DEVICE AND METHOD FOR SEPARATION OF PARTICLES IN A LIQUID, KIT CONTAINING THE DEVICE AND USES OF THE DEVICE

Description

[0001] A device, a method, and a kit for separating particles of different sizes in a liquid are presented. Applications of the device are also proposed. The device and method are based on the principle that particles in a liquid can be bound to solid-phase particles with different diameters. The hydrodynamic diameter of the solid-phase particles determines whether the particles can pass through the pores of a filter element, the diameter of which can be selectively modified (e.g., increased or decreased). This allows for the highly precise separation of particles of the same size (e.g., B cells and T cells) in a liquid. The separation process is simple, fast, and cost-effective. High yields can be achieved, and the particles can be provided in a therapeutically usable liquid.

[0002] Numerous cell-based isolation methods require the use of technical membranes to separate molecules or particles of different sizes. Membranes used to date have a predetermined pore or mesh size. This retains components larger than the pore size, while smaller components can pass through the membrane. However, many technical separation processes, such as the purification of particles from blood, require the separation of more than two differently sized particles (i.e., differently sized blood cells and / or endosomal vesicles) with high purity. Currently, this necessitates expensive and complex multi-stage processes or the series connection of membranes with different pore sizes.

[0003] A method and a device are known in the prior art for separating a specific type of particle from a liquid (e.g., certain cells from whole blood) from other particles in that liquid in a specific manner (see WO 2016 / 092025 A1). The method and the device are based on the specific binding of the target particles (e.g., target cells) via low-affinity Fab fragments to solid-phase particles immobilized between two membranes of a membrane cartridge. The target particles can bind to the modified solid-phase particles, and other particles (e.g., cells) with a smaller diameter than the pore diameter of the two membranes and / or those that do not bind to the solid-phase particles are separated from the target particles.

[0004] This established method allows for the provision of target cells with high purity and yield. However, due to the fixed pore sizes of the membranes used in this method and device (e.g., 45 µm) and the specific affinity of the Fab fragments for the target cells, it is not possible to provide multiple blood cell fractions, each containing a different particle type, in a single step or using a single device (membrane cartridge). Furthermore, only a small blood sample volume is often available. Successfully performing a separation process after dividing this small sample volume into multiple membrane cartridges with differently modified solid-phase particles is often not feasible. Therefore, there is a need for a method that enables the separation of a liquid containing particles into multiple fractions, each containing a specific particle type, within a single device.

[0005] A filter element for separating unwanted components from a fluid flow is known in the prior art, in which the pore diameter of the filter element's pores can be changed by applying an electrical voltage to the filter element and / or by exerting a mechanical force on the filter element (see, for example, DE 10 2016 213 565 A1). A disadvantage of this filter element is that changing the pore size simultaneously changes the shape of the pores' cross-sectional area (e.g., from a rectangular cross-section to a rhomboid cross-section or from a round cross-section to an oval cross-section), and thus the selectivity of the filter element, allowing particles of a specific geometry to pass through, cannot be adjusted with high precision.

[0006] Furthermore, membranes containing or consisting of dielectric elastomers are known in the prior art (see, e.g., DE 10 2012 016 375 A1). It is also known that these membranes can be structured by applying electromagnetic radiation (e.g., from a laser) (see, e.g., DE 10 2012 016 378 A1).

[0007] US Patent 2016 / 199561 A1 discloses a device comprising a multilayer membrane, an electroactive polymer in each layer, and a control unit operationally connected to sequentially activate the electroactive polymer to change one or more sizes of the plurality of differently sized pores in one or more layers of the multilayer membrane. The device further discloses a device comprising a multilayer membrane, an actuator operationally connected to the multiple layers, and a control unit operationally activating the actuator to change a relative lateral position of the layers of the multilayer membrane, thereby aligning at least two pores within the layers of the membrane.

[0008] Based on this, the object of the present invention was to provide a device and a method for separating particles in a liquid that does not exhibit the disadvantages of the prior art. In particular, the device and method should make it possible not only to separate particles of different sizes (e.g., different-sized biological cells and / or endosomes) from a liquid with high precision, but also to separate particles of the same size (e.g., B cells and T cells). The separated particles should be available in a simple, fast, and cost-effective manner, even with small liquid volumes, and in high yield. Furthermore, the particles should be available in a therapeutically usable liquid. In addition, uses of the device should be proposed.

[0009] The problem is solved by the device with the features of claim 1, the kit with the features of claim 12, the method with the features of claim 13, and the use with the features of claim 15. The dependent claims describe advantageous embodiments. According to the invention, a device for separating particles in a liquid containing a) a container for holding a liquid (suspension), b) a planar filter element with a top surface and a bottom surface, wherein the filter element has continuous pores with a defined pore diameter, wherein the filter element is arranged in the container such that it divides the container into an upper compartment towards the top surface of the filter element and into a lower compartment towards the bottom surface of the filter element, such that particles of a liquid in the upper compartment can only enter the lower compartment if they pass through the filter element, wherein the upper compartment of the container has an opening for receiving a liquid containing particles, wherein the upper compartment of the container contains at least one group of solid-phase particles having a specific hydrodynamic diameter and exposing at least one molecule on their surface that is capable of binding specifically to a surface molecule of a first type of particle, and wherein the filter element contains or consists of a materiala device suitable for changing the pore diameter of its pores by applying an electrical voltage to the filter element and / or by acting upon the filter element with a mechanical force, wherein the device has an electrical voltage source; wherein the device has at least two electrically conductive layers connected to the electrical voltage source, wherein at least one of the at least two electrically conductive layers contacts at least one electrical insulating layer arranged between the at least one electrically conductive layer and the filter element, characterized in that the filter element is suitable for changing the pore diameter of its through pores in a range of 10 to 200 µm by applying an electrical voltage to the filter element and / or by acting upon the filter element with a mechanical force.wherein at least one of the at least two electrically conductive layers contains a polymer and electrically conductive particles.

[0010] The term "continuous pores" refers to pores that extend from the top to the bottom of the filter element. The term "with a defined pore diameter" means that the pore diameters of all pores in the filter element differ from each other by less than 10 µm, preferably less than 5 µm. The term "solid-state particles" also includes "gel particles."

