Procedures for maximum production of exoerythrocytic stages of Plasmodium fallax in tissue culture

EXPERIMENTAL PARASITOLOGY19, l-8 (1966)

Procedures

for Maximum of Plasmodium

Abbott

G. Davi~,~ Naval

Medical

Production of Exoerythrocytic fallax iu Tissue Culture1

Clay

G. HII&

and

Research Institute,

Timothy

T.

Stages

Palmer3

Bethesda, Maryland

(Submitted for publication, 21 July 1965) DAVIS, A. G., HUFF, C. G., AND PALMER, T. T. 1966. Procedures for maximum production of exoerythrocytic stages of Plusmodium fallax in tissue culture. Experimental Parasitology 19, 1-8. For the first time a method is described for the cultivation of sufficient numbers of exoerythrocytic stages of a malarial parasite to facilitate the study of fine structure, the action of chemotherapeutic agents, the completion of timelapse cinemicrographic studies, and the more precise determination of growth rates and longevity. The method will be useful also for physiological studies, chemical analyses, and immunological studies on the extracellular, exoerythrocytic parasites. The use and modifications of commercially available media are described as well as alterations of procedures and techniques which meet the need for the production of exoerythrocytic stages in quantities not attained by other methods.

scribed the use of Rose multipurpose chambers containing dialysis membranes for longterm culture in cells less dedifferentiated than the usual tissue culture cells. Previous methods of growing cultures containing liquid medium with gas above the free surface developed in this laboratory yielded either moderate numbers of parasites in long-term cultures or localized concentrations in primary cultures with decreased parasitism in subcultures. We are presenting here the procedures which have been developed in our laboratory for growing maximum numbers of exoerythrocytic stages very rapidly. The utility of such methods will be readily apparent. The emphasis in our presentation will be upon the materials and methods employed in producing the parasites. The results on various types of studies on exoerythrocytic stages such as fine structure, physiology, and chemotherapy will appear in separate publications.

Living malarial parasites in large numbers are essential to success in many types of study. Erythrocytic parasites can be obtained from heavily infected animals, but since their host cells do not multiply in vitro there are

limitations to their mental studies. The grow exoerythrocytic about the time of the

use for certain experihistory of attempts to stages in vitro began discovery of such stages

and has progressed

slowly

during

the three

decades since their discovery. Porter (1948) and Hawking ( 1951) reviewed the early history of their cultivation. Later reviews were published by Pipkin and Jensen (1958) and Huff (1964). Jensen et al. (1964) de1 From Bureau of Medicine and Surgery, Navy Department, Research Task MR 005.09.1030.02. The opinions and assertions contained herein are those of the authors and are not to be construed as official or reflecting the views of the Navy Department or the Naval service at large.

2 HMC, USN. 3 ENS, MSC, USNR. @

1966

by Academic Press Inc. I

2

DAVIS, HUFF, MATERIALS

Seven-day embryonated turkey eggs are inoculated with fallax-infected brain by means of chorioallantoic grafting. These eggs are allowed to incubate 6 days, at which time the brain is removed, placed in Hanks’ balanced salt solution in a petri dish and placed in a 4°C refrigerator overnight. The media presently employed in our routine work are Mixture 199 (modified) and a growth medium which we will call Laboratory Diploid Growth Medium (LDGM). The compositions of these media are presented below (Table I). METHOD

OF CULTURE

OF

P. fallax

The entire brain of an infected, 13-day-old turkey embryo is excised aseptically and placed in a petri dish containing Hanks’ balanced salt solution. The brain is then refrigerated overnight, which enhances cell attachment at a later stage. It is then cut into small pieces, placed in a beaker with 12 ml of 0.25% trypsin solution (pH 7.2-7.4) and a magnetic stir-rod, and allowed to trypsinize with moderate stirring for 30-40 minutes at room temperature. The resulting cell suspension is then poured into a centrifuge tube, corked, and spun at 1000 rpm for 1.5 minutes. The supernate is carefully removed and the “huffy layer” of free cells is drawn into a lo-ml syringe fitted with a 6-inch, 18gauge needle. These cells are injected into a T-30 tissue culture flask containing 4 ml of Mixture 199 and 4 ml of LDGM, then flushed through the needle two or three times to insure good cell suspension. Four milliliters are then removed and placed in a second T-30 flask, the flasks are cotton-stoppered and placed in a 37°C incubator containing a 5% CO2 atmosphere. The cultures remain in this status 2-3 days until approximately SOY0 of one side of the flask is covered with a cell sheet, then the medium is changed and the flasks are closed with #0 silicone stoppers. These cultures are primary cultures from

