WO2009133239A1

WO2009133239A1 - System and method for processing particle suspension

Info

Publication number: WO2009133239A1
Application number: PCT/FI2009/050333
Authority: WO
Inventors: Kauko Lehtinen; Petri Kivelä
Original assignee: Wallac Oy
Priority date: 2008-04-30
Filing date: 2009-04-28
Publication date: 2009-11-05
Also published as: FI20085389A0

Abstract

The present invention provides a system for processing a particle suspension. The system comprises a container (100) for holding the particle suspension, and a dispenser (200) for dispensing a portion of the particle suspension from the container (100) into a well (410) of a microplate (400). The dispenser (200) comprises a pump (210) for transferring the particle suspension, the pump (210) having a chamber (211) for holding the portion of the particle suspension, and a valve unit (220) for allowing the particle suspension to be transferred between the container (100) and the chamber (211), and from the chamber (211) into the well (410) of the microplate (400). The system comprises a connecting tube (301) through which the particle suspension is arranged to be transferred between the container (100) and the dispenser (200). The present invention also provides a method for processing a particle suspension.

Description

SYSTEM AND METHOD FOR PROCESSING PARTICLE SUSPENSION

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a system and method for processing a particle suspension according to the preambles of the appended independent claims.

BACKGROUND OF THE INVENTION

Devices for dispensing particle suspensions are widely used in the fields of chemistry and biotechnology. Some of these devices are designed especially for dis- pensing cell suspensions into microplates.

A typical suspension consists of particles which have different sizes and weights, and thereby distinct densities. At a steady state the particles having a large density tend to settle on the bottom of a container holding the suspension. This results in an uneven distribution of particles within the suspension.

In order to dispense doses having equal particle concentrations, it is necessary to process the suspension in the container during dispensing processes or between them so that an even distribution of particles in the container can be maintained.

There exist various devices and methods for mixing particle suspensions. A conventional device is a magnetic stirrer. A magnetic stirrer consists of a rotating magnet or stationary electromagnets creating a rotating magnetic field. The drawback of a magnetic stirrer is that it must be immersed in the suspension, and therefore there is a potential risk of contaminating the suspension. In addition, a magnetic stirrer is not suitable for stirring thick suspensions or viscous liquids, and only a very low rotation speed can be used to avoid breaking apart the particles. There also exist propeller and blade based stirrers, which must also be immersed in the suspension.

Other often used devices for mixing suspensions are a pendulum for swinging the container, and a shaker for agitating the container. Suspensions can also be mixed by bubbling gas into a suspension, or by using a pump through which a suspension is being circulated. The drawback of these devices is that their construction is complex, requiring extra components and thereby increasing expenses of a dispenser system.

DESCRIPTION OF THE INVENTION

It is the main objective of the present invention to reduce or even eliminate prior art problems presented above.

It is an objective of the present invention to provide a system and method enabling to process, and especially to mix a particle suspension. In more detail, it is an objective of the invention to provide a system and method enabling to maintain an even or at least an almost even distribution of particles within the suspension.

It is a further objective of the invention to provide a system and method for mixing a particle suspension in an easy and efficient, but at the same time gentle way. It is still a further objective of the invention to provide a system having a simple structure.

In order to realise the above-mentioned objectives, the system and method according to the invention are characterised by what is presented in the characterising parts of the appended independent claims. Advantageous embodiments of the invention are described in the depending claims.

The exemplary embodiments of the invention presented in this text are not inter- preted to pose limitations to the applicability of the appended claims. The verb "to comprise" is used in this text as an open limitation that does not exclude the existence of also unrecited features. The features recited in the depending claims are mutually freely combinable unless otherwise explicitly stated.

