GB2507944A

GB2507944A - Apparatus for the culture of parasites

Info

Publication number: GB2507944A
Application number: GB1214452.3A
Authority: GB
Inventors: John Rees
Original assignee: JR BIOMEDICAL Ltd
Current assignee: JR BIOMEDICAL Ltd
Priority date: 2012-08-14
Filing date: 2012-08-14
Publication date: 2014-05-21
Anticipated expiration: 2032-08-14
Also published as: GB2507944B; GB201214452D0

Abstract

An apparatus for the continuous culture of Plasmodium species erythrocyte stage parasites in a synthetic culture fluid in the absence of red blood cells. The apparatus comprising a thermostatically controlled culture vessel 3 containing an insoluble, non-toxic, inert porous scaffold, said pores allowing free movement of the parasites and simulating the parasitophorous vacuole found in red blood cells infected with malaria blood stage parasites. Frits retains the scaffold material such as polyethylene 10 micron spheres and the parasite in the vessel 3, valves 6 and 10 respectively may be opened to allow waste culture fluid and plasmodium merizoites to be collected in vessels 7 and 11.

Description

APPARATUS FOR THE CULTURE OF PARASITES

The present invention relates to apparatus for the culture of Plasmodium species on an industrial scale for the manufacture of blood stage vaccines for malaria.

In particular, the present invention relates to the construction of a scaffold in order to simulate the physical and environmental conditions found in the parasitophorous vacuole of infected human erythrocytes by malaria blood stage parasites.

Despite the vast number of candidate vaccines developed with molecular biology techniques such subunit approaches have yielded mixed resufts in clinical trials.

The traditional approach to successful vaccine production is the use of whole organisms for immunisation. However, two major obstacles to the progress of an effective whole parasite vaccine for malaria are the lack of availahility of sufficient quantities of live blood stage parasites and contamination of cultures with human red blood cells.

The life cycle of the malaria parasite is complex. The sexual component of the life cycle is completed within the Anopholes mosquito whilst the asexual proliferation of the parasite occurs firstly within the liver of the human host and secondly with clinical symptoms and pathology evolving during the blood stage of infection. A whole parasite blood stage vaccine would be a candidate for simu'ating the phenomenon known as premunition, a natural, non sterilizing immunity which provides protection from malaria for people living in areas where malaria is endemic.

A significant obstacle to the production of whole blood stage malaria vaccines has been the requirement for the use of human red blood cells as part of the cultivation process. The presence of red blood cells and other human derived tissues in vaccine preparations increases the risk of adverse immune reactions as well as the potential for an increased risk of transmission of human diseases such as CjD, FIIV and hepatitis.

To overcome these limitations, the present invention proposes an apparatus for the industrial cultivation of blood stage malaria parasites that simulates the physical and chemical environment of the parasitophorous vacuole found in Plasmodium infected erythrocytes, thereby eliminating the requirement for the addition of human blood cells to the culture system.

According to a first embodiment of the present invention the apparatus for the cultivation of Plasmodium erythrocyte-stage organisms comprises of a vessel, such as a flask, made of biologically inert materials such as that routinely used in laboratories for the storage and measuring of reagents; for example glass, stainless steel or a synthetic polymer such as polypropylene or polystyrene.

Said vessel contains; a] an inert, non adsorbent, porous scaffold composed of interconnected spaces, wherein said interconnected spaces are formed by an accretion of non toxic material that allows freedom of movement of said Plasmodium organisms from one pore to another without attachment, embedment, complete encapsulation or adsorption to said scaffold, the pore size of said porous scaffold set to be the same order of magnitude in volume as that of a parasitophorous vacuole of approximately 0.5 m3 to 500 im3 assuming a Plasmodium erythrocyte stage parasite occupies a spherical parasitophorous vacuole with a diameter in the range of 1 to lOkm; and b] a culture fluid, similar in composition to that commonly used in cell culture techniques, that fills said interconnected spaces in order to supply nutrients and a means to control the physical and chemical milieu bathing the parasites in said pores.

According to a second embodiment of the current invention, the porous scaffold is constructed from the accretion of inert particulate material. The important feature of any suitable accreted particulate material for said porous scaffold is that when packed together there is a void volume between the particles, wherein each said pore between the packed particles has a void volume the same order of magnitude as the parasitophorous vacuole. Less important, is the origin of the particles save that they are inert or can be made inert to the proliferation requirements of Plasmodium parasites.

It can be shown mathematically that spherical particles when packed form spaces or pores between the particles of the same order of magnitude as the volume of the individual particles used.

