# Cultivation of Asexual Intraerythrocytic Stages of Plasmodium falciparum

Leonardo K Basco

► To cite this version:

Leonardo K Basco. Cultivation of Asexual Intraerythrocytic Stages of Plasmodium falciparum. Pathogens, 2023, 12 (7), pp.900. 10.3390/pathogens12070900. [https://doi.org/10.3390/pathogens12070900]

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# Cultivation of Asexual Intraerythrocytic Stages of Plasmodium falciparum

Leonardo K Basco

<figure><img src="image_2.png" /></figure>

## To cite this version:

Leonardo K Basco. Cultivation of Asexual Intraerythrocytic Stages of Plasmodium falciparum. Pathogens, 2023, 12 (7), pp.900. 10.3390/pathogens12070900. [https://doi.org/10.3390/pathogens12070900]

HAL Id: hal-04543720

https://amu.hal.science/hal-04543720v1

Submitted on 12 Apr 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_3.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Review

# Cultivation of Asexual Intraerythrocytic Stages of *Plasmodium falciparum*

Leonardo K. Basco <fnref n="1" />,<fnref n="2" /> ID

**Abstract:** Successfully developed in 1976, the continuous in vitro culture of *Plasmodium falciparum* has many applications in the field of malaria research. It has become an important experimental model that directly uses a human pathogen responsible for a high prevalence of morbidity and mortality in many parts of the world and is a major source of biological material for immunological, biochemical, molecular, and pharmacological studies. Until present, the basic techniques described by Trager and Jensen and Haynes et al. remain unchanged in many malaria research laboratories. Nonetheless, different factors, including culture media, buffers, serum substitutes and supplements, sources of erythrocytes, and conditions of incubation (especially oxygen concentration), have been modified by different investigators to adapt the original technique in their laboratories or enhance the in vitro growth of the parasites. The possible effects and benefits of these modifications for the continuous cultivation of asexual intraerythrocytic stages of *P. falciparum*, as well as future challenges in developing a serum-free cultivation system and axenic cultures, are discussed.

**Keywords:** antimalarial drug resistance; in vitro assay; in vitro cultivation; malaria; *Plasmodium falciparum*

<figure><img src="image_2.png" /></figure>

check for
updates

**Citation:** Basco, L.K. Cultivation of Asexual Intraerythrocytic Stages of *Plasmodium falciparum*. *Pathogens* **2023**, *12*, 900. https://doi.org/10.3390/pathogens12070900

Academic Editors: Anastasia Diakou, Serafeim Chaintoutis, Mathios Mylonakis, Maria Panopoulou, Georgia Gioula and Georgia Vrioni

Received: 17 May 2023

Revised: 22 June 2023

Accepted: 26 June 2023

Published: 1 July 2023

<figure><img src="image_3.png" /></figure>

**Copyright:** © 2023 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

## 1. Introduction

*Plasmodium* spp. constitute a wide variety of malaria parasites with different vertebrate hosts (reptiles, birds, rodents, non-human primates, humans) and insect vectors [1,2]. They are single-celled organisms in the Apicomplexa phylum, which includes many other obligate intracellular parasitic protozoans of medical and veterinary importance, such as coccidia and *Toxoplasma gondii*. As eukaryotes, malaria parasites, unlike prokaryotic bacteria, have membrane-bound intracellular compartments and no discrete cell wall outside their delicate limiting plasmalemma. *Plasmodium* spp. have a complex metabolism and, being parasites, depend for survival on the cells of the multicellular eukaryotic host (erythrocytes, liver parenchymal, or reticuloendothelial cells in the vertebrate stages or gut and salivary gland cells in the arthropod vector) and the host environment, which supply the basic components for biosynthesis [3].

Malaria parasites undergo several different cell cycles: (i) mitosis and meiosis (i.e., sexual reproduction) during sporogony in the mosquito vector, (ii) asexual hepatic and erythrocytic schizogony in the human host, and (iii) sexual differentiation into gametocytes in the human host [3–5]. After inoculation of the sporozoites by the mosquito vector, sporozoites enter the liver cells. Hepatic schizogony occurs without any clinical symptoms. The merozoites released from ruptured hepatic schizonts initiate intraerythrocytic schizogony, which may be associated with various clinical signs and symptoms. The division cycle in the blood (blood schizogony) is entirely within the erythrocyte. The erythrocytic schizogony goes through different developmental stages during the 48 h period (72 h period for *Plasmodium malariae*) in the human host [6,7]. The rings and trophozoites are the pre-nuclear

<footnote n="1">¹ Aix-Marseille Université, Institut de Recherche pour le Développement (IRD), Assistance Publique—Hôpitaux de Marseille (AP-HM), Service de Santé des Armées (SSA), Unité Mixte de Recherche (UMR) Vecteurs—Infections Tropicales et Méditerranéennes (VITROME), 13005 Marseille, France; lkbasco@yahoo.fr or leonardo.basco@ird.fr</footnote>

<footnote n="2">² Institut Hospitalo-Universitaire—Méditerranée Infection, 19-21 Boulevard Jean Moulin, 13005 Marseille, France</footnote>
Review

# Cultivation of Asexual Intraerythrocytic Stages of *Plasmodium falciparum*

Leonardo K. Basco <fnref n="1" />,<fnref n="2" /> ID

**Abstract:** Successfully developed in 1976, the continuous in vitro culture of *Plasmodium falciparum* has many applications in the field of malaria research. It has become an important experimental model that directly uses a human pathogen responsible for a high prevalence of morbidity and mortality in many parts of the world and is a major source of biological material for immunological, biochemical, molecular, and pharmacological studies. Until present, the basic techniques described by Trager and Jensen and Haynes et al. remain unchanged in many malaria research laboratories. Nonetheless, different factors, including culture media, buffers, serum substitutes and supplements, sources of erythrocytes, and conditions of incubation (especially oxygen concentration), have been modified by different investigators to adapt the original technique in their laboratories or enhance the in vitro growth of the parasites. The possible effects and benefits of these modifications for the continuous cultivation of asexual intraerythrocytic stages of *P. falciparum*, as well as future challenges in developing a serum-free cultivation system and axenic cultures, are discussed.

**Keywords:** antimalarial drug resistance; in vitro assay; in vitro cultivation; malaria; *Plasmodium falciparum*

<figure><img src="image_4.png" /></figure>

check for
updates

**Citation:** Basco, L.K. Cultivation of Asexual Intraerythrocytic Stages of *Plasmodium falciparum*. *Pathogens* **2023**, *12*, 900. https://doi.org/10.3390/pathogens12070900

Academic Editors: Anastasia Diakou, Serafeim Chaintoutis, Mathios Mylonakis, Maria Panopoulou, Georgia Gioula and Georgia Vrioni

Received: 17 May 2023

Revised: 22 June 2023

Accepted: 26 June 2023

Published: 1 July 2023

<figure><img src="image_5.png" /></figure>

**Copyright:** © 2023 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

## 1. Introduction

*Plasmodium* spp. constitute a wide variety of malaria parasites with different vertebrate hosts (reptiles, birds, rodents, non-human primates, humans) and insect vectors [1,2]. They are single-celled organisms in the Apicomplexa phylum, which includes many other obligate intracellular parasitic protozoans of medical and veterinary importance, such as coccidia and *Toxoplasma gondii*. As eukaryotes, malaria parasites, unlike prokaryotic bacteria, have membrane-bound intracellular compartments and no discrete cell wall outside their delicate limiting plasmalemma. *Plasmodium* spp. have a complex metabolism and, being parasites, depend for survival on the cells of the multicellular eukaryotic host (erythrocytes, liver parenchymal, or reticuloendothelial cells in the vertebrate stages or gut and salivary gland cells in the arthropod vector) and the host environment, which supply the basic components for biosynthesis [3].

Malaria parasites undergo several different cell cycles: (i) mitosis and meiosis (i.e., sexual reproduction) during sporogony in the mosquito vector, (ii) asexual hepatic and erythrocytic schizogony in the human host, and (iii) sexual differentiation into gametocytes in the human host [3–5]. After inoculation of the sporozoites by the mosquito vector, sporozoites enter the liver cells. Hepatic schizogony occurs without any clinical symptoms. The merozoites released from ruptured hepatic schizonts initiate intraerythrocytic schizogony, which may be associated with various clinical signs and symptoms. The division cycle in the blood (blood schizogony) is entirely within the erythrocyte. The erythrocytic schizogony goes through different developmental stages during the 48 h period (72 h period for *Plasmodium malariae*) in the human host [6,7]. The rings and trophozoites are the pre-nuclear

<footnote n="1">¹ Aix-Marseille Université, Institut de Recherche pour le Développement (IRD), Assistance Publique—Hôpitaux de Marseille (AP-HM), Service de Santé des Armées (SSA), Unité Mixte de Recherche (UMR) Vecteurs—Infections Tropicales et Méditerranéennes (VITROME), 13005 Marseille, France; lkbasco@yahoo.fr or leonardo.basco@ird.fr</footnote>

<footnote n="2">² Institut Hospitalo-Universitaire—Méditerranée Infection, 19-21 Boulevard Jean Moulin, 13005 Marseille, France</footnote>
division stages and, by definition, contain only one nucleus (under light microscopy, the presence of two nuclei is an artifact of a bilobed nucleus). Rings are flat or concave with a thin rim of organelle-rich cytoplasm encircling a large, flat, and clear center with few organelles. Trophozoites are much larger in size and have a thick cytoplasm and digestive vacuole containing hemozoin. Schizonts are defined morphologically as the developmental stage with >2 nuclei. In the literature, some authors distinguish between "pre-schizonts" and "schizonts" [8]. The term "pre-schizont" refers to schizonts with 3–8 nuclei. Mature schizonts refer to the more advanced stage (>8 nuclei), in which the parasite (especially *Plasmodium vivax*, *Plasmodium ovale*, and *P. malariae*) may occupy most of the intraerythrocytic space and the merozoites are segmented and individualized. When the infective merozoites escape from the erythrocyte at the end of this period, they are free in the plasma for only a brief period (probably seconds) [9]. The merozoite has special organelles (rhoptries, nicronemes, and dense granules) that facilitate its entry into the new host cell. Gametocytogenesis is initiated during the erythrocytic phase in the human host. The maturation of *Plasmodium falciparum* gametocytes takes about 10–12 days and occurs in the deep organs, notably in the bone marrow [10]. Mature gametocytes circulate in the peripheral circulatory system and are taken up by female *Anopheles* mosquitoes during subsequent blood meals. Sexual reproduction occurs in the mosquito gut.

As briefly described above, *Plasmodium* spp. have a complex metabolism. These facts are not ignored in the cultivation of malaria parasites in vitro. Here, the optimal host environment is artificially reproduced with greater or lesser success, depending on the species, in a laboratory setting under controlled conditions. Our knowledge of malaria biology and biochemistry is still incomplete, and precise nutritional requirements are not known, not even for the most successfully cultivated species, *P. falciparum*. The cultivation of *Plasmodium* spp. resembles most in its requirements the cultivation of viruses, which require complex host cells, rather than the cultivation of pathogenic bacteria, most of which will thrive on cell-free food sources such as serum, yeast extract, and meat broth. The development of short-term and long-term cultivation techniques for a blood-stage malaria parasite was more difficult to achieve than for many viruses, bacteria, and eukaryotic cells. Even now, of the four *Plasmodium* species found in humans, blood stages of *P. vivax*, *P. ovale*, and *P. malariae* have still not been continuously cultivated in vitro with any success (note: short-term cultures of *P. vivax* blood forms have been achieved and are proving to be informative on the distribution of drug resistance in blood stages of this species [11]; malaria due to *Plasmodium knowlesi*, the fifth malaria species that may infect humans, is a zoonosis [12]). It is probably best to concentrate on the human *Plasmodium* species with which we have had the most success, *P. falciparum*, when discussing particular features of in vitro cultivation. From the point of view of the most important forms of drug resistance, only the blood stages are of immediate interest, and these are the only stages where facile and routine cultures are achievable in most facilities.

The continuous culture of *P. falciparum* has had a major impact on almost all areas of malaria research [13]. It has become a reliable source of biological material for experiments in any properly equipped laboratory outside endemic regions and a means to obtain various fractions (DNA, RNA, proteins, and organelles) for basic science. More than 45 years after the development of in vitro techniques for long-term malaria parasite propagation, the basic technical procedures remain unchanged. However, a number of factors involved in the cultivation method can be and have been modified in an effort to enhance the efficacy of in vitro propagation rate. Over the years, these modifications have produced variations in recommended protocols in different specialized laboratories in the world.

The objective of the present work is to review the basic principles of the cultivation of the asexual intraerythrocytic stages of *P. falciparum*. This work is a traditional narrative literature review. The initial search for published papers was based on the search term "in vitro culture or cultivation *Plasmodium*" in the PubMed database with the aim to obtain the maximum number of relevant published articles. Other sources included books on malaria, book chapters, and references listed in selected papers. Papers written in English,
division stages and, by definition, contain only one nucleus (under light microscopy, the presence of two nuclei is an artifact of a bilobed nucleus). Rings are flat or concave with a thin rim of organelle-rich cytoplasm encircling a large, flat, and clear center with few organelles. Trophozoites are much larger in size and have a thick cytoplasm and digestive vacuole containing hemozoin. Schizonts are defined morphologically as the developmental stage with >2 nuclei. In the literature, some authors distinguish between “pre-schizonts” and “schizonts” [8]. The term “pre-schizont” refers to schizonts with 3–8 nuclei. Mature schizonts refer to the more advanced stage (>8 nuclei), in which the parasite (especially *Plasmodium vivax*, *Plasmodium ovale*, and *P. malariae*) may occupy most of the intraerythrocytic space and the merozoites are segmented and individualized. When the infective merozoites escape from the erythrocyte at the end of this period, they are free in the plasma for only a brief period (probably seconds) [9]. The merozoite has special organelles (rhoptries, nicronemes, and dense granules) that facilitate its entry into the new host cell. Gametocytogenesis is initiated during the erythrocytic phase in the human host. The maturation of *Plasmodium falciparum* gametocytes takes about 10–12 days and occurs in the deep organs, notably in the bone marrow [10]. Mature gametocytes circulate in the peripheral circulatory system and are taken up by female *Anopheles* mosquitoes during subsequent blood meals. Sexual reproduction occurs in the mosquito gut.

As briefly described above, *Plasmodium* spp. have a complex metabolism. These facts are not ignored in the cultivation of malaria parasites in vitro. Here, the optimal host environment is artificially reproduced with greater or lesser success, depending on the species, in a laboratory setting under controlled conditions. Our knowledge of malaria biology and biochemistry is still incomplete, and precise nutritional requirements are not known, not even for the most successfully cultivated species, *P. falciparum*. The cultivation of *Plasmodium* spp. resembles most in its requirements the cultivation of viruses, which require complex host cells, rather than the cultivation of pathogenic bacteria, most of which will thrive on cell-free food sources such as serum, yeast extract, and meat broth. The development of short-term and long-term cultivation techniques for a blood-stage malaria parasite was more difficult to achieve than for many viruses, bacteria, and eukaryotic cells. Even now, of the four *Plasmodium* species found in humans, blood stages of *P. vivax*, *P. ovale*, and *P. malariae* have still not been continuously cultivated in vitro with any success (note: short-term cultures of *P. vivax* blood forms have been achieved and are proving to be informative on the distribution of drug resistance in blood stages of this species [11]; malaria due to *Plasmodium knowlesi*, the fifth malaria species that may infect humans, is a zoonosis [12]). It is probably best to concentrate on the human *Plasmodium* species with which we have had the most success, *P. falciparum*, when discussing particular features of in vitro cultivation. From the point of view of the most important forms of drug resistance, only the blood stages are of immediate interest, and these are the only stages where facile and routine cultures are achievable in most facilities.

The continuous culture of *P. falciparum* has had a major impact on almost all areas of malaria research [13]. It has become a reliable source of biological material for experiments in any properly equipped laboratory outside endemic regions and a means to obtain various fractions (DNA, RNA, proteins, and organelles) for basic science. More than 45 years after the development of in vitro techniques for long-term malaria parasite propagation, the basic technical procedures remain unchanged. However, a number of factors involved in the cultivation method can be and have been modified in an effort to enhance the efficacy of in vitro propagation rate. Over the years, these modifications have produced variations in recommended protocols in different specialized laboratories in the world.

The objective of the present work is to review the basic principles of the cultivation of the asexual intraerythrocytic stages of *P. falciparum*. This work is a traditional narrative literature review. The initial search for published papers was based on the search term “in vitro culture or cultivation *Plasmodium*” in the PubMed database with the aim to obtain the maximum number of relevant published articles. Other sources included books on malaria, book chapters, and references listed in selected papers. Papers written in English,
French, Spanish, Portuguese, or Japanese were included. Except for the first published
papers on in vitro cultivation of malaria parasites in 1911–1912, the search period spanned
from January 1970 to February 2023. Most of the papers dealing with *Plasmodium* spp.
other than *P. falciparum* were excluded unless they referred to the in vitro cultivation
method developed by Trager and Jensen to illustrate the impact of this technique beyond
*P. falciparum* [13]. Most early studies on cultivation before 1976 have been excluded. Readers
interested in the “early history” of the development of cultivation methods of malaria
parasites are referred to earlier reviews [14–16]. The cultivation of gametocytes, hepatic
stages, and sporogonic stages is beyond the scope of this review.

## 2. Historical Background

Initial attempts to cultivate malaria parasites in vitro date back to the end of the 19th
century, soon after the discovery of malaria parasites (*P. malariae*) in human erythrocytes
by Charles Louis Alphonse Laveran (1845–1922), a French army surgeon, in Constantine,
Algeria, in 1880. During the period between 1880 and 1911, only short-term survival, but
not multiplication, was attained in vitro. The first successful attempts to grow the intraery-
throcytic asexual stages of malaria parasites (*P. falciparum*, *P. malariae*, and *Plasmodium vivax*)
were reported in 1912 [17,18]. The simple method developed by Bass and Johns consisted of
the collection of venous blood from malaria-infected patients, defibrination, and the addi-
tion of D-glucose. The defibrinated whole blood is incubated at 40 °C and allowed to settle
and separate into cellular components and plasma. Viable parasites undergo schizogony in
the thin layer at the top of the red cell pellet for up to three complete generations (usually
only for a single cycle, rarely four cycles) if leukocytes are removed. This method was
adopted by investigators worldwide and became the reference for the short-term cultiva-
tion of human malaria parasites during the first half of the 20th century. The in vitro drug
susceptibility test derived from the technique, called the “macrotest” or “macrotechnique”,
was used until the 1980s [19,20]. However, some investigators were not able to reproduce
Bass and John’s experiments and questioned whether the technique allows reinvasion and
development of new generations of malaria parasites [14].

The report by Bass and Johns was shortly followed by World War I (1914–1917). Heavy
casualties due to malaria among the troops deployed in southern Europe and a shortage
of quinine, the only effective antimalarial drug available at that time, maintained the
interest of governments and pharmaceutical companies in malaria research, notably for
screening and development of new synthetic antimalarial compounds. During the 1920s
and 1930s, German scientists at the Bayer–Meister–Lucius Laboratories synthesized and
developed quinoline-based synthetic drugs, including pamaquine (also known as plasmo-
quine), mepacrine (also known as quinacrine or atebrin), sontoquine, and chloroquine. The
antimalarial activity was screened by the in vivo avian malaria model. During the same
period, many attempts were made by investigators to propagate the parasite blood stages
in vitro, mostly avian, simian, and human malaria parasites, with different culture media
and under various experimental conditions. However, the trial-and-error approach did not
lead to successful continuous in vitro cultivation.

During World War II (1939–1945), malaria was recognized as a potentially serious
medical problem with major repercussions on military operations both in the regions
surrounding the Mediterranean Sea (i.e., southern Europe and North Africa, where malaria
was endemic during that time) and Pacific Ocean. Quinine was again in shortage, partly due
to the Axis occupation of The Netherlands and the Dutch colony of Java, the major producer
of quinine at that time (note: quinine is readily obtained from *Cinchona* spp. tree bark; total
chemical synthesis with the correct spatial configuration, i.e., stereoisomer, was achieved
only at the turn of the twenty-first century but is uneconomical for mass production [21]).
The synthetic drugs available at the outbreak of the war (i.e., mepacrine) were not well-
tolerated by humans. Antimalarial drug screening and development programs were
actively being pursued in Germany and France and were initiated in the United States of
America, the United Kingdom, and Australia. These war-related research efforts led to the
French, Spanish, Portuguese, or Japanese were included. Except for the first published
papers on in vitro cultivation of malaria parasites in 1911–1912, the search period spanned
from January 1970 to February 2023. Most of the papers dealing with *Plasmodium* spp.
other than *P. falciparum* were excluded unless they referred to the in vitro cultivation
method developed by Trager and Jensen to illustrate the impact of this technique beyond
*P. falciparum* [13]. Most early studies on cultivation before 1976 have been excluded. Readers
interested in the “early history” of the development of cultivation methods of malaria
parasites are referred to earlier reviews [14–16]. The cultivation of gametocytes, hepatic
stages, and sporogonic stages is beyond the scope of this review.

