WO2010130310A1

WO2010130310A1 - Pipette head with filter and flushing means

Info

Publication number: WO2010130310A1
Application number: PCT/EP2010/001248
Authority: WO
Inventors: Frank Fretzsch; 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: 2010-03-01
Publication date: 2010-11-18
Also published as: WO2010130310A8

Abstract

Pipette head (1; 86) having, for receiving a suspension by suction, at least one suction channel (3; 87) which can be connected to a suction means (43) and opens into a pipette opening (7; 88), wherein in the direction of suction flow before the pipette opening (7;88) at least one filter module (23, 29; 57) covering at least the pipette opening (7; 88) with an end side (27; 62) is detachably held in such a way that the suspension is passed during suction through a filter element (26, 32; 70) of the filter module (23, 29; 57), the pipette head (1; 86) having at least one flushing channel (14; 90) which can be connected to at least one flushing means (44) and the free end of which is associated with the end side (27; 57) of the filter module (23) in such a way that liquid and/or gaseous flushing medium conveyed through the flushing channel (14; 90) by means of the flushing means (44) flows through the filter element (26, 32; 70) of the filter module (23, 29; 57) counter to the direction of suction flow. Furthermore, the invention relates to a filter module (57), an automated pipetting device, the use of a pipette head (1; 86) and a method for operating a pipette head.

Description

PIPETTE HEAD WITH FILTER AND FLUSHING MEANS

Description

The invention relates to a pipette head having, for receiving a suspension by suction, at least one suction channel which can be connected to a suction means and opens into a pipette opening.

The invention also relates to a filter module for a pipette head.

The invention further relates to an automated pipetting device with at least one movable pipette head.

The invention further relates to a method for operating a pipette head as described above.

In the technical field of tissue engineering and in particular in relation to regenerative medicine, there is a need to automate biological laboratory processes in a GMP-compliant manner under clean room conditions. 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 convenient automated handling operations. A particular challenge presented to the automation is in this case the problem of separating individual cells from a gen- erally liquid suspension comprising various components in order to be able to subsequently cultivate these cells. In manual processes, it is known to detach the cells from the suspension by centrifuging the suspension in centrifuge tubes. For the separation of disruptive or desired tissue remnants from the cell suspension, it is known to use cell screens which have different pore densities, of for example 40 m, 70 m or 100 m, and are attached to the centrifuge tubes. However, these processes are difficult to handle and harbour a high risk of contamination. The suspension is drawn up using a pipette and pipetted into the centrifuge tube through the screen. In doing so disruptive or desired tissue remnants from the suspension remain in the screen (retention). However, this could reduce the amount of tissue obtained if the tissue material adheres to the pipette. This also in- creases the risk of clogging of the pipette.

Furthermore, for automating this process, it is known to use pipetting devices having at least one movable pipette head. A pipette head of this type has a suction channel, one end of which is connected to a suction means and the other end of which opens into a pipette open- ing of the pipette. A reduced pressure can be generated in the suction channel by means of the suction means, and as a result the suspension can be drawn in or conveyed through the pipette opening into the pipette head or into the suction channel. The pipette head is in this case moved automatically, so that a plurality of pipetting proc- esses can be carried out in a short time. However, the known pipette heads have a plurality of drawbacks in relation to contaminations. As in laboratory processes carried out manually, a large number of disposable vessels and disposable instruments are used in order to reduce the risk of contamination, although this increases the process costs, reduces process safety while ultimately still not allowing a risk of contamination to be ruled out.

Further steps to be carried out during tissue preparation or suspension preparation consist in producing individual cell suspensions, in detaching cells from tissue, in metering liquids, in resuspending cells, in transporting cells and also in setting cell concentrations (number of cells per volume fraction) in suspensions in order to seed a defined number of cells for cultivation.

The invention is thus based on the object of providing a pipette head which can be used in a simple manner to carry out the above- mentioned steps and reduces the risk of contamination.

The object underlying the invention is fully achieved by a pipette head such as is presented in claim 1. According to the invention, the pipette head has, for receiving a suspension by suction, at least one suction channel which can be connected to a suction means and opens into a pipette opening, wherein in the direction of suction flow before the pipette opening at least one filter module covering at least the pipette opening with its end side is detachably held in such a way that the suspension is passed during suction through at least one filter element of the filter module, and wherein the pipette head has at least one flushing channel which can be connected to at least one flushing means and the free end of which is associated with the end side of the filter module in such a way that liquid and/or gaseous flushing medium conveyed through the flushing channel by means of the flushing means flows through the filter element counter to the direction of suction flow. The pipette head therefore has in the first place a filter module which is arranged in the direction of suction flow before the pipette opening and covers with its end side at least the pipette opening. The filter module also has a filter element which is embodied and/or arranged on the filter module in such a way that when the filter module is in the above-described position before the pipette opening, the suspension to be taken up through the pipette head is passed or flows through the filter element. In other words, the suspension is drawn through the filter element of the filter mod- ule during taking-up or drawing-in. Thus, the suspension is already filtered during suction or taking-up, so that specific components of the suspension are retained by the filter element (retention). As the filter module is still located before the pipette opening, the filter mod- ule is the frontmost part of the pipette head. An appropriate embodiment of the pipette head and/or of the filter module allows in this case only the filter module to be dipped into the suspension, thus preventing contamination of the pipette head when the suspension is drawn in. If the pipette head is moved with the filter module out of the suspension and the reduced pressure is maintained in the suction channel, then the retained components of the suspension remain held on the filter element of the filter module and can be transported by moving the pipette head. In addition to the suction channel, the pipette head has at least one flushing channel which can be con- nected to a flushing means, the flushing means being suitable for conveying liquid and/or gaseous flushing medium through the flushing channel. For this purpose, one end of the flushing channel can be connected to the flushing means and the other, free end of the flushing channel is associated with the end side with which the filter module covers the pipette opening. Furthermore, the free end of the flushing channel is associated with the filter module in such a way that the flushing medium conveyed by means of the flushing means flows through the filter element counter to the direction of suction flow. By activating the flushing means or by conveying flushing me- dium, the filter element and if appropriate also the filter module can thus be flushed-through counter to the direction of suction, i.e. back- flushed. The fact that the filter element is flowed-through counter to the direction of suction flow, on the one hand, allows the components of the suspension that are located therein and retained by the filter element to be detached from the filter element and dispensed in a metered manner and, on the other hand, allows the pipette head to be cleansed so as to prevent contamination.

By means of the pipette head, it is thus possible to take up a suspension, to retain components contained therein, to transport said components by moving the pipette head and to subsequently dispense them in a metered manner at a desired location by flushing- out. Compared to the processes known in the art, in which the suspension taken up through the pipette is added dropwise to a filter attachment of a centrifuge tube (falcon tube) and filtered therein by means of gravity or centrifugation, in the present case the suspension is filtered as a result of the negative pressure or as a result of the drawing-in and can at the same time be transported through the same apparatus (the pipette head) and also resuspended. This offers a particularly simple and efficient possibility for automating the above-described method to be carried out.

Preferably, the flushing channel is embodied coaxially with the suction channel. The coaxial embodiment leads to the fact that the outlet opening of the flushing channel is positioned coaxially with the inlet opening of the suction channel and in particular surrounds said inlet opening all the way round. This ensures that the filter element is flushed particularly efficiently at the location at which components of the suspension are held during drawing-in. The coaxial embodiment ensures, in particular, that the flushing medium flows uniformly through the filter element. Particularly preferably, means for ho- mogenising the through-flow via the filter element are provided between the filter and the free end of the flushing channel, so that the flushing medium flows through the filter element, viewed over its entire cross section, as uniformly as possible. This ensures that all of the retained components are detached from the filter element. The filter element can thus be flushed-through/cleansed and subsequently reused, wherein the flushing process also allows disinfection of the filter element to be carried out.

Particularly, the flushing channel surrounds the suction channel in a casing-like manner. This refers to an embodiment of the flushing channel in such a way that the outside of the suction channel forms the inside of the flushing channel. This results in a particularly compact design of the pipette head.

Preferably, the flushing channel is formed substantially by a borehole in the pipette head and a tube element which is inserted into the borehole and forms the suction channel. The casing-shaped, coaxi- ally embodied flushing channel is then formed accordingly by the outside of the tube element and the inside of the borehole. The (dead) volume of the flushing medium that is contained or can be held in the flushing channel can be minimised by appropriately selecting the distance from the inner wall of the borehole to the outer wall of the tube element, i.e. preferably by appropriately selecting the respective diameters. Particularly, the volume is selected so as to be low by way of appropriate configuration, thus allowing the amount of flushing medium consumed to be kept low.

