Table Of ContentCulturing and Biological Cloning
of llypanosoma brucei
Mark Carrington
1. Introduction
Any biochemical analysis is usually made easier by the availabil-
ity of large numbers of cells to be analyzed, and one of the reasons
for the position held by Trypanusoma brucei as the best character-
ized parasite is the relative ease with which it can be cultured in the
laboratory. The ability to culture cloned populations derived from
individual trypanosomes before and after an antigenic switch is vital
in investigations into the mechanism of antigenic variation. Genomic
DNA prepared from such cloned populations used to analyze variant
specific glycoprotein (VSG) genes by Southern blotting led to the
discovery of the genomic rearrangements involved in antigenic varia-
tion (I-5).
This chapter will describe the growth of trypanosomes in labora-
tory rodents. The techniques that this involves start with the growth
from a frozen stabilate, which may be a field isolate, and the prepara-
tion of further stabilatesf rom infected blood. The basis of the preparation
of large numbers of trypanosomes (1 x 109-5 x lOto cells) from blood
is the retention of blood cells on a DEAE-cellulose column because
of their surface negative charge, while trypanosomes pass through
(6,7). These cells then provide the basis for further study, such as the
preparation of DNA or RNA (see Chapter 8), the purification of the
VSG or other protein, and metabolic labeling.
From Methods m Molecular Biology, Vol 21’ Protocols m Molecular Parasitology
Edlted by John E Hyde Copynght 01993 Humana Press Inc , Totowa, NJ
1
2 Carrington
Populations that are homogeneous for a single VSG are prepared
by cloning an individual trypanosome. This is achieved by infecting
a mouse with a single trypanosome; the VSG expressed usually remains
homogeneous for several syringe passagest hrough lethally irradiated
mice (8,9).
To investigate antigenic variation it is necessary to establish con-
ditions in which a relapse peak of parasitemia will occur. This can
be achieved by infecting a rabbit, which leads to a chronic relapsing
parasitemia (8), or by infecting a rat with a small number of para-
sites, in which case a second peak will occur (9). Parasites cloned
from a relapse population will express a different VSG from the iso-
late used to infect the ammal.
The metabolic labeling of trypanosomes has been used to investi-
gate the kinetics of synthesis of VSGs (10-12) and the covalent modi-
fication of the mature C-terminus with a glycosylphosphatidylinositol
(GPI) moiety, specifically in this context the identification of the fatty
acyl component as myristate (13,14). VSG metabolically labeled with
[3H]-myristic acid has been used as a substrate to identify GPI-spe-
cific phospholipase C (15-18).
Procyclic form trypanosomes representing the life cycle stage found
in the midgut of the tsetse fly vector can be readily propagated in
culture using the culture medium SDM-79 (19). One aspect not cov-
ered in this chapter is the growth of bloodstream trypanosomes in cul-
ture (see ref. 20 for a recent use of this technique). It is worth noting
that for most purposes growth in rodents is the only practically fea-
sible protocol as bloodstream forms are not as amenable to culture as
procyclics.
2. Materials
2.1. Growth and Maintainance
of Bloodstream Trypanosomes
1. Trypanosome dilution buffer (TDB): 20 mM Na2HP04, 2 mM NaH2P04,
80 mA4 NaCl, 5 mM KCl, 1 mM MgS04, 20 mit4 glucose, pH 7.7.
2. TDB + FCS: TDB containing 10% (v/v) heat inactivated fetal calf serum.
3. TDB containing 20% (v/v) glycerol.
4. Citrate glucose anticoagulant (CGA): 100 mM tri-sodium citrate, 40
mA4 glucose, pH 7.3.
Trypanosoma brucei 3
5. Separation buffer (SB): 57 mMNa2HP0,, 3 rniV NaH,PO,, 44 mMNaC1,
4 mA4 KCl, 5 mit4 glucose, 80 mM sucrose, pH 8.0.
6. DEAE-cellulose (preswollen Whatman DE52): This should be resus-
pended m SB and equilibrated to pH 8.0 with orthophosphoric acid.
7. Disposable 1-mL, 2-mL, and 20-mL syrmges, 15 x 0.5 mm (25-g),
25 x 0.6 mm (23-g), and 25 x 0.8 mm (21-g) needles.
8. Siliconized cavity microscope slides; the slides are placed m a desicca-
tor together with 5 mL 2% (v/v) dichlorodimethylsilane in 1,2,3-
trichloroethane and the desiccator is evacuated for 1 min. The slides
are then removed and baked at 100°C for 1 h.