## 2. Historical Background

Initial attempts to cultivate malaria parasites in vitro date back to the end of the 19th
century, soon after the discovery of malaria parasites (*P. malariae*) in human erythrocytes
by Charles Louis Alphonse Laveran (1845–1922), a French army surgeon, in Constantine,
Algeria, in 1880. During the period between 1880 and 1911, only short-term survival, but
not multiplication, was attained in vitro. The first successful attempts to grow the intraery-
throcytic asexual stages of malaria parasites (*P. falciparum*, *P. malariae*, and *Plasmodium vivax*)
were reported in 1912 [17,18]. The simple method developed by Bass and Johns consisted of
the collection of venous blood from malaria-infected patients, defibrination, and the addi-
tion of D-glucose. The defibrinated whole blood is incubated at 40 °C and allowed to settle
and separate into cellular components and plasma. Viable parasites undergo schizogony in
the thin layer at the top of the red cell pellet for up to three complete generations (usually
only for a single cycle, rarely four cycles) if leukocytes are removed. This method was
adopted by investigators worldwide and became the reference for the short-term cultiva-
tion of human malaria parasites during the first half of the 20th century. The in vitro drug
susceptibility test derived from the technique, called the “macrotest” or “macrotechnique”,
was used until the 1980s [19,20]. However, some investigators were not able to reproduce
Bass and John’s experiments and questioned whether the technique allows reinvasion and
development of new generations of malaria parasites [14].

The report by Bass and Johns was shortly followed by World War I (1914–1917). Heavy
casualties due to malaria among the troops deployed in southern Europe and a shortage
of quinine, the only effective antimalarial drug available at that time, maintained the
interest of governments and pharmaceutical companies in malaria research, notably for
screening and development of new synthetic antimalarial compounds. During the 1920s
and 1930s, German scientists at the Bayer–Meister–Lucius Laboratories synthesized and
developed quinoline-based synthetic drugs, including pamaquine (also known as plasmo-
quine), mepacrine (also known as quinacrine or atebrin), sontoquine, and chloroquine. The
antimalarial activity was screened by the in vivo avian malaria model. During the same
period, many attempts were made by investigators to propagate the parasite blood stages
in vitro, mostly avian, simian, and human malaria parasites, with different culture media
and under various experimental conditions. However, the trial-and-error approach did not
lead to successful continuous in vitro cultivation.

During World War II (1939–1945), malaria was recognized as a potentially serious
medical problem with major repercussions on military operations both in the regions
surrounding the Mediterranean Sea (i.e., southern Europe and North Africa, where malaria
was endemic during that time) and Pacific Ocean. Quinine was again in shortage, partly due
to the Axis occupation of The Netherlands and the Dutch colony of Java, the major producer
of quinine at that time (note: quinine is readily obtained from *Cinchona* spp. tree bark; total
chemical synthesis with the correct spatial configuration, i.e., stereoisomer, was achieved
only at the turn of the twenty-first century but is uneconomical for mass production [21]).
The synthetic drugs available at the outbreak of the war (i.e., mepacrine) were not well-
tolerated by humans. Antimalarial drug screening and development programs were
actively being pursued in Germany and France and were initiated in the United States of
America, the United Kingdom, and Australia. These war-related research efforts led to the
synthesis and development of chloroquine (synthesized in 1934 but developed later for clinical use), proguanil (1944), amodiaquine (1946), primaquine (1950), and pyrimethamine (1952), all of which are still being used today. In vitro cultivation did not play any important role in drug screening and development programs until the post-war era.

As part of the antimalarial drug research program during World War II, which was focused on the development of synthetic drugs to replace quinine, fundamental studies to understand the metabolic needs of the malaria parasites cultivated in vitro were undertaken. A rational approach, as opposed to empirical studies, based on the understanding of the biochemistry of malaria parasites, was required for an optimal in vitro culture system. It was during this time that William Trager, who would later head one of the two American research teams that succeeded in developing the continuous in vitro cultivation method, had initiated in vitro studies using *Plasmodium lophurae*, avian malaria parasites that infect chicks and ducks, as the experimental model [22]. A more detailed history of early attempts to cultivate malaria parasites between 1880 and 1946 has been reviewed elsewhere [23,24].

Between 1946 and 1976, a number of other scientists worked independently to develop in vitro cultivation methods for avian, simian, and human malaria parasites. Most employed the rocker dilution technique, rocker perfusion technique, or their variations. All of these efforts had, at best, resulted in short-term cultivation of various *Plasmodium* species. More success was achieved with continuous cultivation of the exoerythrocytic (reticuloendothelial) stages of avian malaria. For the blood stages in human malaria, the only pathogenic stage and the most important focus for chemotherapy, the “right combination” of synthetic tissue culture media and their supplements, buffers, gas mixtures, and other factors that were necessary for the long-term continuous culture were either not available at that time (e.g., production of synthetic culture media at a commercial scale) or not yet discovered. Nevertheless, short-term cultures were useful tools to gain insight into the metabolic needs of malaria parasites and study the mechanisms of action of antimalarial drugs. The technical problems encountered in the early culture systems, described in earlier reviews [15,25], were not resolved until the advent of the new era for experimental research in malaria starting in 1976.

The major breakthrough for the long-term in vitro cultivation of blood stages of *P. falciparum* was attained by two American research teams from Walter Reed Army Institute of Research (WRAIR; Washington, D.C. (currently in Silver Spring, Maryland)) and Rockefeller University (New York) that published their techniques for the long-term in vitro cultivation of *P. falciparum* in 1976 [26,27]. These two methods are based on similar culture conditions (Table 1). The following basic requirements for successful in vitro cultivation are derived from their works: appropriate culture media with serum supplement and adequate buffer, a thin layer of settled human erythrocytes, regular (i.e., daily or at least every other day) change in culture media, addition of fresh, uninfected human erythrocytes at intervals of 2–4 days, and high CO₂/low O₂ content. Current methods of in vitro cultivation of *P. falciparum* are essentially based on their methods. As in many scientific works, the long-term cultivation methods were made possible due to many other preceding works (including the early works of William Trager) and may be considered to be a culmination of scientific advances contributed by many scientists in various fields.

<table><caption>**Table 1.** Comparison of culture conditions for the long-term cultivation of *P. falciparum*.</caption><thead><tr><th>Culture Conditions</th><th>Haynes et al. [26]</th><th>Trager and Jensen [27]</th></tr></thead><tbody><tr><td>Culture medium</td><td>Medium 199 with Earle's modified salts <sup>1</sup></td><td>Roswell Park Memorial Institute (RPMI) 1640</td></tr><tr><td>Buffer</td><td>10 mM TES <sup>2</sup></td><td>HEPES + NaHCO<sub>3</sub> <sup>3</sup></td></tr><tr><td>Serum supplement</td><td>10% heat-inactivated fetal bovine serum</td><td>15% type AB human serum <sup>4</sup></td></tr><tr><td>Initial source of infected erythrocytes <sup>5</sup></td><td>Chimpanzee (Pan troglodytes)</td><td>Owl monkey (Aotus trivirgatus)</td></tr><tr><td><i>P. falciparum</i> strain</td><td>Malayan Camp</td><td>FVO</td></tr></tbody></table>
synthesis and development of chloroquine (synthesized in 1934 but developed later for clinical use), proguanil (1944), amodiaquine (1946), primaquine (1950), and pyrimethamine (1952), all of which are still being used today. In vitro cultivation did not play any important role in drug screening and development programs until the post-war era.

As part of the antimalarial drug research program during World War II, which was focused on the development of synthetic drugs to replace quinine, fundamental studies to understand the metabolic needs of the malaria parasites cultivated in vitro were undertaken. A rational approach, as opposed to empirical studies, based on the understanding of the biochemistry of malaria parasites, was required for an optimal in vitro culture system. It was during this time that William Trager, who would later head one of the two American research teams that succeeded in developing the continuous in vitro cultivation method, had initiated in vitro studies using *Plasmodium lophurae*, avian malaria parasites that infect chicks and ducks, as the experimental model [22]. A more detailed history of early attempts to cultivate malaria parasites between 1880 and 1946 has been reviewed elsewhere [23,24].

Between 1946 and 1976, a number of other scientists worked independently to develop in vitro cultivation methods for avian, simian, and human malaria parasites. Most employed the rocker dilution technique, rocker perfusion technique, or their variations. All of these efforts had, at best, resulted in short-term cultivation of various *Plasmodium* species. More success was achieved with continuous cultivation of the exoerythrocytic (reticuloendothelial) stages of avian malaria. For the blood stages in human malaria, the only pathogenic stage and the most important focus for chemotherapy, the “right combination” of synthetic tissue culture media and their supplements, buffers, gas mixtures, and other factors that were necessary for the long-term continuous culture were either not available at that time (e.g., production of synthetic culture media at a commercial scale) or not yet discovered. Nevertheless, short-term cultures were useful tools to gain insight into the metabolic needs of malaria parasites and study the mechanisms of action of antimalarial drugs. The technical problems encountered in the early culture systems, described in earlier reviews [15,25], were not resolved until the advent of the new era for experimental research in malaria starting in 1976.

The major breakthrough for the long-term in vitro cultivation of blood stages of *P. falciparum* was attained by two American research teams from Walter Reed Army Institute of Research (WRAIR; Washington, D.C. (currently in Silver Spring, Maryland)) and Rockefeller University (New York) that published their techniques for the long-term in vitro cultivation of *P. falciparum* in 1976 [26,27]. These two methods are based on similar culture conditions (Table 1). The following basic requirements for successful in vitro cultivation are derived from their works: appropriate culture media with serum supplement and adequate buffer, a thin layer of settled human erythrocytes, regular (i.e., daily or at least every other day) change in culture media, addition of fresh, uninfected human erythrocytes at intervals of 2–4 days, and high CO₂/low O₂ content. Current methods of in vitro cultivation of *P. falciparum* are essentially based on their methods. As in many scientific works, the long-term cultivation methods were made possible due to many other preceding works (including the early works of William Trager) and may be considered to be a culmination of scientific advances contributed by many scientists in various fields.

<table><caption>**Table 1.** Comparison of culture conditions for the long-term cultivation of *P. falciparum*.</caption><thead><tr><th>Culture Conditions</th><th>Haynes et al. [26]</th><th>Trager and Jensen [27]</th></tr></thead><tbody><tr><td>Culture medium</td><td>Medium 199 with Earle's modified salts <sup>1</sup></td><td>Roswell Park Memorial Institute (RPMI) 1640</td></tr><tr><td>Buffer</td><td>10 mM TES <sup>2</sup></td><td>HEPES + NaHCO<sub>3</sub> <sup>3</sup></td></tr><tr><td>Serum supplement</td><td>10% heat-inactivated fetal bovine serum</td><td>15% type AB human serum <sup>4</sup></td></tr><tr><td>Initial source of infected erythrocytes <sup>5</sup></td><td>Chimpanzee (Pan troglodytes)</td><td>Owl monkey (Aotus trivirgatus)</td></tr><tr><td><i>P. falciparum</i> strain</td><td>Malayan Camp</td><td>FVO</td></tr></tbody></table>
<table><caption>**Table 1.** *Cont.*</caption><thead><tr><th>Culture Conditions</th><th>Haynes et al. [26]</th><th>Trager and Jensen [27]</th></tr></thead><tbody><tr><td>Successfully tested erythrocytes</td><td>Chimpanzee, owl monkey, human</td><td>Human</td></tr><tr><td>Atmosphere</td><td>3% CO<sub>2</sub>, 6.6% O<sub>2</sub></td><td>7% CO<sub>2</sub>, 1% or 5% O<sub>2</sub>; candle jar <sup>6</sup></td></tr><tr><td>Temperature</td><td>37 °C</td><td>38 °C</td></tr><tr><td>Culture vessel</td><td>5 cm<sup>2</sup> flask, 96-well microtiter plate</td><td>Flow vial, Petri dish</td></tr></tbody></table>

The works of the two American research teams on *P. falciparum* led to the first successful continuous in vitro culture of the blood-stage of any *Plasmodium* species. A few years after the publication of these works, the techniques developed for *P. falciparum* were successfully adopted to maintain in vitro the intraerythrocytic stages of simian malarial species, including *Plasmodium knowlesi*, *Plasmodium fragile*, *Plasmodium inui*, *Plasmodium cynomolgi*, and *Plasmodium gonderi* [28–35]. In these early but successful attempts to cultivate simian parasites, such as *P. knowlesi*, rhesus monkey erythrocytes were used [29,32]. Even in more recent attempts to ameliorate the in vitro cultivation of *P. cynomolgi*, monkey erythrocytes and monkey serum (or serum substitutes, including fetal bovine serum, normal horse serum, and Albumax II™) were used in a modified protocol based on Trager and Jensen's cultivation method for *P. falciparum* [36,37]. Quite recently, a British research group succeeded in adapting a *P. knowlesi* strain entirely to continuous culture in human erythrocytes [38]. However, for the three other human *Plasmodium* spp. (i.e., *P. vivax*, *P. malariae*, and *Plasmodium ovale*), limited progress has been made despite various attempts to propagate these parasites indefinitely. It is in this context that the technical capacity to propagate in vitro the asexual intraerythrocytic stages of simian malaria parasites, in particular *P. cynomolgi*, is important for research purposes, as they may serve as a model for non-*P. falciparum* human malaria parasites.

In the following sections, the various factors and conditions required for successful continuous cultivation of *P. falciparum* are reviewed, with an emphasis on the principles of in vitro cultivation. Cultivation techniques of stages other than the intraerythrocytic stages and malaria parasites that infect non-human hosts under natural conditions lie beyond the scope of this review. Readers requiring detailed laboratory protocols on culture procedure, cryopreservation, cloning, and synchronization should consult laboratory manuals and other review papers [39–45].

### 3. Culture Media and Nutritional Requirements

During the history of the development of eukaryotic cell culture, many types of artificial media and balanced salt solutions have been formulated. Some media have been specifically developed for mammalian cells, while others have been prepared for insect or plant cells. A medium may have been initially formulated to satisfy the nutritional requirements of a particular type of cells, but subsequent studies have shown that it may also support the growth of other types of cells. Despite the plethora of media formulated, developed, and tested over the past 70 years, a large majority of eukaryotic cells are cultivated today in one of the following eight media: basal medium Eagle (BME), Dulbecco's modified Eagle's medium (DMEM), Iscove's modified Dulbecco's medium (IMDM), McCoy's 5A medium, medium 199 (M-199), minimum essential medium (MEM),

<footnote n="1">¹ Enriched with D-glucose (2 mg/mL), 2 mM L-glutamine, 3 × 10⁻⁵ M 2-mercaptoethanol, (±)-α-tocopherol (30 µg/mL) (note: the units of concentration are those of the authors being cited); ² N-tris-(hydroxymethyl)methyl-2-aminoethanesulfonate (TES); ³ 25 mM each, N-(2-hydroxyethyl)piperazine-N'-((2-ethanesulfonic acid) (HEPES); ⁴ type AB was initially used to avoid agglutination when mixing human and simian (*Aotus trivirgatus*) erythrocytes; ⁵ before the development of long-term in vitro cultivation techniques, several *P. falciparum* isolates from patients (tourists or military personnel) were passaged and maintained in splenectomized monkeys (chimpanzees or owl monkeys) in the United States of America. Haynes et al. [26] initiated cultures from aliquots of cryopreserved infected chimpanzee erythrocytes. ⁶ The candle jar produces an atmosphere of approximately 3% CO₂ and 17–18% O₂.</footnote>
<table><caption>**Table 1.** *Cont.*</caption><thead><tr><th>Culture Conditions</th><th>Haynes et al. [26]</th><th>Trager and Jensen [27]</th></tr></thead><tbody><tr><td>Successfully tested erythrocytes</td><td>Chimpanzee, owl monkey, human</td><td>Human</td></tr><tr><td>Atmosphere</td><td>3% CO<sub>2</sub>, 6.6% O<sub>2</sub></td><td>7% CO<sub>2</sub>, 1% or 5% O<sub>2</sub>; candle jar <sup>6</sup></td></tr><tr><td>Temperature</td><td>37 °C</td><td>38 °C</td></tr><tr><td>Culture vessel</td><td>5 cm<sup>2</sup> flask, 96-well microtiter plate</td><td>Flow vial, Petri dish</td></tr></tbody></table>

The works of the two American research teams on *P. falciparum* led to the first successful continuous in vitro culture of the blood-stage of any *Plasmodium* species. A few years after the publication of these works, the techniques developed for *P. falciparum* were successfully adopted to maintain in vitro the intraerythrocytic stages of simian malarial species, including *Plasmodium knowlesi*, *Plasmodium fragile*, *Plasmodium inui*, *Plasmodium cynomolgi*, and *Plasmodium gonderi* [28–35]. In these early but successful attempts to cultivate simian parasites, such as *P. knowlesi*, rhesus monkey erythrocytes were used [29,32]. Even in more recent attempts to ameliorate the in vitro cultivation of *P. cynomolgi*, monkey erythrocytes and monkey serum (or serum substitutes, including fetal bovine serum, normal horse serum, and Albumax II™) were used in a modified protocol based on Trager and Jensen's cultivation method for *P. falciparum* [36,37]. Quite recently, a British research group succeeded in adapting a *P. knowlesi* strain entirely to continuous culture in human erythrocytes [38]. However, for the three other human *Plasmodium* spp. (i.e., *P. vivax*, *P. malariae*, and *Plasmodium ovale*), limited progress has been made despite various attempts to propagate these parasites indefinitely. It is in this context that the technical capacity to propagate in vitro the asexual intraerythrocytic stages of simian malaria parasites, in particular *P. cynomolgi*, is important for research purposes, as they may serve as a model for non-*P. falciparum* human malaria parasites.

In the following sections, the various factors and conditions required for successful continuous cultivation of *P. falciparum* are reviewed, with an emphasis on the principles of in vitro cultivation. Cultivation techniques of stages other than the intraerythrocytic stages and malaria parasites that infect non-human hosts under natural conditions lie beyond the scope of this review. Readers requiring detailed laboratory protocols on culture procedure, cryopreservation, cloning, and synchronization should consult laboratory manuals and other review papers [39–45].

### 3. Culture Media and Nutritional Requirements

During the history of the development of eukaryotic cell culture, many types of artificial media and balanced salt solutions have been formulated. Some media have been specifically developed for mammalian cells, while others have been prepared for insect or plant cells. A medium may have been initially formulated to satisfy the nutritional requirements of a particular type of cells, but subsequent studies have shown that it may also support the growth of other types of cells. Despite the plethora of media formulated, developed, and tested over the past 70 years, a large majority of eukaryotic cells are cultivated today in one of the following eight media: basal medium Eagle (BME), Dulbecco's modified Eagle's medium (DMEM), Iscove's modified Dulbecco's medium (IMDM), McCoy's 5A medium, medium 199 (M-199), minimum essential medium (MEM),

<footnote n="1">¹ Enriched with D-glucose (2 mg/mL), 2 mM L-glutamine, 3 × 10⁻⁵ M 2-mercaptoethanol, (±)-α-tocopherol (30 µg/mL) (note: the units of concentration are those of the authors being cited); ² N-tris-(hydroxymethyl)methyl-2-aminoethanesulfonate (TES); ³ 25 mM each, N-(2-hydroxyethyl)piperazine-N'--(2-ethanesulfonic acid) (HEPES); ⁴ type AB was initially used to avoid agglutination when mixing human and simian (*Aotus trivirgatus*) erythrocytes; ⁵ before the development of long-term in vitro cultivation techniques, several *P. falciparum* isolates from patients (tourists or military personnel) were passaged and maintained in splenectomized monkeys (chimpanzees or owl monkeys) in the United States of America. Haynes et al. [26] initiated cultures from aliquots of cryopreserved infected chimpanzee erythrocytes. ⁶ The candle jar produces an atmosphere of approximately 3% CO₂ and 17–18% O₂.</footnote>
nutrient mixture F-12 Ham, and RPMI 1640. The formulations of these common media were
developed and published between 1955 and 1967. These products have become standard
media for eukaryotic cell culture and are available from many commercial suppliers.

3.1. RPMI 1640

In many cases, the medium of choice for a given type of eukaryotic cell has been
determined through numerous empirical trials. For established cell lines, the general rule
is to pursue cultivation procedures using the same medium unless there are reasons to
change the type of medium. RPMI 1640 is the most widely used culture medium for
*P. falciparum* and is the medium of choice since the development of continuous cultivation
of this *Plasmodium* species by Trager and Jensen [27]. RPMI 1640 was not "custom-made" to
optimize the in vitro growth of *P. falciparum* based on its nutritional requirements. William
Trager reasoned that RPMI 1640 may be suitable for maintaining *P. falciparum* in vitro
based on the observation that this medium was specifically developed for the cultivation
of human leukocytes [46]. In fact, RPMI 1640, usually supplemented with serum from
human or animal sources, has wide applications for maintaining the growth of different
cell cultures, including parasites other than *P. falciparum*. As in other culture media,
RPMI 1640 consists of D-glucose, amino acids, inorganic salts, vitamins, and glutathione
(the principal reducing agent in erythrocytes that protects hemoglobin from oxidation),
which are essential nutrients required by eukaryotic cells. Mature, enucleated, uninfected
human red blood cells maintain functional biosynthetic pathways, ion transport, and
buffer systems, including when they are placed in in vitro culture conditions in RPMI
1640 medium [47,48]. The importance of the major components of the culture medium is
presented below.

