual response properties of single neurons in the owl's visualWulst suggest that this forebrain structure is an analog ofthe mammalian visual cor-

tex. Features in common with the cat and the monkey visual cortex i1cluhde a precistitopographic organization, a high degree ofbinocular interaction, and selectivity fororientation, direction ofmovement, and binocular disparity ofstraight-litle clontours.

The frontal eyes and prey-catchingskills of the owl suggest that the birdachieves stereopsis, the highly precise,binocular depth sense (1). However,some investigators (2) have arguedagainst this possibility because owls lackpartial decussation at the optic chiasm;instead, all the fibers from one eye crossto the opposite side of the brain (Fig. 1).

Partial decussation allows informationfrom each eye to be compared by thebrain, as Newton first pointed out (3),and is a prominent feature of the visualsystem of all the binocular mammals in-cluding man. Since owls lack a partial de-cussation, it was inferred that they alsolack neurons that can be influenced byboth eyes and, therefore, the disparity-sensitive binocular neurons thought tomediate the first stages of stereoscopicvisual processing in the cat and monkeyvisual cortex (4).

Neuroanatomical studies on the owl(5) suggest that such inferences were pre-mature; despite a totally crossed path-way from the eye to the thalamic relaynucleus, the owl has a bilateral projec-tion from the thalamus to the Wulst, aprominent bulge on the surface of theforebrain (Figs. I and 2). The finding of a

representation of each eye within thisstructure and other cytoarchitectonicsimilarities to the visual cortex of mam-mals, led Karten and co-workers (5) tohypothesize that the Wulst might playthe role for the owl of the visual cortex inthe monkey and the cat.We have studied the visual response

properties of 260 single neurons in theWulst of the barn owl (Tyto alba). Ourfindings confirm the recent ideas of theneuroanatomists, for the physiologicalsimilarities of the Wulst to the visual cor-tex of the cat and monkey are more strik-ing than the differences. In common withthe mammalian cortex are a precise reti-notopic organization, a high degree of bi-nocular interaction, and neurons with re-quirements for stimulus orientation, di-rection of movement, and binoculardisparity.We used tungsten-in-glass micro-

electrodes and conventional extra-cellular recording techniques from aclosed chamber. Anesthesia was inducedwith ketamine (12 mg per kilogram ofbody weight, injected intramuscularly)and maintained by intermittent injectionsthrough a Teflon catheter placed in thepectoral muscle. As the degree of eye

Fig. 1. Schematic re- vcpresentation of the or- SNW_

ganization of forebrain ~\ 1fvisual pathways in thecat and owl. Both ofthese vertebrates dem- soconstrate binocular in-tegration within lami- /nated forebrain struc- trn

tures (visual cortex of trnthe cat and visual Wulstof owl). In the cat(right) binocular con-vergence occurs at thethalamic level because ocof partial decussation of /fibers from the retina(r) at the optic chiasm(oc). In contrast, theowl (left) has a total op-tic decussation, and tha-lamic fibers represent-ing the temporal retina Owl Catmust recross in thesupraopic chiasm (soc) for binocular convergence to take place. Consequently the thalamicrelay nucleus (trn) of the owl represents the whole visual field of the contralateral eye while thecorresponding relay nucleus of the cat represents the contralateral hemifield of both eyes. De-spite these differences, both cat visual cortex (vc) and owl visual Wulst (vw) represent thecontralateral hemifield, and both have binocular neurons with similar functional characteristics.

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