WO2010130303A1

WO2010130303A1 - Automated separation of particles from a suspension

Info

Publication number: WO2010130303A1
Application number: PCT/EP2010/001078
Authority: WO
Inventors: Frank Pretzsch; Ulrike Koropp; Heike Walles; Michaela Kaufmann; Jan Hansmann
Original assignee: Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V.
Priority date: 2009-05-15
Filing date: 2010-02-20
Publication date: 2010-11-18
Also published as: DE102009022353A1

Abstract

The invention relates to processes for separating particles suspended in suspensions, wherein preferably at the same time a predetermined particle concentration can be set in a suspension and a particle suspension having a set concentration can be obtained. The invention relates to means for carrying out this process. A particular field of application are suspensions of biological cells and the GMP-compliant fully automatic processing thereof.

Description

Automated separation of particles from a suspension

Description

The invention relates to a process for separating particles suspended in suspensions, wherein preferably concurrently a predetermined particle concentration in a suspension can be set and a particle suspension having a set concentration can be obtained. The invention also relates to means for carrying out this process. A particular field of application is automated tissue processing.

In the technical field of tissue engineering, particularly in relation to regenerative medicine, there is a need to automate biological laboratory processes in a GMP-compliant manner under clean room condi- tions. This is intended to provide a higher yield, higher process safety and also standardisable process optimisation and process control.

One difficulty consists above all in converting the large number of different previously known manual steps and operational sequences of cell isolation and cell cultivation into expediently automated handling operations. A particular challenge presented to the automation of sequences of this type is in this case the separation of individual cells from a suspension of various components in order to be able to subsequently cultivate these cells. In the manual process, this is conventionally achieved by centrifuging the suspension in centrifuge tubes and pipetting off the pellet from the supernatant. For the separation of tissue remnants from a cell suspension, it is known to use cell sieves which have different pore densities of, for example, 40, 70 or 100 μm, and are put on centrifuge tubes. The suspension with isolated cells and tissue remnants are for this purpose drawn up manually using a pipette and pipetted into the centrifuge tube through the sieve. Thereby, the tissue remnants remain in the screen. This method has a plurality of drawbacks in relation to steril- ity, as particularly the putting of the sieves onto the centrifuge tube and the filtering through the small-volume screen present risks of contamination. High loss or wastage occurs during tissue recovery.

In addition, this method is too expensive and complex in its implementation for automated handling under clean room conditions, for example using automatic robot systems.

Further steps in tissue preparation consist in producing individual cell suspensions, in separating cells from tissues, in metering liquids, in resuspending cells and in transporting cells and also in setting or adjusting cell concentrations (number of cells per volume fraction) in suspensions in order to seed a defined number of cells for cultivation. In known processes, each of these steps is carried out separately, manually and routinely by means of glass or plastic pipettes. There is a high risk of contamination here.

In addition, these laboratory processes carried out manually disad- vantageously use a large number of disposable vessels and disposable instruments; this increases the process costs and reduces process safety. One object of the invention is to carry out in an automated manner the sequence of all the necessary process steps at as low cost as possible. Above all, the aim is to provide a process which can be carried out by means of a single "automated pipette" which can be used as the central handling system for each of these steps, i.e. in particular: separating off the cells or particles from a suspension, separating off any further components of the suspension, adjusting the particle concentration, resuspending the particles and metering determined quantities of particles.

Attempted solutions known in the art that can be carried out using automated pipettes are limited to the precise positioning and metering of liquids or suspensions.

The invention was based on the technical problem to provide a process for isolating particles suspended in a suspension, particularly biological cells, that can be directly carried out within a single automated pipetting means, wherein particularly a predetermined particle concentration of the isolated particles in a further particle suspension obtained therefrom can be set.

The technical problem is fully solved by a process for the isolation of particles suspended in a first suspension from this suspension such as is presented in claim 1. Thereby, the first suspension may, if appropriate, contain further components suspended therein. Particular configurations emerge from the sub-claims.

According to the invention, the process comprise the following se- quence of steps, the steps preferably being carried out in the specified order, preferably immediately in succession, without further intermediate steps: In a first step (step 1), the particles to be isolated are separated from the first suspension containing the particles in a frist suspending liquid, which preferably may be present in a receiver vessel, using at least two filters which are preferably arranged in a filter carrier of the pipetting means and are preferably arranged in series in the direction of suction, i.e. hereinafter in the downstream direction. According to the invention, said first filter is suitable and preferably specifically configured for retaining any further components that may be contained in the first suspension, i.e. retention or retaining, and for allow- ing said particles to be isolated, which are suspended within the first suspension, to pass through the filter, i.e. filtration or filtering. According to the invention, said second filter is suitable and preferably specifically configured for retaining thereon the particles to be isolated (retention) while the fist suspending liquid of the first suspen- sion being filtered (led pass) through the second filter. According to the invention it is accomplished that by suction of the first suspension, any further components contained therein are retained on the first filter and the particles to be isolated therefrom are retained on the second filter.