A typical system for processing a particle suspension according to the invention comprises a container for holding the particle suspension, and a dispenser for dispensing a portion of the particle suspension from the container into a well of a mi- croplate. The dispenser comprises a pump for transferring the particle suspension, the pump having a chamber for holding the portion of the particle suspension, and a valve unit for allowing the particle suspension to be transferred from the con- tainer into the chamber and from the chamber into the well of the microplate. The system also comprises a connecting tube through which the particle suspension is arranged to be transferred between the container and the dispenser, the first end of the connecting tube being connected to the container and the second end of the connecting tube being connected to the valve unit. In a typical system according to the invention the valve unit is arranged to allow the particle suspension to be transferred from the chamber into the container through the connecting tube.

In other words, the present invention provides a dispenser that is suitable for both dispensing and mixing of a particle suspension. Typically, the system according to the invention operates in such a way that dispensing and mixing procedures are performed at different times. That is to say, typically the valve unit is arranged to allow particle suspension to be transferred from the chamber to either the con- tainer or the microplate at a time. However, in some cases the valve unit can be adapted to allow simultaneous transfer.

The present invention is generally suitable for processing particle suspensions. Especially, the present invention is suitable for processing suspensions containing viable cells. Viable cells are fragile and need to be constantly agitated in order to maintain a homogeneous suspension in the container. The present invention provides a gentle mixing technique for these cells. The present invention provides a system wherein no mechanical components are used in the container for agitating the suspension.

Objectives of the present invention are achieved by transferring the particle sus- pension back and forth between the container and the dispenser through the connecting tube.

A container can be any member that is suitable for holding particle suspensions. A container can be, for example, a bottle or a decanting glass. Preferably, the container is an upside down bottle. A container can have an open or closed structure, depending on whether the suspension is in direct contact with ambient air or not.

A pump is used for transferring particle suspension. The pump comprises a chamber having a volume which can be variable or invariable. When dispensing a dose into a well of a microplate, suspension is drawn from the container into the chamber by using the pump. Then the dose is dispensed from the chamber into the well. Typically, the volume of the chamber is small, for example, in the range of 10 to 500 millilitres, or in the range of 50 to 250 millilitres. In some applications the volume can, however, be larger, for example, in the range of 100 to 1000 millilitres, or even in the range of 100 to 10000 millilitres. According to an embodiment of the invention the pump comprises a piston arranged to be movable inside the chamber, whereupon the volume of the chamber is variable. In a preferred embodiment of the invention the pump is a syringe.

The system according to the invention can comprise more than one dispenser. The system can comprise, for example, 2-5, 6-10, 1 1-30 or 31-50 dispenser units. These units can be operated independently or they can be operated in one or more groups.

A purpose of the valve unit is to control suspension flow between the container and the chamber, and between the chamber and the microplate. The valve unit regulates flow of a suspension by opening, closing or partially obstructing ports of the valve unit.

The body of the valve unit can be manufactured from metal, such as brass, bronze, cast or ductile iron, steel, alloy steels and stainless steels. The valve body can also be plastic, such as PVC, PP, PVDF or glass reinforced nylon. Typically, plastic bodies are used for relatively low pressure and temperature applications.

A microplate or microtiter plate is a flat plate with a plurality of wells used as small test tubes. Typically, a microplate has 6, 24, 96, 384 or 1536 sample wells, which are conventionally arranged in a 2:3 rectangular matrix. In some cases, a microplate can even have thousands of wells, for example 9600 wells. Each well of a microplate typically has a volume of somewhere between a few to a few hundred microlitres, for example in the range of 1 to 5000 microlitres.

A connecting tube can be manufactured from materials such as plastic or metal. Typically, an inner diameter of the connecting tube is in the range of 0,5 to 3 mm, preferably in the range of 0,5 to 1 ,5 mm. Typically, the first end of the connecting tube is arranged in connection with the bottom part of the container.

Mixing efficiency depends, for example, on the way the first end of the connecting tube has been arranged relative to the bottom of the container, and on the suspension flow rate and the inner diameter of the connecting tube. Mixing efficiency can be changed, for example, by arranging the first end of the connecting tube at different angles relative to the plane of the bottom of the container.

The system can also comprise another connecting tube through which the particle suspension is arranged to be transferred from the dispenser to the microplate, the first end of the connecting tube being connected to the valve unit. According to an embodiment of the invention the valve unit comprises a first port through which the particle suspension can be transferred between the container and the chamber, and a second port through which the particle suspension can be transferred from the chamber to the well of the microplate.

