Cultivation of bloodstream Trypanosoma brucei*

Bulletin of the World Health Organization, 55 (2-3): 405-409 (1977) Cultivation of bloodstream Trypanosoma brucei * H. HIRUMI,1 J. J. DOYLE,2 & K. HIRUMI 3 Animal-infective forms of Trypanosoma brucei (Strain 427) were successfully propa- gated in HEPES-buffered RPMI 1640 medium in the presence of bovine fibroblast-like cells for over 310 days. The organisms grown in this system were morphologically identical to the long slender bloodstream forms, retained their infectivity for mammalian hosts, and displayed variant-antigen on their surface. Technical details for establishing such blood- stream form cultures are described in the present paper. Salivarian trypanosomes undergo a complex life cycle, involving final (mammalian) and intermediate (arthropod) hosts. In the mammalian hosts, blood- stream forms of the polytypic subgenus Trypanozoon, such as of Trypanosoma brucei, develop from the long slender form to the intermediate form and sub- sequently to the stumpy form as parasitaemia in- creases. This transformation is often periodic with successive populations of parasites, each being recognized by the host's immune response as anti- genically different. In the arthropod vectors, Glossina spp., the blood- stream forms ingested with blood meals rapidly lose their infectivity for mammals and undergo further development. The bloodstream forms first transform to midgut trypomastigote forms (insect midgut forms) and then to proventricular trypomastigote forms (insect proventricular forms) from which they develop to epimastigote forms after entering the salivary glands. The epimastigotes finally transform into metatrypanosomes (metacyclic forms) and regain their infectivity for mammalian hosts. In earlier attempts at in vitro propagation of the Salivaria, bloodstream forms of T. brucei rapidly transformed into the insect midgut and/or pro- ventricular forms in cultures and became non- infective for mammalian hosts. They rarely retained the characteristics of the bloodstream forms and then only for a short time (1, 2, 3, 4, 5). Despite numerous attempts by many investigators over the last seventy years, it has not previously * From the International Laboratory for Research on Animal Diseases (ILRAD), P.O. Box 30709, Nairobi, Kenya. This paper has been approved for publication as ILRAD Journal Series No. 5. 1 Scientist. ' Associate Scientist. ' Research Assistant. proved possible to cultivate the animal-infective bloodstream form of T. brucei for long periods. This inability to grow the animal-infective trypanosomes has been one of the major hindrances preventing faster advances in the control of African trypano- somiasis. Recently, we have developed a laboratory method that supports the long-term in vitro cultivation of bloodstream T. brucei (Strain 427) in the presence of bovine fibroblast-like cells (6). The organisms continued to grow in this system with HEPES- buffered RPMI 1640 medium for over 310 days and were morphologically indistinguishable from blood- stream forms grown in animal hosts. They retained their infectivity for mammals for long periods (Fig. 1). The latest infectivity test revealed that the organisms propagated in vitro for 315 days were still infective for mice. The ability to grow the animal-infective blood- stream forms of the parasite in the laboratory makes it possible to obtain large numbers of suitable organisms for use in the development of potential vaccines. Furthermore, the ability to manipulate the organisms outside of host animals under controlled laboratory conditions makes it possible to undertake 0 A 0 30 60 90 120 150 80 210 230 DAYS AFTER THE INITIATION OF THE ORIGINAL CULTURE 0 30 55 a Infective for lethally irradiated C57BL mice DAYS A Infective for yearling Boron Cross steer 0 Negative for normal C57BL mice * Infective for normal C57BL mice Fig. 1. Ability of bloodstream T. brucei propagated in vitro (370C) to infect mammals. 