Plasmodium falciparum: Continuous cultivation in a semiautomated apparatus

EXPERIMENTAL

PARASITOLOGY

48, 36-41

Plasmodium

JAMES

(19%)

Continuous Cultivation a Semiautomated Apparatus

in

falciparum:

B. JENSEN, WILLIAM

The Rockefeller

University,

(Accepted

TRAGER,

AND JOHN

DOHERTY

New York, New York 10021, U.S.A.

for publication

20 February

1979)

JENSEN, J. B., TRAGER, W., AND DOHERTY, J. 1979. Plasmodium faZciparum: Continuous cultivation in a semiautomated apparatus. Experimental Parasitology 48, 36-41. A semiautomated apparatus for the continuous cultivation of the maIaria1 parasite, Plasmodium falciparum, was developed. It changes the culture medium and redistributes the infected erythrocytes at preselected intervals. Parasitemias between 2 and 16% can be maintained by adding fresh erythrocytes every 2 or 3 days. This apparatus produces approximately 10 ml of packed erythrocytes per week with parasitemias between 12 and 16% INDEX DESCRIPTORS: Plasmodium fulciparum; Protozoa, parasitic; Malaria; Continuous culture.

INTRODUCTION

The first continuous cultures of the human malaria parasite, PZu.smo&um fu!ciparum (Trager and Jensen 1976) were initiated using the flow-vial method (Trager 1971). Soon thereafter a greatly simplified method, the petri dish-candle jar technique (Jensen and Trager 1977) was developed. This method facilitated examination of the different culture parameters, allowing us to improve growth rates and parasite production. In experiments using the petri dish-candle jar technique parasite increases of seven- to eight fold per cycle could be achieved when parasitemias were below 67c, and peak parasitemias of 15 to 20% if the culture medium was renewed every 8 to I2 hr. With the improved continuous flow method (Trager 1979) average increases in parasite numbers of seven to eightfold per cycle with parasitemias reaching 10 to

0014-4894/79/040036-06$02.00/O Copyright Q 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

15% have been obtained. We here describe a different approach to the continuous cultivation of P. fulciparum. It involves a mechanized intermitten change of medium by an apparatus based in part on a method used for prolonged extracellular cultures of P. lophurae (Trager and Jernberg 1961). PI/IATERIALS

AND

WTH~DS

The apparatus is composed of three basic modules; the culture vessels, the manifold feeding module, and the tipping platform and control module. The construction and general function of each module will be described first and then their coordinated operations discussed. The culture vessels. The culture vessels ( Figs. l-3), made in the glass-blowing shop at Rockefeller University, consist of upper and lower chambers connected by a U-shaped side arm. The upper chamber

&SmOdiW?2

fdCijXZ?‘W?Z:

CONTINUOUS

CULTIVATION

37

FIGS. l-3. Schematic drawings of malaria parasite culture vessels in their different operational positions. FIG. 1. Culture vessel consisting of upper chamber, A; connected by U-shaped side arm to lower chamber, B; upper and lower chambers have chimneys fitted with silicone rubber stoppers containing glass tubes E, F, G, H; upper chamber contains a settled layer of Plasmodium-infected erythrocytes with a covering layer of culture medium. Cells can be added or removed through side port, D. FIG. 2. Forward tipping of vessel permits exhausted medium to spill into side arm, C, while erythrocytes flow into small anterior recess. FIG. 3. When vessel is reversed from forward position the exhausted medium flows to a position below the lower-chamber chimney where it can be removed through tube E. Fresh culture medium can be added through tube H, stirring the cells as it enters.