The port can be obstructed or opened up by a valve member such as a valve disc. A valve member can have various shapes, although preferably it is disc-shaped. The disc can be arranged in a moveable manner with respect to the port so that the flow through the port may be partly or fully blocked. The disc can be operated in a gradual change between two or more positions. When the port is closed, the disc is in a leak-tight contact with the interior surface of the valve body, i.e. with the valve seat. The valve body and the valve seat can come in one piece of solid material, or the valve seat can be a separate piece attached or fixed to the inside of the valve body, depending on the valve design.

In the system according to the invention, the first port has a two-way design allow- ing fluid flow in both directions, whereas the second port has preferably a one-way design. Operating positions for the ports can be either closed so that no flow at all goes through it, fully open for maximum flow, or sometimes partially open to any degree in between.

According to an embodiment of the invention the valve unit comprises an actuator for controlling the operation of the valve unit, so that at a time only one of the ports of the valve unit is arranged to allow transfer of the particle suspension through it when the chamber is being emptied.

An actuator can be an electromechanical actuator comprising an electric motor or a solenoid, a pneumatic actuator controlled by air pressure, or a hydraulic actuator controlled by the pressure of a liquid such as oil or water.

According to an embodiment of the invention the actuator is adapted to control the valve discs. The actuator can comprise stems, like rods, for transmitting motion to control the discs. In some cases, the stem and the disc can be combined in one piece. The motion transmitted by the stem can be a linear push or pull motion, a rotating motion, or some combination of these.

In some applications, the pressure of the flow itself or pressure difference of the flow between the ports automatically controls flow through the port. In a preferred embodiment of the invention the actuator comprises a two-position connector for connecting one of the ports at a time in connection with the chamber. In this case, the valve unit, preferably, does not comprise valve discs. By turning the two-position connector between two possible operating positions, suspen- sion can flow either through the first port or the second port. The advantage of a two-position connector is that it has a simple structure, and therefore durable.

According to an embodiment of the invention the container is a closed container comprising a pipe for controlling gas pressure inside the container. In a preferred embodiment of the invention the pipe is arranged in connection with an upper part of the container. Preferably, the gas pressure inside the container is kept balanced with the ambient pressure via the pipe.

According to an embodiment of the invention the first end of the pipe is connected to a gas space inside the container and the second end of the pipe is connected to a gas space outside the container. The gas provided inside and/or outside the container can be, for example, carbon dioxide (CO2).

The present invention also concerns a method for processing a particle suspension. A typical method according to the invention comprises transferring a first portion of the particle suspension from a container holding the particle suspension to a dispenser. A typical method according to the invention also comprises transfer- ring a second portion of the particle suspension from the dispenser to the container whereupon the particle suspension in the container is being mixed.

According to an embodiment of the invention the first portion and the second portion of the particle suspension are in the range of 1 to 1000 microlitres, preferably in the range of 10 to 300 microliters. The amount of these proportions depends on the application, and therefore can vary substantially.

In a preferred embodiment of the invention the first portion of the particle suspension is equal to the second portion of the particle suspension.

According to an embodiment of the invention the method comprises repeating the transferring steps at a rate of 1 to 100 times per minute, preferably at a rate of 5 to 20 times per minute, for a time in the range of 1 to 100 seconds, preferably in the range of 5 to 20 seconds.

Dispensing and mixing procedures can be performed successively, one after another, or they can be carried out according to a predetermined sequence of opera- tions. For example, two doses can be dispensed and then the suspension is mixed by performing the above-mentioned mixing steps, and so on. Or, for example, four doses can be dispensed and after that the mixing steps could be carried out two times. The optimal sequence of operations depends on the application.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features which are considered as characteristic of the invention are set forth in particular in the appended claims. The invention itself, however, both as to its construction and its method of operation, together with additional objects and advantages thereof, will be best understood from the following description of specific embodiments when read in connection with the accompanying drawings.

