Apparatus and method for separating particles of different sizes in a liquid, and use of the apparatus.
The apparatus with adjustable pore diameters addresses the inefficiencies of existing separation methods by enabling high-selectivity, efficient, and cost-effective separation of particles in a liquid, particularly for blood cell products, using centrifugal force or voltage control.
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Patents
- Current Assignee / Owner
- FRAUNHOFER GESELLSCHAFT ZUR FORDERUNG DER ANGEWANDTEN FORSCHUNG EV
- Filing Date
- 2021-12-23
- Publication Date
- 2026-07-24
Smart Images
Figure 0007894869000001 
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Abstract
Description
Technical Field
[0001] Provided are an apparatus and a method for separating particles of different sizes in a liquid. Also provided is the use of the apparatus according to the present invention. The apparatus according to the present invention and the method according to the present invention are based on the fact that the pore diameter of the pores of at least one filter element of the apparatus can be intentionally changed (for example, increased or decreased). The apparatus and the method can separate particles of different sizes in a liquid (for example, biological cells and / or endosomes) with high selectivity, can separate the particles easily, quickly and cost-effectively, achieve a high yield, and have the advantage of being able to provide the separated particles in a therapeutically usable liquid (for example, plasma).
Background Art
[0002] The separation of particles of different sizes in a liquid (for example, a bio-suspension containing biological cells and / or pathogens) is of great concern for many problems in medical technology (for example, the availability of blood cell products, the clinical laboratory analysis of individual components, and / or the processing of cell products for cell therapy measures).
[0003] Two basic principles for separation have been particularly established. The first basic principle is to separate the particles present in a liquid based on their different specific gravities or on their different mechanical properties (for example, deformability and / or their orientation in the flow of the liquid). Generally, for this purpose, filtration and / or centrifugation are used. The second basic principle is to separate the particles based on their different surface properties. In the case of cells, viruses, and / or endosomes as particles, these different surface properties are caused by different molecules (for example, proteins, lipids, and / or sugars) being exposed on the surfaces of these particles.
[0004] Apparatuses and methods for separating particles of different sizes in a liquid from each other are already known in the prior art.
[0005] Firstly, centrifugation is known. This is used, for example, to purify whole blood and is essentially used to produce therapeutic blood products. Different types of separation are distinguished, and these always consist of a sequence of multiple centrifugation steps. Typically, whole blood is centrifuged directly in a blood collection bag and then distributed in a separate machine through hoses into multiple bags connected to this bag. The final products are red blood cells (so-called "concentrated RBCs"), platelet-rich or platelet-poor plasma, platelets, and peripheral blood mononuclear cells (so-called "PBMCs"). The biggest drawback of this established method is the low purity of the final products achieved, particularly the PBMCs present in the so-called "buffy coat".
[0006] Secondly, density gradient centrifugation is known. For example, density gradient centrifugation of whole blood is primarily used in the diagnostic field. This is because the separation medium used in this method cannot be completely separated at the end of the method, and the provided separated cell suspension becomes unsuitable for therapeutic use. Generally, the method is carried out similarly to centrifugation. However, prior to this method, the separation medium is in the lower layer of blood, and its density is between that of PBMCs and that of erythrocytes and granulocytes. As a result, a separation phase is generated during the separation between the erythrocyte / granulocyte phase and the PBMC phase, which can enhance the separation of PBMCs. Usually, the desired final product of the separation is PBMCs. The disadvantages of this method are low yield, as well as low purity of the final product and complicated handling when pipetting the cells.
[0007] Thirdly, microfluidic separation using microarrays is known. In these methods, the separation of blood cells is achieved by the flow of a blood cell suspension through a micromesh integrated into a microfluidic cartridge. High purity can be achieved with respect to the separation of PBMCs using such cartridges. However, the drawbacks of this method are the complex activation and the very long process time (the separation time by microfluidics is about 3 hours for 400 mL of whole blood).
[0008] Fourth, plasma apheresis is known as a continuous centrifugation process. During blood collection, the blood is continuously mixed with an anticoagulant and pumped into a rotating centrifuge. Cells separated from the plasma are collected, isotonically balanced with saline solution, and then returned to the patient via a further pump. The biggest drawback of this method is that it can only continuously separate two fractions of blood, and therefore cannot perform fine division into three or more cell fractions.
[0009] Fifth, filtration is known. These methods typically use filter membranes for plasma separation or filter membranes for separating cell fractions such as PBMCs. The filter membranes used are ideally placed directly in a container to collect the liquid containing particles passing through the membrane. Furthermore, separation containers are known that use a cascade of filters to separate different fractions from each other. There is also a known method of introducing porous microcubes into a suspension and encapsulating cells in the microcubes. The filtration methods currently known have the drawback that they can only separate liquid particles very coarsely, meaning that they can ultimately only separate into two types of particles: particles with a diameter smaller than the pore size of the filter membrane and particles with a diameter larger than the pore size of the filter membrane. Achieving higher selectivity requires further measures (e.g., absorption of specific particles in the microcubes) which are time-consuming, costly, and reduce the yield of separated cells. [Overview of the project] [Problems that the invention aims to solve]
[0010] Moving forward, the object of the present invention is to provide an apparatus and method for separating particles of different sizes in a liquid that does not have the drawbacks of the prior art. In particular, the apparatus and method can separate particles of different sizes in a liquid (e.g., living cells and / or endosomes) from each other with high selectivity, and provide the separated particles in a therapeutically usable liquid easily, quickly, and cost-effectively, in high yield. Furthermore, the use of the apparatus is provided.