In a particular embodiment the first step is carried out in at least two separate consecutive partial steps: In a first partial step of the first step (step 1a) the further components are separated off from the first suspension by sucking the first suspension through the first filter, preferably into a first filter chamber or compartment that is formed between the first and the subsequent second filter. Thereby, the further components are retained on said first filter and the particles to be isolated are filtered (led pass) through the first filter and preferably into the first filter chamber. In a preferred variant thereof the first suspension containing the particles to be isolated are then retained in said first filter chamber. This is preferably accomplished by applying a pressure gradient in the direction of suction, i.e. downstream, across the first filter such as to prevent the suspension from leaking out of the first filter chamber, in particular through the first filter screen. In a second partial step of the first step (step 1b) the particles are separated off from the first suspension by sucking the first suspension containing the particles to be isolated from said first filter chamber through the subsequent second filter, whereby the particles to be isolated are retained on said second filter. In particular, the suspending liquid of the first suspension is drawn through the second filter and preferbaly into a second filter chamber that is formed behind - in the direction of suction, i.e. downstream - the second filter. Preferably, the suspending liquid of the first suspension is retained in the second filter chamber. This is preferably accomplished by apply- ing a pressure gradient in the direction of suction, i.e. downstream, across the second filter such as to prevent the suspending liquid from leaking out of the second filter chamber, in particular through the second filter screen.

In a second step (step 2) the particles retained on the second filter in step 1 are secured or fixed to the second filter. This is preferably accomplished by applying a pressure gradient, running in the direction of suction, i.e. downstream, across the second filter such as to prevent the particles from detaching from the second filter screen.

In a third step (step 3) the first filter, with any further components of the first suspension attached thereto, is separated from the second filter, i.e. preferably uncoupled from a preferably provided filter carrier, and removed. Step 2 and step 3 are preferably carried out concurrently or substantially at the same time in a single integrated process step (step 2, 3).

In a particular embodiment the invention additionally provides, after the isolating of the particles from the first suspension and the sepa- rating of any further, in particular solid components of the suspension, the subsequent resuspending and automatic setting or adjustment of the particle concentration (number of particles per unit of volume of the suspension):

In a fourth step (step 4) which is then preferably carried out therefore in addition, the particles that are secured to the second filter are re- suspended, in a second suspending liquid of a predetermined or defined first volume V-i, as a result of which a second suspension of the isolated particles is obtained. The particles to be isolated are thereby resuspended preferably by what is known as backflushing of the second filter, i.e. having the filter flowed through in the upstream direction.

Preferably, the particles resuspended in this way are flushed into an enclosed volume positioned upstream for receiving the second suspension. Particularly preferred, this enclosed volume is a pipette tip placed onto the second filter, preferably a pipette tip, positioned on or placed onto the second filter or preferably the filter carrier and preferably replaces the first filter separated in step 3.

In a fifth step (step 5) which is then preferably carried out consecutively in addition, the total number N of particles to be isolated in the second suspension obtained in step 4 is assessed. In an alternative variant, the number N_τ of a partial volume V_τ is determined and back-calculated therefrom to the total number N in the total volume V₁. Preferably, the total number N of particles present in the second suspension is calculated from the partial number Nj, the total volume of the second suspension \Λ and the partial volume V_τ in accordance with:

In a sixth step (step 6) which is then preferably carried out consecutively in addition, the particles to be isolated are separated from the second suspension. This takes place preferably by again retaining, i.e. re-retaining, the particles on the same second filter; alternatively on another filter which replaces the second filter. Preferably, the separating is carried out by applying a pressure gradient, which is directed downstream across the second filter, for suction the second suspending liquid off of the second suspension.

In a seventh step (step 7) which is then preferably carried out con- secutively in addition, the particles which are re-retained on the second filter after separating the second suspension are resuspended in a third suspending liquid having a volume V₂, preferably by back- flushing the second filter, particularly in a manner similar to step 4, and a third suspension of the particles is thus obtained.

The invention provides for the second volume V₂ for producing the third suspension to be automatically calculated and/or selected as a function of the total number of cells N determined in step 5 in order to set a predetermined concentration K of the isolated particles in the third suspension. Preferably, the volume V₂ is calculated for this pur- pose accordancing to: ^v-£ (2)

The invention will be described hereinafter primarily in relation to its application to biological cells as the particles to be isolated. The invention is however not restricted to particles of this type; the basic principle of isolating particles from a suspension presented herein, can be applied to other technical areas.

The particles to be isolated are particularly biological cells, especially singled-out eukaryotic cells from tissues, particularly mammalian cells. In another embodiment of the invention the particles are fungal cells. In another embodiment the particles are prokaryotic cells, particularly bacterial cells. The further components of the first suspension that may be contained therein are particularly solid impurities, especially biological impurities such as cell agglomerates, tissue remnants and the like. In relation to the recovery/isolation of individ- ual cells from tissues, preferably biopsates, enzymatic treatments are in particular carried out in order to detach the cells from the cell and tissue cluster. A suspension is thus obtained containing, in addition to free individual cells which are to be isolated using the process of the present invention, also cell agglomerates, fibres and also un- wanted tissue remnants.