3.1.1. Glucose

Most animal cell lines require medium changes once or twice a week, depending on
the growth rate and cell density. For malaria cultivation, daily medium change is the norm.
The frequency of medium change may have to be increased to twice or thrice a day for
special experiments in which a high yield of parasites (therefore, high parasitemias and/or
high hematocrit) is required. Conversely, the frequency of medium change may be reduced
to once every 2 or 3 days under certain conditions by lowering hematocrit or starting
parasitemia, increasing the buffering capacity or glucose concentration, adding "cold"
hypoxanthine (i.e., a naturally occurring purine derivative, as distinguished from tritium-
labeled hypoxanthine that may be used as an indicator of parasite growth in drug sensitivity
assays [49,50]), or the combination of these factors (temperature can also be reduced).

Glucose is the primary source of energy for most cells. Some investigators have
attempted to improve the growth of parasites by increasing D-glucose concentration and
adding supplements to RPMI 1640. In one study, in which the medium was changed daily
(10% erythrocyte suspension and 0.8-1.5% starting parasitemia), the addition of 2 g/L of
extra glucose (i.e., a total of 4 g/L of glucose, including glucose already present in RPMI
1640), reduced glutathione (600 µg/mL) and hypoxanthine (50 µg/mL) to the standard
RPMI 1640 buffered with 40 mM (9.15 g/L) TES (RPMI 1640-TES-NaHCO3 medium),
resulted in a three-fold increase in parasite growth over 4 days in one reference clone
compared to the growth in the standard RPMI 1640-HEPES-NaHCO3 medium (note:
increased glucose needs to be balanced by better buffering or more frequent medium
change because of the greater production of lactate) [51]. The growth rate of three other
uncloned Thai isolates was not affected by hypoxanthine and glutathione supplements. In
another study, it has been shown that laboratory-adapted P. falciparum strains (0.5% initial
parasitemia, 5% hematocrit, and 5% O2-5% CO2-90% N2 gas mixture) can be cultivated
without medium change for three days using a similar glucose-enriched (4 g/L) RPMI
1640-TES-NaHCO3 mixture without hypoxanthine supplement [52]. A slight decrease
in pH (from 7.4 to 7.1) after three days was not detrimental to parasite growth. This
observation was confirmed in another study in which a modified cultivation technique in
nutrient mixture F-12 Ham, and RPMI 1640. The formulations of these common media were
developed and published between 1955 and 1967. These products have become standard
media for eukaryotic cell culture and are available from many commercial suppliers.

3.1. RPMI 1640

In many cases, the medium of choice for a given type of eukaryotic cell has been
determined through numerous empirical trials. For established cell lines, the general rule
is to pursue cultivation procedures using the same medium unless there are reasons to
change the type of medium. RPMI 1640 is the most widely used culture medium for
*P. falciparum* and is the medium of choice since the development of continuous cultivation
of this *Plasmodium* species by Trager and Jensen [27]. RPMI 1640 was not "custom-made" to
optimize the in vitro growth of *P. falciparum* based on its nutritional requirements. William
Trager reasoned that RPMI 1640 may be suitable for maintaining *P. falciparum* in vitro
based on the observation that this medium was specifically developed for the cultivation
of human leukocytes [46]. In fact, RPMI 1640, usually supplemented with serum from
human or animal sources, has wide applications for maintaining the growth of different
cell cultures, including parasites other than *P. falciparum*. As in other culture media,
RPMI 1640 consists of D-glucose, amino acids, inorganic salts, vitamins, and glutathione
(the principal reducing agent in erythrocytes that protects hemoglobin from oxidation),
which are essential nutrients required by eukaryotic cells. Mature, enucleated, uninfected
human red blood cells maintain functional biosynthetic pathways, ion transport, and
buffer systems, including when they are placed in in vitro culture conditions in RPMI
1640 medium [47,48]. The importance of the major components of the culture medium is
presented below.

3.1.1. Glucose

Most animal cell lines require medium changes once or twice a week, depending on
the growth rate and cell density. For malaria cultivation, daily medium change is the norm.
The frequency of medium change may have to be increased to twice or thrice a day for
special experiments in which a high yield of parasites (therefore, high parasitemias and/or
high hematocrit) is required. Conversely, the frequency of medium change may be reduced
to once every 2 or 3 days under certain conditions by lowering hematocrit or starting
parasitemia, increasing the buffering capacity or glucose concentration, adding "cold"
hypoxanthine (i.e., a naturally occurring purine derivative, as distinguished from tritium-
labeled hypoxanthine that may be used as an indicator of parasite growth in drug sensitivity
assays [49,50]), or the combination of these factors (temperature can also be reduced).

Glucose is the primary source of energy for most cells. Some investigators have
attempted to improve the growth of parasites by increasing D-glucose concentration and
adding supplements to RPMI 1640. In one study, in which the medium was changed daily
(10% erythrocyte suspension and 0.8-1.5% starting parasitemia), the addition of 2 g/L of
extra glucose (i.e., a total of 4 g/L of glucose, including glucose already present in RPMI
1640), reduced glutathione (600 µg/mL) and hypoxanthine (50 µg/mL) to the standard
RPMI 1640 buffered with 40 mM (9.15 g/L) TES (RPMI 1640-TES-NaHCO3 medium),
resulted in a three-fold increase in parasite growth over 4 days in one reference clone
compared to the growth in the standard RPMI 1640-HEPES-NaHCO3 medium (note: increased glucose needs to be balanced by better buffering or more frequent medium change because of the greater production of lactate) [51]. The growth rate of three other
uncloned Thai isolates was not affected by hypoxanthine and glutathione supplements. In
another study, it has been shown that laboratory-adapted P. falciparum strains (0.5% initial
parasitemia, 5% hematocrit, and 5% O2-5% CO2-90% N2 gas mixture) can be cultivated
without medium change for three days using a similar glucose-enriched (4 g/L) RPMI
1640-TES-NaHCO3 mixture without hypoxanthine supplement [52]. A slight decrease
in pH (from 7.4 to 7.1) after three days was not detrimental to parasite growth. This
observation was confirmed in another study in which a modified cultivation technique in
agitated culture flasks (1% hematocrit and 1% initial parasitemia) with high glucose (4 g/L) concentration and a TES buffer (32 mM) in RPMI 1640 allowed a high yield of parasites without changing the culture medium for up to 3 days [53]. Ofulla et al. also reported that the addition of an extra 2 g/L of glucose increases growth rates in short-term cultures [54].

For routine cultures with daily medium changes, hematocrit and initial parasitemia should be adjusted and kept relatively low. The same principle applies to in vitro drug susceptibility assays in which the medium is not changed for 24-72 h. This is also important from the point of view of the inoculum effect on 50% inhibitory concentration (IC50) endpoints in drug susceptibility assays. The concentrations of glucose and buffer do not need to be increased for most experiments and assays. It should be kept in mind that the addition of high concentrations of glucose to the culture medium may result in a hypertonic solution (normal osmolarity in human plasma, 290 mOsM/kg), which is deleterious for parasite growth. For routine cultivation of *P. falciparum* parasites maintained at relatively low parasitemia (< 5%), D-glucose contained in RPMI 1640 is sufficient for optimal parasite growth if the culture medium is changed daily.

### 3.1.2. Amino Acids

The most thorough analysis of exogenous amino acid and vitamin requirement of *P. falciparum* maintained in vitro was performed by Divo et al. [55]. In their study, an RPMI 1640 medium containing hypoxanthine was entirely reconstituted, and parasite growth of culture-adapted *P. falciparum* strains in reconstituted RPMI 1640 media (supplemented with dialyzed human serum) without one amino acid or one vitamin was assessed over 48 to 96 h and compared with a reconstituted RPMI 1640 medium with a complete set of amino acids and vitamins. Dialyzed human serum lacks small water-soluble molecules (including amino acids, peptides, vitamins, purines) but retains macromolecules, such as proteins. When extensively dialyzed, human serum does not support the growth of parasites unless it is supplemented with hypoxanthine. The study demonstrated that exogenous amino acids in the culture medium are essential for parasite growth, implying that other potential sources of amino acids, including hemoglobin degradation by the parasites and serum, are not sufficient to sustain optimal parasite growth. Among 20 amino acids present in RPMI 1640, isoleucine and methionine are the most important for short-term cultures of laboratory-adapted *P. falciparum* strains. The absence of one of these two amino acids would result in a marked decrease (i.e., >75%) in parasite growth after 96 h. The absence of cystine, glutamate, glutamine, proline, or tyrosine would also lead to growth inhibition, but to a much lesser extent. Deletion of other amino acids has no apparent effect on parasite growth over 96 h.

A series of "follow-up" in vitro experiments confirmed that RPMI 1640 with isoleucine (20-147.5 µM), without any other amino acids, supported the growth of a reference clone (3D7) in long-term continuous cultivation (>2 months) to the same extent as the standard RPMI 1640 medium containing all 20 amino acids [56]. The parasite seems to be dependent on an exogenous supply of isoleucine because this amino acid is absent from human hemoglobin. An additional requirement for exogenous methionine (present at a low level in hemoglobin) may be strain-dependent.

The RPMI 1640 medium, as well as other synthetic culture media, contains amino acids at much higher concentrations than in human plasma [57]. High concentrations of amino acids have been thought by some malaria researchers to be beneficial for sustaining high parasite multiplication rates in vitro. Others have questioned whether malaria parasites actually require an exogenous source of amino acids. The asexual parasites ingest erythrocyte cytosol and catabolize hemoglobin in the food vacuole, where several enzymes (i.e., aspartic proteases (plasmepsins) and cysteine proteases (falcipains)) necessary to digest globin are found [58]. Based on these observations, it has been suggested that hemoglobin digestion may be the major source of amino acids for the erythrocytic stages of malaria parasites [47,48,59].
agitated culture flasks (1% hematocrit and 1% initial parasitemia) with high glucose (4 g/L) concentration and a TES buffer (32 mM) in RPMI 1640 allowed a high yield of parasites without changing the culture medium for up to 3 days [53]. Ofulla et al. also reported that the addition of an extra 2 g/L of glucose increases growth rates in short-term cultures [54].

For routine cultures with daily medium changes, hematocrit and initial parasitemia should be adjusted and kept relatively low. The same principle applies to in vitro drug susceptibility assays in which the medium is not changed for 24-72 h. This is also important from the point of view of the inoculum effect on 50% inhibitory concentration (IC50) endpoints in drug susceptibility assays. The concentrations of glucose and buffer do not need to be increased for most experiments and assays. It should be kept in mind that the addition of high concentrations of glucose to the culture medium may result in a hypertonic solution (normal osmolarity in human plasma, 290 mOsM/kg), which is deleterious for parasite growth. For routine cultivation of *P. falciparum* parasites maintained at relatively low parasitemia (< 5%), D-glucose contained in RPMI 1640 is sufficient for optimal parasite growth if the culture medium is changed daily.

### 3.1.2. Amino Acids

The most thorough analysis of exogenous amino acid and vitamin requirement of *P. falciparum* maintained in vitro was performed by Divo et al. [55]. In their study, an RPMI 1640 medium containing hypoxanthine was entirely reconstituted, and parasite growth of culture-adapted *P. falciparum* strains in reconstituted RPMI 1640 media (supplemented with dialyzed human serum) without one amino acid or one vitamin was assessed over 48 to 96 h and compared with a reconstituted RPMI 1640 medium with a complete set of amino acids and vitamins. Dialyzed human serum lacks small water-soluble molecules (including amino acids, peptides, vitamins, purines) but retains macromolecules, such as proteins. When extensively dialyzed, human serum does not support the growth of parasites unless it is supplemented with hypoxanthine. The study demonstrated that exogenous amino acids in the culture medium are essential for parasite growth, implying that other potential sources of amino acids, including hemoglobin degradation by the parasites and serum, are not sufficient to sustain optimal parasite growth. Among 20 amino acids present in RPMI 1640, isoleucine and methionine are the most important for short-term cultures of laboratory-adapted *P. falciparum* strains. The absence of one of these two amino acids would result in a marked decrease (i.e., >75%) in parasite growth after 96 h. The absence of cystine, glutamate, glutamine, proline, or tyrosine would also lead to growth inhibition, but to a much lesser extent. Deletion of other amino acids has no apparent effect on parasite growth over 96 h.

A series of "follow-up" in vitro experiments confirmed that RPMI 1640 with isoleucine (20-147.5 µM), without any other amino acids, supported the growth of a reference clone (3D7) in long-term continuous cultivation (>2 months) to the same extent as the standard RPMI 1640 medium containing all 20 amino acids [56]. The parasite seems to be dependent on an exogenous supply of isoleucine because this amino acid is absent from human hemoglobin. An additional requirement for exogenous methionine (present at a low level in hemoglobin) may be strain-dependent.

The RPMI 1640 medium, as well as other synthetic culture media, contains amino acids at much higher concentrations than in human plasma [57]. High concentrations of amino acids have been thought by some malaria researchers to be beneficial for sustaining high parasite multiplication rates in vitro. Others have questioned whether malaria parasites actually require an exogenous source of amino acids. The asexual parasites ingest erythrocyte cytosol and catabolize hemoglobin in the food vacuole, where several enzymes (i.e., aspartic proteases (plasmepsins) and cysteine proteases (falcipains)) necessary to digest globin are found [58]. Based on these observations, it has been suggested that hemoglobin digestion may be the major source of amino acids for the erythrocytic stages of malaria parasites [47,48,59].
Malaria parasites can also selectively take up host serum proteins and digest them [60,61]. How the parasites obtain direct access to macromolecules is still not known. Endocytosis through cytostome, tubovesicular membranes, and/or the controversial parasitophorous duct may be involved [62–66]. The currently available data are not adequate to determine the role and contribution of exogenous (RPMI 1640 medium for in vitro cultivation) and endogenous (human plasma and hemoglobin in vivo and in vitro cultivation) sources of amino acids. It has also been suggested that *P. falciparum* may maintain several sources and pathways, including multiple proteases with overlapping functions in its food vacuole (plasmepsins and falcipains), to “guarantee” the supply of essential amino acids [56].

It may be questioned whether parasite physiology, in particular its basal metabolism, can be studied when the parasites are placed in an artificial environment with high amino acid concentrations, whether the medium is compatible with the identification of candidate biochemical targets and drug screening, and to what extent individual parasites constituting a field or clinical isolate are selected during adaptation to in vitro culture conditions, possibly limiting parasite diversity [57]. For example, an experimental study has suggested that high concentrations of L-cysteine (i.e., L-cysteine in RPMI 1640 plus additional exogenous L-cysteine) may inhibit parasite growth [67]). The significance and implications of that experimental study are not clear at present. Further experiments are clearly needed to define the basic nutritional needs of *P. falciparum* parasites.

### 3.1.3. Inorganic Salts

The balanced salt solution provides inorganic salts required for basal metabolism at sufficient and balanced concentrations that produce osmotic pressure similar to in vivo conditions. Early studies had shown that an addition of inorganic salts that are not in RPMI 1640, such as $FeSO_4$, $CuSO_4$, and $ZnSO_4$, had no beneficial effect on parasite growth [51]. In sharp contrast to amino acids and vitamins, the ionic composition of RPMI 1640 and similar culture media is within or near the physiological values of human plasma [57]. This is probably due to the assumption that osmoregulation is one of the important mechanisms that characterize a complex organization of mammalian cells, tissues, and organs, to maintain physiological levels of ions, including a narrow range of pH values, for optimal growth.

The intraerythrocytic malaria parasite is thought to modify the ionic composition of the host erythrocyte through an ion channel called “plasmodial surface anion channels (PSAC)”, which is expressed and exported by the parasite to the host erythrocyte membrane [68–72]. The ion channel induces a non-specific permeability, allowing various solutes, such as amino acids, sugars (except for disaccharides), glutathione, purines, pantothenic acid, and anions, but not cations (e.g., $Na^+$), to enter and accumulate in the host erythrocyte. However, in vitro cultivation of reference strains of *P. falciparum* in sucrose (impermeant disaccharide)-based RPMI 1640 with modified $Na^+$ or $K^+$ concentrations and dialyzed human serum suggested that low $Na^+$ (as low as 6.8 mM present in the serum supplement, compared to 142.8 mM in the standard RPMI 1640 medium) and high $K^+$ concentrations (up to 148 mM, compared to 5.4 mM in the standard RPMI 1640 medium) support parasite growth to a similar extent as the standard medium [73].

Among essential ions that are critically important for malaria parasites, calcium ($Ca^{2+}$) plays various roles to maintain normal cell functions and growth. $Ca^{2+}$ concentration in the cytosol of mature *P. falciparum* trophozoite has been estimated to be 0.1 µM [74]. The parasite accumulates $Ca^{2+}$ (through increased parasite membrane permeability, erythrocyte $Ca^{2+}$ ATPase, and possibly $Ca^{2+}$ channel) and stores $Ca^{2+}$ in its organelles (endoplasmic reticulum, food vacuole, mitochondrion, and/or acidocalcisome) [75–78]. The mechanisms involved in $Ca^{2+}$ transport in the intraerythrocytic parasite remain poorly understood, and the concentrations of $Ca^{2+}$ in different compartments and organelles are technically difficult to determine.

For $Mg^{2+}$ (0.41 mM in RPMI 1640 and 0.55 mM in human plasma [57]), concentrations between 0.5 and 3 mM have no effect on the in vitro growth of laboratory-adapted *P. falciparum* strains over several asexual intraerythrocytic cycles, but both high $Mg^{2+}$ con-
Malaria parasites can also selectively take up host serum proteins and digest them [60,61]. How the parasites obtain direct access to macromolecules is still not known. Endocytosis through cytostome, tubovesicular membranes, and/or the controversial parasitophorous duct may be involved [62–66]. The currently available data are not adequate to determine the role and contribution of exogenous (RPMI 1640 medium for in vitro cultivation) and endogenous (human plasma and hemoglobin in vivo and in vitro cultivation) sources of amino acids. It has also been suggested that *P. falciparum* may maintain several sources and pathways, including multiple proteases with overlapping functions in its food vacuole (plasmepsins and falcipains), to “guarantee” the supply of essential amino acids [56].

It may be questioned whether parasite physiology, in particular its basal metabolism, can be studied when the parasites are placed in an artificial environment with high amino acid concentrations, whether the medium is compatible with the identification of candidate biochemical targets and drug screening, and to what extent individual parasites constituting a field or clinical isolate are selected during adaptation to in vitro culture conditions, possibly limiting parasite diversity [57]. For example, an experimental study has suggested that high concentrations of L-cysteine (i.e., L-cysteine in RPMI 1640 plus additional exogenous L-cysteine) may inhibit parasite growth [67]). The significance and implications of that experimental study are not clear at present. Further experiments are clearly needed to define the basic nutritional needs of *P. falciparum* parasites.

### 3.1.3. Inorganic Salts

The balanced salt solution provides inorganic salts required for basal metabolism at sufficient and balanced concentrations that produce osmotic pressure similar to in vivo conditions. Early studies had shown that an addition of inorganic salts that are not in RPMI 1640, such as $FeSO_4$, $CuSO_4$, and $ZnSO_4$, had no beneficial effect on parasite growth [51]. In sharp contrast to amino acids and vitamins, the ionic composition of RPMI 1640 and similar culture media is within or near the physiological values of human plasma [57]. This is probably due to the assumption that osmoregulation is one of the important mechanisms that characterize a complex organization of mammalian cells, tissues, and organs, to maintain physiological levels of ions, including a narrow range of pH values, for optimal growth.

The intraerythrocytic malaria parasite is thought to modify the ionic composition of the host erythrocyte through an ion channel called “plasmodial surface anion channels (PSAC)”, which is expressed and exported by the parasite to the host erythrocyte membrane [68–72]. The ion channel induces a non-specific permeability, allowing various solutes, such as amino acids, sugars (except for disaccharides), glutathione, purines, pantothenic acid, and anions, but not cations (e.g., $Na^+$), to enter and accumulate in the host erythrocyte. However, in vitro cultivation of reference strains of *P. falciparum* in sucrose (impermeant disaccharide)-based RPMI 1640 with modified $Na^+$ or $K^+$ concentrations and dialyzed human serum suggested that low $Na^+$ (as low as 6.8 mM present in the serum supplement, compared to 142.8 mM in the standard RPMI 1640 medium) and high $K^+$ concentrations (up to 148 mM, compared to 5.4 mM in the standard RPMI 1640 medium) support parasite growth to a similar extent as the standard medium [73].

Among essential ions that are critically important for malaria parasites, calcium ($Ca^{2+}$) plays various roles to maintain normal cell functions and growth. $Ca^{2+}$ concentration in the cytosol of mature *P. falciparum* trophozoite has been estimated to be 0.1 µM [74]. The parasite accumulates $Ca^{2+}$ (through increased parasite membrane permeability, erythrocyte $Ca^{2+}$ ATPase, and possibly $Ca^{2+}$ channel) and stores $Ca^{2+}$ in its organelles (endoplasmic reticulum, food vacuole, mitochondrion, and/or acidocalcisome) [75–78]. The mechanisms involved in $Ca^{2+}$ transport in the intraerythrocytic parasite remain poorly understood, and the concentrations of $Ca^{2+}$ in different compartments and organelles are technically difficult to determine.