According to a development of the invention, provision is made for the suction channel to end set apart from the end side of the filter module. The end of the suction channel that faces the filter module or the filter element thus does not abut the filter element or the filter module, but is positioned so as to be set (axially) apart therefrom. This allows the flushing medium to be distributed over the entire cross section of the filter element. During the flushing process the suction means is preferably deactivated, so that the flushing medium supplied to the filter element does not escape through the suction channel, instead of flowing through the filter element.

Furthermore, the pipette head preferably comprises at least one switchable metering valve associated with the flushing channel. The amount of flushing medium is metered by means of the metering valve, so that the components of the suspension that are retained on the filter element can be dispensed again in a metered manner. Dead volumes in the supply lines of the flushing means are reduced by providing the metering valves on the pipette head.

According to a development of the invention, the pipette head comprises at least one, preferably a plurality of supply lines of the flushing means that open into the flushing channel and expediently, the respective supply line comprises the switchable metering valve. The different supply lines serve to guide different flushing media of the flushing means. Particularly, a buffer solution is conveyed through one of the supply lines, a sterilising solution is conveyed through another supply line and air is conveyed through a further supply line. The different flushing media can be metered in a simple manner by means of the respective metering valves. Thus, after a drawing-in process, for example, firstly the buffer solution is metered in order to detach the components of the suspension that are retained on the filter element, so that these components can be further processed, and subsequently the filter element or filter module is flushed or cleansed by means of a suitable flushing medium in order to prevent cross-contaminations. Preferably, the supply lines can be interconnected in such a way as to allow the sterilising solution to be conveyed through all of the supply lines.

According to a preferable development of the invention, provision is made for the filter module to be held on the pipette head in a force- fitting and/or form-fitting manner. In particular, force-fitting connection allows simple attachment of the filter module to and simple removal of the filter module from the pipette head. Furthermore, this allows the filter module to be exchanged in a simple manner with low risk of contamination in order to be able to use, for example, different types of filter elements. According to a particular embodiment, the filter module can be attached to the pipette head. For this purpose, the filter module is preferably embodied in such a way as to surround the pipette head at its front region all the way round, so that merely the filter module comes into contact with the suspension when the pipette head is dipped into the suspension.

According to a preferred embodiment, provision is made for the pipette opening to open into a filter module receptacle. The filter module receptacle is preferably embodied as a borehole coaxially with the suction channel or the flushing channel and adjoins said suction channel or flushing channel. The filter module can be inserted into the filter module receptacle, for which purpose said filter module is particularly embodied as a filter insert. The embodiment of the filter module receptacle and the filter insert makes orienting and/or posi- tioning the filter module on the pipette head simple and secure.

Furthermore, provision is made for the filter insert to be embodied in a cup-shaped manner. The filter insert thus has a bottom surface from which a side wall extending over the entire circumference juts out substantially perpendicularly. Particularly, the filter element is arranged in an aperture in the bottom of the cup-shaped filter insert, the underside of the bottom forming the end side of the filter module that is associated with the flushing channel and the suction channel. Preferably, the filter insert abuts the filter module receptacle with its side walls in a sealing manner. As the suspension is drawn into the pipette head through the filter module, the sealing abutment prevents contamination of the filter module receptacle by the suspension. Of course, an oblique side wall can also be provided, the filter module receptacle then preferably also having correspondingly obliquely embodied side walls.

Particularly preferably, the side wall of the cup-shaped filter insert protrudes from the pipette head to form a pipette tip to be dipped into the suspension. The filter insert thus forms with its side wall the tip of the pipette head that is to be dipped into the suspension.

Advantageously, the filter element is embodied for retaining biological material. Advantageously, the filter element is embodied as a coarse filter for tissue components or as a fine filter for individual cells.

According to a preferred development, provision is made for a further filter module, which can be embodied like the above-described filter module, to be detachably held before the filter insert in the direction of suction flow. This provides two filter modules which are connected in series or serially. On the one hand, this allows the overall filter effect to be increased. On the other hand, it is possible to provide dif- ferent filter elements in order to carry out step-by-step filtering or step-by-step filtration.

Furthermore, it is advantageous if the filter element positioned in front in the direction of suction flow is embodied as a coarse filter, in particular for tissue components, and the filter element positioned thereafter in the direction of suction flow is embodied as a fine filter, in particular for individual cells. It is thus possible to filter tissue components and, subsequently or at the same time, individual cells from the suspension in a single working step, namely the drawing-in of the suspension by means of the pipette head according to the invention. Subsequently, it is conceivable firstly to detach the filter module with the tissue filter, i.e. the filter module positioned in front in the direction of suction flow, and subsequently to backflush or to resuspend in a metered manner by means of the flushing means and the metering valves the individual cells held on the fine filter.

Preferably, the filter module receptacle is embodied in a stepped manner, a cup-shaped filter insert being disposed at least partially in each step respectively. The filter insert positioned at the front then protrudes with its side walls from the pipette head, while the internal filter insert abuts the other filter insert with the end side of its side walls. Alternatively, it is conceivable for the filter module positioned in front in the direction of suction flow to be detachably held in a force- fitting and/or form-fitting manner not on the basic element of the pi- pette head, but on the filter insert arranged in the filter module receptacle. Thus, the filter module positioned in front in the direction of suction can, for example, be attached to the filter insert positioned at the back. Contamination of the pipette head itself is effectively prevented in both cases. Particularly, the filter modules abut one an- other in the direction of flow in a sealing manner, so that the filter module receptacle as a whole can no longer come into contact with the suspension. Preferably, each filter insert abuts on the filter module receptacle with its respective side wall in a sealing manner. In doing so, a contamination of the pipette opening and the pipette head itself can be prevented.

Furthermore, provision is made for a pipetting tip to be detachably held in the filter module receptacle and/or on one of the filter modules. The pipetting tip is characterized by a pipetting opening, the dimensions of which are such that a suspension located in the pipet- ting tip in the pressureless state is kept counter to gravity. Dropwise dispensing takes place preferably only after application of a metered pressure in the interior of the pipette head by means of a flushing medium of the flushing means. Thus, for example, after the coarse filter has been discarded, the pipetting tip, which can be embodied as a pipetting tip insert or alternatively as a pipetting tip attachment, can for example be accordingly fastened to the pipette head in a force-fitting and/or form-fitting manner in order subsequently to be able to dispense in a metered and targeted manner the filtered sus- pension or the individual cells/components of the suspension that are retained on the fine filter.

In another embodiment, the filter module or filter insert preferably comprises at least two filter chambers which are fluidically connected in series. In contrast to the above-described embodiment with at least two individual filter modules each comprising a filter element now one filter module is formed in such a way that it forms at least two filter chambers. The at least two filter chambers are fluidically connected such that suspension can be conveyed or transferred from one filter chamber to the other. This serial arrangement of the filter chambers requires for the suspension to be drawn into the second filter chamber that the suspension is drawn into the first filter chamber first. By providing the at least two filter chambers in the filter module or filter insert a further filter module or filter insert is not required.

Preferably, a first filter chamber of the filter chambers is separated from a second filter chamber of the filter chambers by the aforementioned filter element, the second filter chamber being arranged in the direction of suction flow behind the first filter chamber. The filter chambers of the filter module are thus separated by the filter ele- ment, whereby the suspension can be transferred through the filter element.

For providing the first filter chamber a pre-filter element is arranged on a front side of the filter module opposite to the end side of the filter module preferably in an overlapping manner. The pre-filter element is preferably arranged opposite to where the suction channel and the flushing channel communicate with the filter module. For further reducing the risk of contamination, the filter element is preferably arranged on the filter module in an overlapping manner such that the filter element forms the front side of the filter module. Due to this arrangement, the first filter chamber is provided between the pre- filter element and the filter element that is preferably arranged inside of the filter module. The second filter chamber is arranged between the filter element and the end side of the filter module which is as- signed to the suction channel. This filter module is preferably designed as a filter insert in a cup-shaped manner as described above. In contrast to the above described first embodiment, the filter element is arranged spaced apart from the bottom so as to form the second filter chamber between the filter element and the bottom of the filter module/insert.