FIG. 1 illustrates a system for processing a particle suspension according to an embodiment of the invention,

FIGS. 2A-2B illustrate examples of a valve unit that may be used in embodi- ments of the present invention, and

FIG. 3 illustrates a flow diagram of a method for processing a particle suspension according to an embodiment of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

FIG. 1 illustrates a system for processing a particle suspension according to an embodiment of the invention. The system comprises a container 100 for holding the particle suspension. The container 100 is an upside down bottle having a closed structure. A pipe 110 for controlling gas pressure inside the container 100 has been arranged in connection with an upper part of the container 100.

The system also comprises a dispenser 200 for dispensing a portion of the particle suspension from the container 100 into a well 410 of a microplate 400. The dispenser 200 comprises a pump 210 for transferring the particle suspension, the pump 210 having a chamber 211 for holding the portion of the particle suspension. The pump 210 comprises a piston 212 which is arranged to be movable inside the chamber 211. This movement is indicated by a double arrow. The dispenser 200 also comprises a valve unit 220 for allowing the particle suspension to be transferred between the container 100 and the chamber 211 , and from the chamber 211 into the well 410 of the microplate 400. The valve unit 220 comprises a first port 221 having a two-way design for allowing suspension flow in both directions, and a second port 222 having a one-way design for allowing flow in one direction only. The valve unit 220 comprises an actuator 223 for controlling the operation of the valve unit 220, so that at a time only one of the ports 221 , 222 of the valve unit 220 is arranged to allow transfer of the particle suspension through it when the chamber 211 is being emptied.

The system comprises a connecting tube 301 through which the particle suspension is arranged to be transferred between the container 100 and the dispenser 200. The first end of the connecting tube 301 is connected to the container 100 and the second end of the connecting tube 301 is connected to the valve unit 220. The system also comprises another connecting tube 302 through which the parti- cle suspension is arranged to be transferred from the dispenser 200 to the microplate 400.

FIGS. 2A-2B illustrate examples of a valve unit that may be used in embodiments of the present invention. Referring to FIG. 2A, there is shown a first example of a valve unit 220. As shown in FIG. 2A, the first port 221 and the second port 222 are arranged to be opened or closed by the first valve disc 224 and the second valve disc 225, respectively. When the port 221 , 222 is closed, the disc 224, 225 is in a leak-tight contact with the interior surface of the valve body. The actuator (not shown) is adapted to control the valve discs 224, 225 for example by transmitting motion via stems (not shown) which are attached to the valve discs 224, 225. In FIG. 2A, there are shown the connecting tubes 301 , 302 which are connected to the ports 221 , 222, respectively. The valve unit 220 is connected to the pump 210.

Referring to FIG. 2B, there is shown a second example of a valve unit 220. A two- position connector 226 for connecting one of the ports 221 , 222 at a time in connection with the pump 210 is illustrated. By turning the two-position connector 226 between two possible operating positions (indicated by the dotted lines), suspension can flow either through the first port 221 or the second port 222.

FIG. 3 illustrates a flow diagram of a method for processing a particle suspension according to an embodiment of the invention. At step 501 , the procedure for processing a particle suspension is started. At step 502, a portion of the particle suspension is transferred from a container holding the suspension to a dispenser. This is performed by aspirating the portion by using a pump. At step 503, the portion is transferred back to the container whereupon the particle suspension in the container is being mixed.

At step 504, it is determined whether or not the suspension in the container needs to be mixed more before dispensing a dose. If a homogeneous suspension is achieved, then the procedure continues at step 505. Otherwise, the suspension will be mixed more and the procedure is continued at step 502.

At step 505, a dose of the particle suspension is transferred from the container to the dispenser. The dose is dispensed from the dispenser to a well of a microplate at step 506.

At step 507, it is determined whether or not more doses will be dispensed. If more doses will be dispensed and the suspension is homogeneous enough, the proce- dure is continued at step 505. If the suspension needs to be mixed, the procedure is continued at step 502. If no more doses need to be dispensed, the procedure is completed at step 508.