AND PALMER

which subcultures may be obtained when the cell sheet has completely covered the side of the flask. Subculturing is carried out as follows. The medium is removed from the culture and replaced with 1 ml of 0.25% trypsin solution. The culture is then returned to the incubator for lo-20 minutes until the cells have peeled from the side of the flask. Four milliliters each of 199 and LDGM are then added to the culture, the suspension is flushed through the needle two or three times to insure good mixture; then the total volume (9 ml) is removed and equally divided between two new T-30 flasks. These are cotton-stoppered, returned to the incubator for 2-3 days, then silicon-stoppered and changed (50-807, cell sheet). After the second or third subculture, we have found that the cultures tend to become overparasitized, thus damaging the cells and causing failure of the culture on the next passage. As the parasite load increases, we have found that vibrating the culture for lo-20 seconds prior to changing on a VortexGenie mixer tends to free most of the merozoites from the cell surface, as well as to free numerous heavily parasitized cells which are weakly attached to the glass. This reduces the ratio of parasites to cells, which gives the cultures greater longevity. Overparasitism can be reduced also by cooling the cultures by overnight refrigeration at 4°C and by keeping them at room temperature (21-24°C) from 1 to 4 days. The former is used on cultures which are in advanced stages of overparasitism, the latter to control cultures prior to overparasitism. Refrigeration produces varying degrees of success, but culture cells tend to suffer drastically in some cases. Room temperature control is very useful in early stages of parasitism, but ineffective in heavily parasitized cultures (greater than 50%). Parasites from any culture apparently may be carried on through an indefinite number of subcultures, in spite of the overpara-

BME amino acids BME vitamins MEM non-ess. AA Glutamine Pyridoxine Cysteine Glucose Ascorbic acid Glutathione Hypoxanthine Hydroxy-L-proline PABA Niacin Vit B,, Vit A Vit E Vit D Vit K Tween 80 Adenine Sodium acetate Aminobutyric acid Ornithine Taurine Guanine Sodium pyruvate Galactose Folinic acid o-ribose Deoxyadenosine Deoxycytidine 1

+ + +

+

z f + + + + + + + + + + + + + + + + + + -I+

+ +a

BME 2

+ +

NCTC-109 1*

+

+ + + + + + +

-I+ +f + -I+ + + + + + + +

Mixture 199 3

+

+

+ +a

Spinners

+

+

+ +c +b + +

Puck’s 4

+ +

+e +a,c +d + + +

Liebowitz 5

TABLE I Components of the Various Media Used (Balanced salt solution not included)

+

+ +c +b + + + + + + +

Waymoutb 6

+

+ +a

MEM 7

-I-

+

+

+

+

+

Scherer’s 8

+

z +

+

DGM 9

G,

a E; d E

z

2 Y t:

I

it

3 i; ‘a F s

2 E z 2 g

:: + + + + + -i+ -I +

+ + + + + + +

BME 2

Spinners

Puck’s 4

1 (Coniinued) Liebowitz 5

Waymouth 6

MEM 7

DGM 9

b = no alanine, asfrom MEM only in

+

Scherer’s 8

Explanatory Notes for Table I: + = presence of component in the medium; lack of + means no component; a = no biotin; paragine, serine; c = no pyridoxal-HCl; d = no aspartic acid, glutamine, proline; e = no cystine; f = no asparagine; g = differs balanced salt composition. * = the numbers refer to publications listed in references.

S-Methyl cytosine Deoxyguanosine Cocarboxylase Co-A DPN TPN Flavin adenine dinucleotide Uridine triphosphate Glucosamine Glucuronolactone Na glucuronate Thymine Uracil Xanthine Adenylic acid Cholesterol ATP Z-Deoxy-o-ribose Pyridoxamine-2HCl

NCTC-109 1*

TABLE Mixture 199 3

% cd $ K !l

5 :

u 5 ;f

EXOERYTHROCYTIC Plasmodium IN TISSUE CULTURE

sitism problem. Many infected cells float free from the cultures, due to an increase in surface tension on the host cells with increasing size of the parasites. The medium containing these floating cells may be removed and placed directly on a culture of uninfected, normal turkey brain. Removal of this medium in 6 hours generally results in a very low infection rate of the new culture. Thus the culture may be carried through a number of subcultures before the parasite load becomes critical.