[0011] The device according to the invention makes it possible to separate different particles in a liquid (or suspension) using a single device. Furthermore, by specifically coupling certain types of particles in the liquid (e.g., blood cell types and / or vesicle types in blood) to solid-phase particles of different sizes, it is also possible to separate particles of different types that have essentially the same size (i.e., essentially the same hydrodynamic diameter). The device according to the invention thus has the advantage that not only can particles of different sizes (e.g., different-sized biological cells and / or endosomes) in a liquid (suspension) be separated with high precision, but also particles of the same size (e.g., B cells and T cells).The separation of particles of the same size is achieved by coupling the respective particle type to be separated to solid-phase particles, which "imprint" a defined particle size on each particle type. The device also allows for the simple, rapid, and cost-effective distribution of separated particles in high yields, even with small liquid volumes. This is because the pore diameter of the device's filter element can be varied, thus eliminating the need for sequential separation of the liquid particles in multiple devices, each containing filter elements with different, fixed pore diameters. Furthermore, the separated particles can be provided in a therapeutically usable liquid (for example, their source liquid, such as blood plasma).

[0012] The device according to the invention can be designed as a membrane cartridge.

[0013] The device can be characterized in that the upper compartment of the container contains at least a second group of solid-phase particles with a second hydrodynamic diameter, wherein the second hydrodynamic diameter differs from the first hydrodynamic diameter, and wherein the at least second group of solid-phase particles exposes a molecule on its surface that is capable of specifically binding to a surface molecule of a second type of particle. This embodiment has the advantage that not only can a first type of particle be selectively separated from other particles in the liquid, but also a second type of particle can be separated from the first type of particle and from other particles in the liquid.

[0014] Furthermore, the device can be characterized in that the upper compartment of the container contains at least a third group of solid-phase particles with a third hydrodynamic diameter, wherein the third hydrodynamic diameter differs from the first and second hydrodynamic diameters, and wherein the third group of solid-phase particles exposes a molecule on its surface which is suitable to bind specifically to a surface molecule of a third type of particle.Preferably, the upper compartment contains a fourth, fifth, sixth, seventh, eighth, ninth, and / or tenth group of solid-phase particles, each with a hydrodynamic diameter that differs from other groups of solid-phase particles in the upper compartment, and wherein the solid-phase particles of each group expose a molecule on their surface that is capable of specifically binding to a surface molecule of a different particle type. An advantage of this embodiment is that at least three groups of particles in a liquid can be selectively separated from each other and from other particles in the liquid.

[0015] The at least one molecule exposed on the surface of the solid-phase particles and capable of specifically binding to a surface molecule of a first particle type may contain or consist of a polypeptide chain, wherein the polypeptide chain is preferably selected from the group consisting of antibody, antibody fragment and derivatives thereof, and is particularly preferably selected from the group consisting of Fab fragment or derivative thereof.

[0016] Furthermore, this molecule may contain or consist of a polynucleotide, wherein the polynucleotide is preferably selected from the group consisting of DNA, RNA and derivatives thereof.

[0017] Furthermore, this molecule may contain or consist of an oligosaccharide.

[0018] It is preferred that this molecule is reversibly bound to the surface of the solid-phase particles via non-covalent interactions, preferably in such a way that the bond can be dissolved by a measure selected from the group consisting of changing the concentration of a substance, changing the temperature, changing the pH value and combinations thereof.

[0019] Furthermore, it is preferred that this molecule is suitable for specifically binding to a surface molecule of a particle type that has a smaller hydrodynamic diameter than the solid-phase particles, preferably a hydrodynamic diameter that is at most 10%, preferably at most 8%, particularly preferably at most 6%, most preferably at most 4%, particularly at most 2%, of the hydrodynamic diameter of the solid-phase particles.

[0020] The device can have at least two electrically conductive layers that are connected to an electrical voltage source, preferably to an electrical voltage source of the device.

[0021] At least one of the at least two electrically conductive layers can be applied to the top side of the filter element or to the bottom side of the filter element. Preferably, one of the two electrically conductive layers is applied to the top side of the filter element and the other of the two electrically conductive layers is applied to the bottom side of the filter element.

[0022] The filter membrane can act as an electrically insulating layer. An additional electrical insulating layer between the at least two electrically conductive layers may then be unnecessary.

[0023] According to the invention, at least one of the at least two electrically conductive layers, optionally the at least two electrically conductive layers, contacts at least one electrical insulating layer arranged between the at least one electrically conductive layer and the filter element, wherein optionally the at least two electrically conductive layers each contact at least one electrical insulating layer arranged between the at least two electrically conductive layers and the filter element. In this embodiment according to the invention, an electrical short circuit or a momentary flashover can be prevented between the first and the further electrically conductive layer. The electrical insulating layer can be located only on one side of the filter element (i.e.,its top or bottom) or an electrical insulation layer may be arranged on both sides of the filter element.

[0024] Furthermore, at least one of the at least two electrically conductive layers, or optionally both of the at least two electrically conductive layers, can be arranged in a peripheral region of the pores of the filter element. This arrangement can be configured as a point-like feature around the pores of the filter element.

[0025] Furthermore, at least one of the at least two electrically conductive layers, or optionally both, can be arranged completely around the pores of the filter element. In this case, the electrically conductive layer has continuous pores with a defined pore diameter at the same locations as the filter element.

[0026] According to the invention, at least one of the at least two electrically conductive layers, optionally the at least two electrically conductive layers, contains a polymer, optionally an electrically conductive polymer.

[0027] Furthermore, according to the invention, at least one of the at least two electrically conductive layers, or optionally both of the at least two electrically conductive layers, contains electrically conductive particles, preferably carbon particles, and particularly preferably single-walled or multi-walled carbon nanotubes. Optionally, the proportion of electrically conductive particles is in the range of 0.001 to 30 wt.%, preferably in the range of 0.01 to 3 wt.%, based on the total weight of the electrically conductive layer. An advantage of this embodiment is that the electrically conductive layer remains electrically conductive even if it consists (mainly) of a non-electrically conductive polymer.

[0028] Furthermore, at least one of the at least two electrically conductive layers, optionally both of the at least two electrically conductive layers, can contain a metal, the metal preferably being arranged on the surface of the electrically conductive layer. If it contains a metal, this can be in the form of particles.

[0029] It is preferred that at least one of the at least two electrically conductive layers, optionally the at least two electrically conductive layers, is / are connected to the filter element or an electrically insulating layer by means of a force-fit and / or material-fit connection.