For $Mg^{2+}$ (0.41 mM in RPMI 1640 and 0.55 mM in human plasma [57]), concentrations between 0.5 and 3 mM have no effect on the in vitro growth of laboratory-adapted *P. falciparum* strains over several asexual intraerythrocytic cycles, but both high $Mg^{2+}$ con-
centrations (5 mM) and the absence of Mg²⁺ in the medium (i.e., cultivation in Mg²⁺-free RPMI 1640 medium) result in growth retardation by 35–43% after 48 h of cultivation [79].

Further experiments are required to determine the optimal extracellular and intraerythrocytic ionic concentrations that support in vitro parasite growth. Other transport mechanisms or organelles, such as passive diffusion, host erythrocyte channels, cystostome, tubovesicular membranes, and/or the controversial parasitophorous duct, may also possibly be involved in the acquisition of various nutrients and the modification of the host environment [62–66,80].

An early experimental study showed that once the complete RPMI 1640 medium containing 10% human serum (or animal serum) is used to cultivate malaria-infected erythrocytes or even uninfected erythrocytes for up to 12 h and subsequently stored at –20 °C, the spent media do not support parasite growth of the same parasite strain, even when fresh serum (final serum concentration, 20% v/v) is added [29]. The spent medium incubated with uninfected erythrocytes supports parasite growth better than the spent medium exposed to infected erythrocytes, but not to the extent that fresh medium supports parasite growth. This inhibitory effect is not due to the buffering capacity of the medium since the pH was not altered compared with the fresh medium. The study did not identify which RPMI 1640 component was altered when the culture medium was exposed to the erythrocytes.

It has been shown that any change in the concentrations of inorganic salts, D-glucose (for example, increasing from 2 g/L supplied in the medium to 3 g/L), glutathione, adenosine, and amino acids (such as isoleucine and methionine) leads to either no improvement in parasite growth or, worse, poor parasite growth [55,81]. Despite the fact that there is no specific culture medium designed for malaria cultivation and some modified RPMI 1640 media have been shown to support the growth of laboratory-adapted strains of *P. falciparum* in various experimental studies, it can still be said today, in 2023, that, by and large, among commercially available culture media, in the words of Jensen and Trager [81], “the RPMI 1640 medium is so well balanced that alterations are generally deleterious”. A possible exception to this general rule may be the beneficial effect of “cold” hypoxanthine added to the RPMI 1640 medium, especially when Albumax®, instead of human serum, is used to supplement the medium.

### 3.1.4. Vitamins

Vitamins are present at supraphysiological concentrations in RPMI 1640 [57]. In an early study, pantothenate (also referred to as D-pantothenic acid or vitamin B5, a precursor of coenzyme A) was shown to be the only vitamin necessary for optimal parasite growth for 96 h [55]. Whereas uninfected human erythrocytes are impermeable to pantothenate, *P. falciparum* develops its own pantothenate uptake system through a specific transporter [82,83]. The biosynthetic pathway of the transformation of pantothenate to coenzyme A has been characterized in *P. falciparum* [84]. The dependence of the parasite on an exogenous source of pantothenate is further supported by experiments, which demonstrated the inhibitory effects of pantothenate analogs against a laboratory-adapted *P. falciparum* strain [85].

In the study conducted by Divo et al. [55], the “minimal medium” consisting of 10% dialyzed human serum and the seven amino acids and pantothenate in the “basal medium” (inorganic salts, hypoxanthine, glutathione, HEPES buffer) was successfully used to maintain culture-adapted *P. falciparum* strains for up to 6 weeks. The “basal medium” supplemented with 10% undialyzed human serum and pantothenate but without any additional amino acids supported parasite growth to the level comparable to normal RPMI 1640 supplemented with 10% undialyzed human serum for only 48 h. Despite the major insight into the parasite’s nutritional requirement brought about by the study conducted by Divo et al. [55], the exact role of the erythrocyte stores of vitamins (also amino acids) in supplying nutrients to *P. falciparum* is still not well-known [86]. Further experiments on the
centrations (5 mM) and the absence of Mg²⁺ in the medium (i.e., cultivation in Mg²⁺-free RPMI 1640 medium) result in growth retardation by 35–43% after 48 h of cultivation [79].

Further experiments are required to determine the optimal extracellular and intraerythrocytic ionic concentrations that support in vitro parasite growth. Other transport mechanisms or organelles, such as passive diffusion, host erythrocyte channels, cystostome, tubovesicular membranes, and/or the controversial parasitophorous duct, may also possibly be involved in the acquisition of various nutrients and the modification of the host environment [62–66,80].

An early experimental study showed that once the complete RPMI 1640 medium containing 10% human serum (or animal serum) is used to cultivate malaria-infected erythrocytes or even uninfected erythrocytes for up to 12 h and subsequently stored at –20 °C, the spent media do not support parasite growth of the same parasite strain, even when fresh serum (final serum concentration, 20% v/v) is added [29]. The spent medium incubated with uninfected erythrocytes supports parasite growth better than the spent medium exposed to infected erythrocytes, but not to the extent that fresh medium supports parasite growth. This inhibitory effect is not due to the buffering capacity of the medium since the pH was not altered compared with the fresh medium. The study did not identify which RPMI 1640 component was altered when the culture medium was exposed to the erythrocytes.

It has been shown that any change in the concentrations of inorganic salts, D-glucose (for example, increasing from 2 g/L supplied in the medium to 3 g/L), glutathione, adenosine, and amino acids (such as isoleucine and methionine) leads to either no improvement in parasite growth or, worse, poor parasite growth [55,81]. Despite the fact that there is no specific culture medium designed for malaria cultivation and some modified RPMI 1640 media have been shown to support the growth of laboratory-adapted strains of *P. falciparum* in various experimental studies, it can still be said today, in 2023, that, by and large, among commercially available culture media, in the words of Jensen and Trager [81], “the RPMI 1640 medium is so well balanced that alterations are generally deleterious”. A possible exception to this general rule may be the beneficial effect of “cold” hypoxanthine added to the RPMI 1640 medium, especially when Albumax®, instead of human serum, is used to supplement the medium.

### 3.1.4. Vitamins

Vitamins are present at supraphysiological concentrations in RPMI 1640 [57]. In an early study, pantothenate (also referred to as D-pantothenic acid or vitamin B5, a precursor of coenzyme A) was shown to be the only vitamin necessary for optimal parasite growth for 96 h [55]. Whereas uninfected human erythrocytes are impermeable to pantothenate, *P. falciparum* develops its own pantothenate uptake system through a specific transporter [82,83]. The biosynthetic pathway of the transformation of pantothenate to coenzyme A has been characterized in *P. falciparum* [84]. The dependence of the parasite on an exogenous source of pantothenate is further supported by experiments, which demonstrated the inhibitory effects of pantothenate analogs against a laboratory-adapted *P. falciparum* strain [85].

In the study conducted by Divo et al. [55], the “minimal medium” consisting of 10% dialyzed human serum and the seven amino acids and pantothenate in the “basal medium” (inorganic salts, hypoxanthine, glutathione, HEPES buffer) was successfully used to maintain culture-adapted *P. falciparum* strains for up to 6 weeks. The “basal medium” supplemented with 10% undialyzed human serum and pantothenate but without any additional amino acids supported parasite growth to the level comparable to normal RPMI 1640 supplemented with 10% undialyzed human serum for only 48 h. Despite the major insight into the parasite’s nutritional requirement brought about by the study conducted by Divo et al. [55], the exact role of the erythrocyte stores of vitamins (also amino acids) in supplying nutrients to *P. falciparum* is still not well-known [86]. Further experiments on the
modified composition of RPMI 1640 did not lead to any improvement in maintaining the
parasites in vitro.

More recent evidence from biochemical and genomic studies suggested that intraerythrocytic *P. falciparum* may have functional vitamin B1 (thiamine) and B6 (pyridoxine) biosynthetic pathways and/or uptake systems [87,88]. However, there seem to be no plasmodial genes that encode enzymes of the biosynthetic pathways of vitamin C, D₃, E, and biotin [84].

Folic acid (vitamin B9) is one of the molecules collectively referred to as “folates”. Folates play several important roles in cellular functions as a donor of a one-carbon group, including nucleotide (purine) synthesis, synthesis of some amino acids (e.g., methionine), and amino acid metabolism. *Plasmodium falciparum* takes up exogenous folic acid and *para*-aminobenzoic acid (PABA), which are both present in RPMI 1640. Unlike humans, who cannot synthesize folate and require folate in their diet, malaria parasites synthesize folate de novo through intricate pathways that have not yet been fully elucidated [89,90]. Nonetheless, the essential features of the biosynthetic pathway have been known for decades, and two of its key enzymes (note: they are bifunctional enzymes in *P. falciparum*), hydroxymethyldihydropterin pyrophosphokinase-dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR)-thymidylate synthase, have been used as drug targets of sulfones/sulfa drugs and DHFR inhibitors (pyrimethamine, cycloguanil (biologically active metabolite of proguanil)), respectively [91].

3.2. *Alternative Culture Media*

Many other culture media contain similar components as RPMI 1640, with the same amino acids and vitamins and similar inorganic salts, usually, but not always, at slightly different concentrations. Some media contain more vitamins and other components, such as cholesterol, fatty acids, purines, and pyrimidines, compared with RPMI 1640. The following are some commercially available media that support the in vitro growth of asexual intraerythrocytic stages of *P. falciparum*: M-199 with either Hank's or Eagle's salt solution, Ham's nutrient mixture F-12, DMEM, IMDM, Waymouth medium, and NCTC 135 (medium formulated at the National Cancer Institute, Tissue Culture section) [26,30,92–99]. In addition to these media, RPMI 1630 was reported to support parasite growth in vitro [96], but it is not available from commercial sources.

The GIT medium (Wako Pure Chemical Industries Ltd., Osaka, Japan) is a 1:1 (v/v) mixture of IMDM and Ham's F-12 nutrient medium supplemented with an ammonium sulfate fraction of adult bovine serum [100]. It has been used almost exclusively in Japanese research laboratories for cell cultures, including experimental studies on *P. falciparum* cultivation [101,102]. Outside Japan, GIT was used successfully to perform ex vivo drug susceptibility assays (i.e., using clinical isolates directly for assays without prior adaptation to in vitro culture) and to initiate cultivation of fresh clinical isolates and maintain them in culture for up to 14 days in Cameroon [103].

A single study has shown that in vitro cultivation of fresh clinical isolates of *P. falciparum* can be maintained for several weeks using plant tissue culture media, Nitsch medium, and White's medium, supplemented with 10% human serum and 2 mg/mL D-glucose and buffered with 25 mM HEPES and 29 mM NaHCO₃, without prior adaptation to these media [97]. The authors reported that the multiplication rate in these plant tissue culture media was similar to RPMI 1640. Although further experiments are required to validate these results, especially for the long-term cultivation of a panel of reference *P. falciparum* strains beyond four weeks, at present, there seems to be no particular advantage in using plant tissue culture media for the cultivation of *P. falciparum*.

Before 1976, experimental studies on short-term *P. falciparum* culture were generally conducted with the Harvard medium (prepared on the basis of a chemical analysis of monkey plasma; not available from commercial sources), CMRL-1066 (medium developed at the Connaugh Medical Research Laboratories), or M-199, buffered with glycylglycine and/or sodium bicarbonate and supplemented with D-glucose and fetal calf serum or human
modified composition of RPMI 1640 did not lead to any improvement in maintaining the
parasites in vitro.

More recent evidence from biochemical and genomic studies suggested that intraerythrocytic *P. falciparum* may have functional vitamin B1 (thiamine) and B6 (pyridoxine) biosynthetic pathways and/or uptake systems [87,88]. However, there seem to be no plasmodial genes that encode enzymes of the biosynthetic pathways of vitamin C, D₃, E, and biotin [84].

Folic acid (vitamin B9) is one of the molecules collectively referred to as “folates”. Folates play several important roles in cellular functions as a donor of a one-carbon group, including nucleotide (purine) synthesis, synthesis of some amino acids (e.g., methionine), and amino acid metabolism. *Plasmodium falciparum* takes up exogenous folic acid and *para*-aminobenzoic acid (PABA), which are both present in RPMI 1640. Unlike humans, who cannot synthesize folate and require folate in their diet, malaria parasites synthesize folate de novo through intricate pathways that have not yet been fully elucidated [89,90]. Nonetheless, the essential features of the biosynthetic pathway have been known for decades, and two of its key enzymes (note: they are bifunctional enzymes in *P. falciparum*), hydroxymethyldihydropterin pyrophosphokinase-dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR)-thymidylate synthase, have been used as drug targets of sulfones/sulfa drugs and DHFR inhibitors (pyrimethamine, cycloguanil (biologically active metabolite of proguanil)), respectively [91].

3.2. *Alternative Culture Media*

Many other culture media contain similar components as RPMI 1640, with the same amino acids and vitamins and similar inorganic salts, usually, but not always, at slightly different concentrations. Some media contain more vitamins and other components, such as cholesterol, fatty acids, purines, and pyrimidines, compared with RPMI 1640. The following are some commercially available media that support the in vitro growth of asexual intraerythrocytic stages of *P. falciparum*: M-199 with either Hank's or Eagle's salt solution, Ham's nutrient mixture F-12, DMEM, IMDM, Waymouth medium, and NCTC 135 (medium formulated at the National Cancer Institute, Tissue Culture section) [26,30,92–99]. In addition to these media, RPMI 1630 was reported to support parasite growth in vitro [96], but it is not available from commercial sources.

The GIT medium (Wako Pure Chemical Industries Ltd., Osaka, Japan) is a 1:1 (v/v) mixture of IMDM and Ham's F-12 nutrient medium supplemented with an ammonium sulfate fraction of adult bovine serum [100]. It has been used almost exclusively in Japanese research laboratories for cell cultures, including experimental studies on *P. falciparum* cultivation [101,102]. Outside Japan, GIT was used successfully to perform ex vivo drug susceptibility assays (i.e., using clinical isolates directly for assays without prior adaptation to in vitro culture) and to initiate cultivation of fresh clinical isolates and maintain them in culture for up to 14 days in Cameroon [103].

A single study has shown that in vitro cultivation of fresh clinical isolates of *P. falciparum* can be maintained for several weeks using plant tissue culture media, Nitsch medium, and White's medium, supplemented with 10% human serum and 2 mg/mL D-glucose and buffered with 25 mM HEPES and 29 mM NaHCO₃, without prior adaptation to these media [97]. The authors reported that the multiplication rate in these plant tissue culture media was similar to RPMI 1640. Although further experiments are required to validate these results, especially for the long-term cultivation of a panel of reference *P. falciparum* strains beyond four weeks, at present, there seems to be no particular advantage in using plant tissue culture media for the cultivation of *P. falciparum*.

Before 1976, experimental studies on short-term *P. falciparum* culture were generally conducted with the Harvard medium (prepared on the basis of a chemical analysis of monkey plasma; not available from commercial sources), CMRL-1066 (medium developed at the Connaugh Medical Research Laboratories), or M-199, buffered with glycylglycine and/or sodium bicarbonate and supplemented with D-glucose and fetal calf serum or human
serum and incubated in 5% CO₂/95% air [104,105]. Except for M-199, these aforementioned culture media are no longer used for malaria parasite cultivation.

Modified M-199 had been used for the continuous cultivation of *P. falciparum*. In one study [26], M-199 containing Eagle's salts was supplemented with D-glucose (2 mg/mL), L-glutamine (2 mM), 2-mercaptoethanol (0.3 µM), DL-α-tocopherol (30 µg/mL), and 10% heat-inactivated fetal bovine serum. Cryopreserved parasites were thawed and successfully maintained in continuous culture for more than three weeks. In another study [30], M-199 was supplemented with adenosine triphosphate (ATP), adenosine, inositol, coenzyme A, creatinine, glutathione, glyglycine, D-glucose, vitamin C, 15–20% pooled, and heat-inactivated calf serum to initiate the long-term cultivation of cryopreserved isolates and maintain the strain for over a year. The growth of a laboratory-adapted strain routinely maintained in RPMI 1640 was compared in Ham's F12, M-199 with Earle's salts, and M-199 with Hank's salts after a 96 h incubation [93]. Parasite growth in Ham's F12 was comparable, or slightly less, with RPMI 1640 when the media were supplemented with 5% or 10% human serum. Parasite growth in M-199 with Earle's salts or Hank's salts was two-to-three-fold less than either RPMI 1640 or Ham's F12. The use of unsupplemented M-199 resulted in suboptimal parasite growth. In short, there seems to be no advantage in using Ham's F12 or M-199 instead of RPMI 1640 for the continuous culture of *P. falciparum*.

A series of experiments on the short-term ex vivo cultivation of fresh clinical isolates was performed with various commercially prepared culture media [103]. DMEM and IMDM consistently supported the in vitro growth of parasites during the first intraerythrocytic cycle and were usually better than RPMI 1640 in most isolates. However, these media may not be suitable to establish a continuous culture or even for a short-term culture from and beyond the second life cycle. GIT was another medium that supported the growth of fresh isolates for both short-term and long-term cultivation. Unmodified Ham's nutrient mixtures F-12 and M-199 did not support the in vitro growth of fresh isolates, as well as RPMI 1640.

In other studies on laboratory-adapted strains [98,99], it has been shown that various 1:1 (v/v) combinations of two of the following media, RPMI 1640, NCTC 135, and IMDM, supplemented with 10% human serum, do not improve parasite growth over the control medium (RPMI 1640 with 10% human serum) during a 7-day cultivation period. A 2:1:1 mixture of three media resulted in about a 50% increase in parasitemia. The media with serum substitutes, such as Albumax® and some animal sera (fetal calf, horse, rabbit, and goat), also supported the short-term culture with prior adaptation. Furthermore, the authors reported that this combination of three media, supplemented with 10% fetal bovine serum or 10% horse serum, may also be suitable for long-term cultivation.

Although parasite growth using these media may be comparable with RPMI 1640 for short-term and/or long-term culture, most do not seem to offer any distinct advantage over the latter medium. The capacity of some other media to support parasite development for a short-term period has been evaluated, but none were comparable to RPMI 1640 [103,106]. Therefore, after more than 45 years of experience with different malaria researchers, RPMI 1640 remains the standard medium of choice for the in vitro culture of *P. falciparum*. Future studies may possibly reveal that serum-free media are useful for routine cultivation of *P. falciparum*, but more short-term and long-term cultivation experiments will be needed to ascertain whether the majority of laboratory-adapted strains and fresh isolates adapt to alternative media.

## 4. Buffer

### 4.1. pH Regulation in the Human Host

Acid-base regulation is one of the key metabolic activities that characterize a living system. Cell metabolism involves the biosynthesis of many substances as well as the excretion of waste by-products that are generally acidic. In the human host, buffer systems, lungs, and kidneys regulate H⁺ concentration. Under physiological conditions, CO₂, the major metabolic product that yields H₂CO₃, and lactic acid produced due to muscular
serum and incubated in 5% CO₂/95% air [104,105]. Except for M-199, these aforementioned culture media are no longer used for malaria parasite cultivation.

Modified M-199 had been used for the continuous cultivation of *P. falciparum*. In one study [26], M-199 containing Eagle's salts was supplemented with D-glucose (2 mg/mL), L-glutamine (2 mM), 2-mercaptoethanol (0.3 µM), DL-α-tocopherol (30 µg/mL), and 10% heat-inactivated fetal bovine serum. Cryopreserved parasites were thawed and successfully maintained in continuous culture for more than three weeks. In another study [30], M-199 was supplemented with adenosine triphosphate (ATP), adenosine, inositol, coenzyme A, creatinine, glutathione, glyglycine, D-glucose, vitamin C, 15–20% pooled, and heat-inactivated calf serum to initiate the long-term cultivation of cryopreserved isolates and maintain the strain for over a year. The growth of a laboratory-adapted strain routinely maintained in RPMI 1640 was compared in Ham's F12, M-199 with Earle's salts, and M-199 with Hank's salts after a 96 h incubation [93]. Parasite growth in Ham's F12 was comparable, or slightly less, with RPMI 1640 when the media were supplemented with 5% or 10% human serum. Parasite growth in M-199 with Earle's salts or Hank's salts was two-to-three-fold less than either RPMI 1640 or Ham's F12. The use of unsupplemented M-199 resulted in suboptimal parasite growth. In short, there seems to be no advantage in using Ham's F12 or M-199 instead of RPMI 1640 for the continuous culture of *P. falciparum*.

A series of experiments on the short-term ex vivo cultivation of fresh clinical isolates was performed with various commercially prepared culture media [103]. DMEM and IMDM consistently supported the in vitro growth of parasites during the first intraerythrocytic cycle and were usually better than RPMI 1640 in most isolates. However, these media may not be suitable to establish a continuous culture or even for a short-term culture from and beyond the second life cycle. GIT was another medium that supported the growth of fresh isolates for both short-term and long-term cultivation. Unmodified Ham's nutrient mixtures F-12 and M-199 did not support the in vitro growth of fresh isolates, as well as RPMI 1640.

