Experimental Parasitology 116 (2007) 171–174

www.elsevier.com/locate/yexpr

In vitro cultivation of Plasmodium falciparum: Studies

with modified medium supplemented with ALBUMAX II
and various animal sera
Kumkum Srivastava *, Shubhra Singh, Pratibha Singh, S.K. Puri
Division of Parasitology, Central Drug Research Institute, Lucknow, India

Received 4 May 2006; received in revised form 11 December 2006; accepted 15 December 2006
Available online 30 December 2006

Abstract

RPNI, a combination of three commercially available growth media (RPMI-1640, NCTC-135 and IMDM) has been found to support
long term continuous cultivation of 3D7 strain of Plasmodium falciparum in the presence of 10% bovine calf serum. During the present
study, the suitability of this medium was evaluated for the development of P. falciparum in the presence of horse, goat and rabbit sera as
well as various concentrations of ALBUMAX II. RPNI medium supplemented with 10% bovine calf serum (RPNI-BCS) was used as
control. The cultures were maintained in candle jars protocol and parasitaemia was monitored daily up to day 7. Horse, goat and rabbit
sera all supported the development of P. falciparum. Horse serum gave best results in RPNI medium and supported continuous culture
up to day 100. The parasitaemia in the presence of ALBUMAX was significantly higher in RPNI than in RPMI-1640. Addition of hypo-
xanthine in RPMI-1640 caused an increase in parasitaemia whereas no obvious advantage could be observed in RPNI. The findings
exhibited that medium RPNI has an edge over conventional RPMI-1640 medium for in vitro cultivation of P. falciparum.
 2006 Elsevier Inc. All rights reserved.

Index Descriptors and Abbreviations: Malaria; Protozoa; Horse serum; Rabbit serum; Goat serum; RPNI; RPMI- 1640; Hypoxanthine

1. Introduction supplementation with ALBUMAX and different animal

In vitro cultivation of malaria parasites (Trager and Jen-
sen, 1976) has become an easy method to screen com-
pounds against the target parasite. Recent findings have
shown that RPNI, a combination of three commercially
available growth media (RPMI-1640, NCTC-135 and
IMDM) supplemented with 10% bovine calf serum (BCS)
supports long term continuous cultivation of Plasmodium
falciparum (Srivastava and Puri, 2004) and overcomes the
risks associated with use of human sera. As RPNI is a
new combination of media, no information is available
about its utility when used with ALBUMAX or other
serum supplements. We have therefore investigated the
potential of this media for cultivation of P. falciparum after
*
Corresponding author. Fax: +91 522 223405.
E-mail address: kumkum1110@Yahoo.com (K. Srivastava).
sera.
2. Materials and methods
Studies were carried out mainly using laboratory main-
tained CHQ sensitive 3D7 strain of P. falciparum; however,
on a few occasions, different parasite lines (NF-54, JDP-8
and RKL-9) were also used. JDP-8 and RKL-9, the Indian
field isolates (Okoyeh et al., 1999) were obtained from
International Centre for Genetic Engineering and Biotech-
nology, New Delhi, India and maintained at C. D. R. I.
Lucknow. RPNI medium was prepared by combining
RPMI-1640, NCTC-135 and IMDM media (Sigma) as
described by Srivastava and Puri (2004). These three media
were prepared separately. RPMI-1640 powdered medium
was supplemented with Hepes buffer (25 mM), 0.2%
NaHCO3, gentamycin at 40 lg/ml; (Sigma) and Fungizone