Particularly in relation to the cultivation of biological cells, the invention therefore preferably provides for the cells to be separated from a suspension having generally undefined contents of individual cells and tissue remnants.

An advantage of the present invention consists in the fact that complex process steps of preparing tissue samples as well as recovering individual cells, which previously had to be carried out by hand, are replaced by an automatic procedure which can be carried out using an automatically controlled multifunctional pipette. The process according to the invention allows a large number of process steps in the handling of cell suspensions to be integrated in a single functional unit. This primarily allows a linearisation and a marked reduction in the complexity of the process chain for cell processing. Also achieved are higher precision and reproducibility in the adjustment of cell suspensions and in the dispensing of defined numbers of cells. The latter factors are particularly important in defined seeding in large-scale bioreactors for cell cultivation. A multifunctional pipette operated in accordance with the invention can be configured in a GMP-compliant manner, and the risk of contamination or loss of sterility in the preparation of the cell cultures is reduced. Thereby, the use of disposable elements is reduced to a minimum without significantly increasing the risk of contamination.

The solution according to the invention provides for a fully automated separating process which can be carried out in what is known as a multifunctional pipette, advantageously under GMP-compliant condi- tions. This makes possible the fully automatic GMP-compliant isolating of particles, particularly of biological cells, from a suspension and the simultaneous or concomitant setting or adjustment of the particle concentration in a suspension in an integrated process sequence. This also allows the automatic controlled dispensing of defined quan- tities of particles or numbers of cells from the pipette.

The main area of application of the process according to the invention in conjunction with the pipette carrying out this process are, in particular, fully or partly automated systems for the preparation of tissue layers and also for the isolation and further processing of individual cells of a specific cell type in cell cultures. In addition, the process according to the invention provides a stand-alone system as an effective laboratory tool for particle separation and particle meter- ing.

For carrying out the process according to the invention, preferably an automated pipette is preferably dipped into a vessel (receiver vessel) containing the cell suspension and the suspension contained therein is suction-extracted, preferably completely. The suspension is drawn through a filter carrier, which is provided in accordance with the invention, of the pipette with at least two filters arranged in series. In this case, any fibre and tissue remnants contained in the suspension are retained in a first filter and the singled-out cells of the suspension are retained in the downstream second filter. The liquid of the sus- pension (suspending liquid) is discarded through at least one suction-extraction channel which is preferably in fluid connection with the filter carrier.

Preferably, the particles to be isolated and any further components contained in the first suspension are separated by applying a drop in pressure, i.e. pressure gradient, which is directed downstream, i.e. in the flow path of the first suspension, from the receiver vessel into the filter carrier, the first suspension following the pressure gradient. Preferably, this pressure gradient is generated by applying a reduced pressure within the pipette. In the context of the invention, the term "downstream" direction therefore refers to the direction of suction of the first suspension into the filter carrier and pipette body. The term "upstream" direction refers to the direction running out of the pipette body or filter carrier, such as it runs out of the pipette during the metering of the suspension.

In the second step, the cells are secured to the second filter, preferably by maintaining a reduced pressure in the filter carrier; tissue remnants remain in the first filter. Preferably, the first filter stage, including the tissue remnants retained thereon, is now removed, separated or ejected with the aid of an automated device suitable for this purpose. For this purpose, the filter carrier is preferably configured in such a way that the first filter, as an easy-change filter in a separate filter attachment or filter insert, can be coupled to the filter carrier or inserted into the filter carrier. The connection between the filter carrier and filter attachment or filter insert is preferably detachable in its configuration. With the ..ejecting" of the easy-change filter carrier from the filter carrier, any further components contained in the first suspension are separated from the further process sequence. The first filter is preferably separated automatically as a result of design measures carried out on the filter carrier. Alternatively, the easy- change filter carrier is separated with the first filter from the filter carrier outside the automated pipetting means by way of a separately provided device.

Preferably, the first filter is automatically replaced by a pipetting insert which forms a pipette tip instead of the first filter. Depending on the area of application, the separated first filter stage with the tissue remnants clinging thereto is discarded.

In a particular embodiment the invention provides for step 2 and step 3 also to be able to be carried out, depending on the specific configuration of the second filter and/or the filtering process, as a single common step. This is the case especially when no particular additional measures are required to secure there the cells retained on the second filter. This takes place preferably by way of a particular configuration of the pipetting device and the filter carrier, so that the re- tained cells come to be positioned on the second filter in such a way that they are secured there on account of gravity. In another alternative configuration the cells are secured to the underside of the second filter counter to gravity. In an alternative variant this takes place, in addition to the aforementioned preferred process measure of ap- plying a reduced pressure, on account of the capillary forces acting between the filter surface and the cells retained thereon.

The indeterminate number, secured to the second filter, of cells to be isolated is backflushed with a second suspending solution via the pipetting means, preferably via a specific supply channel, so that the cells become detached from the second filter and are resuspended in the second suspending solution. The invention provides for this second suspending solution to have a defined volume which is preset in accordance with the issue at hand or number of cells to be expected.