Preferably, the suction channel and the flushing channel are assigned to the second filter chamber so that suspension can be drawn through the filter element and so that the filter element can be back- flushed with the flushing medium. Particular preferably, the suction channel and/or the flushing channel abut the end side of the filter module or the bottom of the filter insert in a sealing manner, respectively. Preferably, the filter module and/or the filter receptacle comprise means for connecting the flushing channel and/or the suction channel to the filter module in a sealing manner. Preferably, such means are accordingly shaped sealing elements or connectors, which are preferably embodied with the filter module or the filter receptacle in one piece.

Furthermore, a further suction channel which can be connected to a further suction means is preferably assigned to every other filter chamber, respectively. This means that to the first filter chamber and for example a third filter chamber with a further filter element positioned in the direction of suction flow before the first filter chamber a further suction channel is assigned, such that in each filter chamber of the filter module an individual negative pressure can be produced by the suction means. Preferably, each suction channel can be connected to an individual suction means. The suction means are designed so as to produce an adjustable negative pressure in the respective filter chamber of the filter module, so that in each filter chamber a different (negative) pressure can be produced.

Preferably, the filter chambers are arranged/designed such that they are at least essentially concentric. The concentric design allows the filter chambers, which are fluidically connected in series, to be located essentially parallel or side by side. In this way the filter module can be designed rather small or in particular rather short in the axial direction. With the concentric design the filter element between the first and the second filter chamber can be designed as a part of the radial side wall of the first filter chamber, which lies within the second filter chamber. In this case the filter chambers can be designed com- pletely concentric to each other, while featuring the same axial length.

In a preferred embodiment the filter element and the pre-filter element are arranged at least essentially parallel to and spaced apart from each other. In this case the concentric filter chambers are de- signed such that the first filter chamber encloses the second filter chamber on a side and on one front side, whereby the front side of the first filter chamber is preferably provided by the filter element separating the filter chambers from each other. The pre-filter element is arranged parallel to the filter element such that the pre-filter element and the filter element are arranged in the direction of suction flow directly behind each other. The spaced apart arrangement of the filter element and the pre-filter element allows suspension to be conveyed from the first filter chamber through the gap between the filter element and the pre-filter element and through the filter element into the second filter chamber.

Furthermore, the filter element is preferably held on a mounting ring, which is detachably held on the filter module in a force-fitting and/or form-fitting manner. In particular, the mounting ring is preferably held on the free end of the side wall of the second filter chamber in the force-fitting and/or form-fitting manner. In this way the filter element can easily be changed or renewed and in accordance to the suspension to be filtered an adequate filter element can be chosen. Particular preferably, the filter element is designed in on piece with the mounting ring.

Preferably, the filter element and/or the mounting ring comprise multiple protrusion pointing in the direction of the pre-filter element and being distributed over its (filter element and/or mounting ring) circumference. The multiple protrusions act as spacer for guaranteeing a minimum distance between the filter element and the pre-filter element such that the suspension can be drawn from the first filter chamber into the second filter chamber.

In a further development an extraction channel, which can be connected to an extraction means for extraction of suspension from the second filter chamber by suction, is assigned to the second filter chamber. The extraction channel abuts on the end side of the filter module or the bottom of the filter insert preferably in a sealing manner. Preferably, the extraction channel cooperates with an extraction nozzle of the filter module, which extends from the end side of the filter module close to the filter element. The extraction nozzle is preferably designed in one piece with the filter module and arranged concentric to the filter chambers. The closer the extraction nozzle ends to the filter element, the more suspension can be drawn/extracted from the second filter element. In a particular preferably development, the extraction nozzle extends with its free end up to the filter element, whereby the free end comprises one or more recesses in its side wall for assuring the extraction of the whole suspension held in the second filter chamber.

Preferably, in the filter module at least one plunger element is arranged in a movable manner for pushing off the pre-filter element from the filter module. The plunger element is preferably designed as a cylindrical-shaped member, which is arranged in an accordingly cylindrical-shaped receptacle formed in the filter module. The recep- tacle is preferably designed as a through whole, which extends from the end side of the filter module to the front side. Means can be provided for retaining the member within the receptacle even if the pre- filter element is not attached to the filter module. The pipette head preferably further comprises means for moving, in particular for pushing the member against the backside of the pre-filter element. During operation activating the means for pushing the member will cause the member to push the pre-filter element off the filter module.

The advantageous filter module for a pipette head according to the invention comprises at least two filter chambers, which are fluidically connected in series, whereby a first filter chamber (of the filter chambers) is separated from a second filter chamber (of the filter chambers) by a first filter element, and whereby a second filter element is assigned to the first filter chamber in the direction of suction flow be- fore the first filter element, whereby each filter chamber is designed to be connected to a suction means, respectively, for applying individual pressure in each filter chamber, and whereby at least the second filter chamber is designed to be connected to a flushing means. The filter module is preferably made of plastic and particularly pref- erably designed as a disposable part. Particularly, the filter module and the pipette head comprise alignment-means for the adjustment of the filter module in and/or on the pipette head such that the suction channels and the flushing channel, and if applicable the extraction channel, can be brought into connection with correspondent openings in the filter module automatically. Furthermore, the filter module preferably comprises an extraction nozzle, which extends from the end side of the filter module at least close to the exchangeable filter element, as described above. Moreover, the filter module may comprise any other feature which has been described above with respect to the filter module of the pipette head, such for example the movable plunger element and/or the spacers for providing a sufficient space between the filter element and the pre-filter element.

The automated pipetting device according to the invention is distinguished by the embodiment of the pipette head such as has been described above. By means of a pipetting device of this type, it is easily possible to efficiently carry out the above-described process steps in a partly and/or fully automatic manner and without contaminating the pipette head. Preferably, the pipetting device has means for exchanging the filter modules and/or the pipetting tip. Preferably, the filter modules and/or the pipetting tip are embodied as dispos- able components or as exchangeable components. If the filter module or modules and/or the pipetting module is/are detachably held, preferably by means of clamping, in a force-fitting manner on the pipette head and/or on one of the filter modules, exchanging and swapping-over or discarding can be carried out in a particularly simple manner. Of course, it is also conceivable to provide the pipetting device with a plurality of pipette heads of this type in order to achieve a higher yield and productivity. Particularly, the pipetting device furthermore has a suction means which is directly or indirectly con- nected to the suction channel and a flushing means which is directly or indirectly connected to the flushing channel. Preferably, the pipetting device furthermore has a further suction means, which is directly or indirectly connected to the further suction channel, respectively.

Preferably, the pipetting device further comprises an extraction means connected to the extraction channel for extracting suspension from the filter module by suction. Furthermore, the pipetting device particularly comprises pushing means for moving or pushing the plunger element movable held in the filter module against the filter element.

The pipette head according to the invention is preferably used for isolating components of a suspension that are suspended in the suspension, for transporting the isolated components and/or for dispensing the isolated components in a metered manner.

The method for operating a pipette head according to the invention is described below: Firstly, a first negative pressure is applied to the first filter chamber by the further suction means for drawing a suspension into the first filter chamber by passing through the pre-filter element. The first negative pressure can be applied before or after the pipette head is dipped at least partially into the suspension with its pipette tip provided by the filter module. Secondly, a second negative pressure is applied to the second filter chamber by the suction means, whereby the second negative pressure is lower than the first negative pressure such that the suspension is drawn from the first filter chamber into the second filter chamber by passing through the filter element. During this operation the first negative pressure is preferably maintained so that components of the suspension retained by the pre-filter element are still held on the pre-filter element. Subsequently, the suspension is extracted from the second filter chamber by use of the extraction suction means while maintaining at least the second negative pressure in the second filter chamber, for retaining the filtered components of the suspension on the filter element. Then the pre-filter element is discharged by use of the plunger element which can be pushed against the filter module by activating the pushing means of the pipetting device. Afterwards, a predetermined amount of flushing medium is discharged into the second filter chamber by the flushing means. The still applied second negative pressure keeps the flushing medium from unintentionally dripping through the filter element and thus from unintentional releasing of components retained by the filter element. Finally, a release pressure higher than the second negative pressure is applied to the second filter chamber for back-flushing the filter element with the predetermined amount of flushing medium, thereby detaching components of the suspension retained by the filter element in a metered manner. By use of the above-described method firstly larger components of the suspension, such as tissue components, are retained by the pre- filter element. Secondly, smaller components such as individual cells of the suspension are retained by the filter element. After pushing off the pre-filter element, the retained individual cells can be re- suspended in a metered manner by use of the flushing medium and the applied pressures at any time. The invention will be described hereinafter in greater detail based on a plurality of exemplary embodiments. In the drawings:

Figure 1 is a longitudinal section through a pipette head according to the invention;

Figure 2 is a plan view of the pipette head;

Figure 3 is a longitudinal sectional illustration of the pipette head with a pipetting tip;

Figure 4 is a longitudinal sectional illustration of the pipette head with an alternative embodiment of the pipet- ting tip;

Figures 5A to I are schematic illustrations of process steps which can be carried out by means of the pipette head;

Figure 6 is a longitudinal section through another embodiment of the filter module for the pipette head in a perspective illustration;

Figure 7 is a top view of the filter module according to the other embodiment;

Figure 8 is a longitudinal section through the filter module of the other embodiment;

Figure 9 is a first longitudinal section through a pipette head with the filter module of the other embodiment;

Figure 10 is a second longitudinal section through the pipette head of figure 9 and Figure 11A to G are schematic illustrations of process steps which can be carried out by means of the pipette head according to the other embodiment.