5

have found that it has a much more favorable effect on the cultures. Using DGM-Mixture 199 (modified) exclusively, we found that the DGM has a shelf-life of 10 weeks (from date of production), after which the parasites are no longer supported in culture. The reason for this failure has not been determined. To overcome this difficulty, DGM is now made up biweekly in the laboratory by using the following components: Basal Medium Eagle’s as a base, 10% fetal calf serum, 1% folinic acid, 1s glutamine, 1% penicillin-streptoRESULTS mycin (5000 units each), 1% MEM nonesSeveral media have been employed to sential amino acid (100x) and 1% NaHCOa determine which medium or combination of (7.5% ) . This medium, LDGM (Laboratory media would produce rapidly growing cul- prepared DGM), in conjunction with the modified Mixture 199, gives excellent cell and tures and parasites. They may be divided into three groups, based upon the results parasite growth. Numerous cultures have obtained: (1) little or no growth, (2) fair been carried through 15-16 subcultures on this medium combination and were in good growth, and (3) good growth. Group 1 includes Liebowitz L-15, Way- condition prior to being lost by bacterial mouth MB 752/l, Puck’s N-16, Scherer’s contamination. The rapid increase of paraMaintenance Medium, Minimum Essential sites compared to the relatively slow increase Medium (Eagle), NCTC-109, Mixture 199, in host cells is the major drawback to the and MEM Spinner Medium. The first three extension of the longevity of these cultures. With respect to the shelf-life of the LDGM, media were used as commercially prepared, this medium has been stored at 4’C for 15 but the last six media were employed with folinic acid and calf serum in all combina- weeks prior to use, then used in routine work. Alone, and in combination with modified tions. Mixture 199, this medium has supported the In group 2 a combination of equal parts growth of cells and parasites, though not as of Waymouth’s and Diploid Growth Medium well as the freshly prepared medium. ( DGM)4 (a commercially prepared culture In an attempt to quantify the degree of medium) gave fair cultures, as did Mixture parasitism in cultures which we consider to 199 with 1% folinic acid, 10% fetal calf be overparasitized, several fifth passage culserum, and 1% BME vitamins added. tures grown in plastic T-30 flasks were fixed In Group 3 DGM alone, DGM with 1% with methanol and Giemsa-stained. In fields sodium pyruvate, DGM with equal parts of selected at random, counts indicated that the modified Mixture 199, and equal parts of number of cells parasitized ranged from 48LDGM and modified Mixture 199 have 67%, with an average of six parasites (range proved to be highly successful. The modified Mixture 199 contains 10% fetal calf serum 1-18) per infected cell, in each of the cultures and 1% folinic acid. Fetal calf serum has (Fig. I). Approximately 33% of the paranow been substituted for calf serum, as we sitized cells showed evidence of reinfection, namely, the presence of mature and nearly Microbiological 4 Produced by Associates, mature schizonts as well as uninucleate trophozoites. Bethesda, Maryland.

6

DAVIS,

HUFF,

A$i i:3 usually the case in tissue culture studi Lesin which a gas overlays the free surface of the liquid medium, the cells in our cultu Ire:; were undifferentiated. It was, there-

AND

PALMER

fore, impossible to determine which 1LYl)es of cells were parasitized. Success in culturing P, gallinaceum in T- 30 flasks, equal to that of P. fallax, has Ilot ; beLen

of turkey brain culture containing exoerythrocytic stages of Plasmodiu m fall’ ax. FIG . 1. Photomicrograph Parasi ites shown range from immature to mature schizonts. Free merozoites are visible on the surfa Se of 1the cell skleet . This figure was taken with a 3S-mm camera. X 1180.

EXOERYTHROCYTIC

PhSmOctiUm

attained. Primary cultures from infected 14day chick embryos may be obtained, especially when chick serum is substituted for fetal calf serum in preparing the LDGM. However, we have been unable to subculture from these cultures, to date, with any success. Turkey embryos have been infected with gallinaceum and the turkey brain subsequently subcultured in LDGM with chick serum. At the time of this writing, these cultures are 8 weeks old and in their third subculture. Parasites are sparse, but present. Further modifications are apparently necessary in the components of the medium. DISCUSSION