3622 -405- 406 H. HIRUMI ET AL. research on the make-up of the organisms, the mechanisms of " antigenic variation " and other important problems such as the development of effective drugs for control of the disease. The present paper is not intended to be a review article but provides technical details that have proved of value in establishing long-term cultures of the bloodstream forms of T. brucei. MATERIALS Donor mice If antigenically homogeneous populations of trypanosomes are needed, the parasites originating from a clone should be prepared in lethally irradiated inbred mice; otherwise, healthy inbred mice may be used as the donors. Trypanosomes Parasitaemia in donors. In order to establish successful cultures of the bloodstream forms, it was essential to initiate the original culture with a try- panosome population of which the majority were long slender bloodstream forms, and which con- tained a number of actively dividing forms. Such populations were obtained from an infected mouse with rising parasitaemia. Since the course of infection depends upon the virulence of the strain used, the susceptibility of the host animals, the number of infective parasites inoculated, as well as many other factors, the degree of parasitaemia was examined every 24 hours after inoculation. Twenty consecutive microscope fields of a wet blood-film prepared from a drop of tail blood on a slide glass and covered with a cover glass (24 mm x 24 mm) were usually examined at x 400 magnifica- tion. The most successful long-term cultivation of bloodstream T. brucei was generally achieved with parasite populations prepared from a rising parasi- taemia containing 20-50 trypanosomes/field. Para- site populations obtained from an animal with high parasitaemia (100 or more trypanosomes/field) usually gave poor results, although it was not impossible to establish cultures from such a popula- tion. The number of parasites obtained from an animal with lower parasitaemia (less than 10 try- panosomes/field) was found to be inadequate to initiate cultures. Separation. Blood materials (0.2-0.5 ml/mouse) were collected from the hearts of infected mice, using 1-ml tuberculin syringes with a 25 x 3/8-inch (approx. 0.95 cm) hypodermic needle. Prior to blood collection, the inside of the syringe was rinsed with a heparin solution (1000 units/ml in Hanks' Balanced Salt Solution, HBSS). The heparin solution was discarded except for a drop that remained in the neck of the syringe. The blood samples were mixed with cold HBSS (1:1). The trypanosomes were separated either by density gradient centrifugation (Lymphoprep density 1.077 g/ml) at 350 g for 15 min at 4°C or by forming a buffy coat. No significant differences were seen between the two methods of preparation with respect to the ability of the organisms to grow in culture. Following separation, the trypanosomes were washed three times with cold HBSS by sequential centrifugation at 850 g for 5 min at 4°C. HBSS used for the separation and washing steps could be re- placed with the culture medium described below but without a fetal bovine serum supplement. Bovine fibroblast-like cells A cell-free medium system that supports the in vitro propagation of bloodstream forms of the Salivarian trypanosomes would, of course, have great advantages over that employing " host-cell layer" systems. However, at least at present, the presence of mammalian fibroblast-like cells a is essential to grow the bloodstream forms of T. brucei for extended periods. Healthy bovine fibroblast-like cell lines b were derived from freshly collected blood (Fig. 2). A pri- mary bovine cell culture was initiated in a T-75 Falcon plastic culture flask by resuspending the buffy coat cells obtained from 20 ml of blood in 20 ml of Roswell Park Memorial Institute (RPMI) 1640 medium containing 20% heat-inactivated fetal bovine serum and incubated at 37°C. During the first 18 h, most of the macrophages adhered to the surface of the original culture flask while other cell types remained suspended in the medium. The cell suspension was transferred to a new T-75 culture flask after 18 h and an additional a Fibroblast-like cells: both the histological origin and the function of this cell-type in culture is unknown, but it resembles a fibroblast cell in form and characteristic ap- pearance. b Cell line: a cell line arises from a primary culture at the time of the first subculture. The term cell line implies that cultures from it consist of numerous lineages of the cells originally present in the primary culture. The terms " finite " and " continuous " should be used as prefixes if the status of the culture is known. If not, the term " line " should suffice. The term " continuous line " replaces the term " established line ". (TCA News Letter, Vol. 10 (4), Sept.