According to a yet further preferred embodiment of the present invention said particulate materials are microspheres, made of glass or plastic, such as those commonly used in immunoassay agglutination tests and in immunochromatographic medical tests. Commercially available glass and plastic microspheres are provided with specific diameters and spherical shape, the size ol the space formed between packed microspheres being of the same order as the volume of each microsphere. It is the space between accreted particles, such as the said microspheres, that is important to the function of the said invention and not the material or characteristics of said particles, save that the material for the particles should be as far as possible inert, non toxic and easily separable from the culture fluid containing the parasites when harvested. Hence, it will be obvious to the reader that other particles of different three dimensional shape to microspheres could serve equally well to form said porous scaffold. For example; materials commonly used in liquid chromatography for separation of biomolecules including cross-linked polysaccharides, gels such as those used in electrophoresis and immunodiffusion techniques, soluble synthetic polymers, soluble natural polymers such as starch, minerals such as clay or silica particles, dead micro-organisms or organism parts, dead cells, naturally porous materials such as synthetic and natural sponges, filter papers, liposomes and insoluble powders such as carbon particles.

In a further embodiment of the current invention the said porous scaffold may be manufactured by prefabricated bound fibrous material such as porous plastics, glass fibres or porous polymers in the form of a mesh such materials are commonly used as wicks in single step immunochromatographic self test kits such as pregnancy tests.

According to a further embodiment of the present invention it is desirable to have a simple way to add and control the delivery of culture fluid and organisms to the vessel in order to fill the pores of the porous scaffold via a variably open and closable valve to control the feed from a reservoir upstream of the vessel.

According to a further embodiment of the present invention it is desirable to have a simple means to collect parasites in a batch wise or continuous way when sufficient numbers of parasites have been produced for the intended purpose by way of a variable open and closable valve downstream of said culture vessel fitted with a frit of pore size of 2 to 10 im which will retain the porous scaffold hut allow parasites to pass through, facilitating control over the harvesting of parasites at the completion of, or during the cultivation process.

According to yet a further embodiment of the present invention it is desirable to have a simple means of removing waste products produced by the parasites during the cultivation process from the culture vessel without removing the parasites by way of a variably open and closable valve fitted with an inline filter) between the vessel and said valve, with a pore size of less than 1im which will retain the Plasmodium parasites but allow soluble waste products excreted into the culture fluid to pass through.

According to yet another further embodiment of the present invention the apparatus incorporates a means of controlling p1-I, temperature, pressure and atmospheric composition by way of appropriately buffered culture fluid) as is standard practice in cell culture techniques; For example, RPMI 1640 cell culture media buffered with HEPES and or bicarbonate, maintaining temperature at an optimum level for maximum proliferation of Plasmodium parasites in the culture

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system by means of a thermostatic jacket surrounding the culture vessel and maintaining pressure and atmospheric composition through an optimised mix of gas supplied from one or more pressurised gas cylinders through a controllable valve.

According to a further embodiment of the present invention each vessel may be attached to one or more further similar vessels in series, parallel or combination of series and parallel.

According to a preferred embodiment of the present invention the combination of vessels are arranged to allow a primary feeder culture vessel to continually donate nutrients and organisms in a controlled way to a number of further secondary culture vessels downstream of the original feeder culture vessel, and said further secondary culture vessels themselves being feeder culture vessels for even further culture vessels further downstream of said secondary culture vessels, and so on, allowing automated continuous culture in a geometric progression to provide maximum yield and reducing the need br physical manipulation of the culture vessels once the primary culture vessel has been inoculated with Plasmodium organisms.

It is a further embodiment of the current invention that both the vessel containing the scaffold and the scaffold itself is preferentially perfused with culture fluid and growth factors prior to the inoculation with Plasmodium parasites to help reduce non specific adsorption of nutrients to the said vessel interior, valves, filters and porous scaffold that may be needed by the parasites for optimum growth and proliferation, in order that the parasites are free to move, grow and multiply asexually within interconnected pores that form the void volume of the scaffold but are explicitly not encouraged to be non specifically adsorbed or attached in any way such as that found in scaffolds found in tissue engineering applications.

It is a further essential embodiment of the current invention that the Plasmodium parasites are free to move between interconnecting spaces within the scaffold and are explicitly not encouraged to become encapsulated in any way similar to that used for the complete encapsulation of cells as is used in biotechnology for immobilization techniques, for example in the production of hormones from immobilized cells.

It is an important embodiment of the current invention that the particles forming the porous scaffold are packed tightly so that the void volume between said particles is of the same magnitude as the parasitophorous vacuole) this may be achieved by allowing more dense particles to settle by gravity, by centrifugation or floatation of less dense particles in the culture fluid.

Examples of the invention will now be described with reference to the accompanying drawings: Figure 1 shows a typical way in which microparticle spheres [1) can be packed dthin a vessel creating interconnecting pores (2].

Figure 2 shows an apparatus for the continuous culture of Plasmodium blood stage parasites.