In other studies on laboratory-adapted strains [98,99], it has been shown that various 1:1 (*v*/*v*) combinations of two of the following media, RPMI 1640, NCTC 135, and IMDM, supplemented with 10% human serum, do not improve parasite growth over the control medium (RPMI 1640 with 10% human serum) during a 7-day cultivation period. A 2:1:1 mixture of three media resulted in about a 50% increase in parasitemia. The media with serum substitutes, such as Albumax® and some animal sera (fetal calf, horse, rabbit, and goat), also supported the short-term culture with prior adaptation. Furthermore, the authors reported that this combination of three media, supplemented with 10% fetal bovine serum or 10% horse serum, may also be suitable for long-term cultivation.

Although parasite growth using these media may be comparable with RPMI 1640 for short-term and/or long-term culture, most do not seem to offer any distinct advantage over the latter medium. The capacity of some other media to support parasite development for a short-term period has been evaluated, but none were comparable to RPMI 1640 [103,106]. Therefore, after more than 45 years of experience with different malaria researchers, RPMI 1640 remains the standard medium of choice for the in vitro culture of *P. falciparum*. Future studies may possibly reveal that serum-free media are useful for routine cultivation of *P. falciparum*, but more short-term and long-term cultivation experiments will be needed to ascertain whether the majority of laboratory-adapted strains and fresh isolates adapt to alternative media.

## 4. Buffer

### 4.1. pH Regulation in the Human Host

Acid-base regulation is one of the key metabolic activities that characterize a living system. Cell metabolism involves the biosynthesis of many substances as well as the excretion of waste by-products that are generally acidic. In the human host, buffer systems, lungs, and kidneys regulate H⁺ concentration. Under physiological conditions, CO₂, the major metabolic product that yields H₂CO₃, and lactic acid produced due to muscular
activities are neutralized by buffering substances in the blood to maintain blood pH within a narrow range (normal range, pH 7.36–7.44 or 36–44 nM H⁺). The principal buffers in the blood are the carbonic acid/bicarbonate system (H₂CO₃/HCO₃⁻) in plasma, hemoglobin, and plasma proteins. The bicarbonate buffer system is the most important pH regulatory system in the extracellular compartment. An increased H⁺ concentration in the extracellular fluid is buffered by HCO₃⁻, resulting in increased CO₂ and H₂O production. Increased OH⁻ concentration in the extracellular fluid results in a reaction with H₂CO₃ to form HCO₃⁻. Excessive CO₂ and HCO₃⁻ are eliminated by the lungs and kidneys, respectively. The bicarbonate buffer system regulates the pH of the extracellular fluid and is jointly coordinated by the lungs and the kidneys. The protein component of hemoglobin and plasma proteins function as bases (i.e., proton acceptors) due to some amino acids with negative charges. The buffering capacity of hemoglobin is as important as the bicarbonate buffer system. The plasma protein buffer system plays a limited role. There are other buffer systems in the human body, such as the phosphate buffer system, but they do not have a direct bearing on in vitro cell culture.

## 4.2. Buffers in Malaria Cultivation

In a closed in vitro system in which the medium is not continuously replaced, acidic by-products accumulate rapidly in the extracellular environment and decrease the pH of the medium unless a buffer system neutralizes H⁺, and the spent medium is removed and replaced with a fresh medium regularly. The combination of NaHCO₃ added to RPMI 1640 medium, erythrocytes, plasma (or serum) supplement, and gaseous CO₂ (usually 5% in a CO₂ incubator, 3% in a candle jar versus 5–6% CO₂ in human arterial and venous blood), simulates the in vivo conditions.

Sodium bicarbonate is used as one of the buffering systems in the method developed by Trager and Jensen [27]. In fact, most types of cells are cultivated using the bicarbonate/CO₂ buffering system. This system was originally designed to maintain the pH of culture media within the physiological range in gas mixtures containing 5% CO₂ and 95% air. Carbon dioxide gas dissolves in liquid media and affects pH (CO₂ + H₂O ⇌ H⁺ + HCO₃⁻). At a constant NaHCO₃ concentration, a lower percentage of CO₂ (1–4%) in the gas mixture increases pH [107]. Increasing or decreasing the bicarbonate concentration results in lower rates of parasite growth [54].

Since the in vitro culture of many types of cells is performed in 5% CO₂, the addition of NaHCO₃ to commercial media has become a standard practice today. The use of NaHCO₃ is appropriate for malaria cultures in a candle jar (approximately 3% CO₂ and 17–18% O₂) or in a 5% CO₂ incubator. The main disadvantage of bicarbonate is suboptimal buffering capacity within the range of normal physiological pH due to its pKa of 6.3 at 37 °C. In addition to sodium bicarbonate, most culture media, including RPMI 1640, contain phosphate salts, which act as buffers. However, NaHCO₃/gaseous CO₂ systems, phosphates, erythrocytes (hemoglobin), and plasma proteins have a limited buffering capacity to maintain the pH of malaria cultures beyond 24 h, especially at high (>1%) parasitemias.

During the early development of cultivation techniques, one of the major technical challenges of developing cultivation methods for malaria parasites was the maintenance of the pH of the medium within a narrow range of about 7.3–7.5. Two technical solutions were possible to maintain the physiological pH: (i) a perfusion or continuous flow culture system that constantly removes lactic acid and adds fresh culture medium and (ii) the supplementation of the culture medium with zwitterion buffers in dilution systems. Perfusion or flow systems require sophisticated devices and are not suitable for field use. The use of zwitterionic buffers is a practical solution that is applicable even in field laboratories.

The choice of buffers was found to be one of the keys to successful cultivation. Zwitterions have both positive (secondary and tertiary amino groups) and negative (sulfonic and carboxylic groups) ions within the same molecule. Zwitterionic buffers were introduced toward the late-1960s. The buffers that were evaluated for in vitro cultivation of malaria parasites in earlier studies include N-glycylglycine (pKa 7.9 at 37 °C), N,N'-
activities are neutralized by buffering substances in the blood to maintain blood pH within a narrow range (normal range, pH 7.36–7.44 or 36–44 nM H⁺). The principal buffers in the blood are the carbonic acid/bicarbonate system (H₂CO₃/HCO₃⁻) in plasma, hemoglobin, and plasma proteins. The bicarbonate buffer system is the most important pH regulatory system in the extracellular compartment. An increased H⁺ concentration in the extracellular fluid is buffered by HCO₃⁻, resulting in increased CO₂ and H₂O production. Increased OH⁻ concentration in the extracellular fluid results in a reaction with H₂CO₃ to form HCO₃⁻. Excessive CO₂ and HCO₃⁻ are eliminated by the lungs and kidneys, respectively. The bicarbonate buffer system regulates the pH of the extracellular fluid and is jointly coordinated by the lungs and the kidneys. The protein component of hemoglobin and plasma proteins function as bases (i.e., proton acceptors) due to some amino acids with negative charges. The buffering capacity of hemoglobin is as important as the bicarbonate buffer system. The plasma protein buffer system plays a limited role. There are other buffer systems in the human body, such as the phosphate buffer system, but they do not have a direct bearing on in vitro cell culture.

## 4.2. Buffers in Malaria Cultivation

In a closed in vitro system in which the medium is not continuously replaced, acidic by-products accumulate rapidly in the extracellular environment and decrease the pH of the medium unless a buffer system neutralizes H⁺, and the spent medium is removed and replaced with a fresh medium regularly. The combination of NaHCO₃ added to RPMI 1640 medium, erythrocytes, plasma (or serum) supplement, and gaseous CO₂ (usually 5% in a CO₂ incubator, 3% in a candle jar versus 5–6% CO₂ in human arterial and venous blood), simulates the in vivo conditions.

Sodium bicarbonate is used as one of the buffering systems in the method developed by Trager and Jensen [27]. In fact, most types of cells are cultivated using the bicarbonate/CO₂ buffering system. This system was originally designed to maintain the pH of culture media within the physiological range in gas mixtures containing 5% CO₂ and 95% air. Carbon dioxide gas dissolves in liquid media and affects pH (CO₂ + H₂O ⇌ H⁺ + HCO₃⁻). At a constant NaHCO₃ concentration, a lower percentage of CO₂ (1–4%) in the gas mixture increases pH [107]. Increasing or decreasing the bicarbonate concentration results in lower rates of parasite growth [54].

Since the in vitro culture of many types of cells is performed in 5% CO₂, the addition of NaHCO₃ to commercial media has become a standard practice today. The use of NaHCO₃ is appropriate for malaria cultures in a candle jar (approximately 3% CO₂ and 17–18% O₂) or in a 5% CO₂ incubator. The main disadvantage of bicarbonate is suboptimal buffering capacity within the range of normal physiological pH due to its pKa of 6.3 at 37 °C. In addition to sodium bicarbonate, most culture media, including RPMI 1640, contain phosphate salts, which act as buffers. However, NaHCO₃/gaseous CO₂ systems, phosphates, erythrocytes (hemoglobin), and plasma proteins have a limited buffering capacity to maintain the pH of malaria cultures beyond 24 h, especially at high (>1%) parasitemias.

During the early development of cultivation techniques, one of the major technical challenges of developing cultivation methods for malaria parasites was the maintenance of the pH of the medium within a narrow range of about 7.3–7.5. Two technical solutions were possible to maintain the physiological pH: (i) a perfusion or continuous flow culture system that constantly removes lactic acid and adds fresh culture medium and (ii) the supplementation of the culture medium with zwitterion buffers in dilution systems. Perfusion or flow systems require sophisticated devices and are not suitable for field use. The use of zwitterionic buffers is a practical solution that is applicable even in field laboratories.

The choice of buffers was found to be one of the keys to successful cultivation. Zwitterions have both positive (secondary and tertiary amino groups) and negative (sulfonic and carboxylic groups) ions within the same molecule. Zwitterionic buffers were introduced toward the late-1960s. The buffers that were evaluated for in vitro cultivation of malaria parasites in earlier studies include N-glycylglycine (pKa 7.9 at 37 °C), N,N'-
bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES; pKa 7.1 at 25 °C, 6.9 at 37 °C), N-
tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES; pKa 7.4 at 25 °C, 7.2 at 37 °C),
and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; pKa 7.5 at 25 °C, 7.3 at
37 °C) [108]. The use of TES and HEPES is associated with the improved buffering capacity
of the culture medium and increased parasite growth. These two buffers are characterized
by similar useful pH ranges (6.8–8.2) and pKa values closer to the useful pH range for
cell culture media. Haynes et al. [26] and Trager and Jensen [27] have adopted TES and
HEPES for their cultivation techniques, respectively. Zwitterions provide an additional
advantage of being minimally influenced by changes in the gas phase, maintaining the pH
of the medium when cultures are placed in normal air, i.e., outside the CO₂ incubator or
candle jar, during experiments, or a manual change in the culture medium.

Most, if not all, researchers today use HEPES, instead of TES, in addition to NaHCO3
as buffers for P. falciparum cultivation. The buffering capacity of HEPES and TES for malaria
cultivation has not been compared in many studies. In one study, the multiplication rate of
the reference strain FCR-3 was higher at the end of a 72 h cultivation period in the RPMI
1640 medium buffered with 25 mM HEPES and supplemented with glucose (4 g/L) and
hypoxanthine (44 mg/L) compared to the multiplication rate in the medium buffered with
40 mM TES and supplemented with glucose (2 g/L) [109]. However, it is not clear whether
this difference was due to the seemingly higher concentration of glucose provided in the
RPMI 1640 medium buffered with HEPES and the absence of hypoxanthine supplement in
the RPMI 1640 medium buffered with TES in this experiment. Since there seems to be no
advantage to using TES instead of HEPES for malaria cultivation, the latter seems to be the
best choice.

In addition to the choice of buffers, the number of parasites in relation to the volume
of the culture medium should be kept low to limit the accumulation of lactic acid. Buffers
in the culture medium can maintain neutral pH over 24 h, and even for one complete
erythrocytic cycle of *P. falciparum*, provided that hematocrit (usually <12%, preferably
much lower) and initial parasitemia (usually <1%) are kept low. Cultivation at higher
hematocrit and/or parasitemia is possible by either increasing the frequency of medium
change (i.e., instead of once daily, increase to twice or thrice daily), increasing the buffering
capacity of the medium, or using special cultivation devices.

Cultures that are growing well show high glucose consumption by the parasites and
the accumulation of high lactic acid concentrations in the extracellular medium (two moles
of lactate per mole glucose), resulting in decreasing pH over time. Depending on the
developmental stage of asexual parasites, infected erythrocytes may produce 5–100 times
more lactic acid than uninfected erythrocytes [110]. Trophozoites and schizonts excrete
2–20 times more lactic acid than rings [110,111]. Optimal parasite growth occurs when the
parasites are maintained at relatively low parasitemias (<2.5%) with extracellular lactic
acid concentration <12 mM and the medium (RPMI 1640–40 mM TES–NaHCO3 with
50 µg/mL hypoxanthine and a total of 4 g/L glucose) is changed every 12 h [110]. At
an extracellular lactic acid concentration of 12–25 mM, more frequent medium changes
and/or the diminution of parasitemia are necessary to avoid growth inhibition. When the
parasites are exposed to extracellular lactic acid concentrations >25 mM for more than 6 h,
irreversible damage and parasite death occur.

In Trager and Jensen's culture method, buffering with 25 mM HEPES seems to be optimal for the routine maintenance of cultures. Increasing the concentration to 50 mM HEPES, with or without a 50% decrease in NaHCO3 concentration, does not improve parasite growth [81]. However, the addition of 35 mM HEPES, instead of 25 mM, and a reduction in hematocrit may allow cultivation without medium change for up to 3 days [112,113]. At 37 °C, the pKa values of NaHCO3 and HEPES are 6.3 and 7.3, respectively. The combined HEPES-NaHCO3 buffer system brings the buffering range close to the optimal physiological pH range (7.2–7.4).
bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES; pKa 7.1 at 25 °C, 6.9 at 37 °C), N-
tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES; pKa 7.4 at 25 °C, 7.2 at 37 °C),
and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; pKa 7.5 at 25 °C, 7.3 at
37 °C) [108]. The use of TES and HEPES is associated with the improved buffering capacity
of the culture medium and increased parasite growth. These two buffers are characterized
by similar useful pH ranges (6.8–8.2) and pKa values closer to the useful pH range for
cell culture media. Haynes et al. [26] and Trager and Jensen [27] have adopted TES and
HEPES for their cultivation techniques, respectively. Zwitterions provide an additional
advantage of being minimally influenced by changes in the gas phase, maintaining the pH
of the medium when cultures are placed in normal air, i.e., outside the CO₂ incubator or
candle jar, during experiments, or a manual change in the culture medium.

Most, if not all, researchers today use HEPES, instead of TES, in addition to NaHCO3
as buffers for P. falciparum cultivation. The buffering capacity of HEPES and TES for malaria
cultivation has not been compared in many studies. In one study, the multiplication rate of
the reference strain FCR-3 was higher at the end of a 72 h cultivation period in the RPMI
1640 medium buffered with 25 mM HEPES and supplemented with glucose (4 g/L) and
hypoxanthine (44 mg/L) compared to the multiplication rate in the medium buffered with
40 mM TES and supplemented with glucose (2 g/L) [109]. However, it is not clear whether
this difference was due to the seemingly higher concentration of glucose provided in the
RPMI 1640 medium buffered with HEPES and the absence of hypoxanthine supplement in
the RPMI 1640 medium buffered with TES in this experiment. Since there seems to be no
advantage to using TES instead of HEPES for malaria cultivation, the latter seems to be the
best choice.

In addition to the choice of buffers, the number of parasites in relation to the volume
of the culture medium should be kept low to limit the accumulation of lactic acid. Buffers
in the culture medium can maintain neutral pH over 24 h, and even for one complete
erythrocytic cycle of *P. falciparum*, provided that hematocrit (usually <12%, preferably
much lower) and initial parasitemia (usually <1%) are kept low. Cultivation at higher
hematocrit and/or parasitemia is possible by either increasing the frequency of medium
change (i.e., instead of once daily, increase to twice or thrice daily), increasing the buffering
capacity of the medium, or using special cultivation devices.

Cultures that are growing well show high glucose consumption by the parasites and
the accumulation of high lactic acid concentrations in the extracellular medium (two moles
of lactate per mole glucose), resulting in decreasing pH over time. Depending on the
developmental stage of asexual parasites, infected erythrocytes may produce 5–100 times
more lactic acid than uninfected erythrocytes [110]. Trophozoites and schizonts excrete
2–20 times more lactic acid than rings [110,111]. Optimal parasite growth occurs when the
parasites are maintained at relatively low parasitemias (<2.5%) with extracellular lactic
acid concentration <12 mM and the medium (RPMI 1640–40 mM TES–NaHCO3 with
50 µg/mL hypoxanthine and a total of 4 g/L glucose) is changed every 12 h [110]. At
an extracellular lactic acid concentration of 12–25 mM, more frequent medium changes
and/or the diminution of parasitemia are necessary to avoid growth inhibition. When the
parasites are exposed to extracellular lactic acid concentrations >25 mM for more than 6 h,
irreversible damage and parasite death occur.

In Trager and Jensen's culture method, buffering with 25 mM HEPES seems to be optimal for the routine maintenance of cultures. Increasing the concentration to 50 mM HEPES, with or without a 50% decrease in NaHCO3 concentration, does not improve parasite growth [81]. However, the addition of 35 mM HEPES, instead of 25 mM, and a reduction in hematocrit may allow cultivation without medium change for up to 3 days [112,113]. At 37 °C, the pKa values of NaHCO3 and HEPES are 6.3 and 7.3, respectively. The combined HEPES-NaHCO3 buffer system brings the buffering range close to the optimal physiological pH range (7.2–7.4).
**5. Erythrocytes**

Human erythrocytes are required for optimal in vitro growth of *P. falciparum*. Unless the complete life cycle is being initiated experimentally from sporozoites, the usual source of asexual erythrocytic *P. falciparum* culture is previously infected erythrocytes that have been cryopreserved, passaged in suitable animal hosts, or freshly obtained from patients. The most convenient and usual procedure, especially in the field, is to use the patients' own infected erythrocytes to initiate in vitro culture. If the initial parasitemia is less than 1%, the patient's infected erythrocytes can be directly used for culture. If the initial parasitemia is >1% and cultivation is performed in the field, it is recommended to dilute the infected erythrocytes with uninfected erythrocytes from a donor who has not taken an antimalarial drug (or antibiotics with antimalarial activity) in the recent past. To maintain the parasites in vitro beyond the first or second intraerythrocytic cycle, a regular addition of uninfected human erythrocytes, at least once every two intraerythrocytic cycles (i.e., every 4 days), is required.

The ability of fresh isolates of *P. falciparum* to adapt to in vitro cultivation, as well as the growth rates, is variable [114]. Several factors may explain these variations, including initial parasitemia, frequency, and the extent of red cell dilution, nutritional qualities of serum, and recent exposure to drug treatment before cultivation. In the experience of Trager and Jensen [115], the major factor underlying these variations is the still undefined innate characteristic of the donor's erythrocytes to support parasite growth. The results of a recent study tend to support the intuitive conclusion of Trager and Jensen. The multiplication rate of the 3D7 clone varied from 64% to 136% (compared to the mean multiplication rate in normal erythrocytes set to 100%) in different samples of erythrocytes of healthy blood donors with recent African ancestry without any blood disorders (e.g., hemoglobin S, hemoglobin C, thalassemia, glucose-6-phosphate dehydrogenase (G6PD) deficiency, hereditary elliptocytosis) [116]. Parasites grew at a higher multiplication rate over 48 h in the erythrocytes with higher membrane deformability, larger mean volume (i.e., mean corpuscular volume, MCV), a higher mass of hemoglobin per cell (i.e., mean corpuscular hemoglobin, MCH), and less fragile membranes. The genotypic profiles of the erythrocytes that predict a higher multiplication rate of *P. falciparum* have not been established in that study, but may include polymorphisms in ion channel proteins, red blood cell membrane backbone, and/or red cell plasma membrane proteins (which include receptors required for merozoite invasion).

**5.1. *Sources of Uninfected Erythrocytes and Anticoagulants*

In the late-1970s and early-1980s, some researchers used erythrocytes from simian sources to cultivate *P. falciparum* for experimental purposes. Haynes et al. [26] initiated the continuous in vitro culture from a cryopreserved, chimpanzee-adapted *P. falciparum* strain in erythrocytes from chimpanzees (*Pan troglodytes*), owl monkeys (*Aotus trivirgatus*), and humans. This parasite strain failed to develop in erythrocytes from rhesus monkeys (*Macaca mulatta*) and guinea pigs. Peterson et al. [117] also successfully cultivated several strains of *P. falciparum* in the erythrocytes of *Aotus trivirgatus*. Attempts to propagate *P. falciparum* strains in vitro using *Saimiri sciureus* (squirrel monkey) erythrocytes were met with limited success, i.e., up to three weeks of continuous culture [118]. By contrast, few laboratory-adapted *P. falciparum* strains (Palo Alto I/Uganda, FCB/Colombia) have been adapted to propagate in vivo in splenectomized *Saimiri sciureus* and adapted back to in vitro propagation in human erythrocytes [119,120]. Except for specific experimental purposes, human erythrocytes are used for malaria cultivation today.