In a variant of the invention the second suspending liquid and the first suspending liquid filtered from the first suspension are the same or of the same composition or type. In another variant the second suspending liquid is different from the first suspending liquid filtered from the first suspension, preferably of a different composition and type.

The metering, that is to say the conveyed volumes of media, is preferably regulated by way of the ratio of the excess pressure in the feed line and the opening time of a valve connected in the feed line. In an alternative embodiment the amount metered is regulated by a defined piston stroke in a closed pipetting system known per se. The person skilled in the art is familiar with further alternative embodi- ments of automated pipettes in order to dispense a defined volume.

In order to determine the number of particles or cells, the number of cells is counted in the volume of the second suspension. In a preferred variant the number of cells is counted in a partial volume of the second suspension and back-calculated to the total volume and thus to the total number of cells. Processes known per se are used for determining the number of cells. Preferably, the number of cells is determined by introduction into a counting chamber in that the cells in the counting chamber are counted using microscope optics, preferably via automatic image analysis. The person skilled in the art is familiar with alternative processes such as inductive measuring processes and also fluorescence measurement for determining the number of cells in a suspension. These processes can be applied in a similar manner.

The invention preferably provides that, for metering the partial vol- ume of the second suspension, in which the separated particles or cells are resuspended, a metered volume of air is introduced for the count, so that the internal pressure in the collection volume of the pipetting attachment is increased, so that a defined amount of the cell suspension contained can be dispensed into the counting appa- ratus. Alternative processes for the metering of cell suspensions can be applied in a similar manner (see above). The total number of cells in the collection volume of the pipetting attachment is preferably determined from the ratio of the number of cells in the partial volume (counting sample) to the partial volume removed (volume of the cell sample), which is precisely as high as the ratio of the total number of cells to the total volume. The number of cells removed for the cell count is in this case regarded as being negligibly small and is not taken into account in the calculation.

Furthermore, the invention preferably provides that, after the number of cells in the pipette has been determined in this way, the buffer solution used for the count is, again, sucked off and discarded. For this purpose, cells are again retained on the second filter. The second suspending solution is sucked off, preferably by applying a reduced pressure via the suction-extraction channel provided in the pipette.

The invention thereby preferably provides that, in order to obtain a cell suspension of the isolated cells having a defined cell concentration (number of cells per unit of volume) which is preferably selected in advance, a defined volume of a third suspending solution is introduced into the multifunctional pipette above the inflow channel which is preferably provided in order to backflush the second filter. As a result, the cells which are again secured to the second filter are re- suspended in the third suspending solution, so that a third suspension is obtained, this third suspension containing a defined number of cells per unit of volume, i.e. a defined cell concentration. For this purpose, the invention preferably provides that the volume of the third suspending solution to be supplied is adjusted in accordance with the ratio of the volume of the third suspending liquid to be supplied to the total number of cells in the pipette, which is to corre- spond to the ratio of the unit of volume of the cell concentration to the number of cells per unit of volume of the cell concentration. The volume of the third suspending liquid thereby corresponds to the quotient from the determined total number of cells of the isolated cells and the concentration to be set (number of cells per unit of volume).

In a variant of the invention, the third suspending liquid and the first suspending liquid filtered from the first suspension are the same or of the same composition or type. In a further variant of the invention, the third suspending liquid and the second suspending liquid are the same or of the same composition of type. In another variant, the third suspending liquid is different from the first suspending liquid filtered from the first suspension, preferably of differing composition and type. In further variant, the third suspending liquid is different from the second suspending liquid, preferably of differing composition and type.

The invention provides, preferably in additional process steps, that after the third suspension having a defined particle concentration or cell concentration has been produced in accordance with the inven- tion, said suspension is if appropriate maintained in a homogeneous blend in order then to dispense, that is to say to meter, by metering in a targeted manner specific partial volumes from the suspension, a defined quantity of particles or cells suspended therein. The metering is carried out preferably in partial doses, preferably repeatedly and particularly preferably as aliquoting. The metering is carried out in relation to the metering of biological cells, for example on membranes for the cultivation thereof. In a preferred embodiment of the invention, the invention therefore also allows consistently thorough blending of the particle suspension, particularly cell suspension, contained in the collection volume of the pipetting attachment even over relatively long processing periods such as occur, for example, in the point-by-point seeding of cells in bioreactors for cell cultivation. A repeated sequence of a pumping-off process with subsequent drawing-back-in of the cell suspension is provided for this purpose.

Preferably, in a first step the cell suspension is conveyed from the pipetting attachment, preferably with the aid of compressed air/excess pressure, into a sterile vessel arranged below the pipetting attachment. In a second step the suspension conveyed into the vessel is again drawn up or pumped into the collection volume of the pipetting attachment. This procedure is repeated preferably at least once, particularly several times, in order to obtain a homogeneous blending of the suspension. The number of repetitions and also the time intervals at which the repetitions take place are preferably selected as a function of the cell type, the suspension medium and also the cell concentration.