0014-4894/$ - see front matter  2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.exppara.2006.12.003
172 K. Srivastava et al. / Experimental Parasitology 116 (2007) 171–174

at 0.25 lg/ml; (GIBCO). The NCTC-135 and IMDM

media were prepared as described by Franke et al. (1987).
Finally all three media were mixed in the ratio of 2:1:1
(RPMI-1640:NCTC:IMDM) to prepare RPNI and supple-
mented with either 10% commercially available (Gibco/Sig-
ma) goat/horse/rabbit serum or ALBUMAX II (Gibco) at
0.5%, 0.1%, 0.05% or 0.01% concentrations. Two different
batches of each serum source were used during the study.
The growth profiles of parasites cultured in conventional
(RPMI-1640) as well as modified (RPNI) medium supple-
mented with respective supplements were monitored. The Fig. 2. Effect of hypoxanthine on Plasmodium falciparum (3D7) in RPNI
parasitaemia was observed for seven consecutive days i.e. (NI) and RPMI-1640 (MI) supplemented with bovine calf serum (BCS).
at least for three asexual cycles.
Experiments were carried out in 24-well plates and each
well received 2 ml volume. Asynchronous culture main-
tained in RPNI-BCS was diluted by adding washed unin-
fected human erythrocytes and the corresponding
complete medium to achieve 0.5% parasitaemia and 6%
haematocrit. Cultures were maintained static in candle jars
at 37 C. The culture medium was replaced every 48 h. In
each test three wells were run for each supplement and
three such replicates were carried out on separate
occasions.
The parasitaemia was monitored daily up to day 7. A
drop of erythrocytes from the settled layer of the culture
was spread into thin film on a clean grease free glass slide.
It was air dried, fixed in methanol, stained with Giemsa stain
and examined under a light microscope (100· oil immer-
sion). Approximately 10,000 cells per smear were scanned
and percent parasitaemia was calculated as following
No: of parasitized erythrocytes  100
%Parasitaemia ¼
No: of erythrocytes

3. Results

Growth profiles of P. falciparum in both RPNI and
RPMI-1640 media supplemented with different concentra-
tions of ALBUMAX, hypoxanthine and different animal
Fig. 3. (a) Growth pattern of Plasmodium falciparum (3D7) in RPNI (NI)
medium supplemented with horse (HR), rabbit (Rb), goat (G) and bovine
calf serum (BCS) (b) Growth pattern of Plasmodium falciparum (3D7) in
RPMI-1640 (MI) medium supplemented with horse (HR), rabbit (Rb),
goat (G) and bovine calf serum (BCS).