[0030] At least one of the at least two electrically conductive layers, optionally the at least two electrically conductive layers, can be applied to the filter element or to an electrically conductive layer by a process selected from the group consisting of pad printing, squeegee printing, screen printing, inkjet printing, jetting, spraying, vapor deposition and combinations thereof, optionally combined with a laser structuring process.

[0031] According to the invention, the device comprises an electrical voltage source that is electrically conductively connected to at least two electrically conductive layers of the device, preferably to at least two electrically conductive layers that are applied to the top and / or bottom of the filter element. An advantage of this is that the pore diameter of the pores of the filter element can be changed by applying an electrical voltage to the filter element.

[0032] In a further preferred embodiment, the device includes a means suitable for changing the pore diameter of the pores of the filter element by applying a mechanical force. The means is selected from the group consisting of a piston for exerting pressure on the filter element, a pneumatic device for exerting pressure on the filter element, a bimetallic wire for exerting pressure on the filter element, a NiTiCu alloy for exerting pressure on the filter element, and combinations thereof. The advantage here is that the pore diameter of the pores of the filter element can be changed by applying a mechanical force to the filter element.

[0033] The device can include a means suitable for applying an oscillating fluid flow to the upper surface of the filter element, preferably a fluid flow that oscillates in a direction perpendicular to the upper surface of the filter element. The advantage of this is that it prevents possible clogging of the filter element during particle separation.

[0034] Furthermore, the device may include a control unit configured to control an electrical voltage from a voltage source and / or a mechanical force on the filter element.

[0035] The control unit preferably operates by gradually changing the electrical voltage and / or mechanical force applied to the filter element during particle separation in a liquid. This change preferably occurs automatically over time or manually via user input. Specifically, the electrical voltage is reduced, and the mechanical force is increased. The aim in this case is to widen the pore size accordingly.

[0036] Furthermore, the control via the control unit can be carried out in such a way that the pore diameter of the pores of the filter element is changed in a range of 10 to 200 µm, preferably in a range of 20 to 180 µm, particularly preferably in a range of 30 to 160 µm, and especially in a range of 40 to 120 µm.

[0037] Apart from that, the control via the control unit can be carried out in such a way that the pore diameter of the pores of the filter element is automatically changed over time or manually by input(s) from a user of the device in increments to a larger pore diameter, preferably in steps of 5 to 15 µm, particularly preferably in steps of 9 to 11 µm, most preferably from a pore diameter of 10 µm to a pore diameter of 200 µm, in particular from a pore diameter of 40 µm via a pore diameter of 50 µm, a pore diameter of 60 µm, a pore diameter of 70 µm, a pore diameter of 80 µm, a pore diameter of 90 µm, a pore diameter of 100 µm, a pore diameter of 110 µm up to a pore diameter of 120 µm.One advantage of a DEA membrane as a filter element is that the pore size is not necessarily limited to predetermined levels, but can be continuously changed by an electrical voltage and / or a mechanical force.

[0038] The device may include a means suitable for moving the liquid containing particles through the filter element.

[0039] Preferably, this device includes or consists of a plunger that is arranged in a liquid-tight and movable manner within the first compartment of the container. The advantage here is that a user can manually control the flow of liquid through the filter element by moving the plunger.

[0040] Furthermore, this means can include or consist of a conveying device, preferably a pump, and particularly preferably a pump configured to move a liquid containing particles bidirectionally through the filter element. The advantage here is that a user can control the conveyance of the liquid through the filter element either manually by adjusting the pump activity or automatically (via a control unit of the device).

[0041] The filter element can contain or consist of an electroactive material. Furthermore, the filter element can contain or consist of a piezoelectric material. Additionally, the filter element can contain or consist of a dielectric material. The filter element can be a dielectric elastomer actuator (DEA). The advantage of these materials, or of the DEA, is that the pore diameter of the filter element can be adjusted by changing an electrical voltage applied to the filter element, without significantly altering the cross-sectional shape of the pores (e.g., the cross-sectional shape remains circular). This allows for more precise adjustment of the filter element's selectivity than with other filter elements where changing the pore diameter also alters the cross-sectional shape of the pores (e.g.,...).(changed from rectangular to rhomboid or from circular to oval). The operating principle of a DEA is based on the Maxwell voltage tensor, which forms between two separate, oppositely charged electrodes, similar to that of a capacitor. When an electrical voltage is applied, a potential difference arises between the electrodes, leading to a mechanical stress in the dielectric. The Maxwell voltage causes an equi-biaxial, linear movement and thus achieves the actual actuator effect, resulting in a change in the pore diameter of the filter element's pores.

[0042] It is preferred that the filter element contains or consists of an elastic material. Such a material allows for better adjustment of the pore diameter by applying a mechanical force to the filter element. It is also advantageous for adjusting the pore diameter by applying an electrical voltage if the filter element contains an elastic material (e.g., an elastomer). The stepless adjustability of the electrical voltage thus allows the macroscopic or mesoscopic actuator action to be influenced. DEAs often contain or consist of an incompressible elastomer. Consequently, the filter element can also contain or consist of an incompressible material.

[0043] In a preferred embodiment, the filter element contains or consists of a polymer, preferably an elastomer, particularly preferably a thermoplastic elastomer. In particular, the polymer is selected from the group consisting of silicone elastomer, liquid rubber elastomer, and combinations thereof.

[0044] The filter element can have a thickness from the top to the bottom of ≤ 250 µm, preferably ≤ 200 µm, particularly preferably ≤ 150 µm, and especially ≤ 100 µm. The advantage of the small thickness of the filter element is that the device can be made lighter, more compact, and more cost-effective.

[0045] In a preferred embodiment, the filter element is prestressed, preferably by an equibiaxial pre-tension in the range between 50% and 150%. The pre-tensioning can be achieved by connecting the filter element to a fixed frame of the device. An advantage of the pre-tensioning is that the original pore size of the filter element, i.e., the pore diameter of the pores in an unstressed state (relaxed state), can be smaller than a pore size required for carrying out a process for separating particles in a liquid. This can reduce the manufacturing costs of the filter element.

[0046] In a preferred embodiment, the filter element has continuous pores with a substantially round cross-section. This is advantageous if the solid-phase particles in the upper compartment of the device are spherical (which is preferred), as the opening provided by the pores can then be precisely matched to the spherical diameter of the solid-phase particles.