The invention preferably provides that, in order to convey the cell suspension, preferably sterile compressed air is supplied in a me- tered manner into the filter carrier and into the pipetting attachment in order to increase the internal pressure in the collection volume of the pipetting attachment in such a way that the entire amount of the cell suspension contained is conveyed into the vessel. As a result of the subsequent preferred metered applying of a reduced pressure in the intake channel of the pipette described in the present document, the cell suspension is preferably drawn back into the collection vol- ume of the pipetting attachment, allowing no solution to be suction- extracted or, as a result, the cell concentration in the suspension to be altered.

The invention provides in further steps the controlled dispensing of defined numbers of particles or cells. After the setting according to the invention of a suspension having a defined concentration and if appropriate after ensuring a consistent homogeneous blending based on the procedures described hereinbefore, the invention provides the dispensing of precisely defined numbers of cells. The number of cells is in this case set by monitoring the drop volume during dispensing from the pipetting attachment. For this purpose, the drop volume is determined based on the cell concentration and preferably dispensed by the metered supply of sterile compressed air. The drop volume is determined from the law which states that the ratio of the drop volume to the total volume of the solution in the pipette corresponds to the number of cells to be dispensed in relation to the total number of cells in the pipette.

The invention provides for the process steps described hereinbefore to be carried out in an automated manner, and without the interven- tion of human reasoning or manual manipulations, in a single automated pipetting means. For this purpose, the pipetting means is specifically embodied to be able to carry out in an automated manner the process according to the invention as described hereinbefore. The pipetting means has appropriate control elements for carrying out the flushing procedures and suction-extraction procedures and also means for generating drops in pressure (excess pressure or reduced pressure) or is fluidically connected and operatively connected to means of this type. Furthermore the automatic pipetting means preferably has at least one computing unit which is data- connected to at least one counting means for calculating the defined volumes to be metered, in particular V₂, and also, if appropriate, for calculating the total number N of particles, for determining the num- ber of particles in a suspension and is connected to at least one means for controlling the metering of the volumes, at least of the volume V₂, and also preferably to means for controlling the overall sequence of the process, but preferably at least to means for controlling the separating of the first filter in step 3 and preferably in addi- tion for receiving a bounded volume which is coupled-on instead of the first filter, in particular the pipette tip/pipetting attachment.

The invention accordingly also relates to a pipetting device which is specifically embodied for carrying out the process according to the invention. The device preferably consists of a filter carrier with at least two filters connected in series in the direction of flow, at least the first filter being detachably connected to the filter carrier. Preferably, the filter is configured in such a way that any further components found in a suspension of particles to be isolated are retained thereon, although the particles to be isolated can be filtered through the filter. The second filter provided in the filter carrier is preferably configured in such a way that the particles to be isolated from the suspension are retained thereon and the suspending liquid can be filtered through said second filter. In this way the filter carrier allows the particles to be isolated to be separated from the suspending liq- uid of the suspension and from further components contained in the suspension in a single intake stroke.

In relation to the cultivation and preparation of biological cells, the first filter insert for retaining cell agglomerates and tissue compo- nents has a pore size of 20 μm and more, preferably of from 20 μm to 100 μm. For the filtration of cell aggregates, the pore size is particularly preferably from 20 to 60 μm, preferably from 20 to 40 μm and alternatively preferably from 40 to 60 μm. For the filtration of tis- sue components, the pore size is preferably from 80 to 100 μm, preferably about 80 μm, and alternatively preferably about 100 μm.

For the filtration on the second filter of the individual cells/particles to be isolated, the pore size is preferably 10 μm or less, preferably 8 μm or less, particularly preferably 6 μm or less or 4 μm or less. The pore size of the second filter is preferably from 2 to 8 μm, particularly preferably from 2 to 6 μm or from 2 to 4 μm, alternatively preferably from 4 to 8 μm.

The filters are embodied in a manner known per se. Filter nets are preferred, particularly made of filter cloths or meshes. Preferred ma- terials are PTFE, PET, glass, ceramic, cellulose, polyethylene, polypropylene, polystyrene, polyamide or composites thereof; PET membranes are particularly preferred.

The filters are preferably embodied, in the filter carrier which is preferably provided in accordance with the invention, as filter inserts which are held, preferably by clamping, in the filter carrier. The filter inserts are preferably configured in such a way that the cells to be separated and the suspension and its further components enter into contact exclusively with the sterilisable filter inserts, but preferably not with the body/wall of the filter carrier itself, thus allowing cross- contaminations between cells and the suspension within the filter carrier of the automatic pipette to be prevented. The filter carrier which is preferably provided is part of an automatic pipette which is designed in accordance with the invention and allows especially the setting of the cell concentration in a liquid, the holding of the cells in suspension and also the resuspending and, in addition, the controlled dispensing of defined numbers of cells. An automatic pipette according to the invention consists especially of the following assemblies: the filter carrier described in the present document and also at least one valve body coupled thereto. The valve body is fluidically connected to the filter carrier. For the basic operability of the process according to the invention, at least the following components are formed on the valve body and/or on the filter carrier: at least one supply channel for media and at least one suction-extraction channel.