Fig. 1. Growth pattern of Plasmodium falciparum (3D7) in RPMI-1640

and RPNI media supplemented with different concentrations of Fig. 4. Growth pattern of different parasite lines of Plasmodium falcipa-
ALBUMAX. rum in RPNI medium supplemented with bovine calf serum (BCS).
 K. Srivastava et al. / Experimental Parasitology 116 (2007) 171–174
sera are shown in Figs. 1–3a and b, respectively. Fig. 4 shows
growth profile of different parasite lines in RPNI medium
supplemented with bovine calf serum (BCS). It is evident
from Fig. 1 that parasitaemia increased continuously up
to day 7 in both RPNI and RPMI-1640 medium supple-
mented with different concentrations of ALBUMAX. The
maximum parasitaemia in RPNI medium ranged between
10 and 12% where as the corresponding values in RPMI-
1640 were 6 and 7%. Fig. 2 depicts the mean % parasitaemia
as observed in RPNI and RPMI-1640 (with bovine calf
serum) with or without hypoxanthine. It was observed that
in all four media combinations parasitaemia increased con-
tinuously up to day 7. The maximum parasitaemia as
observed on day 7 in RPNI medium with or without hypo-
xanthine was 6.9 (±1.3) and 7.0 (±1.3), respectively whereas
the respective values in RPMI-1640 were 5.5 (±0.44) and 4.0
(±0.7). Figs. 3a and b show the growth profiles of 3D7 strain
of P. falciparum in medium RPNI and RPMI-1640 supple-
mented with horse, rabbit, goat and bovine calf sera. In all
the cases except in RPMI-goat serum parasitaemia
increased continuously up to day 7. The best parasite
growth was observed with horse serum in RPNI medium
whereas in medium RPMI-1640, rabbit serum was found
most supportive. It was also interesting to note that horse
serum supplemented in RPNI medium could support long
term continuous culture for up to 100 days of observation
(data not shown). As horse serum was found to be the best
of the animal sera used, the same media combination was
further evaluated for growth and development of three more
parasite lines (Fig. 5). Figs. 4 and 5 depict the mean parasita-
emia of four parasite lines as observed in RPNI with bovine
calf serum and horse serum respectively. It is seen that in
both media combinations parasitaemia increased continu-
ously up to day 7. The pattern of parasitaemia of all the par-
asite lines was very similar.
4. Discussion
It has been established that RPNI medium has an
advantage over conventional medium RPMI-1640 as the
former supported the development of P. falciparum in the
presence of BCS (Srivastava and Puri, 2004). This medium
Fig. 5. Growth pattern of different parasite lines of Plasmodium falcipa-
rum in RPNI medium supplemented with horse serum (HR).
173
is rich in nutrients such as co-enzymes, nucleic acid deriva-
tives, ascorbic acid, glutathione, glucose, amino acids and
vitamins. The present study was under taken to observe
the suitability of RPNI medium for the development of
P. falciparum in the presence of horse, goat, or rabbit sera
as well as various concentrations of ALBUMAX. Horse,
goat and rabbit sera supported the growth and develop-
ment of P. falciparum irrespective of the media used but
better results were obtained with RPNI medium. The best
parasite growth was observed in RPNI medium supple-
mented with horse serum where as goat serum was found
least supportive. Earlier studies had also shown that horse
serum (Ramose et al., 1986) and individually collected rab-
bit serum (Sax and Rieckmann, 1980) supported for the
development of P. falciparum in RPMI-1640 medium after
prior adaptation for 2–4 weeks. In the present study, the
period of observation was only 7 days and thus no conclu-
sion can be drawn regarding prior adaptation.
ALBUMAX which is used at 0.5% concentration for
routine cultivation of P. falciparum (Ofulla et al., 1993;
Cranmer et al., 1997) was tried at lower concentrations.
The results revealed that of ALBUMAX concentration as
low as 0.01% can also be used for short term cultivation
of P. falciparum. Another investigation during present
study was to observe the effect of hypoxanthine, a purine,
used to improve parasitaemia in the presence of human
serum as well as ALBUMAX (Asahi and Kanazawa,
1994; Asahi et al., 1996; Cranmer et al., 1997). Malaria par-
asites need purines for their development and RPNI medi-
um contains purines other than hypoxanthine as one of the
original constituents. Cranmer et al. (1997) have reported
enhanced parasite growth after addition of hypoxanthine
in RPMI-1640. During the present study, addition of hypo-
xanthine in RPNI medium did not have any additional
effect on parasite growth whereas in medium RPMI-1640
a definite improvement in % parasitaemia was seen. The
findings including growth of different parasite lines in
medium RPNI with BCS as well as horse serum show that
use of RPNI medium is advantageous for long term culti-
vation of P. falciparum and commercially available FBS/
BCS or other animal sera overcome, the risk of exposure
to potentially bio-hazardous human blood. ALBUMAX
another alternative to human serum is reported to interfere
with chemosensitivity of known antimalarials (Basco,
2003). Further studies need to be carried out to investigate
which ingredients of RPNI are critical for development of
P. falciparum and whether functional characteristics of the
parasites cultured in RPNI medium are changed.
Acknowledgments
The CDRI communication no. is 6905. The authors are
grateful to the Director CDRI for continued encourage-
ment and support. The financial assistance in the form of
research scholar to Ms Shubhra Singh and Research intern
to Ms Pratibha Singh by DOD and CSIR, New Delhi, is
acknowledged.
174 K. Srivastava et al. / Experimental Parasitology 116 (2007) 171–174
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