The principal source of uninfected human erythrocytes, kept at +4 °C, is blood banks. Several blood storage protocols have been developed with different preservatives added at the time of blood collection from healthy donors. Blood preservatives are usually predosed in an aseptic collection pouch so that venous blood being collected is immediately mixed with the preservative. Blood preservatives that have been or are commonly used at blood banks include acid-citrate-dextrose (ACD), citrate-phosphate-dextrose (CPD) modified
**5. Erythrocytes**

Human erythrocytes are required for optimal in vitro growth of *P. falciparum*. Unless the complete life cycle is being initiated experimentally from sporozoites, the usual source of asexual erythrocytic *P. falciparum* culture is previously infected erythrocytes that have been cryopreserved, passaged in suitable animal hosts, or freshly obtained from patients. The most convenient and usual procedure, especially in the field, is to use the patients' own infected erythrocytes to initiate in vitro culture. If the initial parasitemia is less than 1%, the patient's infected erythrocytes can be directly used for culture. If the initial parasitemia is >1% and cultivation is performed in the field, it is recommended to dilute the infected erythrocytes with uninfected erythrocytes from a donor who has not taken an antimalarial drug (or antibiotics with antimalarial activity) in the recent past. To maintain the parasites in vitro beyond the first or second intraerythrocytic cycle, a regular addition of uninfected human erythrocytes, at least once every two intraerythrocytic cycles (i.e., every 4 days), is required.

The ability of fresh isolates of *P. falciparum* to adapt to in vitro cultivation, as well as the growth rates, is variable [114]. Several factors may explain these variations, including initial parasitemia, frequency, and the extent of red cell dilution, nutritional qualities of serum, and recent exposure to drug treatment before cultivation. In the experience of Trager and Jensen [115], the major factor underlying these variations is the still undefined innate characteristic of the donor's erythrocytes to support parasite growth. The results of a recent study tend to support the intuitive conclusion of Trager and Jensen. The multiplication rate of the 3D7 clone varied from 64% to 136% (compared to the mean multiplication rate in normal erythrocytes set to 100%) in different samples of erythrocytes of healthy blood donors with recent African ancestry without any blood disorders (e.g., hemoglobin S, hemoglobin C, thalassemia, glucose-6-phosphate dehydrogenase (G6PD) deficiency, hereditary elliptocytosis) [116]. Parasites grew at a higher multiplication rate over 48 h in the erythrocytes with higher membrane deformability, larger mean volume (i.e., mean corpuscular volume, MCV), a higher mass of hemoglobin per cell (i.e., mean corpuscular hemoglobin, MCH), and less fragile membranes. The genotypic profiles of the erythrocytes that predict a higher multiplication rate of *P. falciparum* have not been established in that study, but may include polymorphisms in ion channel proteins, red blood cell membrane backbone, and/or red cell plasma membrane proteins (which include receptors required for merozoite invasion).

**5.1. *Sources of Uninfected Erythrocytes and Anticoagulants*

In the late-1970s and early-1980s, some researchers used erythrocytes from simian sources to cultivate *P. falciparum* for experimental purposes. Haynes et al. [26] initiated the continuous in vitro culture from a cryopreserved, chimpanzee-adapted *P. falciparum* strain in erythrocytes from chimpanzees (*Pan troglodytes*), owl monkeys (*Aotus trivirgatus*), and humans. This parasite strain failed to develop in erythrocytes from rhesus monkeys (*Macaca mulatta*) and guinea pigs. Peterson et al. [117] also successfully cultivated several strains of *P. falciparum* in the erythrocytes of *Aotus trivirgatus*. Attempts to propagate *P. falciparum* strains in vitro using *Saimiri sciureus* (squirrel monkey) erythrocytes were met with limited success, i.e., up to three weeks of continuous culture [118]. By contrast, few laboratory-adapted *P. falciparum* strains (Palo Alto I/Uganda, FCB/Colombia) have been adapted to propagate in vivo in splenectomized *Saimiri sciureus* and adapted back to in vitro propagation in human erythrocytes [119,120]. Except for specific experimental purposes, human erythrocytes are used for malaria cultivation today.

The principal source of uninfected human erythrocytes, kept at +4 °C, is blood banks. Several blood storage protocols have been developed with different preservatives added at the time of blood collection from healthy donors. Blood preservatives are usually predosed in an aseptic collection pouch so that venous blood being collected is immediately mixed with the preservative. Blood preservatives that have been or are commonly used at blood banks include acid-citrate-dextrose (ACD), citrate-phosphate-dextrose (CPD) modified
CPD with 0.2 g/L adenine (CPD-A), and saline-adenine-glucose (SAG; or saline-adenine-glucose-mannitol, SAGM). CPD or CPD-A is the most commonly used anticoagulant. Judging from transfusion malaria occurrences, *P. falciparum*-infected cells in CPD retain their infectivity well in the refrigerator. Whole blood may be initially collected on CPD and centrifuged, and their plasma may be removed for other medical uses. The remaining blood is stored as packed cells.

In an early study, Jensen and Trager recommended the use of uninfected erythrocytes stored at +4 °C in citrated anticoagulants (ACD or CPD) for 2 to 4 weeks, rather than using erythrocytes freshly drawn from donors, to obtain an optimal growth of culture-adapted laboratory strains [81]. In a later study, it was reported that fresh blood collected on CPD and blood stored in CPD for 16–20 days resulted in up to a 2.3-fold increase in parasite growth compared with blood stored for 4–16 days [121]. The type of anticoagulants (CPD, CPD-A, and protein-poor preservatives) did not influence parasite growth when blood was stored for up to 30 days. Erythrocytes stored as packed cells were not suitable for culture beyond 21 days. These authors also noted that erythrocytes stored in CPD and protein-poor preservatives containing adenine supported parasite growth even after 84 days. In practice, most malaria research laboratories procure donors’ blood on a monthly basis so that any leftover blood beyond five weeks is discarded. In a more recent study, it was reported that high parasitemia and high multiplication rates of laboratory-adapted *P. falciparum* strains were consistently attained when erythrocytes stored at 4 °C in CPDA (as whole blood) for less than two weeks were used in cultures [122]. Another recent study confirmed that the growth of reference strains of *P. falciparum* was comparable in fresh erythrocytes collected into ACD anticoagulant and those that were stored as packed erythrocytes at 4 °C for two weeks, but was significantly diminished in erythrocytes stored in the same condition (packed erythrocytes at 4 °C) for four weeks (35–58% growth) and six weeks (<10% growth) [123]. Similar results were obtained with different storage buffers and anticoagulants, including ACD and CPDA. Contrary to some of the aforementioned early studies [81,121], a South Korean research group reported that, compared to the growth rate in freshly collected erythrocytes, the 96 h multiplication rates of a reference strain (FCR-3) cultivated under standard conditions (RPMI 1640 supplemented with 10% type AB⁺ human serum; 5% hematocrit; starting parasitemia 0.1% in suspension culture with constant shaking, in 5% CO₂, 5% O₂, and 90% N₂, and medium change every 12 h) decreased as the age of stored erythrocytes increased from 1 to 4 weeks [124]. The final parasitemias in cultures with 4-week-old erythrocytes collected in ACD or EDTA were 70% and 50% of those attained with freshly collected erythrocytes, respectively. The underlying reasons for these apparently discordant results have not been explored, but they may be partly due to the short cultivation period (i.e., two cycles) in the latter study.

From a practical viewpoint, fresh erythrocytes are usually not available on a daily or weekly basis in most malaria research laboratories. Although it would be ideal if a new batch of fresh erythrocytes could be obtained every two weeks [122], erythrocytes stored for up to 4 weeks are suitable for routine cultivation procedures. For optimal storage, erythrocytes should be kept in plasma until use. Erythrocytes are washed with RPMI 1640 before use.

Parasite growth was also reported to be comparable in erythrocytes from whole blood collected on CPD-A, packed cells stored in CPD-A, and packed cells stored in SAGM preservative (plasma from a 500 mL donor’s blood is removed and replaced with 100 mL of SAGM) [125]. Likewise, the use of fresh erythrocytes or erythrocytes stored in CPD for 4 weeks before use resulted in similar parasite growth during the 14-day cultivation period of a laboratory-adapted strain [29]. However, hemolysis tends to occur in red cells stored for over 5 weeks. Such outdated erythrocytes are not suitable for cultures. When the growth of several culture-adapted *P. falciparum* strains was compared over more than 1 month in the uninfected erythrocytes stored as whole blood in CPD and those stored as packed red cells without plasma in SAG, higher parasitemia was consistently observed with the erythrocytes stored in SAG [51].
CPD with 0.2 g/L adenine (CPD-A), and saline-adenine-glucose (SAG; or saline-adenine-glucose-mannitol, SAGM). CPD or CPD-A is the most commonly used anticoagulant. Judging from transfusion malaria occurrences, *P. falciparum*-infected cells in CPD retain their infectivity well in the refrigerator. Whole blood may be initially collected on CPD and centrifuged, and their plasma may be removed for other medical uses. The remaining blood is stored as packed cells.

In an early study, Jensen and Trager recommended the use of uninfected erythrocytes stored at +4 °C in citrated anticoagulants (ACD or CPD) for 2 to 4 weeks, rather than using erythrocytes freshly drawn from donors, to obtain an optimal growth of culture-adapted laboratory strains [81]. In a later study, it was reported that fresh blood collected on CPD and blood stored in CPD for 16–20 days resulted in up to a 2.3-fold increase in parasite growth compared with blood stored for 4–16 days [121]. The type of anticoagulants (CPD, CPD-A, and protein-poor preservatives) did not influence parasite growth when blood was stored for up to 30 days. Erythrocytes stored as packed cells were not suitable for culture beyond 21 days. These authors also noted that erythrocytes stored in CPD and protein-poor preservatives containing adenine supported parasite growth even after 84 days. In practice, most malaria research laboratories procure donors’ blood on a monthly basis so that any leftover blood beyond five weeks is discarded. In a more recent study, it was reported that high parasitemia and high multiplication rates of laboratory-adapted *P. falciparum* strains were consistently attained when erythrocytes stored at 4 °C in CPDA (as whole blood) for less than two weeks were used in cultures [122]. Another recent study confirmed that the growth of reference strains of *P. falciparum* was comparable in fresh erythrocytes collected into ACD anticoagulant and those that were stored as packed erythrocytes at 4 °C for two weeks, but was significantly diminished in erythrocytes stored in the same condition (packed erythrocytes at 4 °C) for four weeks (35–58% growth) and six weeks (<10% growth) [123]. Similar results were obtained with different storage buffers and anticoagulants, including ACD and CPDA. Contrary to some of the aforementioned early studies [81,121], a South Korean research group reported that, compared to the growth rate in freshly collected erythrocytes, the 96 h multiplication rates of a reference strain (FCR-3) cultivated under standard conditions (RPMI 1640 supplemented with 10% type AB⁺ human serum; 5% hematocrit; starting parasitemia 0.1% in suspension culture with constant shaking, in 5% CO₂, 5% O₂, and 90% N₂, and medium change every 12 h) decreased as the age of stored erythrocytes increased from 1 to 4 weeks [124]. The final parasitemias in cultures with 4-week-old erythrocytes collected in ACD or EDTA were 70% and 50% of those attained with freshly collected erythrocytes, respectively. The underlying reasons for these apparently discordant results have not been explored, but they may be partly due to the short cultivation period (i.e., two cycles) in the latter study.

From a practical viewpoint, fresh erythrocytes are usually not available on a daily or weekly basis in most malaria research laboratories. Although it would be ideal if a new batch of fresh erythrocytes could be obtained every two weeks [122], erythrocytes stored for up to 4 weeks are suitable for routine cultivation procedures. For optimal storage, erythrocytes should be kept in plasma until use. Erythrocytes are washed with RPMI 1640 before use.

Parasite growth was also reported to be comparable in erythrocytes from whole blood collected on CPD-A, packed cells stored in CPD-A, and packed cells stored in SAGM preservative (plasma from a 500 mL donor’s blood is removed and replaced with 100 mL of SAGM) [125]. Likewise, the use of fresh erythrocytes or erythrocytes stored in CPD for 4 weeks before use resulted in similar parasite growth during the 14-day cultivation period of a laboratory-adapted strain [29]. However, hemolysis tends to occur in red cells stored for over 5 weeks. Such outdated erythrocytes are not suitable for cultures. When the growth of several culture-adapted *P. falciparum* strains was compared over more than 1 month in the uninfected erythrocytes stored as whole blood in CPD and those stored as packed red cells without plasma in SAG, higher parasitemia was consistently observed with the erythrocytes stored in SAG [51].
An unconventional source of uninfected human blood for *P. falciparum* cultivation is biopreserved erythrocytes. It was reported that parasite growth in biopreserved erythrocytes was comparable to fresh erythrocytes [123]. For cryopreservation, fresh erythrocytes mixed with human plasma were added to freezing media (28% glycerol, 3% sorbitol, 0.65% NaCl). Biopreserved erythrocytes have not been used extensively for malaria cultivation but may be useful for experimental purposes.

Another source of human erythrocytes is human hematopoietic stem cells [126]. Culture systems of various protozoans, including *Plasmodium* spp., based on induced pluripotent stem cells are opening up new horizons [127]. One of the applications of human erythrocytes derived from pluripotent stem cells is humanized mice, which may hold promise in different research areas [128–132]. There is hope that this new experimental model will replace non-human primate models. Hematopoietic stem cells can also be used to study parasite biology in future in vitro experiments [133].

## 5.2. Blood Types

In the initial attempts to develop continuous cultivation of *P. falciparum*, malaria-infected erythrocytes of *Aotus* monkeys were mixed with human-type $AB^+$ erythrocytes and diluted in an RPMI 1640 medium containing 15% human-type $AB^+$ serum to avoid incompatibility with the *Aotus* blood [27]. After several cycles, established cultures were maintained in human-type $A^+$ erythrocytes and serum due to their wide availability. Type $A^+$ serum is compatible with both types A and O erythrocytes, which represent the dominant blood types in a large majority of Caucasian populations.

Early studies had established that all four human blood groups are suitable for culture [29,134]. If type AB human serum is used to supplement the RPMI 1640 medium, erythrocytes belonging to any of the four blood groups are compatible. This approach is convenient for ex vivo drug susceptibility assays on fresh isolates and the initial cultivation of clinical isolates in the field. Regardless of the initial blood type in which the parasites were present in the original human host, the subsequent in vitro intraerythrocytic cycle can be maintained in any blood type.

It has been hypothesized that *P. falciparum*-infected humans with group O+ have a survival advantage and that this selection pressure exerted by *P. falciparum* during the evolutionary history of *Homo sapiens* may explain the current global distribution of the ABO blood group, notably the high frequency of the group O+ in sub-Saharan Africa and South America [135]. Several studies have also suggested that type O+ is associated with a decreased risk of severe malaria [136,137]. One of the possible implications of this hypothesis is that *P. falciparum* may have adapted better to type O+ erythrocytes compared to other blood types. The results of a recent experimental study tend to support this viewpoint. Several laboratory strains of *P. falciparum* incubated simultaneously in the presence of a mixture of $A^+$, $B^+$, $O^+$, and $AB^+$ erythrocytes showed a preference for $O^+$ erythrocytes [138]. In another experiment, the parasitemias of two reference strains attained after two cycles when cultivated separately in $A^+$, $B^+$, and $O^+$ erythrocytes were higher in $O^+$ erythrocytes [139]. There was only a slight (but statistically significant) preference for $O^+$ erythrocytes, and the parasites also invaded erythrocytes of the other blood types at lower proportions or parasitemia.

In practice, many malaria research laboratories use type O+ and/or $A^+$ erythrocytes, which are the most common (i.e., close to ¾ of the human population) blood groups in the world. There seems to be no compelling reason at present to opt for a specific blood type. Any available ABO blood types can be used for *P. falciparum* in vitro cultivation. However, the use of erythrocytes from donors with known hemoglobinopathy (thalassemia, hemoglobin C, sickle cell anemia) should be avoided because *P. falciparum* growth may be suboptimal [116,139–146].
An unconventional source of uninfected human blood for *P. falciparum* cultivation is biopreserved erythrocytes. It was reported that parasite growth in biopreserved erythrocytes was comparable to fresh erythrocytes [123]. For cryopreservation, fresh erythrocytes mixed with human plasma were added to freezing media (28% glycerol, 3% sorbitol, 0.65% NaCl). Biopreserved erythrocytes have not been used extensively for malaria cultivation but may be useful for experimental purposes.

Another source of human erythrocytes is human hematopoietic stem cells [126]. Culture systems of various protozoans, including *Plasmodium* spp., based on induced pluripotent stem cells are opening up new horizons [127]. One of the applications of human erythrocytes derived from pluripotent stem cells is humanized mice, which may hold promise in different research areas [128–132]. There is hope that this new experimental model will replace non-human primate models. Hematopoietic stem cells can also be used to study parasite biology in future in vitro experiments [133].

## 5.2. Blood Types

In the initial attempts to develop continuous cultivation of *P. falciparum*, malaria-infected erythrocytes of *Aotus* monkeys were mixed with human-type $AB^+$ erythrocytes and diluted in an RPMI 1640 medium containing 15% human-type $AB^+$ serum to avoid incompatibility with the *Aotus* blood [27]. After several cycles, established cultures were maintained in human-type $A^+$ erythrocytes and serum due to their wide availability. Type $A^+$ serum is compatible with both types A and O erythrocytes, which represent the dominant blood types in a large majority of Caucasian populations.

Early studies had established that all four human blood groups are suitable for culture [29,134]. If type AB human serum is used to supplement the RPMI 1640 medium, erythrocytes belonging to any of the four blood groups are compatible. This approach is convenient for ex vivo drug susceptibility assays on fresh isolates and the initial cultivation of clinical isolates in the field. Regardless of the initial blood type in which the parasites were present in the original human host, the subsequent in vitro intraerythrocytic cycle can be maintained in any blood type.

It has been hypothesized that *P. falciparum*-infected humans with group O+ have a survival advantage and that this selection pressure exerted by *P. falciparum* during the evolutionary history of *Homo sapiens* may explain the current global distribution of the ABO blood group, notably the high frequency of the group O+ in sub-Saharan Africa and South America [135]. Several studies have also suggested that type O+ is associated with a decreased risk of severe malaria [136,137]. One of the possible implications of this hypothesis is that *P. falciparum* may have adapted better to type O+ erythrocytes compared to other blood types. The results of a recent experimental study tend to support this viewpoint. Several laboratory strains of *P. falciparum* incubated simultaneously in the presence of a mixture of $A^+$, $B^+$, $O^+$, and $AB^+$ erythrocytes showed a preference for $O^+$ erythrocytes [138]. In another experiment, the parasitemias of two reference strains attained after two cycles when cultivated separately in $A^+$, $B^+$, and $O^+$ erythrocytes were higher in $O^+$ erythrocytes [139]. There was only a slight (but statistically significant) preference for $O^+$ erythrocytes, and the parasites also invaded erythrocytes of the other blood types at lower proportions or parasitemia.

In practice, many malaria research laboratories use type O+ and/or $A^+$ erythrocytes, which are the most common (i.e., close to ¾ of the human population) blood groups in the world. There seems to be no compelling reason at present to opt for a specific blood type. Any available ABO blood types can be used for *P. falciparum* in vitro cultivation. However, the use of erythrocytes from donors with known hemoglobinopathy (thalassemia, hemoglobin C, sickle cell anemia) should be avoided because *P. falciparum* growth may be suboptimal [116,139–146].
### 5.3. Hematocrit

For continuous culture, parasites are usually maintained in 8–12% erythrocyte suspension (i.e., hematocrit). According to Jensen and Trager [81], 10–12% erythrocyte suspension in a complete RPMI 1640 medium yielding a depth of 3.5 to 4.5 mm in a 35 mm diameter Petri dish results in optimal parasite growth (note: based on the total volume of 1.5 mL of the RPMI human serum mixture with 10% erythrocyte suspension in the Petri dish, the hematocrit layer depth would be approximately 0.16 mm). In addition to the depth of the culture medium, the surface area of the medium is probably just as important to optimize gas exchange between the medium (i.e., O₂ and CO₂ dissolved in the medium) and the atmosphere in the incubator.