The supply channel is preferably blockably fluidically connected to one or more lines, particularly to at least one further line which passes a buffer solution suitable for determining the number of cells, at least one line which passes a cell further processing solution, at least one line which passes a sterilising solution and at least one line for gas or air for purging the supply channel and for applying pres- sure to the interior of the filter carrier for metering media/suspensions from the pipette. Lines for further media, such as for example cell culture medium, can be added, depending on the intended use.

The further element is the at least one suction-extraction channel, which is spatially separated from the supply channel, for suction- extracting liquid media and also for building up a drop in pressure, preferably a reduced pressure, which is directed into the filter carrier or valve body, that is to say downstream, and which can be used to hold and handle the particles or cells to be isolated on the second filter, if appropriate counter to gravity. The invention preferably provides for suction-extraction and supply to be configured so as to be completely spatially separated from each other in the pipette, substantially in order to prevent soiling or contamination of fresh media or cells.

The automatic pipette preferably has at least one pipetting insert (pipette tip) configured as an exchangeable insert, the pipette having at least one volume for storing cell solution and a pipetting opening for dispensing the solution from the pipetting insert. According to the invention, the dimensions of the opening in the pipetting insert are in this case preferably such that the cell solution is kept in the pressure- less state counter to gravity. Dropwise dispensing takes place preferably only after application of a metered pressure within the pipette through the supply channel of air or gas into the interior of the filter carrier via the valve body.

Preferably, the pipetting insert, like the filter insert, is held on the filter carrier by clamping. The insert is configured in such a way that the cells to be treated enter into contact exclusively with the sterile exchangeable inserts and not with the filter carrier.

The process sequence according to the invention is controlled on the pipette preferably via valves which can open or close the one or more supply lines, which are fluidically connected to the supply channel of the pipette and to which excess pressure is preferably applied, and also valves which can open and close the preferably at least one suction-extraction channel, to which reduced pressure is applied, with the supply channel. For reducing the dead volumes, the valves of the supply lines are arranged preferably directly on the valve body on the inflow channel and preferably in direct proximity to the filter carrier.

Preferably, the conveyed volumes of media are regulated by way of the ratio of excess pressure in the feed line and the opening time of the valve. The metering of the dispensing volume per drop of cell solution, and thus the number of cells dispensed for controlled dispensing, is carried out preferably via the metering of the supplied volume of air which is also monitored by way of the combination of the opening time of the air valve with the excess pressure applied.

Further advantages of the invention will become apparent from the figures which are described hereinafter and in which:

Figure 1 is a schematic illustration of the sequence of steps of the process according to the invention for isolating particles from a suspension:

A first suspension (100) with particles (110) to be isolated and any other components (120) also contained is located in a receiver (150). The pipette used for carrying out the process has at the beginning of the process a filter carrier (400) and a second filter (430), which is arranged therein and is preferably embodied as an individual cell filter, and also a filter attachment (410) with a first filter (420) which is preferably embodied as a tissue filter. The filter carrier (400) and the pipette interior (440) are fluidically connected to a pressure line (300) with a first pump unit or pressure receiver (310). The filter carrier (400) and the pipette interior (440) are fluidically connected to an inflow channel (320) with a second pump unit or liquid reservoir (330). Figure 1a: In step 1 a reduced pressure is generated in the pipette interior (440) via the pressure line (300), as a result of which the suspension (100) is drawn up from the receiver (150) into the interior (440). In this case, the first suspension (100) passes through the first filter (420) and subsequently the second filter (430) which is positioned downstream, the suspending liquid of the suspension (100) being filtered through both filters (420, 430) and if appropriate discarded, preferably via the pressure line (300). The particles (110) to be isolated are retained on the second filter (430). Any further com- ponents (120) of the suspension are retained on the first filter (420).

Figure 1b: In step 2 the particles (110) are in contact with the second filter (430) and are secured thereto. The remaining components (120) are if appropriate in contact with the first filter (420) and are preferably secured thereto. In the embodiment illustrated in this fig- ure, the securing takes place by way of reduced pressure applied via the pressure line (300).

Figure 1c: In step 3 the filter attachment (410), with the first filter (420) and the remaining components (120) retained thereon, is separated from the filter carrier (400). In the filter carrier (400) of the pi- pette, on the second filter (430), the particles (110) are now isolated from the remaining components (120) and the suspending liquid of the suspension (100) and can be used for further subsequent process steps. Figure 2 shows a preferred embodiment of the invention, the separating steps according to the invention as illustrated in Figure 1 being followed by further steps for resuspending the isolated particles and setting the particle concentration in a suspension:

Figure 2a and 2b show process steps 1 to 3 illustrated in Figures 1a, 1b and 1c. Subsequently, a pipetting insert (500) is attached to the filter holding unit (400) instead of the filter attachment (410) (Figure 1c).