[0047] According to the invention, the filter element is suitable for changing the pore diameter of its through pores by applying an electrical voltage to the filter element and / or by applying a mechanical force to the filter element in a range of 10 to 200 µm, preferably in a range of 20 to 180 µm, particularly preferably in a range of 30 to 160 µm, and most preferably in a range of 40 to 120 µm. In particular, the suitability is such that the change in pore diameter is isotropic in all directions along a cross-sectional area of ​​the pores.

[0048] The particles to be separated can be selected from the group consisting of vesicles and biological cells, preferably selected from the group consisting of vesicles and biological cells from blood, particularly preferably selected from the group consisting of endosomal vesicles, exosomal vesicles, platelets, erythrocytes, leukocytes and combinations thereof.

[0049] The solid-phase particles suitable for specifically binding to a surface molecule of a first particle type of the particles to be separated may contain or consist of a polymer, wherein the polymer is preferably selected from the group consisting of plastic, agarose and combinations thereof.

[0050] The solid-phase particles can be solid-phase spheres and preferably have a substantially round shape (spherical shape). The (substantially) round shape is advantageous because the solid-phase particles thus have a (substantially) uniform spatial extent (i.e., the same diameter) in all directions of expansion, and therefore their suitability to pass through a pore with a defined pore diameter can be predicted more accurately and is more precisely defined.

[0051] Furthermore, according to the invention, a kit is provided which contains a device according to the invention and a device which has at least one, preferably all, of the following means: A means suitable for applying an electrical voltage to the filter element, wherein the means preferably includes or consists of an electrical voltage source; a means suitable for applying an oscillating fluid flow to the top surface of the filter element, preferably a fluid flow oscillating in a direction perpendicular to the top surface of the filter element; and a means suitable for moving the liquid containing particles through the filter element, wherein the means is preferably selected from the group consisting of a piston that is arranged in a liquid-tight and movable manner in the first compartment of the container, a conveying device, preferably a pump, particularly preferably a pump configured to move a liquid containing particles bidirectionally through the filter element, and combinations thereof.

[0052] Furthermore, a method for separating particles in a liquid is provided, comprising the steps a) Providing at least one group of solid-phase particles having a specific hydrodynamic diameter and exposing at least one molecule on their surface capable of binding specifically to a surface molecule of a first particle type of the liquid particles; b) Incubating the liquid with the at least one group of solid-phase particles until the solid-phase particles of the at least one group of solid-phase particles have bound specifically to a surface molecule of a first particle type of the liquid particles; c) Providing a filter element containing or consisting of a material capable of changing the pore diameter of its pores by applying an electrical voltage to the filter element and / or by acting upon the filter element with a mechanical force;d) Adjusting the pore diameter of the pores of the filter element by applying an electrical voltage to the filter element and / or by applying a mechanical force to the filter element, such that only particles up to a desired particle diameter can pass through the filter element, wherein the adjusted particle diameter is smaller than a hydrodynamic diameter of the at least one group of solid-phase particles; e) Moving the liquid through the filter element; f) Isolating the liquid; g) Increasing the pore diameter of the pores of the filter element by reducing the strength of the electrical voltage and / or by increasing the mechanical force on the filter element, such that particles up to a desired, now larger particle diameter can pass through the filter element, wherein these are preferably the particles of the first type of particle that are bound to the group of solid-phase particles;h) Optionally, add a liquid, preferably free of particles, to the liquid containing the unfiltered particles; i) move the liquid through the filter element; j) isolate the liquid containing the particles of the first particle type; k) Optionally, repeat steps g) to j) until all particles of the liquid are separated according to their size in separate liquids.

[0053] The method can be characterized by the fact that it is carried out with a device according to the invention.

[0054] Preferably, the procedure then includes the following steps: i) Adjusting the pore diameter of the pores of the filter element of the device by applying an electrical voltage to the filter element and / or by acting upon the filter element with a mechanical force, so that only particles up to a desired particle diameter can pass through the filter element, wherein the adjusted particle diameter is smaller than a hydrodynamic diameter of the at least one group of solid-phase particles; ii) Filling the upper compartment of the container of the device with a liquid containing particles of different sizes; iii) Incubating the liquid in the upper compartment of the container of the device with the at least one group of solid-phase particles until at least solid-phase particles of the at least one group of solid-phase particles have specifically bound to a surface molecule of a first type of particle (e.g., a first type of blood particles);iv) Moving the liquid through the filter element of the device into the lower compartment of the container; v) Isolating the liquid containing the passed particles from the lower compartment of the container of the device; vi) Increasing the pore diameter of the pores of the filter element of the device by reducing the strength of the electrical voltage and / or by increasing a mechanical force on the filter element, so that particles up to a desired, now larger particle diameter can pass through the filter element, preferably being particles of the first type of particle that are bound to the group of solid-phase particles; vii) Optionally filling the upper compartment of the container of the device with a liquid that preferably does not contain particles; viii) Moving the liquid through the filter element of the device into the lower compartment of the container;ix) Isolate the liquid containing the particles of the first particle type from the lower compartment of the device's container; x) Optionally, repeat steps vi) to ix) until all particles of the liquid are separated according to their size in separate liquids.

[0055] Furthermore, the use of the device and / or kit according to the invention for separating particles of different sizes present in a liquid is proposed. This use can involve isolating one or more blood cell fractions from blood, preferably for providing the blood cell fractions for diagnostic purposes and / or for the production of blood products, particularly for the production of cell therapeutics. In addition, the use can involve isolating bacterial cells from blood. Furthermore, the use can involve isolating endosomal or exosomal vesicles from blood, blood serum, or biosuspensions, preferably for providing endosomal vesicles for diagnostic purposes and / or for the production of vaccines. Beyond this, the use can involve isolating tissue cells from mixed tissue cell fractions.Furthermore, the use may involve the isolation of cells from mixed cell suspensions originating from bioreactors, wherein the cells are preferably selected from the group consisting of plant cells, animal cells, human cells, bacterial cells, yeast cells and combinations thereof.

