Malaria culture
Encyclopedia
Malaria culture is the method to grow malaria
parasites
outside the body i.e. in an ex vivo
environment.
Plasmodium falciparum
is currently the only human malaria parasite that has been successfully cultured continuously ex vivo. Although attempts for propagation of the parasites outside of humans or animal models reach as far back as 1912, the success of the initial attempts was limited to one or just a few cycles. The first successful continuous culture was established in 1976. Initial hopes that the ex vivo culture would lead quickly to the discovery of a vaccine
were premature. However, the development of new drugs
was greatly facilitated.
s are incubated in a culture dish or flask at 37°C together with a nutrient
medium and plasma
, serum
or serum substitutes. A special feature of the incubation is the special gas mixture of mostly nitrogen (93% nitrogen
, 4% carbondioxide, 3% oxygen
) allowing the parasites to grow at 37°C in a cell incubator. An alternative to gasing the cultures with the exact gas mixture, is the use of a candlejar. The candlejar is an airtight container in which the cultures and a lit candle
are placed. The burning candle consumes some of the oxygen
and produces carbon dioxide
, which acts as a fire extinguisher
. Carbon dioxide content in fresh air varies between 0.036% and 0.039%, at an app. 5% concentration the candle stops burning. The number of parasites increased by a factor 5 approximately every 48 hours (= one cycle). The parasitemia
can be determined via blood film
, to keep it within the wanted limits, the culture can be thinned out with healthy red blood cells.
The original method for the successful ex vivo propagation of P. falciparum described culture of the parasite under static conditions (Trager-Jensen method). James B. Jensen joined Trager’s laboratory as a post-doctoral fellow in 1976. He decided to employ a candlejar instead of the incubator. In the summer of 1976 Milton Friedman, a graduate student in the Trager lab who was working in the MRC laboratories in The Gambia
, arranged for a sample of human blood infected with P. falciparum to be sent to New York
. This was diluted with RPMI 1640
(which turned out to be the best of the commercial media) in Petri dish
es, placed in a candlejar and incubated. The line grew very well and became FCR-3/Gambia, one of the most widely used strains. Later, other lines would be established using similar methods and the impact of continuous cultivation of P. falciparum was phenomenal especially for the testing of putative antimalarials and for deciphering its genes. A number of subsequent reports (from as far back as the early 1980s), showed that cell suspension (using a shaking-incubator) significantly increased culture growth. Continuous agitation has also been shown to improve other parameters of culture growth relevant to researchers, such as the prolongation of culture synchrony after synchronization procedures, and a reduction of the rate of multiple infections. Despite this, the practice of culturing the parasite under static conditions remains widespread. The greatest value of the candlejar method is that it can be used in laboratories almost anywhere in the world where there is an incubator, a candle
and a desiccator
. Around 60% parasitized cells can be obtained using optimized culturing conditions. Recent studies of P. falciparum isolated directly from infected patients indicate that alternative parasite biological states occur in the natural host that are not observed with ex vivo cultivated parasites.
gradient
procedure can be used to isolate
infected red blood cells because red cells containing plasmodia are less dense than normal ones. Young trophozoites
coincided with erythrocytes in a broad band corresponding to densities
from 1.075 to 1.100 g/ml, whereas schizonts were concentrated at a density approximating 1.062 g/ml. There are studies, however, that suggest that some strains of P.falciparum are affected in their capacity of invasion after being exposed to this chemical.
The difference between diamagnetic low-spin oxyhemoglobin in uninfected red blood cells and paramagnetic hemozoin
in infected red blood cells can also be used for isolation. Magnetic columns have shown to be less harmful for the parasite and are simple and adjustable to the needs of the researcher. The column is mounted in a potent magnet holder and the culture flowed through it. The column traps the erythrocytes infected with the latest stages of the parasites, which can then be eluted when the column is removed from the magnet. It is a simple method that does not need expensive equipment and it does not seem to affect the parasites as to their invasion capabilities afterwards.
Malaria
Malaria is a mosquito-borne infectious disease of humans and other animals caused by eukaryotic protists of the genus Plasmodium. The disease results from the multiplication of Plasmodium parasites within red blood cells, causing symptoms that typically include fever and headache, in severe cases...
parasites
Parasitism
Parasitism is a type of symbiotic relationship between organisms of different species where one organism, the parasite, benefits at the expense of the other, the host. Traditionally parasite referred to organisms with lifestages that needed more than one host . These are now called macroparasites...
outside the body i.e. in an ex vivo
Ex vivo
Ex vivo means that which takes place outside an organism. In science, ex vivo refers to experimentation or measurements done in or on tissue in an artificial environment outside the organism with the minimum alteration of natural conditions...
environment.