9. Laboratory rats and mice.
10. A facility to lethally irradiate laboratory mice (850-900 rads); the mtce
must be irradiated during the 24 h prior to infection.
11 Glass capillary tubes (50-100 pL vol), Crystaseal (glass sealant)
(Hawksley Ltd, Lancing, Sussex, UK), and plastic cryopreservatron
tubes mto which the capillaries will fit.
12. Neubauer improved hemocytometera nd microscope to estimatep arasiterma.
2.2. Metabolic Labeling
of Bloodstream Trypanosomes
1. Modified Eagle’s Minimal Essential Medium minus methionine: Eagle’s
Minimal Essential Medium (Gibco 320- 190 formulation) without methion-
me and without vitamms. To this are added: Eagle’s Minimal Essential
Medium nonessenttal ammo acids to 1X concentration, 0.03% (w/v)
glucose, 0.1% (w/v) bovine serum albumin, 1% (w/v) glucose, 25 mM
HEPES, pH 7.4 (see ref. II).
2. [35S]-methionme, >lOOO Ci/mmol.
3. RPM1 1640 medium supplemented with 1% (w/v) fatty acid free bovine
serum albumin, 25 mM HEPES, pH 7.4.
4. [9, 10-3H]-myristic acid, >50 Ci/mmol.
5. 2% (w/v) sodmm dodecyl sulfate (SDS).
6. 1 mg/mL bovine serum albumin.
7 20% (w/v) trichloroacetic acid.
8. 10% (w/v) trichloroacetic acid.
2.3. Culture of Procyclic Trypanosomes
1. SDM-79 medium (19). Unfortunately this is not commercially avail-
able so the components are given in Table 1.
2. Hemm stock: 2.5 g/L hemm dissolved in 50 mM NaOH; autoclave to
ensure sterility
Carrington
Table 1
Components of SDM-79 Medium
for the Culture of Procychc Trypanosomes
Component Amount/L
MEM (Earle’s) powder (Gibco 072-1400A) 7.0 g
Medium 199 powder (Gibco 071-l 100A) 2.0 g
50X MEM essential amino acids
(Gibco 043- 1130H) 80mL
100X MEM nonessentral amino acids
(Gibco 043- 1140H) 60mL
Glucose log
HEPES (sodium salt) 8.0 g
MOPS (free acid) 5og
Sodium hydrogen carbonate 2.0 g
Sodmm pyruvate 100 mg
L-Alanme 200 mg
L-Argmine 100 mg
L-Glutamine 300 mg
L-Methronine 70 mg
L-Phenylalanine 80 mg
L-Proline 600 mg
L-Serine 60 mg
Taurine 160 mg
L-Threonine 350 mg
L-Tyrosine 100 mg
Guanosine 10 mg
Folic acid 4mg
Glucosamine hydrochloride 50 mg
p-Aminobenzoic acrd 2w
Biotin 02mg
All components should be tissue culture grade After all the components
have dissolved, adjust the pH to 7 3 with 2M NaOH and filter sterilize. Add 3
mL/L hemm stock and 10% (v/v) heat inactrvated fetal or newborn calf serum
to complete the medmm The medtum has a shelf life of about 2-3 mo at 4°C
before addltlon of serum and <1 mo after
3. Fetal or newborn calf serum that has been screened to ensure it sup-
ports the growth of procyclic trypanosomes.
4. Sterile plastic tissue culture flasks, 25-225 cm* size, depending on the
volume of culture needed.
5. 27°C incubator and facilities for sterrle manipulatrons.
6. 1%mL cryopreservation vials.
7. 60% (v/v) glycerol (tissue culture grade).
Trypanosoma brucei 5
3. Methods
3.1. Growth of Trypanosomes
in Laboratory Rodents
The proper methods and instructions on exsanguination of mice
and rats should be determined by consultation with the appropriate
local authority. Permission and training to perform the other proce-
dures on animals outlined here should also be obtained.
The doses of parasites given in the methods have been used for the
following mice and rats: (BALBK x CBA) Fl mice 20-25 g, CFLP
mice 20-30 g, and CFY rats 300400 g. It is not necessary to use these
strains, but the initial inoculum of trypanosomes may have to be varied
in order to achieve the required growth rate, because even within one
strain of rats the growth rate of trypanosomes expressing different
VSGs varies.
3.1.1. Infection of a Mouse
with Trypanosomes from a Frozen Stabilate
1. Remove the stabrlatef rom liquid nitrogen, thaw rapidly in a 37°C water
bath, break off the sealede nd, add to 1 mL TDB, and mix.