[0056] The following figures and examples are intended to explain the subject matter of the invention in more detail, without limiting it to the specific embodiments shown here. Figure 1Figure 1 schematically shows an adjustment of the pore size of a filter element 1 containing or consisting of an electroactive material. When a high electrical voltage (in the kV range) is applied to the filter element via two electrically conductive layers 4, 4' located on the side wall of a pore 2 of the filter element and separated by an insulating layer 5, the pores 2 of the filter element 1 contract, and their diameter becomes relatively small. When the voltage of the electrical voltage source 3 is reduced to zero, the pores 2 of the filter element 1 expand to their maximum size. The pore diameter of the pores 2 of the filter element 1 can be precisely and continuously adjusted between the high voltage (e.g., several kV) and zero. Figure 2Figure 1 shows a macroscopic image of the expansion of a single pore 2 of a filter element 1 containing or consisting of an electroactive material, as a function of the applied electrical voltage. Here, the filter element is a DEA membrane. It can be seen that the pore diameter of this pore 2 can be reduced from a diameter of 4.6 mm at an applied electrical voltage of zero to a pore diameter of 4.35 mm at an applied electrical voltage of 3 kV (electric field applied to the filter element). The resulting reduction in the diameter of the depicted pore 2 is approximately 250 µm in this case. Figure 3Figure 1 schematically shows the preparation of three groups with solid-phase particles 6, 6', 6" of different sizes, wherein the solid-phase particles 6, 6', 6" of each group have a specific binding molecule 7, 7', 7" reversibly bound to their surface. In a first step, three groups of solid-phase particles 6, 6', 6" are provided, each group having solid-phase particles 6, 6', 6" with a different hydrodynamic radius among the groups ( Figure 3A In a second step, the surface of the solid-phase particles 6, 6', 6" undergoes a specific chemical functionalization, such that the solid-phase particles 6, 6', 6" of each group expose a specific binding molecule 7, 7', 7" on their surface, and the specific binding molecule 7, 7', 7" differs among the three groups ( Figure 3B). In a third step, particles 8, 8', 8" (e.g., blood cells) from the liquid containing the particles to be separated are bound to the solid-phase particles 6, 6', 6" of the respective groups. This binding occurs in such a way that each of the three groups of solid-phase particles 6, 6', 6" binds only specific particles 8, 8', 8" (e.g., specific blood cells such as B cells or T cells). This allows even particles 8, 8', 8" of the same size (such as B cells and T cells) to be separated from one another, since they are bound to significantly larger solid-phase particles 6, 6', 6" that determine the respective hydrodynamic radius of the resulting complexes. Thus, a specific hydrodynamic radius is "imprinted" on each of the equally sized particles 8, 8', 8" that determines their separation properties by the filter element. Figure 4Figure 1 schematically shows the penetration of a pore 2 of the filter element 1 by the respective solid particles 6, 6', 6". Since the solid particles 6, 6', 6" are each bound via a specific binding molecule 7, 7', 7" to a respective particle type of the particles to be separated 8, 8', 8" and have a significantly larger diameter than the particles to be separated 8, 8', 8", the possibility of the particles to be separated 8, 8', 8" passing through the pore 2 of the filter element 1 is determined by the solid particle 6, 6', 6" to which they are bound. If a high electrical voltage is applied to the filter element 1 (left figure), the pore diameter of its pores 2 is relatively small. Consequently, only the particles 8 that are bound to the relatively smallest solid particles 6 can pass through the pore 2. If a lower electrical voltage is applied to the filter element 1 (middle figure), the pore diameter of its pores is 2 times larger.Consequently, the 8' particles, which are bound to larger solid particles 6', can now pass through pore 2. If no electrical voltage is applied to the filter element 1 (right-hand figure), the pore diameter of its pores 2 adjusts to its largest value (maximum value). Consequently, the 8" particles, which are bound to the largest solid particles 6", can now also pass through pore 2. Figure 5Figure 1 schematically shows a device according to the invention for separating particles in a liquid, which is here designed in the form of a cartridge. The device comprises a container 9 for holding a liquid (suspension) and a planar filter element 1 with a top surface 10 and a bottom surface 11, wherein the filter element 1 has continuous pores 2 with a defined pore diameter. The filter element 1 is arranged in the container 9 such that it divides the container 9 in the direction of the top surface 10 of the filter element 1 into an upper compartment 12 with an opening 14 and in the direction of the bottom surface 11 of the filter element 1 into a lower compartment 13, so that particles of a liquid in the upper compartment 12 can only enter the lower compartment 13 if they pass through the filter element 1, wherein the upper compartment 12 of the container 9 has an opening 14 for holding a liquid containing particles.The device is characterized in that the upper compartment 12 of the container 9 contains at least one group of solid-phase particles 6, 6', 6" having a specific hydrodynamic diameter and exposing at least one molecule 7, 7', 7" on their surface (not shown separately), which is capable of specifically binding to a surface molecule of a first particle type 8, 8', 8" (not shown separately). The filter element 1 contains or consists of a material capable of changing the pore diameter of its pores 2 by applying an electrical voltage to the filter element 1 and / or by acting upon the filter element 1 with a mechanical force. In the lower part of . Figure 5The figure illustrates how, in the case of a small pore diameter of the pores 2 of the filter element 1, initially only the smaller bonding complexes 6, 7, 8 can pass through the pores 2 of the filter element 1 and move from the upper compartment 12 of the container 9 to the lower compartment 13 of the container 9. If the pore diameter of the pores 2 of the filter element 1 is subsequently increased by applying an electrical voltage and / or a mechanical force, the larger bonding complexes 6', 7', 8' can now also pass through the pores 2 of the filter element and move from the upper compartment 12 of the container 9 to the lower compartment 13 of the container. Example 1 - Production of suitable solid-state particles

[0057] The solid-phase particles used in the device can be produced effectively, quickly adaptable, potentially highly scalable and resource-efficiently in water-in-oil emulsions.

[0058] In this process, for example, water-based agarose in liquid phase is applied to an oil phase, causing the agarose to deform into droplets or beads. After subsequent polymerization of the agarose, the beads can be washed and functionalized. This method yields solid-phase agarose particles with a polydisperse size distribution. The size distribution can be precisely controlled by changing the production parameters, such as the agarose-to-oil ratio or the oil viscosity.

[0059] It is advantageous if the produced solid-phase particles exhibit a size variation of less than 10 µm, i.e., are relatively homodisperse. Furthermore, it is advantageous if several different size fractions of solid-phase particles are produced, wherein the difference in the mean diameters of the solid-phase particles between the individual fractions is preferably at least 10 µm.