Plasmodium falciparum
Plasmodium falciparum
Plasmodium falciparum is a protozoan parasite, one of the species of Plasmodium that cause malaria in humans. It is transmitted by the female Anopheles mosquito. Malaria caused by this species is the most dangerous form of malaria, with the highest rates of complications and mortality...
is currently the only human malaria parasite that has been successfully cultured continuously ex vivo. Although attempts for propagation of the parasites outside of humans or animal models reach as far back as 1912, the success of the initial attempts was limited to one or just a few cycles. The first successful continuous culture was established in 1976. Initial hopes that the ex vivo culture would lead quickly to the discovery of a vaccine
Malaria vaccine
Malaria vaccines are an area of intensive research. However, there is no effective vaccine that has been introduced into clinical practice.The global burden of P. falciparum malaria increased through the 1990s due to drug-resistant parasites and insecticide-resistant mosquitoes; this is illustrated...
were premature. However, the development of new drugs
Antimalarial drug
Antimalarial medications, also known as antimalarials, are designed to prevent or cure malaria. Such drugs may be used for some or all of the following:* Treatment of malaria in individuals with suspected or confirmed infection...
was greatly facilitated.
Method
Infected human red blood cellRed blood cell
Red blood cells are the most common type of blood cell and the vertebrate organism's principal means of delivering oxygen to the body tissues via the blood flow through the circulatory system...
s are incubated in a culture dish or flask at 37°C together with a nutrient
Nutrient
A nutrient is a chemical that an organism needs to live and grow or a substance used in an organism's metabolism which must be taken in from its environment. They are used to build and repair tissues, regulate body processes and are converted to and used as energy...
medium and plasma
Blood plasma
Blood plasma is the straw-colored liquid component of blood in which the blood cells in whole blood are normally suspended. It makes up about 55% of the total blood volume. It is the intravascular fluid part of extracellular fluid...
, serum
Blood serum
In blood, the serum is the component that is neither a blood cell nor a clotting factor; it is the blood plasma with the fibrinogens removed...
or serum substitutes. A special feature of the incubation is the special gas mixture of mostly nitrogen (93% nitrogen
Nitrogen
Nitrogen is a chemical element that has the symbol N, atomic number of 7 and atomic mass 14.00674 u. Elemental nitrogen is a colorless, odorless, tasteless, and mostly inert diatomic gas at standard conditions, constituting 78.08% by volume of Earth's atmosphere...
, 4% carbondioxide, 3% oxygen
Oxygen
Oxygen is the element with atomic number 8 and represented by the symbol O. Its name derives from the Greek roots ὀξύς and -γενής , because at the time of naming, it was mistakenly thought that all acids required oxygen in their composition...
) allowing the parasites to grow at 37°C in a cell incubator. An alternative to gasing the cultures with the exact gas mixture, is the use of a candlejar. The candlejar is an airtight container in which the cultures and a lit candle
Candle
A candle is a solid block or cylinder of wax with an embedded wick, which is lit to provide light, and sometimes heat.Today, most candles are made from paraffin. Candles can also be made from beeswax, soy, other plant waxes, and tallow...
are placed. The burning candle consumes some of the oxygen
Oxygen
Oxygen is the element with atomic number 8 and represented by the symbol O. Its name derives from the Greek roots ὀξύς and -γενής , because at the time of naming, it was mistakenly thought that all acids required oxygen in their composition...
and produces carbon dioxide
Carbon dioxide
Carbon dioxide is a naturally occurring chemical compound composed of two oxygen atoms covalently bonded to a single carbon atom...
, which acts as a fire extinguisher
Fire extinguisher
A fire extinguisher or extinguisher, flame entinguisher is an active fire protection device used to extinguish or control small fires, often in emergency situations...
. Carbon dioxide content in fresh air varies between 0.036% and 0.039%, at an app. 5% concentration the candle stops burning. The number of parasites increased by a factor 5 approximately every 48 hours (= one cycle). The parasitemia
Parasitemia
Parasitemia is the quantitative content of parasites in the blood. It is used as a measurement of parasite load in the organism and an indication of the degree of an active parasitic infection...
can be determined via blood film
Blood film
A blood film or peripheral blood smear is a thin layer of blood smeared on a microscope slide and then stained in such a way to allow the various blood cells to be examined microscopically...
, to keep it within the wanted limits, the culture can be thinned out with healthy red blood cells.
The original method for the successful ex vivo propagation of P. falciparum described culture of the parasite under static conditions (Trager-Jensen method). James B. Jensen joined Trager’s laboratory as a post-doctoral fellow in 1976. He decided to employ a candlejar instead of the incubator. In the summer of 1976 Milton Friedman, a graduate student in the Trager lab who was working in the MRC laboratories in The Gambia
The Gambia
The Republic of The Gambia, commonly referred to as The Gambia, or Gambia , is a country in West Africa. Gambia is the smallest country on mainland Africa, surrounded by Senegal except for a short coastline on the Atlantic Ocean in the west....