Figure 1 is a plan view of a movable pipette head 1 of an automated pipetting device 2 (not shown here in greater detail). The pipette head 1 has a suction channel 3 formed in the present case by a tube element 5 inserted into a borehole 4. At one of its ends, the tube element has a widened cross section which forms a receptacle 6 and serves to receive a connector of a suction means (not shown here in greater detail). Its other end opens into a pipette opening 7 through which an, in particular liquid, suspension can be conveyed by suction into the suction channel by means of the suction means, provided that the pipette head 1 is at least partially dipped into the suspension, with its end having the pipette opening 7.

The pipette opening 7, in turn, opens into a filter module receptacle 8. The filter module receptacle 8 is embodied in a stepped manner, the two steps 9 and 10 provided in the present case having cross sections of different size. In this case, the step 9 adjoins the pipette opening 7 and has a larger diameter than the borehole 4. The step 10, which adjoins the step 9, has a larger diameter than the step 9. Overall, the filter module receptacle 8 is or the steps 9 and 10 are arranged/embodied coaxially with the extension of the bore axis of the borehole 4. Advantageously, the filter module receptacle 8 is embodied as a stepped borehole.

Set apart from the pipette opening 7, the tube element 5 has on its outside a radial projection 11 which extends over the entire circumference and in which a ring seal 12 is disposed, which is embodied in particular as an O-ring, so that the ring seal is braced between the tube element 5 and the inside of the borehole 4 so as to produce a seal. Moreover, the external diameter of the tube element 5 is embodied so as to be less than the internal diameter of the borehole 4 in such a way as to form a casing-shaped free space 13 between the ring seal 12 and the end of the tube element 5 that is associated with the pipette opening 7. The free space 13 forms a flushing channel 14 which likewise opens into the pipette opening 7. The flushing channel 14 is thus embodied coaxially with the suction channel 3. A supply line 15, which extends substantially perpendicularly to the flushing channel 14 and the suction channel 3, opens into the flushing channel 14 below the ring seal 12 of the tube element 5. A switchable metering valve 16, which can be used to completely or partly clear or close the cross section of the supply line 15, is associated with the supply line 15. A further supply line 17, with which a further metering valve 18 is associated, opens into the supply line 15. The supply line 17 also opens into the flushing channel 14 via the supply line 15.

As may be seen from Figure 2. which is a plan view onto the pipette head 1 , the pipette head 1 has further metering valves 19, 20 and 21 which are each associated with a supply line opening into the flush- ing channel. The respective supply lines are connected by their free ends to a flushing means (not shown here in greater detail) which conveys different liquid and/or gaseous flushing media through the supply lines. The flushing media can be metered into the flushing channel 14 and fed out from the pipette opening 7 by means of the metering valves 16, 18 - 21 arranged on the pipette head 1. In particular, at least sterilising liquid, buffer solution and (sterilising) air are provided as flushing media. Dead volumes, in particular, are reduced as a result of the provision of the metering valves on the pipette head 1 or on the basic element thereof, so that the amount of flushing medium consumed can be kept low. Furthermore, according to Figure 1. a filter module 23, which is embodied as a filter insert 22, is inserted into the step 9 of the filter module receptacle 8. The filter insert 22 is embodied in a substantially cup-shaped manner and has for this purpose a bottom 24 and also a side wall 25 which protrudes substantially perpendicularly from the bottom and extends over the entire circumference. The bottom 24 has in this case an aperture in which a filter element 26 is enclosed in a flush manner. The filter element 26 is expediently embodied for retaining biological material. The bottom 24 forms with its free (under)side an end side 27 of the filter module 23. The filter module 23 or the filter insert 22 is arranged in the filter module receptacle 8 in such a way that the end side 27 is associated with and covers the pipette opening 7. With its side wall 25 and its bottom 24, the filter insert 22 or the filter module 23 is enclosed in the filter module re- ceptacle 8 so as to produce a seal, so that suspension cannot reach the inside of the step 9 between the filter module receptacle 8 and the filter insert 22.

A further filter module 29, which is embodied as a filter insert 28, is inserted into the step 10 of the filter module receptacle 8. The filter insert 28 is also embodied in a cup-shaped manner and has for this purpose a bottom 30 and also a corresponding side wall 31. A filter element 32 for retaining biological material is also arranged in an aperture in the bottom 30. While the diameter of the filter element 26 corresponds substantially to the diameter of the borehole 4, the di- ameter of the filter element 32 corresponds substantially to the internal diameter of the cup-shaped filter insert 22. In this case, the filter element 26 is oriented/arranged aligned with the pipette opening 7 and the filter element 32 is oriented/arranged aligned with the filter insert 22. The filter inserts 22 and 28 abut each other (axially) in the direction of flow in a sealing manner. For this purpose, the side wall 25 of the filter insert 22 advantageously protrudes to a certain depth into the step 10 of the filter module receptacle 8, so that the filter insert 28 can abut with its end side 33 the end face of the side wall 25 so as to produce a seal. With its side wall 31 , the filter insert 28 is also enclosed in the filter module receptacle 8 so as to produce a seal. The side wall 31 protrudes in this case well clear of the filter module receptacle 8 or the pipette head 1 and thus forms a pipette tip 34 of the pipette head 1 that serves for dipping into the suspension. By controlling in a targeted manner the depth of dipping into the suspension, it is also possible to separate floating tissue layers and cells from ones which have sunk.

In relation to the cultivation and preparation of biological cells, the filter element 32, which is positioned in front in the direction of suction flow, for retaining cell agglomerates and tissue components has a pore size of 20 μm and more, preferably of from 20 μm to 100 μm. Particularly preferably, the pore size is, for the filtration of cell aggregates, 20 to 60 μm, preferably 20 to 40 μm and alternatively preferably 40 to 60 μm. Preferred for the filtration of tissue components is a pore size of from 80 to 100 μm, preferably of approximately 80 μm, and alternatively preferably of approximately 100 μm.

Preferably, the filter element 26 of the filter module 23 that is positioned at the rear in the direction of suction flow has, for the filtration of individual cells/particles to be isolated from the suspension, a pore size of 10 μm or less, preferably 8 μm or less, particularly preferably of 6 μm or less or 4 μm or less. The pore size of the filter element 26 is preferably 2 to 8 μm, particularly preferably 2 to 6 μm or 2 to 4 μm, alternatively preferably 4 to 8 μm. The filter elements 26 and 32 are embodied in a manner known per se. Preferably, filter nets or filter cloths or meshes are provided as the filter elements 26, 32. Pre- ferred materials are in this case PTFE, PET, glass, ceramic, cellulose, polyethylene, polypropylene, polystyrene, polyurethanes, poly- amide or composites thereof. PET and/or PTFE membranes are particularly preferred. Combinations of pore sizes and/or materials are also conceivable.

The end of the tube element 5 that is associated with the end side 27 of the filter module 23 ends set apart from the end side 27, so that the suction channel 3 also ends set apart from the end side 27 or from the filter module 23. This produces a (small) intermediate space between the bottom 24 or the filter element 26 of the filter module 23 and the tube element 5, which intermediate space can contain both drawn-in suspension and suspension filtered by means of the filter modules 23 and 29 as well as flushing medium conveyed into the flushing channel 14 by means of the flushing means.