[0060] For example, fractions are provided in which the solid-phase particles within a fraction have a size variation of less than 10 µm and in which the mean diameter of the solid-phase particles of a first fraction is 40 µm, a second fraction 50 µm, a third fraction 60 µm, a fourth fraction 70 µm, a fifth fraction 80 µm, a sixth fraction 90 µm, a seventh fraction 100 µm, an eighth fraction 110 µm and a ninth fraction 120 µm. Example 2 - Attachment of a binding molecule to the solid-phase particles

[0061] To attach a specific molecule suitable for binding to a surface molecule of a first type of particle, Strep-Tactin®< is chemically covalently attached to the solid-phase particles (e.g., agarose particles) (e.g., via a chemical coupling process). An antibody fragment (e.g., Fab fragment) that has chemically covalently attached to a Strep-tag®< can be used as the binding molecule (e.g., via microbiological production of a protein containing both the antibody fragment and the Strep-tag®<). When the modified solid-phase particles and the modified antibody fragments are combined in an aqueous solution, they bind to each other via a non-covalent (and reversible) Strep-Tactin®<-Strep-tag®< bond, i.e., a binding complex is formed.

[0062] If the antibody fragment is selected to bind only T cells, for example, the T cells can be bound to the solid-phase particles via binding to the Fab fragment, which in turn is immobilized on the solid-phase particle via non-covalent interactions through the Strep-Tactin®< - Strep-tag®< linkage. The non-covalent Strep-Tactin®< - Strep-tag®< linkage is reversible and can be broken by adjusting the biotin concentration in the aqueous solution. In other words, after separation, the T cells bound to the solid-phase particles can be separated from them again by adding biotin. Example 3 - Production of a filter membrane with selective pore size

[0063] The production of a membrane containing a dielectric elastomer is known in the art (see, for example, DE 10 2012 016 375 A1). Such membranes can be used to produce a filter element, as used in the inventive method and device.

[0064] It is also known that such membranes can be structured by the action of electromagnetic radiation (e.g. a laser) (see e.g. DE 10 2012 016 378 A1).

[0065] To produce a filter membrane suitable for the device and method according to the invention, an array of continuous pores with a defined pore diameter is introduced, for example, into a filter membrane containing or consisting of a dielectric polymer. Pores with a "defined" pore diameter mean that the pore diameter of all pores of the filter element varies by less than 10 µm, preferably less than 5 µm. Pores meeting these requirements can, for example, be introduced into the filter membrane using electromagnetic radiation from a laser. Example 4 - Method for separating particles in a liquid

[0066] In a first step, different fractions of solid-phase particles of varying sizes are produced (e.g., according to Example 1).

[0067] In a second step, these are each functionalized with the different cell-specific binding molecules (e.g. according to example 2).

[0068] In a third step, a filter membrane is provided for separating the particles (e.g. according to example 3).

[0069] In a fourth step, the different-sized solid-phase particles (e.g., agarose particles) are contacted with a liquid containing the particles to be separated (e.g., blood cells and vesicles from blood). Certain types of particles (e.g., specific blood cells) bind specifically to solid-phase particles of a certain size, thereby imposing ("imprinting") the size of the solid-phase particles on them.

[0070] In a fifth step, the particles bound to the respective solid-phase particles (e.g., blood cells) are separated stepwise, starting with the smallest fraction of the solid-phase particles, via the filter membrane, whereby the pore diameter of the filter membrane is gradually increased. Reference symbol list

[0071] 1: Filter element; 2: Pore of the filter element; 3: Electrical voltage source; 4, 4': Electrically conductive layer; 5, 5': Insulating layer; 6, 6', 6": Solid-phase particle; 7, 7', 7": Binding molecule; 8, 8', 8": Particle of solution (suspension) to be separated.

Claims

1. A device (8, 8', 8") for separating particles in a liquid, comprising a) a container for receiving a liquid (suspension), b) an areal filter element (1) having an upper side surface and a lower side surface, wherein the filter element (1) has continuous pores (2) having a defined pore diameter, the filter element (1) being arranged in the receptacle such that it divides the receptacle, in the direction of the upper side surface of the filter element (1), into an upper compartment and, in the direction of the lower side surface of the filter element (1), into a lower compartment, so that particles (8, 8', 8") of a liquid in the upper compartment can only reach the lower compartment if they pass through the filter element (1), the upper compartment of the receptacle having an opening for receiving a liquid with particles (8, 8', 8"), wherein the upper compartment of the container contains at least one group of solid phase particles (6, 6', 6") that have a specific hydrodynamic diameter and that expose at least one molecule (7, 7', 7") at their surface that is suitable to specifically bind to a surface molecule of a first type of particle (8), wherein the filter element (1) comprises or consists of a material that is suitable to change, by application of an electrical voltage to the filter element (1) and / or by the action of a mechanical force on the filter element (1), the pore diameter of its pores (2), wherein the device has an electrical voltage source (3); wherein the device has at least two electrically conductive layers (4, 4') which are connected to the electrical voltage source (3), wherein at least one of the at least two electrically conductive layers (4, 4') contacts at least one electrical insulation layer (5, 5'), which is arranged between the at least one electrically conductive layer (4, 4') and the filter element (1), characterized in that the filter element (1) is suitable for changing the pore diameter of its continuous pores (2) in a range from 10 to 200 µm by the action of an electrical voltage on the filter element (1) and / or by the action of a mechanical force on the filter element (1), wherein at least one of the at least two electrically conductive layers (4, 4) comprises a polymer and electrically conductive particles.

2. A device in accordance with claim 1, characterized in that the upper compartment of the container contains at least one second group of solid phase particles (6, 6', 6") having a second hydrodynamic diameter, with the second hydrodynamic diameter differing from the first hydrodynamic diameter, and with the at least one second group of solid phase particles (6, 6', 6") exposing a molecule (7, 7', 7") at their surface that is suitable to specifically bind to a surface molecule of a second type of particle (8').

3. A device in accordance with claim 2, characterized in that the upper compartment of the container contains at least one third group of solid phase particles (6, 6', 6") having a third hydrodynamic diameter, with the third hydrodynamic diameter differing from the first and second hydrodynamic diameters, and with the third group of solid phase particles (6, 6', 6") exposing a molecule (7, 7', 7") at their surface that is suitable to bind to a surface molecule of a third type of particle (8"), with the upper compartment preferably comprising a fourth, fifth, sixth, seventh, eighth, ninth, and / or tenth group of solid phase particles (6, 6', 6") each comprising a hydrodynamic diameter that differs from other groups of solid phase particles (6, 6', 6") in the upper compartment, and with the solid phase particles (6, 6', 6") of the respective groups exposing a molecule (7, 7', 7") at their surface that is suitable to specifically bind to a surface molecule of a respective other type of particle (8, 8', 8").