2. Estimate the number of trypanosomes/mL using a hemocytometer (see
Note 1).
3. Infect two mice, one with 5 x 105, and one with 1 x lo6 trypanosomes
by intraperitoneal Injection of the appropriate volume using a I-mL
syringe and 15 x 0.5 mm (25-g) needle (see Note 2). If a cloned pop-
ulation of trypanosomes is being propagated, a lethally irradiated (850
rads) mouse should be used to prevent any chance of an immune
response.
4. Follow the mfection by estimatmg the density of trypanosomes in the blood
obtained from tail bleeds. The parasitemia should reach l-5 x lO*/mL
after 3 d if a rodent-adapted laboratory strain is being used.
3.1.2. Preparation of a Frozen Stabilate
1, Infect a mouse as in Section 3.1.1.; if a cloned population of trypano-
somes is being propagated, a lethally irradiated (850-900 rads) mouse
should be used.
2. Allow the parasitemia to develop for 3 d; it should reach l-5 x lO*/mL.
3. Anesthettze the mouse and exsanguinate into a 2-mL syringe contain-
ing 0.2 mL CGA. It should be possible to recover l-2 mL of blood
from a 30-g mouse.
6 Carrington
4. Transfer the blood to a tube on ice and add an equal volume of ice-cold
TDB + 20% glycerol.
5. Fill glass capillaries with this mixture and seal one end with Crystaseal
and place the capillaries into a plastic cryopreservatlon tube on ice.
6. Once sufficient stabilates have been made, cool the tube(s) slowly by
placing them in the gas phase above llquld mtrogen in a Dewar flask.
After at least 3 h submerge the tube(s) for long-term storage.
7. After 1 wk check the viability of the stabllate by infecting a mouse as m
Sectlon 3.1.1.
3.1.3. Large Scale Preparation
of Trypanosomes from Blood
1. Infect a mouse and follow the level of parasltemia as in Section 3.1.1
If a cloned population of trypanosomes 1s bemg propagated, a lethally
irradiated (850-900 rads) mouse should be used. Within 3 d the den-
sity of trypanosomes m blood should be >l x lO*/mL. Exsanguinate
and estimate the density of trypanosomes, then dilute to 3 x 107/mL
with TDB.
2. Rats are anesthetized prior to infection with 3 x lo7 trypanosomes by
mtraperitoneal mjectlon using a l-r& syringe and a 0.6-mm (23-g)
needle (see Note 3). Follow the parasltemla by viewing blood from tall
bleeds under a microscope. Three days after infection the parasltemla
should be >3 x lO*/mL.
3. Prepare a DEAE-cellulose column before collectmg the blood. This can
conveniently be poured m a 50-mL syringe using glass wool to block
the flow of column matrix out of the bottom. The volume of DEAE-
cellulose used will be determined by the number of rats, but as a guide
a 20-mL column is usually sufficient for three rats. Equilibrate the col-
umn by passing through 5-10 vol of SB.
4. The rats are exsangumated using a 20-mL syringe containing 2 mL CGA.
It 1s usually possible to recover lo-15 mL of blood from a 300-g rat.
Ensure that the CGA and blood mix to prevent clotting. Transfer the
blood to a glass centrifuge tube on ice; leave on ice until the blood has
been collected from all of the rats.
5. Centrifuge the blood (750g for 10 mm at 4°C); there should be a discrete
whitish layer comprlsmg mainly trypanosomes overlaymg the sedl-
mented red blood cells. Remove and discard the serum from above the
trypanosomes and carefully layer ice-cold SB on top of the trypano-
some layer. Usmg a Pasteur plpet and a gentle swirling action, resus-
pend the trypanosomes with minimal disturbance of the red blood cells.
Transfer the suspension to a fresh centrifuge tube on Ice.
Trypanosoma brucei 7
6. Repeat the centrlfugation and resuspension. Two cycles are usually
enough to remove most of the erythrocytes and serum protein. Keep the
suspension on ice.
7. Apply the suspension to the DEAE-cellulose column; keep the column
flowing by adding SB to the top as necessary.O nly the trypanosomes will
pass through the column, leukocytes and erythrocytes are retained on
the column. Estimate the yield of trypanosomes using a hemocytometer.