As a result of the embodiment of the advantageous pipette head 1 , the suspension is drawn in during a suction process into the suction channel 3 so as to be conveyed firstly through the filter module 29 and subsequently through the filter module 23, as denoted by an arrow 35 indicating the direction of suction flow. The suction means is in this case embodied in such a way that the drawn-in suspension, which is filtered by means of the filter elements 32 and 26, is ultimately removed or discarded. Furthermore, the suction means of the pipetting device 2 is embodied in such a way that components of the suspension that are retained on the filter elements 32 and 26 can be held on the filter elements 26 or 32 even without the liquid of the suspension. The retained components can thus be transported by means of the pipette head 1.

The filter module receptacle 8 or the steps 9 and 10 and the filter inserts 22 and 28 are advantageously embodied in such a way that the filter inserts 22 and 28 are held in a force-fitting manner in the filter module receptacle 8. This allows simple detaching or exchanging of the filter inserts 22, 28. The pipetting device 2 preferably comprises for this purpose a type of magazine (not shown here) in which different filter inserts are kept in stock. Particularly, for exchanging the filter inserts, the pipette head 1 is moved toward the magazine, where the exchanging process can take place in an automated manner.

It is also possible to provide, in addition to filter modules or filter in- serts, pipetting tips in the form of pipetting inserts which can be used, for example, instead of the filter module 28. Figure 3 shows for this purpose the pipette head 1 of the pipetting device 2, which pipette head corresponds substantially to the pipette head 1 from Figure 1 , wherein like elements are provided with the same reference numer- als and reference is to this extent made to the foregoing description. However, instead of the filter module 29 or the filter insert 28, a pipetting tip 36 in the form of a pipetting insert 37 is provided in the step 10 of the filter module receptacle 8. The pipetting insert 37 is embodied in a substantially funnel-shaped or Y-shaped manner and has a tip 38 which points downward or away from the pipette head 1 and has a pipetting opening 39. The tip 38 protrudes in this case from the pipette head 1 and thus forms the pipette tip 34 of the pipette head 1. The dimensions of the pipetting opening 39 are such that the suspension located in the pipetting tip 36 in the pressureless state is kept counter to gravity in the pipetting tip 36. At its side facing the pipette head 1 , the pipetting insert 37 has an internal diameter corresponding substantially to the internal diameter of the filter insert 22. In addition, the pipetting insert 37 has, at the side facing the pipette head 1 or the filter module receptacle 8, a collar 40 which bridges the spacing of the funnel-shaped pipetting tip 36 to the inside of the step 10 of the filter module receptacle 8, so that the pipetting insert 37, like previously the filter insert 28, abuts in a force-fitting manner and so as to produce a seal in the filter module receptacle 8 or the step 10 and also at the end side of the side wall 25 of the filter insert 22. The collar 40 is in this case embodied in such a way that its free end is flush with the pipette head 1. The provision of the collar allows the pipetting tip 36 or the pipetting insert 37 to be produced with a uniform wall thickness, as illustrated. This offers advantages, in particular in relation to the production of the pipetting tip 36.

Figure 4 shows a further conceivable embodiment of the pipette head 1 and in particular the pipetting tip 36. The pipette head 1 shown in Figure 4 corresponds to the pipette head 1 known from Figure 1 , so that like elements are provided with the same reference numerals and reference is to this extent made to the foregoing de- scription. In contrast to the foregoing exemplary embodiment with the pipetting tip 36 embodied as a pipetting insert 37, the pipetting tip 36 is, according to the exemplary embodiment of Figure 4, embodied as a pipetting attachment 41. For this purpose, the pipetting tip 36 has at its side facing the pipette head 1 a receptacle 42 for receiving the filter insert 28 protruding from the pipette head 1. The receptacle 42 has for this purpose a diameter which corresponds substantially to the external diameter of the side wall 31 of the filter insert 28 and is in particular embodied so as to be somewhat smaller, so that the pipetting tip 36, on the one hand, abuts the filter insert 28 so as to produce a seal and, on the other hand, is detachably held thereon in a force-fitting manner. If appropriate, the receptacle 42 and/or the filter insert 28 can be configured in such a way that the side wall 31 also abuts with its free end side the pipetting tip 36 so as to produce a seal. Of course, it is also conceivable to configure the pipetting attachment 41 in such a way that it can, additionally or alternatively, be slid onto or attached to the pipette head 1. In the present exemplary embodiment, this would allow the provision of the filter insert 28 to be dis- pensed with. Of course, the filter modules 23 and/or 29 can also be embodied as filter attachments. In any case, the embodiment of the pipette head 1 ensures that, during the receiving of suspension by suction, the suspension does not come into contact with the pipette head at least in the region of the filter module receptacle 8, so that contamination of the pipette head 1 and in particular cross- contamination is prevented.

In order to improve the understanding of the invention, the mode of operation of the pipetting device 2 will be described hereinafter in greater detail based on the process steps illustrated in Figures 5A to 5I. Figures 5A to 5I show for this purpose in a greatly simplified illustration the pipetting device 2, with the pipette head 1 , and also with a suction means 43 connected to the suction channel and a flushing means 44 connected to the metering valves 16, 18 - 21 (not shown here). Of the filter inserts 22 and 28, merely the filter elements 26 and 32 are shown.

Figure 5A: In step 1 a reduced pressure is generated in the suction channel 3 by means of the suction means 43, as a result of which a suspension 46, which is located in a receiver 45 and comprises particles 47 to be isolated, such as for example individual cells, and also further, larger components, such as for example tissue components 48, is drawn into the suction channel 3. For this purpose, the pipette head 1 is dipped with the protruding portion of the filter insert 28, i.e. with the pipette tip 34, into the suspension 46. During the suction process the suspension 46 flows through firstly the filter element 32 and subsequently the filter element 26 positioned downstream, the suspending liquid of the suspension 46 being filtered through both filter elements 32, 26 and if appropriate preferably discarded via the suction channel 3. The coarse filter element 32 retains the tissue parts 48 and lets the particles 47 through. The filter element 26, on the other hand, retains the particles 47.

Figure 5B: In step 2 the reduced pressure continues to be maintained and the pipette head is removed from the receiver 45. As a result of the reduced pressure, the particles 47 continue to be held on the filter element 26 and the tissue parts 48 continue to be held on the filter element 32. The pipette head 1 corresponds in this case to the pipette head 1 shown in Figure 1 , so that during the suction process the suspension comes into contact only with the filter modules 23 and 29 and also with the interior of the suction channel 3. If, as indicated in step 2, the filter module 29 or the filter insert 28 is removed in an automated manner from the pipette head 1 , although the step 10 of the filter module receptacle 8 is exposed, the advantageous embodiment has prevented it from becoming contaminated by the suspension. Subsequently, the pipetting tip 36, be it as the pipetting insert 37 or as the pipetting attachment 41 , can, as also indicated in Figure 5B, be fastened, as described above, to the pipette head 1.

Figure 5C: Subsequently, in step 3 a pressure is built up in the interior of the pipette head 1 by means of the flushing means 44, pref- erably by actuating the metering valve which clears the flow cross section of a supply line for a buffer solution or for a second suspending liquid. This allows the particles 47, which are retained on the filter element 26 in isolation from the remaining components of the suspension, to be backflushed or detached, so that the particles 47 are resuspended in the second suspension 49. The second suspension 49 is subsequently located in the volume surrounded by the pipetting tip 36.

Figure 5D: In an advantageous further step 4 an excess pressure is generated by applying an excess pressure by means of the flushing means 44, in particular by actuating the metering valve for air, as a result of which a specific partial volume of the second suspension 49 is transferred to a counting unit 50. The number of particles 47 contained in the partial volume is determined in the counting unit 50.

Figure 5E: In an advantageous further step 5 the second suspension 49 is drawn in through the filter element 26 by generating a negative pressure by means of the suction means 43 and discharged via the suction channel 3. The particles 47 are in this case re-retained on the filter element 26.

Figure 5F: In step 6, which is preferably additionally carried out, the particles 47 retained on the filter element 26 are backflushed or re- suspended by adding a third suspending liquid via the flushing channel 14 by means of the flushing means 44 and the metering valves (not shown here). The third suspension 51 obtained in this way is subsequently located in the volume surrounded by the pipetting tip 36. 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 49.

Figure 5G: In a further advantageous step the third suspension 51 located in the pipetting tip 36 and having a defined cell concentration is dispensed, by backflushing according to Figure 5H by means of the flushing means 44 and the flushing channel 14, into a receiver vessel 52 and subsequently taken up again into the pipetting tip 36, as shown in Figure 51. by generating a reduced pressure by means of the suction means 43. The through-flow through the pipetting opening 39 generates in this case turbulences, 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 tip 36.