Depending on the experimental purpose, cultures may be maintained at a lower hematocrit, which favors parasite growth due to the relatively more abundant nutrients and buffering capacity in relation to the absolute number of parasites in Petri dishes, flasks, or culture plates, provided that the parasitemia is also kept at low levels (<2–3%) and fresh medium is added daily. In such conditions (low starting parasitemia of 0.1% and about 5% hematocrit), a more frequent medium change (i.e., the interval of medium change every 12 h versus every 24 h) does not improve the parasite's multiplication rate over two cycles, although a prolonged delay in medium change (i.e., every 48 h) would result in decreased multiplication rate during the second cycle [124]. In the protocol developed by Radfar et al. to optimize the yield of synchronized reference clones of *P. falciparum* (3D7 and Dd2; RPMI 1640 buffered with 25 mM HEPES and 1.77 mM NaHCO₃ and supplemented with 0.5% Albumax® I and 100 µM hypoxanthine; 5% CO₂ in air or 3% CO₂-1% O₂-96% N₂ gas mixture) [122], the authors recommend hematocrit values within the range of 0.8 to 1.5% and proposed a mathematical formula to calculate the approximate volume of the culture medium necessary to maintain parasite growth for 24 h (i.e., until the next change in the medium). Using this protocol (and fresh human erythrocytes stored <14 days), the authors reported that high parasitemia (20–30%) can be obtained in 150 cm² culture flasks (maximum volume of culture medium, 140 mL). For in vitro drug susceptibility assays that require a 42–72 h incubation, cultures in 96-well plates are performed at low hematocrit, generally at 1.5%, to avoid the labor-intensive task of removing the spent medium and adding the fresh medium after the initial 24 h of incubation. The cultures can also be maintained at a much higher hematocrit in special devices. This will be discussed in another section.

Theoretically, parasite growth is optimal at the interface between the culture medium and the uppermost layer of erythrocytes, where the exchange of gas, nutrients, and waste products occurs freely. By contrast, the exchange is suboptimal at the bottom layer of the erythrocytes. Depending on the protocols of cultivation in different research laboratories, the depth of the hematocrit layer may vary from 0.15 mm to 0.8 mm or more. Mathematical models that attempt to describe the complex interplay between parasite multiplication rate, hematocrit layer depth, the width or diameter of culture devices, and various other factors have been developed, and the effects of the dimensions of the hematocrit layer were analyzed to determine the conditions leading to optimal parasite growth [147,148]. One of the key findings using the model and experimental data obtained from *P. falciparum* (3D7 reference strain) cultivated under standard conditions (RPMI 1640 supplemented with 10% human serum, 5% hematocrit, initial parasitemia 0.5%, 5% O₂, 5% CO₂, and 90% N₂) in 6-well plates (35 mm diameter wells) and the renewal of culture medium and dilution of cultures to 0.5% parasitemia every 48 h was that optimal development (i.e., highest parasitemia after 48 h) occurs in a hematocrit layer depth between 0.18 mm and 0.34 mm. This value is close to the theoretical hematocrit layer depth found empirically by Jensen and Trager (i.e., approximately 0.16 mm) [81]. Parasite growth diminished considerably when the depth of the hematocrit layer exceeded 1 mm.
### 5.3. Hematocrit

For continuous culture, parasites are usually maintained in 8–12% erythrocyte suspension (i.e., hematocrit). According to Jensen and Trager [81], 10–12% erythrocyte suspension in a complete RPMI 1640 medium yielding a depth of 3.5 to 4.5 mm in a 35 mm diameter Petri dish results in optimal parasite growth (note: based on the total volume of 1.5 mL of the RPMI human serum mixture with 10% erythrocyte suspension in the Petri dish, the hematocrit layer depth would be approximately 0.16 mm). In addition to the depth of the culture medium, the surface area of the medium is probably just as important to optimize gas exchange between the medium (i.e., O₂ and CO₂ dissolved in the medium) and the atmosphere in the incubator.

Depending on the experimental purpose, cultures may be maintained at a lower hematocrit, which favors parasite growth due to the relatively more abundant nutrients and buffering capacity in relation to the absolute number of parasites in Petri dishes, flasks, or culture plates, provided that the parasitemia is also kept at low levels (<2–3%) and fresh medium is added daily. In such conditions (low starting parasitemia of 0.1% and about 5% hematocrit), a more frequent medium change (i.e., the interval of medium change every 12 h versus every 24 h) does not improve the parasite's multiplication rate over two cycles, although a prolonged delay in medium change (i.e., every 48 h) would result in decreased multiplication rate during the second cycle [124]. In the protocol developed by Radfar et al. to optimize the yield of synchronized reference clones of *P. falciparum* (3D7 and Dd2; RPMI 1640 buffered with 25 mM HEPES and 1.77 mM NaHCO₃ and supplemented with 0.5% Albumax® I and 100 µM hypoxanthine; 5% CO₂ in air or 3% CO₂-1% O₂-96% N₂ gas mixture) [122], the authors recommend hematocrit values within the range of 0.8 to 1.5% and proposed a mathematical formula to calculate the approximate volume of the culture medium necessary to maintain parasite growth for 24 h (i.e., until the next change in the medium). Using this protocol (and fresh human erythrocytes stored <14 days), the authors reported that high parasitemia (20–30%) can be obtained in 150 cm² culture flasks (maximum volume of culture medium, 140 mL). For in vitro drug susceptibility assays that require a 42–72 h incubation, cultures in 96-well plates are performed at low hematocrit, generally at 1.5%, to avoid the labor-intensive task of removing the spent medium and adding the fresh medium after the initial 24 h of incubation. The cultures can also be maintained at a much higher hematocrit in special devices. This will be discussed in another section.

Theoretically, parasite growth is optimal at the interface between the culture medium and the uppermost layer of erythrocytes, where the exchange of gas, nutrients, and waste products occurs freely. By contrast, the exchange is suboptimal at the bottom layer of the erythrocytes. Depending on the protocols of cultivation in different research laboratories, the depth of the hematocrit layer may vary from 0.15 mm to 0.8 mm or more. Mathematical models that attempt to describe the complex interplay between parasite multiplication rate, hematocrit layer depth, the width or diameter of culture devices, and various other factors have been developed, and the effects of the dimensions of the hematocrit layer were analyzed to determine the conditions leading to optimal parasite growth [147,148]. One of the key findings using the model and experimental data obtained from *P. falciparum* (3D7 reference strain) cultivated under standard conditions (RPMI 1640 supplemented with 10% human serum, 5% hematocrit, initial parasitemia 0.5%, 5% O₂, 5% CO₂, and 90% N₂) in 6-well plates (35 mm diameter wells) and the renewal of culture medium and dilution of cultures to 0.5% parasitemia every 48 h was that optimal development (i.e., highest parasitemia after 48 h) occurs in a hematocrit layer depth between 0.18 mm and 0.34 mm. This value is close to the theoretical hematocrit layer depth found empirically by Jensen and Trager (i.e., approximately 0.16 mm) [81]. Parasite growth diminished considerably when the depth of the hematocrit layer exceeded 1 mm.
### 5.4. Leukocytes

Leukocytes are present in whole blood or packed red blood cells obtained from blood banks or in whole blood collected directly from patients. An early study showed that leukocytes from Gambian children with acute malaria inhibit parasite growth [149]. In another more recent study conducted in Tanzania, blood samples were obtained from malaria-infected children <5 years old, and the isolates were cultivated in vitro after the removal of leukocytes by the filtration of venous blood through the cellulose column [150]. Compared to the control isolates (i.e., unfiltered blood samples), parasites in cellulose-filtered samples grew faster and attained higher parasitemias during the first three cycles. Cultivation was not pursued beyond three cycles to assess the long-term beneficial effect of leukocyte-free blood to adapt fresh clinical isolates from African patients to in vitro conditions. By contrast, studies conducted in non-endemic countries demonstrated that the presence of leukocytes, some of which may be intact while others are in the process of disintegration, does not affect in vitro parasite development [121]. These authors reported that the elimination of leukocytes by passage through Whatman CF-11-powdered cellulose results in a lower multiplication rate of laboratory-adapted *P. falciparum* strains (a 54.4-fold increase in parasitemia over two cycles in untreated blood vs. a 34.5-fold increase in leukocyte-free blood). These observations were made using blood obtained from non-immune donors.

In the present author's opinion and experience, the presence of leukocytes is a minor technical issue in adapting fresh isolates collected from African patients to in vitro conditions, at least for routine procedures that do not require "pure" parasite DNA for genomic studies. An extra step of leukocyte removal, which may expose the parasites to the risk of bacterial contamination, and additional costs incurred do not seem to offset potential gains from obtaining more rapid adaptation to in vitro cultivation or a higher multiplication rate. As for leukocytes in blood obtained from blood banks in non-endemic countries, leukocyte removal is not necessary for parasite cultivation.

## 6. Serum and Serum Substitutes

Serum remains the principal supplement of culture media in animal cell culture, including malaria cultivation. Classical synthetic media (RPMI 1640, BME and its modifications, MEM, Ham's F-12, McCoy's 5A medium) are deficient in some nutrients, such as growth factors, hormones, lipids, and proteins (not amino acids, which are abundant in culture media), which are provided by serum. Depending on the cell line, the following sera have been commonly used: fetal, newborn, or adult bovine serum, human serum, horse serum, and chicken serum. Of these, fetal bovine serum (also referred to as fetal calf serum) is the most widely used in cell culture. Sera from other animals have specialized uses.

In recent years, technical advances have been made to grow animal cells in newly constituted, serum-free, chemically defined media, in modified classical media supplemented with relatively low concentrations (i.e., *v/v* < 5%) of serum, or in unmodified classical media supplemented with serum substitutes. The advantages of serum-free media include a completely defined medium, lack of biological variability, absence of blood-borne pathogens, and for malaria cultivation in developing countries, compatibility with any blood type and ease in ordering and transporting a dry medium. Serum-free media are relatively more expensive than classical media. A particular serum-free medium may also be adapted for a limited number of cell lines due to different requirements for serum supplements and basal medium. There is no universal serum-free medium to cultivate all cell lines. In the coming years, serum-free culture will probably become the standard procedure of cell culture or will at least be one of the objectives to be attained for research.

### 6.1. Human Serum

For malaria cultivation, serum or serum replacement is an essential component of the culture medium. In many, but not all laboratories, human serum is used to supplement the culture medium for continuous culture. Trager and Jensen used 15% (*v/v*) human
### 5.4. Leukocytes

Leukocytes are present in whole blood or packed red blood cells obtained from blood banks or in whole blood collected directly from patients. An early study showed that leukocytes from Gambian children with acute malaria inhibit parasite growth [149]. In another more recent study conducted in Tanzania, blood samples were obtained from malaria-infected children <5 years old, and the isolates were cultivated in vitro after the removal of leukocytes by the filtration of venous blood through the cellulose column [150]. Compared to the control isolates (i.e., unfiltered blood samples), parasites in cellulose-filtered samples grew faster and attained higher parasitemias during the first three cycles. Cultivation was not pursued beyond three cycles to assess the long-term beneficial effect of leukocyte-free blood to adapt fresh clinical isolates from African patients to in vitro conditions. By contrast, studies conducted in non-endemic countries demonstrated that the presence of leukocytes, some of which may be intact while others are in the process of disintegration, does not affect in vitro parasite development [121]. These authors reported that the elimination of leukocytes by passage through Whatman CF-11-powdered cellulose results in a lower multiplication rate of laboratory-adapted *P. falciparum* strains (a 54.4-fold increase in parasitemia over two cycles in untreated blood vs. a 34.5-fold increase in leukocyte-free blood). These observations were made using blood obtained from non-immune donors.

In the present author's opinion and experience, the presence of leukocytes is a minor technical issue in adapting fresh isolates collected from African patients to in vitro conditions, at least for routine procedures that do not require "pure" parasite DNA for genomic studies. An extra step of leukocyte removal, which may expose the parasites to the risk of bacterial contamination, and additional costs incurred do not seem to offset potential gains from obtaining more rapid adaptation to in vitro cultivation or a higher multiplication rate. As for leukocytes in blood obtained from blood banks in non-endemic countries, leukocyte removal is not necessary for parasite cultivation.

## 6. Serum and Serum Substitutes

Serum remains the principal supplement of culture media in animal cell culture, including malaria cultivation. Classical synthetic media (RPMI 1640, BME and its modifications, MEM, Ham's F-12, McCoy's 5A medium) are deficient in some nutrients, such as growth factors, hormones, lipids, and proteins (not amino acids, which are abundant in culture media), which are provided by serum. Depending on the cell line, the following sera have been commonly used: fetal, newborn, or adult bovine serum, human serum, horse serum, and chicken serum. Of these, fetal bovine serum (also referred to as fetal calf serum) is the most widely used in cell culture. Sera from other animals have specialized uses.

In recent years, technical advances have been made to grow animal cells in newly constituted, serum-free, chemically defined media, in modified classical media supplemented with relatively low concentrations (i.e., *v/v* < 5%) of serum, or in unmodified classical media supplemented with serum substitutes. The advantages of serum-free media include a completely defined medium, lack of biological variability, absence of blood-borne pathogens, and for malaria cultivation in developing countries, compatibility with any blood type and ease in ordering and transporting a dry medium. Serum-free media are relatively more expensive than classical media. A particular serum-free medium may also be adapted for a limited number of cell lines due to different requirements for serum supplements and basal medium. There is no universal serum-free medium to cultivate all cell lines. In the coming years, serum-free culture will probably become the standard procedure of cell culture or will at least be one of the objectives to be attained for research.

### 6.1. Human Serum

For malaria cultivation, serum or serum replacement is an essential component of the culture medium. In many, but not all laboratories, human serum is used to supplement the culture medium for continuous culture. Trager and Jensen used 15% (*v/v*) human
serum in the RPMI 1640 medium to establish the first continuous cultures [27]. It has been suggested that 15% (v/v) human serum may yield better results for initiating culture and for reactivating cryopreserved parasites [16,81]. In their later studies, the concentration of serum was reduced to 10%. The experiences from various laboratories suggest that laboratory-adapted strains do not require more than 10% human serum.

Any major modification of the concentration of human serum in the RPMI 1640 medium leads to suboptimal parasite growth. High serum concentrations (25% and 50%) generally result in suboptimal parasite growth [151]. In a study conducted by Golightly and Greenwood on a synchronized laboratory-adapted *P. falciparum* strain over 24–48 h, increasing serum concentration in the RPMI 1640 medium at a 2.5% increment from 2.5% to 12.5% resulted in a proportional increase in parasitemia, while at higher serum concentrations (15–20%), parasitemia declined [152]. In another study, however, serum concentrations of 10%, 50%, and 80% were shown to result in a similar growth rate of a laboratory-adapted *P. falciparum* strain over a 30–45 h incubation [153]. The long-term effect of high (50–80%) serum concentrations was not studied by these latter investigators. Although malaria parasites survive under a relatively high serum concentration in the human host, the reasons for the suboptimal growth in vitro at 15–50% serum concentration are not known.

As for the minimal serum concentration, Jensen and Trager have suggested that the reduction in serum concentration to 5% results in suboptimal parasite growth [81]. In later studies, it was reported that 5% pooled human serum supports optimal parasite growth of a laboratory-adapted strain, as well as 10% serum, provided that the culture medium is changed daily [154]. Even 2.5% pooled human serum, but not lower serum concentrations, supports parasite growth, albeit at a suboptimal level. A laboratory strain of *P. falciparum* can be adapted to decreasing levels of human serum by progressively replacing the latter with peptone until human serum is completely eliminated [155]. However, the growth rate decreases considerably (8–16%) compared with the control parasites in 10% human serum. Based on the experience of various investigators, the addition of 10% (v/v) pooled human serum is recommended for routine culture and in vitro drug susceptibility assays.

The nutritional quality of different batches of non-immune human sera to support *P. falciparum* cultures differs considerably. Therefore, depending on the samples, unpooled serum added to RPMI 1640 may result in considerably lower parasite growth (<60%) at 5% serum concentration compared with 10% serum concentration [156]. Human sera obtained from commercial sources may not support parasite growth as much as sera collected in blood banks [27,156]. Since some commercial suppliers accept sending small samples of pooled human serum batches for testing prior to ordering larger quantities of serum, it would be possible to assess the growth of reference laboratory strains of *P. falciparum* in different serum batches before changing serum for cultivation.

Parasite growth varies even with freshly collected individual serum from blood banks. The nutritional deficiencies leading to variations in parasite growth are less apparent when the concentration of individual, unpooled human serum is set at 10% (v/v). To further optimize the nutritional quality of human serum, it is strongly advised to pool non-immune human serum samples. An adequate pool of human serum is obtained by pooling samples from >10 individual non-immune donors (the more, the better). Some commercial sources of human serum are prepared from a very large number of donors. Even these pooled human serum samples require prior testing to ensure optimal parasite growth. Heat inactivation (56 °C for 30 min) does not improve the nutritional quality of fresh human serum [29,81]. Furthermore, the use of freshly collected human serum stored at +4 °C for more than 2 weeks does not support parasite growth [81]. It is preferable to store aliquots of fresh human serum at –20 °C. Such frozen aliquots can be stored for long periods (i.e., several years) without any loss of their nutritional quality for the parasites.

Serum from the umbilical cord is an alternative source of human serum suitable for cultivation [96,112,113,157,158]. Both laboratory-adapted *P. falciparum* strains and freshly collected clinical isolates grow in a medium supplemented with human umbilical cord serum. Obtaining serum from fresh placenta requires coordination with maternity clinics
serum in the RPMI 1640 medium to establish the first continuous cultures [27]. It has been suggested that 15% (v/v) human serum may yield better results for initiating culture and for reactivating cryopreserved parasites [16,81]. In their later studies, the concentration of serum was reduced to 10%. The experiences from various laboratories suggest that laboratory-adapted strains do not require more than 10% human serum.

Any major modification of the concentration of human serum in the RPMI 1640 medium leads to suboptimal parasite growth. High serum concentrations (25% and 50%) generally result in suboptimal parasite growth [151]. In a study conducted by Golightly and Greenwood on a synchronized laboratory-adapted *P. falciparum* strain over 24–48 h, increasing serum concentration in the RPMI 1640 medium at a 2.5% increment from 2.5% to 12.5% resulted in a proportional increase in parasitemia, while at higher serum concentrations (15–20%), parasitemia declined [152]. In another study, however, serum concentrations of 10%, 50%, and 80% were shown to result in a similar growth rate of a laboratory-adapted *P. falciparum* strain over a 30–45 h incubation [153]. The long-term effect of high (50–80%) serum concentrations was not studied by these latter investigators. Although malaria parasites survive under a relatively high serum concentration in the human host, the reasons for the suboptimal growth in vitro at 15–50% serum concentration are not known.

As for the minimal serum concentration, Jensen and Trager have suggested that the reduction in serum concentration to 5% results in suboptimal parasite growth [81]. In later studies, it was reported that 5% pooled human serum supports optimal parasite growth of a laboratory-adapted strain, as well as 10% serum, provided that the culture medium is changed daily [154]. Even 2.5% pooled human serum, but not lower serum concentrations, supports parasite growth, albeit at a suboptimal level. A laboratory strain of *P. falciparum* can be adapted to decreasing levels of human serum by progressively replacing the latter with peptone until human serum is completely eliminated [155]. However, the growth rate decreases considerably (8–16%) compared with the control parasites in 10% human serum. Based on the experience of various investigators, the addition of 10% (v/v) pooled human serum is recommended for routine culture and in vitro drug susceptibility assays.

The nutritional quality of different batches of non-immune human sera to support *P. falciparum* cultures differs considerably. Therefore, depending on the samples, unpooled serum added to RPMI 1640 may result in considerably lower parasite growth (<60%) at 5% serum concentration compared with 10% serum concentration [156]. Human sera obtained from commercial sources may not support parasite growth as much as sera collected in blood banks [27,156]. Since some commercial suppliers accept sending small samples of pooled human serum batches for testing prior to ordering larger quantities of serum, it would be possible to assess the growth of reference laboratory strains of *P. falciparum* in different serum batches before changing serum for cultivation.

Parasite growth varies even with freshly collected individual serum from blood banks. The nutritional deficiencies leading to variations in parasite growth are less apparent when the concentration of individual, unpooled human serum is set at 10% (v/v). To further optimize the nutritional quality of human serum, it is strongly advised to pool non-immune human serum samples. An adequate pool of human serum is obtained by pooling samples from >10 individual non-immune donors (the more, the better). Some commercial sources of human serum are prepared from a very large number of donors. Even these pooled human serum samples require prior testing to ensure optimal parasite growth. Heat inactivation (56 °C for 30 min) does not improve the nutritional quality of fresh human serum [29,81]. Furthermore, the use of freshly collected human serum stored at +4 °C for more than 2 weeks does not support parasite growth [81]. It is preferable to store aliquots of fresh human serum at –20 °C. Such frozen aliquots can be stored for long periods (i.e., several years) without any loss of their nutritional quality for the parasites.

Serum from the umbilical cord is an alternative source of human serum suitable for cultivation [96,112,113,157,158]. Both laboratory-adapted *P. falciparum* strains and freshly collected clinical isolates grow in a medium supplemented with human umbilical cord serum. Obtaining serum from fresh placenta requires coordination with maternity clinics
and hospitals and aseptic techniques. In some populations in malaria-endemic countries, there may be culture-related practices and taboos concerning childbirth, and the placenta may have to be returned to the consenting woman.