(Fig. 1; A), made from 20 x 30-mm rectanguler glass tubing, is 90 mm long and contains a 4 x 4-mm recess at one end. The U-shaped side arm (Fig. 1; C) originates above this recess and connects the upper chamber with the lower one (Fig. 1; B), which is 10 x 30 x 140 mm. Both chambers have vertical (70 x 22 mm) round chimneys, fitted with silicone rubber stoppers containing two glass tubes each (Fig, 1; E, F, G, H), and a serumstoppered side port (Fig. 1; D). During use the upper chamber contains a settled layer of infected erythrocytes (0.75 ml packed cells) and a covering layer (8 ml) of RPM1 1640 medium (Grand Island) with 10% human A+ serum. To change the culture medium the vessel is slowly tipped forward, allowing the exhausted medium to flow into the U-shaped side arm and the erythrocytes into the anterior recess (Fig. 2). As the vessel is then tipped in reverse, the exhausted medium ilows into position below the chimney of the lower chamber and the erythrocytes to a similar position in the upper chamber (Fig. 3). At this time infected cells can be removed and fresh, uninfected cells added by hypodermic needle via the serum-stoppered side port. Also, while in

the reversed position (Fig. 3) the exhausted medium can be removed from the lower chamber through tube E (Fig. 1) and fresh medium added to the cells in the upper chamber through tube H (Fig. 1). Humidified gas (3% CO,; 10% 0,; 87% Ns) flows continuously through the culture vessel from tube F to tube G (Fig. 1), both of which are cotton plugged to maintain sterility. The manifold feeding module. The premeasured medium for each vessel, contained in the arms of a glass manifold (Fig. 4), is connected by silicone rubber tubing to each of the respective upper chambers via tube H (Figs. 1 and 4). The medium is held in the manifold by a long bar-clamp that constricts the silicone tubing until released by a solenoid valve at a specified time. Opposite the manifold arms are short upward-pointing venting ports filled with sterile cotton. These ports allow air into the manifold when the medium is released. The tipping platform and control module. The tipping platform, with places for eight culture vessels, held in position by strips of spring metal (Fig. 4), has its position controlled by an electrically driven cam (Fig. 5). Most of the time the

3s

JENSEN,

TRACER,

platform is horizontal in the “culture” position. When it is time to change the medium, a roller, connected to the platform, rides over the cam causing the platform to slowly tip forward (7 min for forward movement) until it achieves a 30” angle from the horizontal. Once this forward position is reached the pitch of the cam changes causing the roller to reverse the movement of the platform until it reaches a new position that is now 30” aft of the horizontal culture position ( 2.5 min). Once the rearward position of the platform is achieved, switches, located concentric with the cam, close energizing two solenoid valves that open the barclamp mentioned above and a vacuum aspirator hose to be described below. After these two solenoid valves close the platform returns to its original horizontal position. The platform, with culture vessels, occupies the lower portion of a large incubator (Thelco, Model 6) set at 37 C. The manifold module with the solenoid controlled bar-clamp sits on a shelf above the platform with each of the manifold arms connected to its respective culture vessel by a piece of silicone rubber tubing passing through the bar-clamp (Fig. 4). The

AND

DOHERTY

period between medium changes is controlled by a main timer (Fig. 6; A) which can be set at any interval between 2 and 15 hr. If, for example, a medium changing interval of 12 hr is needed the main timer is set at a IO-hr interval. When 10 hr have elapsed the main timer activates two other timers, one set for 4 min, the other for 2 hr. The 4-min timer turns on a peristaltic pump (Sage, Model 375A; Fig. 6; B) that pumps the culture medium from a 500-ml flask in a small adjacent refrigerator (Fig. 6; C) through Teflon-coated plastic tubing to the manifold (Fig. 6; D). The medium fills the first arm of the manifold and spills over to the second, then to the third until each of the eight are filled. The overflow returns via silicone rubber tubing to the original flask in the refrigerator (not illustrated). The medium in the manifold is warmed to 37 C over a period controlled by the 2-hr timer. At the end of the 2 hr this last timer turns on the electric motor that drives the cam and hence the platform. As already described, the platform first moves forward 30” then reverses to a position aft of horizontal 30” (Fig. 6; E). During this time the exhausted culture medium is spilled from the upper chamber

FIG. 4. Malaria parasite culture apparatus in incubator. The various functions are controlled by timers (T). The medium manifold ( M) is filled by peristaltic pump (PP). The flow of the medium from manifold (M) to culture vessels is controlled by bar-clamp (BC). The culture vessels are moved through the positions illustrated in Figs. l-3 by the tipping platform (TP) which is controIled by an electrically driven cam in control box at left. FIG. 5. Cam that controls the different operations of the malaria culture apparatus. Actual size and dimensions (in.) of the cam that controls the movements of the tipping apparatus.