Figures 6 to 10 relate to another embodiment of the filter module according to the present invention. In this embodiment a filter module 57 is provided, which comprises two filter chambers 58 and 59, which are fluidically connected in series as best shown in figures 6 and 8. The filter module 57 is embodied as filter insert 60 and is shaped in an essentially cylindrical manner. In the present embodiment of the filter module 57 the filter chambers 58 and 59 are designed/arranged essentially concentric to each other. For this the filter module 57 is formed in a cup-shaped manner with a bottom 61 forming an end side 62, which mainly corresponds to the end side 27 of the filter module 23 and with an outer side wall 63 extending perpendicular from the bottom and with an inner side wall 64 extending perpendicular from the bottom 61 at a smaller diameter than the outer side wall 63. The outer side wall 63 is larger (in the axial direction) than the inner side wall 64. On the free end 65 of the side wall 63 a pre-filter element 66 is arranged in a force-fitting manner. For this the pre-filter element 66 comprises a ring like axial protrusion 67, which extends over the circumference of the pre-filter element 66. The free end 65 of the side wall 63 features a recess 68 such that close to the free end 65 the outer side wall 63 has a smaller outer diameter than the main part of the side wall 63. The recess 68 and the protrusion 67 are formed in such a way that the pre-filter element 66 can be put on to the side wall 63 of the filter module 57 in a force- fitting manner, whereby the pre-filter element 66 overlaps the whole front side 69 opposite to the end side 62 of the filter module 57.

In a similar manner a filter element 70 is attached to the free end 71 of the inner side wall 64. For a better overview the filter element 70 itself is not shown in figure 6. The filter element 70 is held on a mounting ring 72, which comprises a protrusion 73, which extends over the circumference of the mounting ring 72. The protrusion 73 is designed to cooperate with a recess 74 on the free end 71 of the side wall 64 such that the mounting ring 72 is held on the side wall 64 in a force-fitting manner.

The mounting ring 72 further comprises multiple protrusions 74, which extend axially (downward) in the direction of the pre-filter element 66. The protrusions 74 are spaced apart in the circumferential direction such that between adjacent protrusions 74 recesses 75 are provided. As shown in figure 6 the length of the side wall 64 and the thickness of the mounting ring 72 with its protrusions 74 is chosen such that the end face of the protrusions 74 rest on the backside of the filter element 66, thereby acting as spacers 76. The spacers 76 ensure that at any time a distance between the filter element 70 and the pre-filter element 66 is provided.

Hence, the filter element 70 separates the first filter chamber 58 from the second (inner) filter chamber 59 such that the filter chambers 58 and 59 are fluidically connected in series through the filter element 70. In an alternative embodiment the filter element 70 could be ar- ranged as part of the side wall 64 while the free end 71 of the side wall 64 or the second filter chamber 59 is closed by an accordingly designed lid. The pre-filter element 66 is preferably embodied like the filter element 32 and the filter element 70 is preferably embodied like the filter element 26, in particular with respect to the respective pore sizes and materials.

Additionally, the filter module 57 comprises an extraction nozzle 77, which extends from the bottom 61 through the second filter chamber 59 close to the filter element 70. The extraction nozzle as well as the side walls 63 and 64 are embodied in one piece with the bottom 61 of the filter module 57. The extraction nozzle 77 can be connected to an extraction channel of the pipette head as described later on.

In its bottom 61 the filter module 57 comprises various through-holes 78, which can be seen best in the top view shown in figure 7. Two through-holes 79 of the through-holes 78 correspond with the first filter chamber 58. Through-holes 80 of the through-holes 78 which are arranged on a smaller diameter correspond with the second filter chamber 59. The function of the through-hole 78 will be described later on. In addition to the above-described embodiment it is also possible to arrange the through-holes 79 on the side wall 63 of the filter module 57.

The filter module 57 further includes a plunger element 81 designed as a cylindrical member 82. For holding the plunger element 81 or the cylindrical member 82 in a movable manner in the filter module 57 the side wall 63 of the filter module 57 comprises a thickened area, which extends from the bottom 61 to the free end 65 and which includes a cylindrical bore hole 83 arranged in the axial direction. The diameter of the bore hole 83 is chosen such that the cylindrical member 82 is held movable in the bore hole 83 in the axial direction. Usually, the plunger element 81 rests with one end on the back side of the pre-filter element 66 as shown in figures 6 and 8. If needed the plunger element 81 can be pushed towards the pre-filter element 66 for pushing the pre-filter element 66 of the filter module 57 as indi- cated by an arrow 84. In the present embodiment three plunger elements 81 are provided and equally distributed over the circumference of the filter module 57.

Close to its end side 62 the outer diameter of the side wall 63 has a reduced diameter or tapered area 84, which functions as fastening mechanism for fastening the filter module 57 on a pipette head 86 as described below with reference to figures 9 and 10. Figure 9 shows a longitudinal cross section of a pipette head 86 along the line A-A shown in figure 7. Figure 10 shows a longitudinal cross section of the pipette head 86 along the line B-B shown in figure 7.

Figures 9 and 10 show the pipette head 86 which comprises the filter module 57. The pipette head 86 equals in many parts to the pipette head 1 described above so that in the following only differences between the pipette head 86 and the pipette head 1 will be described. The pipette head 86 can be used as part of the automatic pipetting device 2 described before. The pipette head 86 has a suction channel 87, which opens into a pipette opening 88 and corresponds with at least one of the through-holes 80. The pipette opening 88 opens into or is designed as a filter module receptacle 89 for the filter mod- ule 57. The filter module receptacle 89 and the area 85 of the filter module 57 are designed such that the filter module 57 is be held on the pipette head 86 in a force-fitting manner. Due to the rather small area 85 inserting and discharging the filter module 57 requires less force than in the previous embodiment. In contrast to the embodi- ment of the pipette head 1 the suction channel 87 of the pipette head 86 is not arranged in a centric manner. The suction channel 87 is arranged such that it corresponds with at least one of the through- holes 80 of the filter module 57. The suction channel 87 is connected to suction means of the pipetting device 2 (not shown here in greater detail) for applying a negative pressure within the filter chamber 59.

Furthermore, the pipette head 86 comprises a flushing channel 90, which corresponds with at least one other through-hole 80 of the fil- ter module 57. The flushing channel 90 can be connected to a flushing means (not shown here in greater detail) by use of at least one supply line comprising a switchable metering valve as described above with reference to pipette head 1. The supply lines are connected with the flushing means, which conveys liquid and/or gaseous flushing media through the supply lines and the flushing channel into the second filter chamber 59 of the filter module 57.

As shown in Figure 9 the pipette head 86 further comprises an extraction channel 91 , which is arranged in a centric manner and cooperates with the extraction nozzle 77 when the filter module 57 is ar- ranged in the pipette head opening 88. The extraction channel 91 is connected to an extraction means, which is used for extraction of suspension from within the second filter chamber 59 by suction.

The pipette 86 includes at least one further suction channel 92, which corresponds with at least one of the through-holes 79 of the filter module 57 and which can be connected to a further suction means for applying a negative pressure within the filter chamber 58. The suction means and the further suction means can be regulated individually so that different pressures can be applied in the filter chambers 58 and 59.

Figure 10 shows the pipette head 86 in another longitudinal cross section. In this view hydraulic or pneumatic pushing means 93 and 94 of the pipette head 86 can be seen. The pushing means 93 comprises a movable piston 95 the free end of which abuts on the free end of the plunger element 81 of the filter module 57. By activating the pushing means 93, the piston 95 is pushed against the plunger element 81 , which in return is pushed against the pre-filter element 66 or against a pipetting tip 96, which can be held on the filter mod- ule 57 instead of the pre-filter element 66. By pushing the plunger element 81 against the backside of the filter element 66 or the pipetting tip 96, either of them can be pushed off the filter module 57. The pipetting tip 96 is preferably designed like the pipetting tip 36 described above.

The pushing means 94 comprise a movable piston 97, which abuts the end side 62 of the filter module 57. By activating the pushing means 94, the piston 97 is pushed against the end side 62 of the filter module 57 so that the filter module 57 can be automatically pushed off the pipette head 86. Preferably three of the pushing means 94 and three of the pushing means 93 are evenly distributed over the circumference of the filter module.