, arranged for a sample of human blood infected with P. falciparum to be sent to New York
New York
New York is a state in the Northeastern region of the United States. It is the nation's third most populous state. New York is bordered by New Jersey and Pennsylvania to the south, and by Connecticut, Massachusetts and Vermont to the east...
. This was diluted with RPMI 1640
Roswell Park Memorial Institute medium
Roswell Park Memorial Institute medium, commonly referred to as RPMI, is a form of medium used in cell culture and tissue culture. It has traditionally been used for growth of Human lymphoid cells...
(which turned out to be the best of the commercial media) in Petri dish
Petri dish
A Petri dish is a shallow glass or plastic cylindrical lidded dish that biologists use to culture cells or small moss plants. It was named after German bacteriologist Julius Richard Petri, who invented it when working as an assistant to Robert Koch...
es, placed in a candlejar and incubated. The line grew very well and became FCR-3/Gambia, one of the most widely used strains. Later, other lines would be established using similar methods and the impact of continuous cultivation of P. falciparum was phenomenal especially for the testing of putative antimalarials and for deciphering its genes. A number of subsequent reports (from as far back as the early 1980s), showed that cell suspension (using a shaking-incubator) significantly increased culture growth. Continuous agitation has also been shown to improve other parameters of culture growth relevant to researchers, such as the prolongation of culture synchrony after synchronization procedures, and a reduction of the rate of multiple infections. Despite this, the practice of culturing the parasite under static conditions remains widespread. The greatest value of the candlejar method is that it can be used in laboratories almost anywhere in the world where there is an incubator, a candle
Candle
A candle is a solid block or cylinder of wax with an embedded wick, which is lit to provide light, and sometimes heat.Today, most candles are made from paraffin. Candles can also be made from beeswax, soy, other plant waxes, and tallow...
and a desiccator
Desiccator
thumb|right|A desiccator and a vacuum desiccator - note the stopcock which allows a vacuum to be applied. The blue [[silica gel]] in the space below the platform is used as the [[desiccant]]....
. Around 60% parasitized cells can be obtained using optimized culturing conditions. Recent studies of P. falciparum isolated directly from infected patients indicate that alternative parasite biological states occur in the natural host that are not observed with ex vivo cultivated parasites.
Concentration of infected cells
To achieve synchronization and/or concentration of the parasites in culture several methods have been developed. A discontinuous PercollPercoll
Percoll was first formulated by Pertoft et al. as a tool for more efficient density separation. It is used for the isolation of cells, organelles, and/or viruses by density centrifugation...
gradient
Differential centrifugation
Differential centrifugation is a common procedure in microbiology and cytology used to separate certain organelles from whole cells for further analysis of specific parts of cells. In the process, a tissue sample is first homogenised to break the cell membranes and mix up the cell contents...
procedure can be used to isolate
infected red blood cells because red cells containing plasmodia are less dense than normal ones. Young trophozoites
Apicomplexan cellular morphology
Apicomplexa life cycle stages have evolved to allow intracellular parasites to survive the wide variety of environments they are exposed to during their complex life cycles. Each stage is typified by a cellular variety with a distinct morphology and biochemistry.Not all apicomplexa develop all the...
coincided with erythrocytes in a broad band corresponding to densities
Density
The mass density or density of a material is defined as its mass per unit volume. The symbol most often used for density is ρ . In some cases , density is also defined as its weight per unit volume; although, this quantity is more properly called specific weight...
from 1.075 to 1.100 g/ml, whereas schizonts were concentrated at a density approximating 1.062 g/ml. There are studies, however, that suggest that some strains of P.falciparum are affected in their capacity of invasion after being exposed to this chemical.
The difference between diamagnetic low-spin oxyhemoglobin in uninfected red blood cells and paramagnetic hemozoin
Hemozoin
Hemozoin is a disposal product formed from the digestion of blood by some blood-feeding parasites. These hematophagous organisms such as Malaria parasites , Rhodnius and Schistosoma digest hemoglobin and release high quantities of free heme, which is the non-protein component of hemoglobin...
in infected red blood cells can also be used for isolation. Magnetic columns have shown to be less harmful for the parasite and are simple and adjustable to the needs of the researcher. The column is mounted in a potent magnet holder and the culture flowed through it. The column traps the erythrocytes infected with the latest stages of the parasites, which can then be eluted when the column is removed from the magnet. It is a simple method that does not need expensive equipment and it does not seem to affect the parasites as to their invasion capabilities afterwards.