3.2. Cloning and Generation
of Antigenic Variants
3.2.1. Cloning of Bloodstream Form Trypanosomes
1. The parasitemia m the infected animal is followed and blood is col-
lected when there are more than 1 x lo* trypanosomes/mL. A fraction
enriched m trypanosomes is prepared by centrifuging 1 mL of blood in
a mlcrofuge (12,OOOg)f or 1 min, removing the serum and resuspendmg
the whitish trypanosome layer in 1 mL TDB with mimmal disturbance
of the red blood cells.
2. Estimate the density of trypanosomes and dilute to 1 x 103/mL with
TDB + FCS. Place one l-pL drop m the cavity of a cavity slide and
inspect using a microscope. The whole drop should be wlthm the field
of view. If two observers agree that there is only a single trypanosome
within the drop, add 0.3 mL TDB + FCS. Recover this into a I-mL
syringe and Inject intraperitoneally mto a lethally irradiated mouse.
3. After 3 d exsangumate the mouse (see Section 3.1.2.) and estimate the
parasitemla. Use the blood to infect a further lethally Irradiated mouse
(see Section 3.1.1.). Continue in this way until the parasitemia is above
1 x lo* on the third day after infection. This usually occurs m the sec-
ond mouse and rarely requires the use of a third serial passage.
4. If antlbodies are available, the homogeneity can be checked by lmmu-
nofluorescence microscopy (ref. 9; see also Chapter 31). The population
is usually more than 99.9% homogeneous with respect to the VSG (9). If
antibodies are not available then they should be raised against VSG puri-
fied from the cloned population (8,21). If homogeneous, the VSG and anti-
serum should produce a single preclpitin arc m an immunodiffusion assay.
3.2.2. Creating Relapse Populations
3.2.2.1. ESTABLISHMENT OF A CHRONIC INFECTION IN A RABBIT
This method was used to generate the cloned antigenic variants first
isolated from the MITaR 1 serodeme (8). When a rabbit is infected a
chronic relapsing parasitemia occurs, and samples of blood taken at
8 Carrington
intervals of more than 1 wk should contain a series of different anti-
genie types. Individual trypanosomes can then be cloned and expanded
in mice.
1. Infect a lethally irradtated mouse, follow the parasitemia and exsan-
guinate after 3 d (see Section 3.1.2.). Determine the density of trypan-
osomes in the recovered blood using a hemocytometer.
2. Use these trypanosomest o inject a rabbit; an inoculum of 1.5 x lo*
trypanosomes has been successfully used with a 2.5-kg New Zealand
White. This should not need to be adjusted for other breeds, however
the time taken for the first peak of parasrtemia to occur may vary.
3. At intervals, collect approx 2 mL of blood from an ear vein and mea-
sure the parasitemia If the parasitemia is >l 10*/n& clone immedi-
x
ately (see Section 3 2 1.) If necessary, the population can be expanded
by infecting a lethally irradiated mouse before cloning mdividuals.
3.2.2.2. INFECTION OF RATS WITH A SMALLI NOCULLJM
This represents an alternative method and has been used to gener-
ate cloned populations in the ILTaR 1 serodeme (9). If a rat is infected
with a small number of trypanosomes it usually survives the first peak
of parasitemia; the second peak is lethal but comprises novel anti-
genie types.
1. Infect a lethally irradiated mouse, follow the parasitemta and exsan-
gumate after 3 d (see Section 3.1.2.). Determine the density of trypano-
somes in the recovered blood using a hemocytometer.
2. Dilute the trypanosomes with TDB to 100 cells/ml and inoculate a rat
intraperitoneally with 0.1 mL.
3. Follow the parasitemia to ensure that one peak occurs and exsangui-
nate the rat when the second peak rises above 1 x lo* trypanosomes/mL
of blood.
4 Clone individual trypanosomes from this blood (see Section 3.2.1.).
3.3. Metabolic Labeling
of BZoodstream Form Trypanosomes
1. The trypanosomes are separated from blood usmg DEAE-cellulose (see
Section 3.1.3.).
2. After passage of the trypanosomes through the DEAE-cellulose, the
cells are washed once in labeling medium; recover the cells from the
column eluate by centrifugation (750g for 10 mm). Discard the super-
natant, resuspend the cells in labeling medium and centrifuge again (750g
Trypanosoma brucei 9
for 10 mm). Resuspend the cells in the relevant labeling medium at the
desired density. The medium is determined by the type of metaboltc
labeling (see Note 5).