4. A device in accordance with one of the preceding claims, characterized in that the at least one molecule (7, 7', 7") that is exposed at the surface of the solid phase particles (6, 6', 6") and that is suitable to specifically bind to a surface molecule of a first type of particle (8); i) comprises or consists of a polypeptide chain, with the polypeptide chain preferably being selected from the group consisting of antibody, antibody fragment, and derivatives thereof, particularly preferably selected from the group consisting or a fab fragment or a derivative thereof; and / or ii) comprises or consists of a polynucleotide, with the polynucleotide preferably being selected from the group consisting of DNA, RNA, and derivatives thereof; and / or iii) comprises or consists of an oligosaccharide; and / or iv) is reversibly bound to the surface of the solid phase particles (6, 6', 6") via non-covalent interactions, preferably such that the binding is releasable via a measure selected from the group consisting of changing a concentration of a substance, changing a temperature, changing the pH, and combinations thereof; and / or v) is suitable to specifically bind to a surface molecule of a type of particle (8) that has a smaller hydrodynamic diameter than the solid phase particles (6, 6', 6"), preferably a hydrodynamic diameter that amounts to a maximum of 10%, preferably a maximum of 8%, particularly preferably a maximum of 6%, very particularly preferably a maximum of 4%, in particular a maximum of 2%, of the hydrodynamic diameter of the solid phase particles (6, 6', 6").

5. A device in accordance with one of the preceding claims, characterized in that at least one of the at least two electrically conductive layers (4, 4'), optionally the at least two electrically conductive layers (4, 4'), i) is applied to the upper side or to the lower side of the filter element (1), with optionally one of the layers (4, 4') being applied to the upper side of the filter element (1) and the other being applied to the lower side of the filter element (1); and / or ii) is arranged in a marginal region of the pores (2) of the filter element (1); and / or iii) is fully arranged around the pores (2) of the filter element (1) and having continuous pores (2) having a defined pore diameter at the same points as the filter element (1); and / or iv) comprises a metal, with the metal preferably being arranged at the surface of the electrically conductive layer (4, 4'); and / or v) is connected to the filter element (1), or to an electrically insulating layer (5, 5'), with a friction-lock and / or a material bond; wherein at least one of the at least two electrically conductive layers (4, 4'), optionally the at least two electrically conductive layers (4, 4'), is / are applied to the filter element (1) or to an electrically conductive layer (4, 4') via a process selected from the group consisting of pad printing, doctor knife coating, screen printing, inkjet printing, jetting, spraying, vaporization, and combinations thereof, optionally combined with a laser structuring process.

6. A device in accordance with one of the preceding claims, characterized in that the device has a means that is suitable to i) vary the pore diameter of the pores (2) of the filter element (1) by the action of a mechanical force on said filter element (1), with the means being selected from the group consisting of a stamp for exerting a pressure on the filter element (1), a pneumatic device for exerting a pressure on the filter element (1), a bimetallic wire for exerting a pressure on the filter element (1), an NiTiCu alloy for exerting a pressure on the filter element (1), and combinations thereof; and / or ii) apply an oscillating fluid flow, preferably a fluid flow that oscillates in a direction perpendicular to the upper side surface of the filter element, to the upper side surface of the filter element (1).

7. A device in accordance with one of the preceding claims, characterized in that the device has a control unit that is configured to control an electrical voltage of a voltage source (3) and / or a mechanical force on the filter element (1), preferably such that i) an electrical voltage and / or a mechanical force on the filter element (1) is changed stepwise in the course of a separation of particles (8, 8', 8") in a liquid, with the variation preferably taking place automatically over the course of time or manually by an input of a user, with the variation of the electrical voltage in particular being a reduction of the electrical voltage, and the variation of the mechanical force on the filter element (1) in particular being a reduction of the mechanical force on the filter element (1); and / or ii) the pore diameter of the pores (2) of the filter element (1) is changed in a range from 10 to 200 µm, preferably in a range from 20 to 180 µm, particularly preferably in a range from 30 to 160 µm, in particular in a range from 40 to 120 µm; and / or iii) the pore diameter of the pores (2) of the filter element (1) is changed automatically over the course of time, or manually by input(s) by a user of the device, stepwise to a larger pore diameter, preferably in steps of 5 to 15 µm, particularly preferably in steps of 9 to 11 µm, very particularly preferably from a pore diameter of 10 µm up to a pore diameter of 200 µm, in particular from a pore diameter of 40 µm via a pore diameter of 50 µm, a pore diameter of 60 µm, a pore diameter of 70 µm, a pore diameter of 80 µm, a pore diameter of 90 µm,, a pore diameter of 100 µm, a pore diameter of 110 µm, up to a pore diameter of 120 µm.

8. A device in accordance with one of the preceding claims, characterized in that the device has a means that is suitable to move the liquid having particles (8, 8', 8") through the filter element (1), with the means preferably i) comprising or consisting of a stamp that is arranged in a liquid tight and movable manner in the first compartment of the container and / or ii) comprising or consisting of a conveying device, preferably a pump, particularly preferably a pump that is configured to move a liquid having particles (8, 8', 8") bidirectionally through the filter element (1).

9. A device in accordance with one of the preceding claims, characterized in that the filter element (1) comprises or consists of a material that i) is electroactive; and / or ii) is piezoelectric; and / or iii) is dielectric; and / or iv) is elastic;, and / or v) is incompressible; and / or vi) is a polymer, preferably an elastomer, particularly preferably a thermoplastic elastomer, with the polymer in particular being selected from the group consisting of silicone elastomer, liquid rubber elastomer, and combinations thereof.