At present, there is no quantitative standardization in our method of culturing cells. It will become increasingly necessary to set up cultures with a known number of cells in order to form a base line against which to measure culture growth, percentage of cells parasitized, and number of parasites introduced. Clones should be derived from the hostcell line and the parasite. Apparently, not all cells of the vertebrate body are susceptible to invasion by the exoerythrocytic stage of malaria. Therefore, we must assume that some cells in any given culture will be resistant or insusceptible to infection. If clones are derived from susceptible cells, then a very reliable foundation will have been established on which to base the percentage of cells parasitized as well as the degrees of infectivity of the merozoites. It should be noted that successful use of this method, over more than short periods of time, still is dependent upon the parallel maintenance of a strain of exoerythrocytic stages in turkey embryos. At the present stage of development of the method it is unrealistic to plan to use subcultures in vitro indefinitely without having the embryo-toembryo line available, in case the subcultures fail to grow. It is hoped that the culture method de-

IN

TISSUE

CULTURE

7

scribed above will provide a useful tool in several areas of malariology. Aikawa et al. (1965) have successfully used cultures produced by this method for descriptive work, utilizing the electron microscope. Studies in chemotherapy are enhanced in that the parasites may be viewed and photographed while under direct influence of drugs. Studies in physiology and genetics should also benefit from these procedures. ACKNOWLEDGMENTS

The authors wish to express their appreciation to Mr. S. J. Marsden of the Poultry Research Branch, Agricultural Research Service, Beltsville, Maryland, for his excellent cooperation in supplying turkey embryos and poults, and to Mr. F. Mitchell, Mr. T. K. Ruebush, and Miss J. Hartgrove of this Institute for their valuable laboratory assistance. REFERENCES M., HEPLER, P. K., HUFF, C. G., AND SPRINZ, H. 1966. The feeding mechanism of avian malarial parasites. Journal of Cell Biology 28, 355-373. needs of mammalian EAGLE, H. 1955. Nutritional cells in tissue culture. Science 123, 501-504 [Ref. 2 in Table 11. EAGLE, H. 1959. Amino acid metabolism in mammalian cell cultures. Science 130, 432-437 [Ref. 7 in Table Il. HAWKING, F. 1951. Tissue culture of plasmodia. British Medical Bulletin 8, 16-21. HUFF, C. G. 1964. Cultivation of the exoerythrocytic stages of malarial parasites. American Journal of Tropical Medicine and Hygiene 13, 171-177. JENSEN, D. V., HUFF, C. G., AND SHIROISHI, T. 1964. Advantages in the use of multipurpose chambers and dialysis membranes in the cultivation of exoerythrocytic stages of avian malarial parasites. American Journal of Tyopical Medicine and Hygiene 13, 653-6.58. LIEBOWITZ, A. 1963. The growth and maintenance of tissue-cell cultures in free gas exchange with the atmosphere. American Journal of Hygiene 78, 173-180 [Ref. 5 in Table Il. MCQUILKEN, W. T., EVANS, V. J., and EARLE, W. R. 1957. The adaptation of additional lines of NCTC, Clone 929 (Strain L) cells to chemically defined protein-free medium NCTC-109. 19, Journal of the National Cancer Institute 885-907 [Ref. 1 in Table Il. AIKAWA,

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DAVIS,

HUFF,

MORGAN, J. F., MORTON, H. J., and PARKER, R. C. 1950. Nutrition of animal cells in tissue culture. I. Initial studies on a synthetic medium (17557). Proceedings of the Society for Experimental Biology and Medicine 73, 1-8. [Ref. 3 in Table

Il. ASSOCIATES, INC. Personal communication to A. G. Davis. [Ref. 9 in Table I]. PIPKIN, A. C., AND JENSEN, D. V. 1958. Avian embryos and tissue culture in the study of parasitic protozoa. I. Malarial parasites. Experimental Parasitology 7, 491-530. PORTER, R. J. 1948. Studies in tissue culture of exoerythrocytic schizogony in avian malarial parasites. Jouvnl of Parasitology 34, 300-305.

MICROBIOLOGICAL

AND

PALMER

T. T., CIECIURA, S. J., AND ROBINSON, A. 1958. Genetics of somatic mammalian cells. III. Long-term cultivation of euploid cells from human and animal subjects. Journal of Experimental Medicine 108, 945-955. [Ref. 4 in Table Il. SCHERER, W. F. 1953. The utilization of a pure strain of mammalian cells (Earle) for the cultivation of viruses in vitro. American Journal of Pathology 29, 133 [Ref. 8 in Table Il. WAYMOUTH, C. 1959. Rapid proliferation of sublines of NCTC Clone 929 (Strain L) mouse cells in a simple chemically defined medium (MB 752/I). Journal of the Xational Cancer Institute 22, 1003-1017 [Ref. 6 in Table I]. PUCK,