The present invention can be applied, for example, to Ca²⁺/Aequorin -based GPCR (G-Protein Coupled Receptor, 7-trans membrane receptor) activation as- says using viable cells expressing an apoaequorin protein. In an agonist assay the cells are dispensed into a well of a microplate containing the assayed compound (possible agonist/activator). In a case of an agonist, the GPCR receptor is activated and a functional reaction chain is initiated inside the cell, which increases the cytoplasmic Ca²⁺ level. Ca²⁺ is released and within 30 seconds captured back by ER inside the cell. Also a screen evaluating both agonists and antagonists requires the same cell dispensing step.

This application is of a kinetic nature. The reaction occurs within 30 seconds and thus a dispenser is required to initiate the reaction. The reaction can be followed in a kinetic mode (a read once a second) or by integrating over the whole time. Dur- ing the processes, the cells need to be constantly agitated to maintain a homogeneous suspension in the container. There is a 30 second interval between wells, meaning that depending on the plate format / instrument detector count, it usually takes 10-60 minutes per plate. This is too long for the suspension to remain and therefore the suspension has to be agitated. The system and method according to the invention can be applied for this application.

The present invention can also be applied to a kinetic evaluation of biomolecule binding characteristics using scintillating particles (SPA, Scintillation Proximity As- say). The binding reaction is activated once the beads have been dispensed into the well. On- and off-rate binding velocity can be used to estimate binding affinity.

Only advantageous exemplary embodiments of the invention are described in the figures. It is clear to a person skilled in the art that the invention is not restricted only to the examples presented above, but the invention may vary within the limits of the claims presented hereafter. Some possible embodiments of the invention are described in the dependent claims, and they are not to be considered to restrict the scope of protection of the invention as such.

Claims

1. A system for processing a particle suspension, the system comprising:

- a container (100) for holding the particle suspension,

- a dispenser (200) for dispensing a portion of the particle suspension from the container (100) into a well (410) of a microplate (400), the dispenser (200) comprising:

- a pump (210) for transferring the particle suspension, the pump (210) having a chamber (211 ) for holding the portion of the particle suspen- sion, and

- a valve unit (220) for allowing the particle suspension to be transferred from the container (100) into the chamber (211 ) and from the chamber (211 ) into the well (410) of the microplate (400); and

- a connecting tube (301 ) through which the particle suspension is arranged to be transferred between the container (100) and the dispenser (200), the first end of the connecting tube (301 ) being connected to the container (100) and the second end of the connecting tube (301 ) being connected to the valve unit (220); characterised in that the valve unit (220) is arranged to allow the particle suspen- sion to be transferred from the chamber (211 ) into the container (100) through the connecting tube (301 ).

2. The system according to claim 1 , characterised in that the valve unit (220) comprises a first port (221 ) through which the particle suspension can be transferred between the container (100) and the chamber (211 ), and a second port (222) through which the particle suspension can be transferred from the chamber (211 ) to the well (410) of the microplate (400).

3. The system according to claim 2, characterised in that the valve unit (220) comprises an actuator (223) for controlling the operation of the valve unit (220), so that at a time only one of said ports (221 , 222) is arranged to allow transfer of the particle suspension through it when the chamber (211 ) is being emptied.

4. The system according to claim 3, characterised in that the actuator (223) comprises a two-position connector (226) for connecting one of said ports (221 , 222) at a time in connection with the chamber (211 ).

5. The system according to any of the preceding claims 1 to 4, characterised in that the pump (210) comprises a piston (212) arranged to be movable inside the chamber (211 ), whereupon the volume of the chamber (211 ) is variable.

6. The system according to claim 5, characterised in that the pump (210) is a syringe.

7. The system according to any of the preceding claims 1 to 6, characterised in that the container (100) is a closed container comprising a pipe (110) for controlling gas pressure inside the container (100).

8. The system according to claim 7, characterised in that the first end of the pipe (110) is connected to a gas space inside the container (100) and the second end of the pipe (110) is connected to a gas space outside the container (100).