The pipette 86 further comprises means for applying a positive pressure in the second filter chamber 59. These means can be provided by the suction means described above or by separate pressure means, which can be connected through a pressure channel in the pipette head 86 to the inner, second filter chamber 59. Preferably, the pressure means apply positive pressure to the second filter chamber 59 by conveying or delivering air into the second filter chamber 59.

With reference to figures 11A to 11G the function of the second embodiment shall be explained. Figures 11A to 11G show the filter module 57 in a simplified manner for better understanding. Figure 11 A: In step 1 the filter module 57 of the pipette head 86 is dipped at least partially into the suspension 43, which is located in the receiver 45 and comprises particles 47 to be isolated, as described above.

Figure 11 B: In step 2 a first negative pressure is applied to the first filter chamber 58 by use of the further suction means as indicated by arrows 97, as a result of which the suspension 46 is drawn into the first filter chamber 58 passing through the pre-filter element 66. The pre-filter element 66 is designed for retaining larger components such as tissue components 48. The pre-filter element 66 therefore retains the tissue components 48 and lets the particles 47 and the remaining suspension 46 pass into the first filter chamber 58.

Figure 11 C: In step 3 a second negative pressure is applied to the second filter chamber 58 by use of the suction means as indicated by arrows 98, after the suspension 46 has been fully drawn into the first filter chamber 58. The second negative pressure is chosen to be lower than the first negative pressure such that the suspension 46 is drawn from the first filter chamber 58 through the filter element 70 into the second filter chamber 59. The first and the second negative pressure are chosen such that the drawing/sucking procedure allows the suspension and certain particles to be drawn through the respective filter element 66 or 70 without destroying components retained by or passing through the respective filter element. During step 2 the particles 47 are retained by the filter element 70. Maintaining the first negative pressure in the outer, first filter chamber 58 keeps the suspension from leaking back into the surroundings.

Figure 11 D: Subsequently, in step 4 the suspension 46, which was drawn into the inner, second filter chamber 59 is extracted from the filter chamber 59 by use of the extraction means as indicated by ar- rows 99 through the extraction nozzle 77. Due to the applied second negative pressure, the particles 47 remain on the front side of the filter element 70.

Figure 11 E: In step 5 the pre-filter element 66 is pushed off the filter module 57 by use of the pushing means and the plunger elements 81. In doing so the retained tissue components 48 are removed. The further suction means can now be deactivated since no negative pressure is necessary in the first filter chamber 58 anymore.

Figure 11 F: In step 6 a flushing medium is discharged into the filter chamber 59 as indicated by an arrow 101 in a metered manner while maintaining the second negative pressure within the second filter chamber 59 so that the flushing medium does not pass through the filter element 70.

Figure 11 G: In step 7 a positive pressure is applied to the second filter chamber 59 as indicated by arrows 102 so that the liquid and/or gaseous flushing medium conveyed through the flushing channel 90 fully passes through the filter element 70 of the filter module 57 counter to the direction of suction flow, thereby back-flushing the filter element 70 and resuspending the particles 47 in a metered man- ner into a second suspension 103. Before the positive pressure (102) is applied, the pipetting tip 96 can be arranged on the filter module 57.

It is obvious that additionally one or more steps described with reference to figures 5C to 5I can be executed also by using the pipette head 86 with the filter module 57.

All in all, it is obvious that the second embodiment of the pipette head 86 and the filter module 57 also features a GMP-complying multifunctional pipette, which reduces the risk of contamination and in particular cross contamination when preparing cell solutions or suspensions. In order to preserve and regain sterility of the pipetting device 2 or in particular of the pipette head 86 after a potential con- tamination, a sterilizing solution - as described above - can be conveyed by means of the flushing means and the corresponding metering valves through all the supply lines within the pipette head 86 and also through the filter module receptacle 86. It is clear that two or more of the above-described features of the two explicitly described embodiments can be combined.

The filter module 57 and the filter module receptacle 89 are preferably designed such that the filter module 57 can only be pushed into the filter module receptacle 89 in a certain alignment such that the flushing and suction channels automatically match with the corre- sponding through-hole 78 of the filter module 57.

As a result of the advantageous embodiment of the pipette heads 1 and 86, the suspension comes into contact exclusively with the ster- ilisable filter inserts 22, 28, 60, and not with the body/wall of the filter module receptacle 8 or 89, so that cross-contaminations or contami- nations of fresh media or cells within the filter module receptacle 8 or 89 of the pipette head 1 or 86 are prevented. Preferably, the pipetting device 2 has at least one computing unit for calculating the defined volumes to be metered, and also if appropriate for calculating the number of particles 47 with the aid of the counting means. Either a desired excess pressure or a desired volume of a flushing medium can be generated or released in a simple manner by means of the metering valves 16, 18, 19, 20, 21.

The fact that the suction channel 3 or 87 is embodied substantially separately from the flushing channel 14 or 90 also prevents contami- nation within the pipette head 1 or 86 beyond the filter module receptacle 8 or 89, respectively. The suspension contained therein is either discarded through the suction channel 3 or the extraction channel 91 or flushed-out or backflushed from the flushing channel 14 or 90 by means of the flushing medium. In order to ensure a particularly uniform through-flow through the filter elements 26 and 32 or 70, the filter modules 23 and 29 or 57 advantageously have appropriate means, such as for example guide ribs and/or webs, for homogenising the through-flow. Although the foregoing discussion has assumed substantially circularly cylindrical shapes with respect to the filter module receptacle 8/89, the filter modules 23 and 29/57, the suction channel 3/87 and also the flushing channel 14/90, it is obvious that any other cross-sectional shape is also possible. If appropriate, ejection means can additionally be provided in the pipette head 1 as ex- changeable means which serve to eject one or more of the filter modules 23, 29. Thus, for example, mechanical, pneumatic and/or hydraulic means can be provided that automatically push out one of the filter inserts 22 or 28 by exerting a force on the corresponding end side 27 and/or 33.

The pipette head 1 is expediently embodied in a plurality of parts. In addition, the tube element 5 has between the receptacle 6 and the radial projection 11 a further, outwardly pointing radial projection 52 which is enclosed in a recess 53 which is embodied as a borehole coaxial with the borehole 4 in the multipart basic element of the pi- pette head 1. An elastically deformable compensating element 54 is in this case arranged below the radial projection 52. During the mounting of the tube element 5, said tube element is firstly introduced into the pipette head 1 or into the borehole 4 counter to the direction of suction flow until the compensating element 54 rests on the end face of the recess 53. Subsequently, a holding plate 55, which has a borehole 56, the diameter of which corresponds substantially to the external diameter of the tube element 5, so that said tube element can be slid through the borehole 56, is slid onto the free end of the tube element 5. The holding plate 55 can be slid up to the radial projection 52. The holding plate 55 is held on the pipette head 1 by means of screwing, the compensating element 54, which at the same time also acts as a sealing element, being braced or elastically deformed until the holding plate 55 rests on the pipette head 1. As a result, the tube element 5 is oriented and held in the borehole 4. In addition, the radial projection 11 , as well as the likewise elastically deformable ring seal 12 arranged therein, serves to further orient and to ensure the free space 13.

It is easily possible to carry out filtering, transportation of the material retained during the filtering and also backflushing of the retained ma- terial at a desired location by means of the advantageous pipette head 1 or 86. The multifunctional pipette or the pipetting device 2 is GMP-compliant and reduces the risk of contamination or loss of sterility when preparing cell solutions or suspensions. In addition, the use of disposable elements, in particular of disposable filter ele- ments, is reduced to a minimum without increasing the risk of contamination. Advantageously, the surfaces in the interior of the pipette head, i.e. all of the surfaces which have entered into contact with the suspension, are hydrophobic. This prevents the possible adhesion of tissue layers and allows the complete transferral of separated com- ponents of the suspension to the receiver vessel.

In order to preserve or restore the sterility of the pipetting device 2 after potential contamination, a sterilising solution is conveyed by means of the flushing means and the corresponding metering valve through all the supply lines within the pipette head 1 or 86 and also through the filter module receptacle 8 or 89, respectively, and disposed of in a collecting vessel suitable for this purpose via said filter module receptacle. The pipette head 1 or 89 can be flushed for completely eliminating residues and sterilising solutions in two ways: ei- ther by conveying sterile flushing solution or by conveying sterile air through all the supply lines and the filter module receptacle by means of the flushing means. Residues are flushed out of the supply lines and the filter module receptacle as a result of the conveyance of sterile flushing solution. The conveyance of sterile air allows the subsequent drying of the supply lines and removal of any remaining flushing solution.