Plasmodium

falciparum:

CONTINUOUS

39

CULTIVATION

FIG. 6. An enlarged view of the components of the malaria culture tipping appartus. The interval between medium changes is controlled by a set of timers (-4). Two hours before the medium is to be changed one of these timers switches on a peristaltic pump (B) that transports the medium, via Teflon-lined plastic tubing, from a reservoir held in an adjacent refrigerator (C) to the warming manifold (D). When the medium in the vessels is to be changed the platform and vessels (E ) tips 30” downward, spilling the exhausted medium from the upper to the lower chambers of the culture vessels (se‘ Figs. l-3). After spilling the exhausted medium into the lower chambers the platform reverses and moves the vessels upwards 30” to activate two solenoid valves; one (F) releases the freshly warmed medium from the manifold (D) into the culture vessels, the other opens a port between two vacuum flasks that suck the exhausted medium from the culture vessels to reservoir H. A special gas mixture is hydrated in distilled water before flowing through the vessels, as illustrated.

other solenoid valve opens a tube between mixing the erythrocytes as it enters; the two aspirator flasks connected to a vacuum line (Fig. 6; H) that sucks out the exhausted medium from the lower chamber through tube E (Fig. 1). Once these two functions are completed the platform re-

to the lower one (Figs. 2 and 3). When the platform reaches the 30” reversed position the switches associated with the cam energize the two solenoid valves. The first valve opens the bar-clamp (Fig. 6; F) allowing the freshly warmed culture medium to flow rapidly into the upper chamber, TABLE Growth

Rates of Plasmodium

falciparum

in Tipping

I

Apparatus

without

Addition

of Fresh Erythrocytesa

Parasites per 10,000 erythrocytes

Mean Mean Mean

“0” time counts 4%hr count@ 96-hr countsd 144-hr countme

Rh

T

2N

>2N

Total

62 364 730 1553

19 184 462 1012

4 16 230 540

1445 408 875

790: (7%) 1830 (18%) 3980 (40%)

Rate increase in parasitemia per continuous cycle

a Maintaining cultures at high paraaitemias requires large quantities of culture medium parasite multiplication per cycle is low is an inefficient method of parasite production. b R, Rings; T, trophoaoites; 2N, binucleated stage; >2N, schizonts. c Interval between medium changes; 12 hr. d Interval between medium changes; 9 hr. e Interval between medium changes; 6 hr.

8X 2.6X

2x and because

40

JENSEN,

TRAGER, AND DOHERTY

This represents an eightfold increase in parasitemia for the first cycle and a 2.6 times and 2 times increase for the second and third cycles, respectively. However, as parasitemias increase to 25 to 4070 the interval between medium changes must be shortened from 12 to 6 hr. Also, due to the consumption of erythrocytes by the growing culture the number of erythrocytes drops so that the 4070 parasitemia actually represents less total parasites after three cycles than the IS% parasitemia achieved after two cycles. Thus, allowing parasitemias to rise to 407; over a 6-day period is an inefficient means of parasite production. Because the greatest rate of parasite multiplication is achieved during the first cycle, after addition of fresh erythrocytes, they are routinely added three times per week, usually Monday, Wednesday, and Friday,

turns to the horizontal and the main timer resets to begin timing another interval. A special gas mixture, consisting of 3% COZ, 10% 02, 87% N2 flows through each vessel, as illustrated (Fig. 6). The apparatus usually runs continuously for 3 months; then the culture vessels and manifold are removed to be washed and resterilized. RESULTS

AND DISCUSSION

The rate of Plasmodium falciparum multiplication in the apparatus depends largely upon the interval between the addition of fresh erythrocytes and the parasitemias after the fresh cells are added. For example, if the parasitemia is around 1% after the addition of fresh cells it will rise to 7 to 8% after the first 4%hr cycle, to 18 to 205% after the second cycle and to 35 to 40% after the third cycle (Table I). TABLE Comparison