10. A device in accordance with one of the preceding claims, characterized in that the filter element (1) i) has an extent from the upper side in the direction of the lower side in the range from ≤ 250 µm, preferably in the range from ≤ 200 µm, particularly preferably in the range from ≤ 150 µm, in particular in the range from ≤ 100 µm; and / or ii) is pretensioned, preferably via an equibiaxial prestretching in the range between 50% and 150%, with the pretension preferably being implemented via a connection of the filter element (1) to a fixed frame of the device; and / or iii) has continuous pores (2) that have a substantially round cross-section; and / or iv) is suitable to change the pore diameter of its continuous pores (2) by the action of an electrical voltage on the filter element (1) and / or by the action of a mechanical force on the filter element (1) in a range from 20 to 180 µm, particularly preferably in a range from 30 to 160 µm, very particularly preferably in a range from 40 to 120 µm, with the variation of the pore diameter in particular taking place isotropically in all the directions along a cross-sectional surface of the pores (2).

11. A device in accordance with one of the preceding claims, characterized in that the particles (8, 8', 8") are selected from the group consisting of vesicles and biological cells, preferably selected from the group consisting of vesicles and biological cells of blood, particularly preferably selected from the group consisting of endosomal vesicles, exosomal vesicles, thrombocytes, erythrocytes, leukocytes, and combinations thereof.

12. A kit comprising i) a device in accordance with one of the claims 1 to 11; and ii) a unit that has at least one, preferably all, of the following means: a means that is suitable to apply an electrical voltage to the filter element (1), with the means preferably comprising or consisting of an electrical voltage source (3); a means that is suitable to apply an oscillating fluid flow to the upper side surface of the filter element (1), preferably a fluid flow that oscillates in a direction perpendicular to the upper side surface of the filter element (1); and a means that is suitable to move the liquid having particles (8, 8', 8") through the filter element (1), with the means preferably being selected from the group consisting of a stamp that is arranged in a liquid tight and movable manner in the first compartment of the container, a conveying device, preferably a pump, particularly preferably a pump that is configured to move a liquid having particles (8, 8', 8") bidirectionally through the filter element (1), and combinations thereof.

13. A method for separating particles (8, 8', 8") in a liquid, comprising the steps a) providing at least one group of solid phase particles (6, 6', 6") that have a specific hydrodynamic diameter and that expose at least one molecule (7, 7', 7") at their surface that is suitable to specifically bind to a surface molecule of a first type of particle (8) of the particles (8, 8', 8") of the liquid; b) incubating the liquid having the at least one group of solid phase particles (6, 6', 6") until the solid phase particles (6, 6', 6") of the at least one group of solid phase particles (6, 6', 6") have been specifically bound to a surface molecule of a first type of particle (8) of the particles (8, 8', 8") of the liquid; c) providing a filter element (1) that comprises or consists of a material that is suitable to change, by application of an electrical voltage to the filter element (1) and / or by the action of a mechanical force on the filter element (1), the pore diameter of its pores (2); d) setting the pore diameter of the pores (2) of the filter element (1) via application of an electrical voltage to the filter element (1) and / or the action of a mechanical force on the filter element (1) so that only particles up to a desired particle diameter can pass through the filter element (1), with the set particle diameter being smaller than a hydrodynamic diameter of the at least one group of solid phase particles (6, 6', 6"); e) moving the liquid through the filter element (1); f) isolating the liquid; g) increasing the pore diameter of the pores (2) of the filter element (1) by reducing the power of the electrical voltage and / or by increasing the mechanical force on the filter element (1) so that particles (8, 8', 8") up to a desired, now larger particle diameter can pass through the filter element (1), with said particles preferably being the particles (8) of the first type of particle that are bound to the group of solid phase particles (6, 6', 6"); h) optionally adding a liquid that preferably does not comprise any particles (8, 8', 8") to the liquid having the particles (8, 8', 8") that have not passed through; i) moving the liquid through the filter element (1); j) isolating the liquid that contains the particles (8) of the first type of particle; k) optionally repeating steps g) to j) until all particles (8, 8', 8") of the liquid are present in separate liquids, separated according to size; characterized in that the method is carried out using an apparatus according to any one of the claims 1 to 11.

14. A method for separating particles (8, 8', 8") in a liquid in accordance with claim 13, characterized in that the method preferably comprises the following steps i) setting the pore diameter of the pores (2) of the filter element (1) of the device via application of an electrical voltage to the filter element (1) and / or the action of a mechanical force on the filter element (1) so that only particles (8, 8', 8") up to a desired particle diameter can pass through the filter element (1), with the set particle diameter being smaller than a hydrodynamic diameter of the at least one group of solid phase particles (6, 6', 6"); ii) filling the upper compartment of the container of the device with a liquid that contains particles having different sizes; iii) incubating the liquid in the upper compartment of the container of the device having the at least one group of solid phase particles (6, 6', 6") until at least solid phase particles (6, 6', 6") of the at least one group of solid phase particles (6, 6', 6") have specifically bound to a surface molecule of a first type of particle (8) of the particles (8, 8', 8"); iv) moving the liquid through the filter element (1) of the device into the lower compartment of the container; v) isolating the liquid, including the passed particles, from the lower compartment of the receptacle of the device; vi) increasing the pore diameter of the pores (2) of the filter element (1) of the device by reducing the power of the electrical voltage and / or by increasing a mechanical force on the filter element (1) so that particles up to a desired, now larger particle diameter can pass through the filter element (1), with said particles preferably being the particles (8) of the first type of particle that are bound to the group of solid phase particles (6, 6', 6"); vii) optionally filling the upper compartment of the container of the device with a liquid that preferably does not comprise any particles; viii) moving the liquid through the filter element (1) of the device into the lower compartment of the container; ix) isolating the liquid that comprises the particles (8) of the first type of particles from the lower compartment of the container of the device; x) optionally repeating steps vi) to ix) until all the particles of the liquid are present separated by their size in separate liquids.

15. Use of the device in accordance with one of the claims 1 to 11 and / or the kit in accordance with claim 12, for separating particles (8, 8', 8") of different sizes that are present in a liquid, preferably for i) isolating one or more blood cell fractions from blood, preferably for providing blood cell fractions for diagnostics and / or for producing blood products, in particular for creating cell therapeutics; and / or ii) the isolation of bacterial cells of blood; and / or iii) the isolation of endosomal or exosomal vesicles of blood, blood serum, or biosuspensions, preferably for providing endosomal vesicles for diagnosis and / or for manufacturing vaccines; and / or iv) the isolation of tissue cells of mixed tissue fractions; and / or v) isolating cells from mixed cell suspensions that originate from bioreactors, with the cells preferably being selected from the group consisting of plant cells, animal cells, human cells, bacteria cells, yeast cells, and combinations thereof.