Claims

1. Pipette head (1 ; 86) having, for receiving a suspension by suction, at least one suction channel (3; 87) which can be connected to a suction means (43) and opens into a pipette opening (7; 88), wherein in the direction of suction flow before the pipette opening (7;88) at least one filter module (23, 29; 57) covering at least the pipette opening (7; 88) with an end side (27; 62) is detachably held in such a way that the suspension is passed during suction through a filter element (26, 32; 70) of the filter module (23, 29; 57), the pipette head (1 ; 86) having at least one flushing channel (14; 90) which can be connected to at least one flushing means (44) and the free end of which is associated with the end side (27; 62) of the filter module (23) in such a way that liquid and/or gaseous flushing medium conveyed through the flushing channel (14; 90) by means of the flushing means (44) flows through the filter element (26, 32; 70) of the filter module (23, 29; 57) counter to the direction of suction flow.

2. Pipette head according to claim 1 , characterized in that the flushing channel (14) is embodied coaxially with the suction channel (3).

3. Pipette head according to one of the preceding claims, characterized in that the flushing channel (14) is formed substantially by a borehole (4) in the pipette head (1 ) and a tube element (5) which is inserted into the borehole (4) and forms the suction channel (3).

4. Pipette head according to one of the preceding claims, char- acterized in that the suction channel (3) ends set apart from the end side (27) of the filter module (23).

5. Pipette head according to one of the preceding claims, characterized by at least one switchable metering valve (16, 18, 19, 20, 21 ) associated with the flushing channel (14; 90).

6. Pipette head according to one of the preceding claims, char- acterized by at least one, preferably a plurality of supply lines (15,

17) which open into the flushing channel (14; 90) and can be connected to the flushing means (44).

7. Pipette head according to one of the preceding claims, characterized in that a buffer solution, a sterilising solution and/or air are provided as the flushing medium.

8. Pipette head according to one of the preceding claims, characterized in that the filter module (23, 29; 57) is held on the pipette head (1 ; 86) in a force-fitting and/or form-fitting manner.

9. Pipette head according to one of the preceding claims, char- acterized in that the pipette opening (7; 88) opens into a filter module receptacle (8; 89).

10. Pipette head according to one of the preceding claims, characterized in that the filter module (23, 29; 57) is embodied as a filter insert (22, 28; 60).

11. Pipette head according to one of the preceding claims, characterized in that the filter insert (22, 28; 60) is embodied in a cup- shaped manner.

12. Pipette head according to one of the preceding claims, characterized in that the filter element (26, 32) is arranged in an aperture in the bottom (24, 30) of the cup-shaped filter insert (22, 28).

13. Pipette head according to one of the preceding claims, characterized in that a side wall (31 ; 63) of the cup-shaped filter insert (28; 60) that juts out substantially perpendicularly from the bottom (30) protrudes from the pipette head (1 ; 86) to form a pipette tip (34) to be dipped into the suspension.

14. Pipette head according to one of the preceding claims, characterized in that the filter element (26, 32; 70, 66) is embodied for retaining biological material, in particular tissue components (48) and/or individual cells (47).

15. Pipette head according to one of the preceding claims, characterized in that at least one further filter module (29) characterised in accordance with one or more of the preceding claims is detach- ably held before the filter insert (22) in the direction of suction flow.

16. Pipette head according to one of the preceding claims, char- acterized in that the further filter module is embodied as a filter attachment and/or as a filter insert (28).

17. Pipette head according to one of the preceding claims, characterized in that the filter element (32; 66) positioned in front in the direction of suction flow is embodied as a coarse filter and the filter element (26; 70) positioned thereafter in the direction of suction flow is embodied as a fine filter.

18. Pipette head according to one of the preceding claims, characterized in that the filter modules (23, 29) abut one another in the direction of flow in a sealing manner.

19. Pipette head according to one of the preceding claims, characterized in that each filter insert (22, 28; 60) abuts the filter module receptacle (8; 89) with its side wall (25, 31 ; 63) in a sealing manner, respectively.

20. Pipette head according to one of the preceding claims, characterized in that a pipetting tip (36; 96) is detachably held in and/or on the filter module receptacle (8; 89) and/or on one of the filter modules (23, 29; 57).

21. Pipette head according to one of the preceding claims, characterized in that the filter module (57) comprises at least two filter chambers (58, 59) which are fluidically connected in series.

22. Pipette head according to one of the preceding claims, characterized in that a first filter chamber (58) is separated from a second filter chamber (59) by the filter element (70), the second filter chamber (59) being arranged in the direction of suction flow behind the first filter chamber (58).

23. Pipette head according to one of the preceding claims, characterized in that for providing the first filter chamber (58) a pre-filter element (66) is arranged on a front side (69) of the filter module (57) opposite to the end side (62) of the filter module (57).

24. Pipette head according to one of the preceding claims, char- acterized in that the suction channel (87) and the flushing channel

(90) are assigned to the second filter chamber (59).

25. Pipette head according to one of the preceding claims, characterized in that a further suction channel (92) which can be connected to a further suction means is assigned to every other filter chamber (58), respectively.

26. Pipette head according to one of the preceding claims, characterized in that the filter chambers (58, 59) are arranged/designed such that they are at least essentially concentric.

27. Pipette head according to one of the preceding claims, char- acterized in that the filter element (70) and the pre-filter element (66) are arranged at least essentially parallel to and spaced apart from each other.

28. Pipette head according to one of the preceding claims, characterized in that the filter element (70) is held on a mounting ring (72) which is detachably held on the filter module (57) in a force-fitting and/or form-fitting manner.

29. Pipette head according to one of the preceding claims, characterized in that the filter element (70) and/or the mounting ring (72) comprise multiple protrusions (74) pointing in the direction of the pre- filter (66) element and being distributed over its circumference.

30. Pipette head according to one of the preceding claims, characterized in that an extracting channel (91 ) which can be connected to an extraction means for extraction of suspension in the second filter chamber (59) by suction is assigned to the second filter cham- ber (59).

31. Pipette head according to one of the preceding claims, characterized in that the extraction channel (91 ) is cooperating with at least one extraction nozzle (77) of the filter module which extends from the end side (62) of the filter module at least close to the filter element (66).

32. Pipette head according to one of the preceding claims, characterized in that in the filter module (57) at least one plunger element (81 ) is arranged in a movable manner for pushing off the pre-filter element (66) or the pipetting tip (96) from the filter module (57).

33. Filter module (57) for a pipette head, in particular according to one or more of the preceding claims, comprising at least two filter chambers (58, 59) which are fluidically connected in series, whereby a first filter chamber (58) is separated from a second filter chamber (59) by a filter element (70), and whereby a pre-filter element (66) is assigned to the first filter chamber (58) in the direction of suction flow before the filter element (70), whereby each filter chamber (58, 59) is designed to be connected to a suction means, respectively, for applying individual pressure in each filter chamber (58, 59), and whereby at least the second filter chamber (59) is designed to be connected to a flushing means.

34. Automated pipetting device with at least one movable pipette head, characterized by the embodiment of the pipette head (1 ; 86) in accordance with one or more of the preceding claims.

35. Automated pipetting device according to claim 34, characterized by a suction means (43) connected to the suction channel (3; 87) and a flushing means (44) connected to the flushing channel (14; 90).

36. Automated pipetting device according to one of the preceding claims, characterized by a further suction means connected to each further suction channel (92), respectively.

37. Use of a pipette head according to one or more of claims 1 to 33 for isolating biological components suspended in a suspension, in particular tissue components or individual cells, for transporting the isolated components and/or for dispensing the isolated components in a metered manner.

38. Method for operating a pipette head according to one of the preceding claims, characterized in that

- firstly a first negative pressure is applied to the first filter chamber by the further suction means for drawing a suspension into the first filter chamber by passing through the pre-filter element,

- secondly a second negative pressure lower than the first negative pressure is applied to the second filter chamber by the suc- tion means for drawing the suspension from the first filter chamber passing into the second filter chamber through the filter element;

- subsequently the suspension is extracted from the second filter chamber by use of the extraction suction means while maintain- ing at least the second negative pressure in the second filter chamber;

- then the pre-filter element is discharged by use of the plunger element;

- afterwards a predetermined amount of flushing medium is dis- charged into the second filter chamber by the flushing means; and

- then a release pressure higher than the second negative pressure is applied to the second filter chamber for back-flushing the filter element with the predetermined amount of flushing medium, thereby detaching components of the suspension retained by the filter element in a metered manner.