3. a. [35S]-Methionme. The labeling medium used is Modified Eagle’s
Minimal Essential Medium minus methionine (see Note 6). After the
wash, resuspend the cells at 3 x 107/mL in this labeling medium. Add
[35S]-methionmet o 100 pCl/mL and incubate at 37OCi n a shaking water-
bath. This mcubation should not exceed 3 h.
b. [9, 10-3H]-Myristic acid: The labeling medium used is RPM1 1640
medium supplemented with 1% bovine serum albumin and 25 mM
HEPES, pH 7.4 (see Note 7). After one wash resuspend the cells at
5 x 107/mL m labeling medium, incubate for 15 min at 37°C in a shak-
ing waterbath, then add the [9, 10-3H]-myristic acid to 100 pCi/mL
and continue the incubation at 37°C m a shaking waterbath. This incu-
bation should not exceed 3 h. The [9, 10-3H]-myristic acid is prepared
by evaporating the solvent (usually toluene) using a stream of nitro-
gen and dissolving the myristic acid in a small volume (1 pIJ10 l&i)
of water. This should contain an amount of fatty acid-free bovine
serum albumin such that there are equal molar amounts of bovine
serum albumin and myristic acid.
c. [3H]-Sugars/nucleotides: The same protocol is used as in 3b above
except that the desired [3H]- sugar, dissolved m water, is added instead
(see ref. 22 for a recent example); in this paper the RPM1 1640, based
labeling medium contained 3 g/L glycerol in addition to the bovine
serum albumin and HEPES.
4. In all cases incorporation of radiolabel mto macromolecules can be
determined by removing 50 pL samples at surtable time points. The
sample is added to 50 pL of 2% SDS and immediately incubated at
100°C for 3 min. Add 50 pL of this lysate to 450 pL of 1 mg/mL bovine
serum albumin (as a carrier), then add 500 pL 20% trichloroacetic acid.
After 10 min at room temperature collect the precipitate by filtration
onto glass fiber disks, wash with 10% trichloroacetic acid, dry the disks,
add scmtillant, and count the incorporated radiolabel.
3.4. Culture of Procyclic Trypanosomes
3.4.1. Routine Maintenance of Procyclic Cultures
The growth of procyclic trypanosomes in culture is straightforward.
The cells are subcultured to a density of 1 x lO%nL and will grow to
approx 3 x 107/mL. Cell density is estimated using a hemocytometer
(seeN ote 1). The cells can be grown in tissue culture flasks (0.4 r&/cm2
10 Carrington
area), spinner flasks, or even conical flasks in orbital incubators. The
growth rate varies between different isolates and growth conditions,
but doubling should occur between 10 and 24 h.
3.4.2. Preparation of Frozen Stabilates
As with bloodstream trypanosomes, procyclic forms can be kept as
frozen stabilates in liquid nitrogen.
1. In a 1.8-mL cryopreservation vial mix 0.2 mL 60% (v/v) glycerol and
0.4 mL SDM-79. Add 0.6 mL of a late log phase culture of procychc
trypanosomes (1 S-2 X 107/mL).
2. Cool to liquid nitrogen temperature as in Sectton 3.1.2.
3 To resuscitate the culture, thaw the vial rapidly at 37OC and add the
contents to 9 mL SDM-79 at 28OC in a 25 cm2 tissue culture flask. The
trypanosomes should be motile immediately when the culture is viewed
wtth an inverted microscope.
3.4.3. Metabolic Labeling of Procyclic Trypanosomes
Procyclic trypanosomes can be metabolically labeled in SDM-79
medium. If the radiolabeled compound is normally presenti n the medium,
for example methionine, then a special batch of SDM-79 should be
made without the relevant component. The trypanosomes are washed
with the depleted medium by centrifugation (600g for 10 min) and resus-
pension in the depleted medium followed by centrifugation again
(600g for 10 min) and resuspensiona t the labeling density. If a depleted
medium is not needed, then the radioactive compound can be added
directly to a culture.
4. Notes
1. Since trypanosomes are motile it is often difficult to obtain a count
using a hemocytometer. An easy way around this is to make dilutions
of the cells m TDB containing 0.1% formaldehyde. A Neubauer
hemocytometer 1su sed to count cells at around 1 x 106/mL so it 1su sual
to make at least one IO-fold dtlution of the trypanosome sample.
2. In some protocols for this step the mouse IS held firmly by the scruff of
the neck, so it is very important to use a short needle to reduce the
chances of the needle passmg through the mouse and into the
experimenter’s finger.
3. A common mistake here is to inject the trypanosomes between the skm
and the muscle wall of the peritoneum. It is important that the trypano-
somes are inJected into the peritoneum.