For field workers, it is more convenient to procure human sera from local donors. The suitability of locally acquired individual serum samples or pooled samples for in vitro culture needs to be evaluated first. Several early studies have suggested that serum from immune human populations (also serum from *Aotus* monkeys experimentally infected with *P. falciparum*) can inhibit the growth of parasites in vitro, presumably due to the presence of antimalarial antibodies [159–164]. In a study on a large number of heat-inactivated and dialyzed sera collected from healthy Sudanese adult volunteers residing in hyperendemic areas, all serum samples inhibited merozoite invasion of a laboratory-adapted reference strain [151,165]. Most sera also produced slower maturation of synchronous parasites as well as morphologically abnormal schizonts (called the “crisis forms”, i.e., a retarded development of asexual intraerythrocytic parasite associated with the host immune response, resulting in the formation of degenerating schizonts containing less than the average number of merozoites per schizont). By contrast, serum samples from healthy Sudanese adults residing in a hypoendemic area did not exhibit any inhibitory effect on parasite growth. In these studies, each test serum was dialyzed to remove about 98% of chloroquine that may eventually have been present in the samples due to self-medication. Thus, any inhibitory effect on parasite growth was attributed to immune factors.

In another study conducted in the field, *P. falciparum* isolates from asymptomatic children in Burkina Faso were initially cultivated for up to 25 h in an RPMI 1640-HEPES-NaHCO₃ medium supplemented with 10% non-immune human serum, and the schizonts were incubated for an additional 20 h in a medium supplemented with immunoglobulin fractions from enrolled children [166]. Compared with the medium containing non-immune serum, merozoite invasion was inhibited by the immunoglobulins from asymptomatic carriers. The inhibitory effect was more pronounced with heterologous immunoglobulin fractions (i.e., parasite-antibody pairs from different donors) than with autologous immunoglobulins (i.e., parasite-antibody pairs from the same donor). The investigators suggested that their observation may be related to the selection of a parasite population with a low expression of the antigens specifically recognized by the host's antibodies or infection with parasite strains that had not been encountered before by the host. In another field study, the in vitro growth of a laboratory-adapted *P. falciparum* strain was compared with (i) serum from healthy semi-immune African donors, (ii) acute-phase serum from African malaria-infected patients, and (iii) serum from European and Asian healthy non-immune donors [167]. The parasite growth over three intraerythrocytic cycles was similar with sera from non-immune donors and patients with malaria but was much lower with serum from semi-immune subjects. For initiating the culture of primary isolates and short-term cultivation ex vivo (i.e., for in vitro drug susceptibility assays), the acute-phase autologous serum, i.e., autologous serum matched with the corresponding isolate obtained from the same patient, was shown to be suitable [168]. These observations in the field suggest that sera obtained from populations residing in malaria-endemic areas may be used for in vitro cultivation, at least for short-term cultures. These findings are also in agreement with the World Health Organization (WHO) in vitro macrotest and microtest, which use the patients' own acute-phase plasma to obtain schizonts for a single intraerythrocytic cycle. However, some studies have suggested that serum from immune and semi-immune humans may be unsuitable for in vitro cultivation [151,165,166]. For long-term *P. falciparum* cultures in malaria-endemic countries, it may be best to avoid the use of sera obtained from local human populations as much as possible and rely on non-immune human serum from donors in malaria-free countries, animal sera, or synthetic serum substitutes. In the following sections, alternative solutions are examined and proposed.
and hospitals and aseptic techniques. In some populations in malaria-endemic countries, there may be culture-related practices and taboos concerning childbirth, and the placenta may have to be returned to the consenting woman.

For field workers, it is more convenient to procure human sera from local donors. The suitability of locally acquired individual serum samples or pooled samples for in vitro culture needs to be evaluated first. Several early studies have suggested that serum from immune human populations (also serum from *Aotus* monkeys experimentally infected with *P. falciparum*) can inhibit the growth of parasites in vitro, presumably due to the presence of antimalarial antibodies [159–164]. In a study on a large number of heat-inactivated and dialyzed sera collected from healthy Sudanese adult volunteers residing in hyperendemic areas, all serum samples inhibited merozoite invasion of a laboratory-adapted reference strain [151,165]. Most sera also produced slower maturation of synchronous parasites as well as morphologically abnormal schizonts (called the “crisis forms”, i.e., a retarded development of asexual intraerythrocytic parasite associated with the host immune response, resulting in the formation of degenerating schizonts containing less than the average number of merozoites per schizont). By contrast, serum samples from healthy Sudanese adults residing in a hypoendemic area did not exhibit any inhibitory effect on parasite growth. In these studies, each test serum was dialyzed to remove about 98% of chloroquine that may eventually have been present in the samples due to self-medication. Thus, any inhibitory effect on parasite growth was attributed to immune factors.

In another study conducted in the field, *P. falciparum* isolates from asymptomatic children in Burkina Faso were initially cultivated for up to 25 h in an RPMI 1640-HEPES-NaHCO₃ medium supplemented with 10% non-immune human serum, and the schizonts were incubated for an additional 20 h in a medium supplemented with immunoglobulin fractions from enrolled children [166]. Compared with the medium containing non-immune serum, merozoite invasion was inhibited by the immunoglobulins from asymptomatic carriers. The inhibitory effect was more pronounced with heterologous immunoglobulin fractions (i.e., parasite-antibody pairs from different donors) than with autologous immunoglobulins (i.e., parasite-antibody pairs from the same donor). The investigators suggested that their observation may be related to the selection of a parasite population with a low expression of the antigens specifically recognized by the host's antibodies or infection with parasite strains that had not been encountered before by the host. In another field study, the in vitro growth of a laboratory-adapted *P. falciparum* strain was compared with (i) serum from healthy semi-immune African donors, (ii) acute-phase serum from African malaria-infected patients, and (iii) serum from European and Asian healthy non-immune donors [167]. The parasite growth over three intraerythrocytic cycles was similar with sera from non-immune donors and patients with malaria but was much lower with serum from semi-immune subjects. For initiating the culture of primary isolates and short-term cultivation ex vivo (i.e., for in vitro drug susceptibility assays), the acute-phase autologous serum, i.e., autologous serum matched with the corresponding isolate obtained from the same patient, was shown to be suitable [168]. These observations in the field suggest that sera obtained from populations residing in malaria-endemic areas may be used for in vitro cultivation, at least for short-term cultures. These findings are also in agreement with the World Health Organization (WHO) in vitro macrotest and microtest, which use the patients' own acute-phase plasma to obtain schizonts for a single intraerythrocytic cycle. However, some studies have suggested that serum from immune and semi-immune humans may be unsuitable for in vitro cultivation [151,165,166]. For long-term *P. falciparum* cultures in malaria-endemic countries, it may be best to avoid the use of sera obtained from local human populations as much as possible and rely on non-immune human serum from donors in malaria-free countries, animal sera, or synthetic serum substitutes. In the following sections, alternative solutions are examined and proposed.
## 6.2. Human Plasma

Serum is plasma minus coagulation factors. Plasma is easier to obtain at blood banks. Whole blood collected in anticoagulant-containing pouches from donors is immediately separated into plasma and cellular components by centrifugation. These fractions are used separately for transfusion, depending on the individual needs of the patients. In contrast, to prepare serum samples, whole blood from donors is collected in pouches without anticoagulant, and coagulated cells are discarded. For blood banks and hospitals, it is more rational to prepare plasma and packed cells, rather than serum, for transfusion.

Human plasma may be used instead of human serum for malaria cultivation [156,169,170]. In the study performed by Hui et al., the growth of laboratory-adapted *P. falciparum* strains in a medium with 10% pooled or unpooled human plasma or 10% human serum was assessed over 5–6 days [171]. There was no difference in parasite growth, and an adaptation period was not necessary for parasites that were previously grown in the medium supplemented with 10% human serum to grow in the medium with 10% human plasma. Read and Hyde compared the rate of parasite growth in media supplemented with untreated pooled human plasma from 2–4-day-old whole blood collected on CPDA, heat-treated plasma (56 °C × 1 h), or serum prepared from plasma [125]. The untreated plasma supported the growth of a reference *P. falciparum* strain slightly better than heat-treated plasma and serum over two schizogonic cycles. Parasitemia was 25–40% higher with fresh, untreated plasma within four weeks of blood donation than plasma stored at –20 °C or –80 °C.

It has already been mentioned in the previous section that sera obtained from semi-immune and immune donors do not seem to support parasite growth [151,165,167]. Likewise, RPMI 1640 supplemented with 10% plasma obtained from malaria-negative Gabonese mothers at the time of delivery or 10% plasma derived from their umbilical cords resulted in a significant decrease (>three-fold decrease) in the growth of a reference strain compared to non-immune plasma [172]. Contrary to the results of these aforementioned studies, heat-inactivated plasma from semi-immune donors residing in endemic areas has been successfully used to initiate a continuous culture of both reference clones and fresh isolates in a study conducted in Nigeria [173]. The growth rate and in vitro drug response of reference clones and newly established strains cultivated in RPMI 1640 supplemented with human serum from non-immune or semi-immune donors were similar. Moreover, the authors claimed to have succeeded in initiating the cultivation of 170 of 250 (68%) fresh isolates and maintaining them in continuous culture by using semi-immune plasma. Although the total number of African donors was not stated in their paper, the authors reported that plasma and erythrocytes from four and three donors, respectively, did not support parasite growth.

If serum is preferred over plasma for malaria cultivation but is not available, serum can be prepared from plasma samples. Clot formation is induced by the addition of CaCl₂ and thrombin to plasma, and after several hours of incubation at 37 °C, the serum is separated from the clot. Defibrinated human plasma supplemented with hypoxanthine (50 µg/mL), with or without reduced glutathione (600 µg/mL), has been shown to support parasite growth for a short-term duration as much as, or better than human serum [51].

## 6.3. Animal Sera

The original culture method developed by Trager and Jensen required the addition of human serum obtained from non-immune donors to the RPMI 1640 medium (v/v, 5–15%) [27]. Some laboratories, in particular those in endemic countries, encounter the problem of procuring a constant supply of type AB⁺ human serum from non-immune donors, which in most human populations is not the predominant blood type compared to the other three blood groups. Because of several other disadvantages in obtaining human serum from non-immune donors, including high cost and the requirement to screen for blood-borne pathogens, there have been efforts to search for suitable replacements.

One of the two initial culture systems for *P. falciparum*, that of Haynes et al., was developed by supplementing the culture medium with fetal calf serum [26]. Soon after
## 6.2. Human Plasma

Serum is plasma minus coagulation factors. Plasma is easier to obtain at blood banks. Whole blood collected in anticoagulant-containing pouches from donors is immediately separated into plasma and cellular components by centrifugation. These fractions are used separately for transfusion, depending on the individual needs of the patients. In contrast, to prepare serum samples, whole blood from donors is collected in pouches without anticoagulant, and coagulated cells are discarded. For blood banks and hospitals, it is more rational to prepare plasma and packed cells, rather than serum, for transfusion.

Human plasma may be used instead of human serum for malaria cultivation [156,169,170]. In the study performed by Hui et al., the growth of laboratory-adapted *P. falciparum* strains in a medium with 10% pooled or unpooled human plasma or 10% human serum was assessed over 5–6 days [171]. There was no difference in parasite growth, and an adaptation period was not necessary for parasites that were previously grown in the medium supplemented with 10% human serum to grow in the medium with 10% human plasma. Read and Hyde compared the rate of parasite growth in media supplemented with untreated pooled human plasma from 2–4-day-old whole blood collected on CPDA, heat-treated plasma (56 °C × 1 h), or serum prepared from plasma [125]. The untreated plasma supported the growth of a reference *P. falciparum* strain slightly better than heat-treated plasma and serum over two schizogonic cycles. Parasitemia was 25–40% higher with fresh, untreated plasma within four weeks of blood donation than plasma stored at –20 °C or –80 °C.

It has already been mentioned in the previous section that sera obtained from semi-immune and immune donors do not seem to support parasite growth [151,165,167]. Likewise, RPMI 1640 supplemented with 10% plasma obtained from malaria-negative Gabonese mothers at the time of delivery or 10% plasma derived from their umbilical cords resulted in a significant decrease (>three-fold decrease) in the growth of a reference strain compared to non-immune plasma [172]. Contrary to the results of these aforementioned studies, heat-inactivated plasma from semi-immune donors residing in endemic areas has been successfully used to initiate a continuous culture of both reference clones and fresh isolates in a study conducted in Nigeria [173]. The growth rate and in vitro drug response of reference clones and newly established strains cultivated in RPMI 1640 supplemented with human serum from non-immune or semi-immune donors were similar. Moreover, the authors claimed to have succeeded in initiating the cultivation of 170 of 250 (68%) fresh isolates and maintaining them in continuous culture by using semi-immune plasma. Although the total number of African donors was not stated in their paper, the authors reported that plasma and erythrocytes from four and three donors, respectively, did not support parasite growth.

If serum is preferred over plasma for malaria cultivation but is not available, serum can be prepared from plasma samples. Clot formation is induced by the addition of CaCl₂ and thrombin to plasma, and after several hours of incubation at 37 °C, the serum is separated from the clot. Defibrinated human plasma supplemented with hypoxanthine (50 µg/mL), with or without reduced glutathione (600 µg/mL), has been shown to support parasite growth for a short-term duration as much as, or better than human serum [51].

## 6.3. Animal Sera

The original culture method developed by Trager and Jensen required the addition of human serum obtained from non-immune donors to the RPMI 1640 medium (v/v, 5–15%) [27]. Some laboratories, in particular those in endemic countries, encounter the problem of procuring a constant supply of type AB⁺ human serum from non-immune donors, which in most human populations is not the predominant blood type compared to the other three blood groups. Because of several other disadvantages in obtaining human serum from non-immune donors, including high cost and the requirement to screen for blood-borne pathogens, there have been efforts to search for suitable replacements.

One of the two initial culture systems for *P. falciparum*, that of Haynes et al., was developed by supplementing the culture medium with fetal calf serum [26]. Soon after
the development of *P. falciparum* cultivation methods, it has been recognized that the use of human serum, which seemed to yield the best results for parasite growth, is a limiting factor for the wide application of this technique, especially in field laboratories. Several investigators have empirically sought to replace human serum with animal sera, which may be easier to obtain from local abattoirs or commercial sources. In early studies using Trager and Jensen's cultivation method, it has been shown that some animal sera may support parasite growth [29,156].

A number of studies on the use of animal sera for parasite cultivation followed in the 1980s. Animal sera tested for *P. falciparum* cultivation include the following [26,29,30,81,93, 98,99,101,155,156,169,174–181]: fetal calf, calf, adult cow, rabbit, goat, pig, horse, and sheep (Table 2). Human sera can be replaced with animal sera, but the success rate varies with different animal sources. An adaptation period lasting several days to weeks of continuous culture may be required for the parasites to grow in non-human sera [155,174,175]. Once the parasites adapt to animal sera, growth may be sustained for several cycles but may decline over time. In such cases, it may be possible to sustain parasite growth by switching to human sera [176].

Mixed results have been obtained with fetal calf serum, often depending on the source or batches [26,81,98,101,106,155,156,182]. Without supplements (e.g., peptones), gradual adaptation, increased number of sera (15–20%), and/or twice daily medium change, sera from newborn calves and adult cows do not seem to be suitable for parasite cultivation. However, using Ham's F12 medium supplemented with various human and animal sera (without a neopeptone supplement), Divo and Jensen found that the medium supplemented with 10% pooled adult bovine serum resulted in fair growth (61% of growth supported by 5% pooled human serum in F12 medium) over 96 h compared to F12 supplemented with 5% human serum, and supports continuous culture (tested for 100 days) [93]. Parallel experiments with pooled sera from pigs and goats showed inferior parasite growth over 96 h and failure of continuous culture.

<table><caption>**Table 2.** Sera from animal sources for the in vitro cultivation of *P. falciparum*.</caption><thead><tr><th>Animal Sera<sup>1</sup></th><th>Parasite Growth<sup>2</sup></th><th>References</th></tr></thead><tbody><tr><td>Fetal calf</td><td>good<sup>3</sup><br>poor to fair</td><td>[26,98,155]<br>[81,101,156]</td></tr><tr><td rowspan="5">Calf</td><td>poor</td><td>[156]</td></tr><tr><td>very good (with peptones)</td><td>[155]</td></tr><tr><td>very good<sup>4</sup></td><td>[30]</td></tr><tr><td>very good<sup>5</sup></td><td>[174]</td></tr><tr><td>very good<sup>6</sup></td><td>[175]</td></tr><tr><td rowspan="4">Adult cow</td><td>poor to no growth (unsupplemented)</td><td>[93,156,169]</td></tr><tr><td>fair (with neopeptone)</td><td>[93,156,169]</td></tr><tr><td>very good (with hypoxanthine)</td><td>[176]</td></tr><tr><td>very good<sup>6</sup></td><td>[175]</td></tr><tr><td>Rabbit</td><td>very good (may require adaptation)</td><td>[99,177,178]<br>[179]<sup>7</sup></td></tr><tr><td rowspan="4">Goat</td><td>fair (for 96 h), poor &gt; 96 h</td><td>[93]</td></tr><tr><td>fair to good up to 30 days</td><td>[169]</td></tr><tr><td>poor for three cycles</td><td>[99]</td></tr><tr><td>comparable to 10% fetal bovine serum</td><td>[180]<sup>8</sup></td></tr><tr><td rowspan="4">Pig</td><td>poor (data not shown)</td><td>[29]</td></tr><tr><td>poor (agglutination and hemolysis)</td><td>[156]</td></tr><tr><td>very good for 96 h<sup>9</sup></td><td>[93,155]</td></tr><tr><td>poor to fair (strain-dependent)</td><td>[169]</td></tr></tbody></table>
the development of *P. falciparum* cultivation methods, it has been recognized that the use of human serum, which seemed to yield the best results for parasite growth, is a limiting factor for the wide application of this technique, especially in field laboratories. Several investigators have empirically sought to replace human serum with animal sera, which may be easier to obtain from local abattoirs or commercial sources. In early studies using Trager and Jensen's cultivation method, it has been shown that some animal sera may support parasite growth [29,156].

A number of studies on the use of animal sera for parasite cultivation followed in the 1980s. Animal sera tested for *P. falciparum* cultivation include the following [26,29,30,81,93, 98,99,101,155,156,169,174–181]: fetal calf, calf, adult cow, rabbit, goat, pig, horse, and sheep (Table 2). Human sera can be replaced with animal sera, but the success rate varies with different animal sources. An adaptation period lasting several days to weeks of continuous culture may be required for the parasites to grow in non-human sera [155,174,175]. Once the parasites adapt to animal sera, growth may be sustained for several cycles but may decline over time. In such cases, it may be possible to sustain parasite growth by switching to human sera [176].

Mixed results have been obtained with fetal calf serum, often depending on the source or batches [26,81,98,101,106,155,156,182]. Without supplements (e.g., peptones), gradual adaptation, increased number of sera (15–20%), and/or twice daily medium change, sera from newborn calves and adult cows do not seem to be suitable for parasite cultivation. However, using Ham's F12 medium supplemented with various human and animal sera (without a neopeptone supplement), Divo and Jensen found that the medium supplemented with 10% pooled adult bovine serum resulted in fair growth (61% of growth supported by 5% pooled human serum in F12 medium) over 96 h compared to F12 supplemented with 5% human serum, and supports continuous culture (tested for 100 days) [93]. Parallel experiments with pooled sera from pigs and goats showed inferior parasite growth over 96 h and failure of continuous culture.

<table><caption>**Table 2.** Sera from animal sources for the in vitro cultivation of *P. falciparum*.</caption><thead><tr><th>Animal Sera <sup>1</sup></th><th>Parasite Growth <sup>2</sup></th><th>References</th></tr></thead><tbody><tr><td rowspan="2">Fetal calf</td><td>good <sup>3</sup></td><td>[26,98,155]</td></tr><tr><td>poor to fair</td><td>[81,101,156]</td></tr><tr><td rowspan="5">Calf</td><td>poor</td><td>[156]</td></tr><tr><td>very good (with peptones)</td><td>[155]</td></tr><tr><td>very good <sup>4</sup></td><td>[30]</td></tr><tr><td>very good <sup>5</sup></td><td>[174]</td></tr><tr><td>very good <sup>6</sup></td><td>[175]</td></tr><tr><td rowspan="4">Adult cow</td><td>poor to no growth (unsupplemented)</td><td>[93,156,169]</td></tr><tr><td>fair (with neopeptone)</td><td>[93,156,169]</td></tr><tr><td>very good (with hypoxanthine)</td><td>[176]</td></tr><tr><td>very good <sup>6</sup></td><td>[175]</td></tr><tr><td>Rabbit</td><td>very good (may require adaptation)</td><td>[99,177,178]<br>[179]<sup>7</sup></td></tr><tr><td rowspan="4">Goat</td><td>fair (for 96 h), poor > 96 h</td><td>[93]</td></tr><tr><td>fair to good up to 30 days</td><td>[169]</td></tr><tr><td>poor for three cycles</td><td>[99]</td></tr><tr><td>comparable to 10% fetal bovine serum</td><td>[180]<sup>8</sup></td></tr><tr><td rowspan="4">Pig</td><td>poor (data not shown)</td><td>[29]</td></tr><tr><td>poor (agglutination and hemolysis)</td><td>[156]</td></tr><tr><td>very good for 96 h <sup>9</sup></td><td>[93,155]</td></tr><tr><td>poor to fair (strain-dependent)</td><td>[169]</td></tr></tbody></table>