Groupsa

A

B

C

Parasites

per 10,000 erythrocytes

T

2x

>2N

ltO”c 48 hrd 96 hrf

25 353 636

16 157 356

1 18 60

7 70 468

49 598 1520

"0" 48 hr

58 423

38 442

7 68

18 114

131 1047

"0" "0" 48 hr

92 832 150 575

103 555 59 541

16 53 10 71

24 183 42 117

235 1623 261 1304

"0" 72 hre "0" 72 hr

77 697 158 1084

21 352 56 603

1 35 6 33

12 282 21 370

111 1366 241 2090

-

Apparatus

Increase of parasitemia

Rb

48 hr

D

II

of Growth Rates of Plasmodium jdciparum in Tipping with Different Starting Parasitemias

Total

12x 31x

8.7X

7x 5x

12.3X 8.7X

a Group A represents typical growth rates when initial parasitemias are approximately 0.5y0; group B when initial par&tern& are approximately 1.0 to 1.570,; and group C when they are approximately 2.0 to 2.57& Group D represents typical growth rates seen over the a-day, Friday-Monday period when initial parasitemias are approximately 1.0 and 2.5y0 respectively. * R, Rings; T, trophozoites; 2N, binucleated stage; >2N, schizonts. c Represents parasitemias after the addition of fresh erythrocytes. d.e,/ Represent parasitemia after 48, 72, and 96 hr, respectively.

Plasmodium

falciparum:

When the parasites are harvested from the culture vessel a small portion are left behind as “seed” for the next cycle. If the parasitemia after the addition of red cells is between 0.5 and 1.0% the rate of parasite growth will usually be S- to N-fold, resulting 48 hr later in parasitemias of 6 to 8% (Table II). If the parasitemia after addition of fresh cells is between 1.0 and 1.5% the parasites will increase six to eight times raising the parasitemias to 10 to 12%, and if it is between 2.0 and 2.5% the resulting parasitemia will be 13 to 16%. This level of parasite multiplication requires medium changes at 12-hr intervals and routinely produces about 10 ml of packed erythrocytes per week with parasitemias of 12 to 16%. The S-day interval between Friday and Monday requires medium changes at 9-hr intervals but usually achieves parasitemias of 18 to 20%. We have now developed three different methods for the cultivation of P. falciparum. The petri dish-candle jar method (Jensen and Trager 1977) is the simplest, but has the disadvantages of requiring daily manual attention. The continuous flow method (Trager and Jensen 1976; Trager 1979), and the tipping apparatus described here, are both semiautomated and could serve as models for some future large-scale production of parasite antigens for diagnostic and immunization purposes. Of these two semiautomated systems the tipping apparatus is currently the most efficient in terms of parasite production per unit of culture medium used and has

CONTINUOUS CULTIVATION

41

the added advantage of being able to handle higher parasitemias when the tipping interval is shortened. The two systems are not directly comparable, however, because in the tipping apparatus the erythrocytes receive a more frequent redistribution, which may be advantageous. The refinement of both of these systems is not complete and further work on improving them is in progress. ACKNOWLEDGMENTS It is Creene of this ported Agency

a pleasure to acknowledge Mrs. Diane D. for her excellent help in the preparation manuscript. This investigation was supby Contract ta-C-1373 from the U.S. for International Development.

REFERENCES JENSEN, J. B., AND TRACER, IV. 1977. PZa.smod’um falciparum in culture: Use of outdated erythrocytes and description of the candle-jar method. Journal of Parasitology 63, 883-886. TRACER, W. 1971. A new method for intraerythrocytic cultivation of malaria parasites ( Plasmodium coatneyi and P. falciparum) . Journal of Protozoology 18, 239-242. TRAGER, W. 1979. Plasmodium falcipawm in culture: An improved continuous flow method. Journal of Protozoology, in press. TRAGER, W., AND JENSEN, J. B. 1976. Human malaria parasites in continuous culture. Science 193, 673-675. TRAGER, W., AND JEFINBERG, R. A. 1961. Apparatus for change of medium in extracellular maintenance in vitro of an intracellular parasite (malaria). Proceedings of the Society for Experimental Biology and Medicine 108, 175178.