- Oct., 1976.) CULTIVATION OF BLOODSTREAM TRYPANOSOMA BRUCEI 407 20 ml of fresh medium was added. Thus, this second flask contained approximately 40 ml of medium. During the next 14 days, fibroblast-like cells, lymphocytes, and some macrophages attached to the surface of the new flask and increased in number. Fibroblast-like cells tended to adhere more firmly to the culture flask than the other cells, which were removed by repeated mild trypsinizations during this period. When fibroblast-like cells became predominant in the culture, a subculture was made using a trypsin-EDTA mixture by conventional means. Following four successive serial subcultures, a homologus fibroblast-like cell line, designated as ILR-BHF-476, was obtained (Fig. 2). Since the life-span of the bovine fibroblast-like cell line is unknown at present, new cell lines have been initiated every 3-4 months. Other continuous vertebrate cell lines, such as a mouse L-cell line and a dog kidney cell line, were also found to be adequate in maintaining the blood- Healthy Bovine Blood Buffy___j Resuspend in Coat . RPMI 1640 Mediun 0Macrophages 37 C for 24 Hrs Transfer ail Culture Fluld Initial Flask New Flask 's 37C 10-14 Days Repeated LZJLYItiTrypsinization Serial Subcultivations ILR-BHF-476 Fig. 2. Establishment of the ILR-BHF-476 cell line. stream forms of T. brucei previously adapted to in vitro conditions in the presence of the bovine fibroblast-like cell line. They were, however, far inferior to the bovine fibroblast-like cell system in their ability to support the initial cultivation of the bloodstream forms. Since many continuous cell lines of vertebrate origin are currently available, it would be worth while to investigate their ability to support the initial cultivation of bloodstream forms of Salivarian trypanosomes in the future. CULTIVATION OF BLOODSTREAM FORMS Culture conditions A total of 96 different culture conditions (12 different media, 4 different concentrations of FBS, and in the presence or absence of bovine fibroblast- like cells) were initially tested at 37°C. The best growth rate of the bloodstream forms was obtained in HEPES-buffered (25 mmol/litre) RPMI 1640 medium with 20% of heat-inactivated FBS, penicillin (100 IU/ml) and streptomycin (50 ,ug/ml), at pH 7.2, a substance concentration of 290 mmol/kg,a in the presence of the bovine fibroblast-like cells (6). The dosages of antibiotics used seemed to have no significant effect on the ability of organisms to grow in culture. Initiation of the primary culture Primary cultures can be initiated in either Disposo- Trays b or T-25 Falcon plastic culture flasks con- taining the bovine fibroblast cells (6). A standard procedure has been established, based on informa- tion obtained from previous successful cultivations c as follows: (1) Collect T. brucei-infected blood from animals with rising parasitaemia (usually a total of 2-3ml from 8 infected mice, see page 406). (2) Separate and wash the bloodstream forms (see page 406 and Fig. 3). (3) Prepare a series of tenfold dilutions at con- centrations of 1-2 x 105, x 104, x 103, and x 102 trypanosomes/ml in cold culture medium. (4) Inoculate T-25 culture flasks containing bovine cells with the parasites (2 flasks/dilution, 6 ml/flask) and incubate the cultures at 370C. a Advanced DigiMatic Osmometer Model 3D, Advance Instruments, Inc., Needham Heights, MA, USA. Calibration was done using Clinitrol 290. b Linbro Chemical Co., New Haven, CT, USA. c Ten cultures of bloodstream T. brucei have been successfully established so far. 408 H. HIRUMI ET AL. Rising -/ Porasitoemia EBSS Buff L Coat Washing Resuspend EBSS Medium ILR-BHF-476 16ml/Flask Incubate at 37½C Cell Layer-.a. }1°/ M /FM 4 OM /FM / mmglm t mlY/3ml 24 Hrs I __________r_r___________f_______ * ~~~5-7 Days) 4 Subcultivation (24 Hrs) Fig. 3. Initiation of the primary culture. (5) Change the culture fluid and select the best culture during the first 7 days of cultivation. (6) Make subcultures from the best culture during this period (1: 1, 1: 2, 1: 3 or 1: 4 splits, depending on the population densities). Keep the density of both new and old cultures at 1-5 x 105/ml. (7) Establish long-term cultures of the blood- stream forms from populations having consistent growth rates 7-10 days after the initial cultivation. In general, 50-70 bovine cell cultures should be prepared during the initial period. Extreme care is necessary in the handling of the initial cultures as regards both the amount and timing of medium replacement and subcultivation. An example of initial cultivation is shown in Table 1. The number of parasites in cultures initiated with a high concentration (1.6 x 105/ml) usually reached a maximum density within 24 hours. In such cultures, trypanosomes became sluggish and large in size. On day 3, only very few dividing forms were seen. The number of parasites con- siderably decreased on day 5. They gradually died out by the 8th day of cultivation. On the other hand, the bloodstream forms in cultures initiated with low densities (1.6 x 102/ml) increased in number during the first 5 days of initial cultivation and reached