Figure 2c: In step 4, which is preferably carried out in addition, the second filter (430) is backflushed by adding a defined amount of a second suspending liquid into the filter carrier (400) via the inflow channel (330), so that the particles (110) secured thereto are resus- pended in a second suspension (200). The second suspension (200) is subsequently located in the volume (510) enclosed by the pipetting insert (500).

Figure 2d: In step 5, which is preferably carried out in addition, an excess pressure is generated by applying an excess pressure via the pressure line (300), as a result of which a specific partial volume (210) of the second suspension (200) is transferred to a counting unit (600). The number of particles contained in the partial volume (210) is determined in the counting unit (600).

Figure 2e: In step 6, which is preferably carried out in addition, the second suspension (200) is drawn up via the second filter by applying a reduced pressure to the pressure line (300) as an outflow channel (300) discharge. The particles (110) are re-retained on the second filter (430). Figure 2f: In step 7, which is preferably carried out in addition, the particles (110) retained on the second filter (430) are resuspended by adding via the inflow channel (330) a third suspending liquid which backflushes the second filter (430). The third suspension (250) obtained in this way is subsequently located in the enclosed space (510) formed by the pipetting insert (500). According to the invention, the amount of solution added is selected in such a way that a predetermined cell concentration is achieved based on the previously counted number of cells in the second suspension (200).

Figure 3 is a schematic illustration of a further process sequence, which is preferably carried out in addition in relation to the invention, in order to obtain or to maintain a homogeneous suspension. For this purpose, the third suspension (250) which is produced in the process according to the invention, has a defined cell concentration and is located (Figure 3a) in the pipetting insert (500, 510) is dispensed (Figure 3b) into a receiver vessel (700) by applying an excess pressure to the pressure line (300) and subsequently taken up again into the pipetting insert (500) by applying a reduced pressure to the pressure line (300). The through-flow through the tip of the pipetting in- sert (530) generates vortices, as a result of which the cells are suspended homogeneously after repeating one or more times the dispensing and taking-back-up into the pipetting insert (Figure 3c).

Figure 4 is a cross-sectional view of a specific design of the pipetting means which can be used in accordance with the invention and has a filter carrier (400), a pressure line (300) embodied as a suction- extraction channel, an inflow channel (330) embodied as a jacket channel around the suction-extraction channel, and a valve body (450) with valves (460, 470). Arranged on the filter carrier (400) is a filter (430) and a pipetting insert (500) arranged therebelow (figure 4b) or, instead of the pipetting insert, a filter attachment (410) with a filter (420) (figure 4a).

Figure 5 is another schematic illustration of the sequence of steps of the process according to the invention performed in another embodiment of the pipette: A first suspension (100) with particles (110) to be isolated, e.g. single cells, from other particulate components (120), e.g. tissue residues, is located in a petridish or receiver (150). Figure 5a is a cross-sectional view of an insert (600) into a pipette system. The insert (600) comprises at least two compartments (640, 650) which are arranged in this embodiment in a basically concentrical, coaxial manner. An inner chamber (640) is surrounded by an annular outer chamber (650). Inner chamber (640) and outer chamber (650) are separated from one another by a sep- turn (660), which is the wall of the inner chamber. Near the base of the insert (600) there is arranged a first filter element (620) and a second filter (630). The filter elements (620, 630) each basically form the bottoms of the chambers (640, 650). The inner chamber (640) is in fluid connection with the outer chamber (650) across the fil- ter (630). In the bottom region the outer chamber (650) is in fluid connection with the outside via the first filter (620) for taking up liquid or suspension into the outer chamber (650). The outer chamber (650) further comprises at least one opening or access (652) for applying positive pressure or vacuum or for media exchange. The inner chamber comprises at least one opening or access (642,646) for applying positive pressure or vacuum or for media exchange. The inner chamber (640) further comprises a riser tube (644) for suction of liquid out of the inner chamber (640). In figure 5a the pipette insert (600) is brought in contact with the receiver (150) containing the suspension (100). Figure 5b shows a first partial step 1 wherein a suspension (100) is drawn across the first filter element (620) into the outer chamber (650) by applying a first vacuum at opening (652). The suspension (100) follows a pressure gradient across the first filter (620). Particulate matter (120) comprised within the suspension (100) cannot pass the first filter element (620) and are retained thereon, whereas the particles (110) to be isolated, in particular single cells, and the suspension liquid enter the outer chamber (650) through the first filter (620). Figure 5c shows a second partial step of step 1 , wherein the suspension provided in the outer chamber (650) in the first partial step is drawn into the inner chamber (640) across the second filter element (630) by applying a second vacuum to the opening (642). To prevent leaking of liquid from the outer chamber out of the pipette (600), the first vacuum at opening (652) may be maintained. In a particular embodiment, the second vacuum applied to opening (642) is higher than the first vacuum applied to opening (652). A pressure gradient is established between the outer chamber (650) and the inner chamber (640) and across the second filter element (630). The second filter (630) is specifically designed as to retain the particles to be isolated and to let the liquid part of the suspension (100) pass into the inner chamber (640). A third partial step of the step 1 is depicted in figure 5d. The liquid part of the suspension (100) is drained through the riser tube (644) of the inner chamber leaving the insert (600) free of liquid. For retaining the particles (110) and other particulate components (120) at the filter elements (620, 630), the vacuum applied to openings (652) and/or (642) may be maintained. In step 2 depicted in figure 5e, the particles to be isolated (110) are retained on the second second filter element (630), preferably by maintaining the vacuum at opening (642).