Example 1

To remove antimicrobials and block non specific binding 20g of solid polyethylene microspheres 10im in diameter and density of 1.3g/ml are washed three times with RPMI 1640 cell culture media by repeated low speed centrifugation and resuspension. After the third wash the microspheres are resuspended in 14m1 of cell culture media and added to a flat 250m1 cell culture flask with screw cap closure, previously rinsed with cell culture media. The flask is then inoculated with lml of viable blood stage Plasmodium parasites previously washed with cell culture media, and placed in an incubator with a means of controlling temperature, typically around 37 degrees centigrade and the atmospheric composition by one or more cylinders of compressed gas. After three to ten days depending on the species of Plasmodium cultivated, fresh cell culture fluid is added to the flask and gently swirled to distribute parasites in the cell culture fluid. The microspheres are then allowed to settle for one hour before the cell culture fluid containing parasites is collected by decantation and the flask re-equilibrated with gas and returned to the incubator for a further three to ten days before repeating the harvesting procedure.

Example 2

With reference to Figure 2, the culture vessel (3) comprises of a wide bore glass chromatography column fitted with polypropylene screw end caps (13 and 14) encased in a thermostatic jacket (15) that allows the temperature within the culture vessel to be controlled for optimum proliferation of the particular species of Plasmodium, typically around 37 degrees centigrade. The culture vessel is packed with polyethylene microspheres prepared as described in Example 1.

When the culture vessel is orientated vertically, the uppermost end cap (13] is perforated by tubing via a stopcock (9) leading to a vented reservoir containing a feed of cell culture fluid (8). Also penetrating the upper end cap [13) is tubing connecting to one or more cylinders (4] of compressed gasses via a control valve (5] including air, oxygen, nitrogen and carbon dioxide, aflowing the atmospheric composition and pH within the reaction vessel to be varied to optimise the growth of different Plasmodium species. A unidirectional variable gas vent (12) maintains neutral atmospheric pressure within the culture vessel.

A variable open and closable valve (6) with proximal filter retains parasites and porous scaffold within the culture vessel (3) whilst waste culture fluid passes freely when the valve is opened into a collection vessel (7].

As fluid with parasites drips through the fritted outlet valve (10] under the influence of gravity the fluid containing parasites is collected in the collection vessel (11) whilst the porous scaffold is retained within the culture vessel by the frit and fresh culture fluid is pulled into the culture vessel (3]. The filtered valve (10) allows said microspheres to be retained within the culture vessel whilst parasites are collected in the vessel (11).

The arrangement described above similarly allows a simple means for the washing of polyethylene microspheres and blocking of non specific binding to the microspheres and culture vessel (3) interior by manipulation of the valves (9 and 6] prior to primary inoculation of the culture vessel with parasites.

The arrangement also allows multiples of the culture vessels to be connected whereby the fritted collection valve (10] of a primaiy culture vessel is connected to the culture fluid inlet valve (9] of a secondary culture vessel in place of the culture fluid reservoir (8). Similarly, the culture vessel (3) may be fitted with more than one fritted outlet valve (10) so that a single primary culture vessel can act as the feed for more than one secondary culture vessels and secondary culture vessels acting as primary culture vessels for further secondary culture vessels.

Combining culture vessels in series or parallel as described above offers the opportunity to scale up production ol Plasmodium parasites on demand.

Claims

Claims: 1. An apparatus for the cultivation of Plasmodium erythrocyte-stage organisms in the absence of erythrocytes, which apparatus comprises of a vessel containing multitudes of interconnected spaces wherein said interconnected spaces are formed by an accretion of non-toxic material forming an inert, non adsorbent, porous scaffold that allows freedom of movement of said organisms from one pore to another without attachment to the scaffold wherein said scaffold pore volume is of the same order of magnitude to the volume of a single Plasmodium organism.
2. An apparatus according to claim 1, wherein said pores in said porous scaffold are formed by the void volumes between accreted insoluble particulate material.
3. An apparatus according to any preceding claims, wherein said accreted insoluble particulate material is composed ofmicrospheres.
4. An apparatus according to any preceding claims, wherein said apparatus incorporates a means of adding nutrients and organisms to said vessel to fill said interconnected spaces.
5. An apparatus according to any preceding claims, wherein said apparatus incorporates a means of recovering the organisms from said vessel.
6. An apparatus according to any preceding claims, wherein said apparatus incorporates a means of removing waste products from the vessel.
7. An apparatus according to any preceding claims, wherein said apparatus incorporates a means of controlling pH, temperature, pressure and atmospheric composition within said vessel.
8. An apparatus according to any preceding claims, wherein said apparatus incorporates a means of connecting multitudes of said vessels together.
9. An apparatus according to any preceding claims, wherein said porous scaffold is formed by a prefabricated binding of fibres.
10. An apparatus according to any preceding claims, wherein said scaffold is a naturally porous material.
11. An apparatus according to any preceding claims, wherein said scaffold is a porous gel.
12. An apparatus according to any preceding claims, wherein said vessel interior surface and scaffold have been rendered inert by washing with culture fluid.
13. A system comprising of one or more apparatus for the cultivation of Plasmodium organisms according to any combination of preceding claims.