concentrations of 4 x 105-1 x 106/ml on day 6 or 7. In these cul- tures, the majority of trypanosomes were long, slender bloodstream forms and numerous dividing forms were seen. Successful subcultures were often made from these cultures. The selection of cultures for subculturing should be based on (a) population density, (b) growth rate, (c) morphology (select cultures in which long, slender forms are predominant), (d) motility, (e) number of dividing forms, and (f) size distribution in the culture. The observations (a) and (b) can be made by counting; (c), (d), and (e) by using an inverted-phase contrast microscope with a long working-distance condenser; and (f) by Coulter Counter Channelizer (aperture 70 ,um). Maintenance of cultures After the establishment of cultures using this system, the bloodstream forms of T. brucei can be easily maintained by replacing a portion of the culture fluid with the fresh medium (usually 1: 1 or 1: 2 every 24 hours). Table 1. Counts of bloodstream Trypanosoma brucei in cultures during an early stage of the in vitro cultivation initiated with various densities (at 370 C) Initial Numbers of trypanosomes (x106/ml) a density at different times (h) after the initiation of culture (per ml) 24 48 72 96 120 144 1.6 x 105 7.5 7.9 5.1 7.0 2.6 D(3.1) (3.3) (2.6) (3.5) (1.3) 1.6 x 104 NC 5.7 5.9 8.7 6.7 4.6 (2.9) (3.0) (4.4) (3.4) (2.3) 1.6 x 103 NC NC 7.3 6.9 5.7 5.2(3.0) (3.5) (2.9) (2.6) 1.6 x 102 NC NC 3.5 NC 6.9 5.0(1.5) (3.5) (2.5) a By Coulter Counter ZeI; aperture: 70 gm; BCT: 10; WW: 40. The figures in parentheses are the numbers of trypanosomes after the medium was changed. D = discontinued; NC = not counted (no medium was changed). CULTIVATION OF BLOODSTREAM TRYPANOSOMA BRUCEI 409 In young cultures (1-7 days old), the majority of the trypanosomes were swimming in the culture medium and often entered into the intercellular spaces of the bovine cell layers in older cultures (8-14 days old). The timing of subcultivation should, therefore, depend on the objectives of the particular experiment. If cultures containing more than 5 x 105 trypa- nosomes/ml remain without a change of medium for more than 3 days, the slender forms often transformed into stumpy forms. If subcultures were initiated with a low density of trypanosomes (102/ml), it took 4-6 days to regain the population density of 105/ml without a change of medium. In these cultures, the trypanosomes retained the morphological characteristics of the long slender form. CRYOPRESERVATION Cultured bloodstream forms of T. brucei have been preserved in liquid nitrogen using either 7.5% dimethyl sulfoxide or 10% glycerol in RPMI 1640 medium with 20% FBS. TRANSPORT Cultures of the bloodstream forms have been successfully transported by air to two laboratories in England and Switzerland. Prior to shipment, fresh cultures were prepared in T-25 culture flasks and filled with fresh culture medium, avoiding any air bubbles. The cultures were carried by passengers in their hand luggage and reached the laboratories within 30 hours. Frozen cultures have not yet been tested for long distance shipment. ACKNOWLEDGEMENTS The authors would like to thank N. Bhaiji and J. Wandow for their technical assistance, and the Dean of the Faculty of Medicine of the University of Nairobi for laboratory facilities. We would also like to thank Dr K. Brunner of the Swiss Cancer Research Institute, Lausanne, Switzerland, for the supply of lethally irradiated mice, and Dr I. Schneider of the Walter Reed Army Institute of Research, Washington, DC, USA, for her critical review of the manuscript. RESUMIE CULTURE DE FORMES SANGUICOLES DE TRYPANOSOMA BRUCEI On a obtenu la multiplication, pendant plus de 310 jours, de formes de Trypanosoma brucei (souche 427) infectantes pour les animaux dans du RPMI 1640 tamponne a 1'HEPES en presence de cellules bovines de type fibroblastique. Les micro-organismes qui se sont d6veloppes dans ce milieu etaient morphologiquement identiques aux formes sanguicoles allongees, conservaient leur pouvoir infectant a 1'egard des mammiferes hotes, et laissaient apparaitre un antigene variant A leur surface. Le present article contient une description technique d6taillee des methodes de culture de ces formes sangui- coles. REFERENCES 1. CROSS, G. A. M. & MANNING, J. C. Parasitology, 67: 315 (1973). 2. TRAGER, W. Annals of tropical medicine and parasit- ology, 53: 473 (1959). 3. LEPAGE, R. W. F. Nature, 16: 1141 (1967). 4. MENDEZ, Y. & HONIGBERG, B. M. Journal ofparasit- ology, 58: 1122 (1972). 5. CUNNINGHAM, I. Experimental parasitology, 33: 34 (1973). 6. HIRUMI, H. ET AL. Science, 196: 992 (1977).