In step 3, also depicted in figure 5e, the first filter element (620) comprising the remaining components (120) attached thereto is removed.

In the following step depicted in figures 5f and 5g, the particles (110) are removed from the second filter element (630) by backflushing the filter by providing a second suspending liquid into the inner cham- ber (640) through another opening or access (646), and optically by support of a positive pressure applied through opening (642). Before back-flushing, the inner chamber (640) may be filled with a preferably defined amount of the suspending liquid through opening (646) while a vacuum is optionally maintained at opening (642) to prevent the second suspending liquid from leaving the inner chamber (640) via the second filter element (630). By that, the particles (110) are flushed from the second filter element (630) and re-suspended in the second suspending liquid yielding a second suspension (200) comprising only the isolated particles (110) in a receiver (150), and pref- erably in a defined amout. In an alternative embodiment, the receiver (150) is replaced by a pipette tip (500) as depicted in figures 2 and 3, and the second suspension (200) may be processed further as depicted in figures 2 and 3.

Claims

1. Process for isolating particles suspended in a first suspension from said suspension, wherein the first suspension optionally further contains suspended larger components, comprising the sequence of steps of:

(1) separating off the particles from the first suspension with at least two filtering means, the first filter being suitable for retaining the further components and for filtering (letting pass) the particles to be isolated from the first suspension and the second filter being suitable for retaining the particles to be isolated, whereby the fur- ther components are retained on said first filter and the particles to be isolated are retained on said second filter;

(2) securing the retained particles on the second filter; and

(3) removing the first filter with said further components of the first suspension bound thereto.

2. Process according to claim 1 with the subsequent further steps:

(4) resuspending the particles secured to the second filter in a defined first volume Vi of a second suspending liquid to obtain a second suspension of the particles; (5) determining the total number N of particles in said second suspension or the number of particles N_τ in a partial volume V_τ thereof;

(6) separating off the particles to be isolated from said second suspension by again retaining the particles on said second filter; and

(7) resuspending the particles re-retained on said second filter in a second volume V₂ of a third suspending liquid to obtain a third suspension of the particles, said second volume V₂ of the third suspending liquid being determined as a function of the number of particles N determined in step 5, and adjusting a predetermined concentration K of the particles in the third suspension being set therefrom.

3. Process according to claim 2, wherein the volume V₂ is calcu- lated according to:

K <1)

4. Process according to one of the preceding claims, wherein in step (1) the suspending liquid of the first suspension is filtered through the at least two filters by applying a pressure gradient.

5. Process according to one of the preceding claims, wherein first and second filters are arranged in series in the direction of suction.

6. Process according to one of the preceding claims, wherein step (1) is performed in subsequent partial steps: (1a) separating off the further components from the first suspension by suction of the first suspension through the first filter into a first filter chamber formed in between the first and the second filter, whereby the further components are retained on said first filter and the particles to be isolated are filtered (led pass) through the first filter, and retaining the first suspension containing the particles to be isolated in said first filter chamber, and

(1b) separating off the particles from the first suspension by suction of the first suspension from said first filter chamber through the second filter, whereby the particles to be isolated are retained on said second filter.

7. Process according to one of the preceding claims, wherein in step (2) the particles to be isolated are secured to the second filter by applying a pressure gradient across said filter.

8. Process according to one of claims 2 to 7, wherein in step (4) and/or (7) the particles are resuspended by backflushing the second filter with the second suspending liquid into an enclosed volume for receiving the second suspension.

9. Process according to claim 8, wherein the enclosed volume is a pipette tip.

10. Process according to one of claims 2 to 9, wherein step (5) is performed in subsequent partial steps:

(5a) transferring a partial volume V_τ of the second suspension to a counting means and (5b) counting the number N_τ of particles in the partial volume V_T of the second suspension, the total number of particles N being calculated from

11. Process according to one of claims 2 to 10, wherein in step (6) the second suspending liquid of the second suspension is filtered through the second filter by applying a pressure gradient.

12. Process according to one of the preceding claims, wherein the pore size of the second filter is 10 μm or less.

13. Process according to one of the preceding claims, wherein the pore size of the first filter is 20 μm or more.

14. Process for preparing a particle suspension having a predetermined particle concentration, wherein a first suspension of unknown particle concentration with the particles suspended therein is provided and steps (1) to (7) of the process according to one of claims 2 to 13 are carried out and a particle suspension of predetermined particle concentration is obtained.