[0011] The objective is achieved by an apparatus having the features of claim 1, a method having the features of claim 13, and a use having the features of claim 15. Dependent claims illustrate advantageous embodiments. [Means for solving the problem]
[0012] The present invention provides an apparatus for separating particles of different sizes in a liquid. The apparatus is a) A container for receiving liquid, b) At least one filter element having a top surface, a bottom surface, and at least one side connecting the top surface and the bottom surface, Equipped with, The filter element includes through holes having a defined hole diameter. The container is divided into an upper section in the direction of the top surface of the filter element and a lower section in the direction of the bottom surface of the filter element, and the filter element is positioned in the container such that if liquid particles in the upper section pass through the filter element, only the liquid particles in the upper section can reach the lower section. The upper compartment of the container has an opening for receiving liquid containing particles. The device is characterized by comprising means for changing the pore diameter of the pores of the filter element.
[0013] The apparatus according to the present invention has the advantage of being able to separate particles of different sizes in a liquid (e.g., living cells and / or exosomes / endosomes) from one another with high selectivity. High selectivity is provided by means of the apparatus suitable for changing the pore size of the filter element. By using this means, the pore size can be intentionally changed (i.e., increased) during the separation process so that particles of different particle sizes (i.e., in particular, smaller particles first, then larger particles) pass through the filter element in a continuous manner. Thus, the apparatus can sequentially release different particles into the lower compartment of the apparatus's container. Each time the pore size of the filter element changes, each liquid containing the passed particles can be removed from the lower compartment so that at the end of the separation process, there are several separated liquids containing particles of different particle sizes. The pore size can be changed not only stepwise but also linearly so that the apparatus can achieve very high selectivity.
[0014] Furthermore, the particles of the starting liquid can be easily, quickly, and cost-effectively separated from each other with high yield. The ease, speed, and low cost stem from the fact that only one apparatus is used to separate the liquid particles, i.e., there is no need to use multiple different apparatuses successively to achieve particle separation. This also results in a higher particle yield because there are fewer contact surfaces on which particles can adsorb during the particle separation process. In addition, the separation method can be performed very quickly using the apparatus of the present invention because a further cleaning step of the apparatus is unnecessary. Moreover, since it is composed of inexpensive parts, the apparatus can be provided in a cost-effective manner. The expandability of the apparatus is another advantage, namely, the suitability of the apparatus for receiving very large volumes of liquid and separating particles according to size when the container (especially its upper compartment) is expanded accordingly.
[0015] A further advantage of the device is that, after separation, the particles can remain in the soluble fraction of the starting fluid. In other words, the device allows the soluble fraction of the starting fluid, which contains particles of different sizes at the start of the separation process, to remain unchanged at the end of the separation process. This is a significant advantage if blood serves as the starting fluid containing the particles—that is, if plasma is the starting fluid containing blood cells and exosomes as particles—because the separated particles can remain in the plasma. Plasma is a therapeutically usable fluid, for example, because it is suitable for transfusion.
[0016] The apparatus may be characterized in that the means for changing the diameter of the holes in the filter element is suitable for applying a force to the filter element from the center of the surface of the filter element toward the edge of the surface of the filter element, or in the opposite direction. The diameter of the holes in the filter element can be increased or decreased by the action of such force.
[0017] Means for changing the hole width of a filter element may include a centrifuge and include at least one body connected to the outside of at least one side of the filter element in a force-fit manner, or at least two bodies, each connected to the outside of two opposing sides of the filter element in a force-fit manner. The at least one body is characterized by being suitable for applying a compressive or tensile force to at least one side of the filter element by changing the rotational speed of the centrifuge so that the holes of the filter element are compressed or expanded. Thus, the mechanism of operation of this means is based on a tensile or compressive force acting on the side of the filter element that occurs as a function of gravity, which is successively adjustable by the rotational speed of the centrifuge. Such means make it possible to adjust the hole diameter more quickly and easily than when using means to achieve any hole diameter, for example, by applying a mechanically adjustable pressure to at least one filter element (e.g., by applying a mechanically adjustable force to at least one filter element by a screw of a clamping device).
[0018] Furthermore, the means for changing the pore width of the filter element's pores may include a centrifuge and may include at least one body, preferably several bodies. The bodies are positioned on the upper surface of the filter element and / or within the filter element and, particularly preferably, have a higher specific density and / or higher charge than the particles to be separated. Preferably, at least one body, particularly preferably several bodies, are embodied as nanoparticles. Here, in particular, the nanoparticles are positioned around the pores of the filter element. At least one body is characterized by being suitable for applying compressive or tensile force to the pores of the filter element by changing the rotational speed of the centrifuge to compress or expand the pores of the filter element. Thus, the mechanism of action of this means is based on a tensile or compressive action expressed as a function of gravity acting locally on the pores of the filter element. Here, the level of gravity can also be adjusted by changing the rotational speed of the centrifuge. By such means, for example, by applying a mechanically adjustable pressure to at least one filter element (e.g., by applying a mechanically adjustable force to at least one filter element using a screw in a clamping device), the hole diameter can be adjusted more quickly and easily than when using means to achieve any hole diameter. An advantage of this embodiment compared to the embodiments described above is that at least one body is partially positioned within the container, thereby allowing the device to have a more compact design than when at least one body is connected to the outside of at least one side of the filter element by a force-fitting method.
[0019] Separately, the means for changing the hole width of the holes in the filter element may include a voltage source and may include at least one, optionally at least two, conductive layers. Here, the voltage source is connected by electrical conduction to at least one, optionally at least two conductive layers, preferably the at least two conductive layers are arranged on two opposing surfaces of at least one side of the filter element and electrically insulated from each other. The voltage source is characterized by being suitable for applying compressive or tensile force to at least one, optionally at least two conductive layers by changing the voltage to compress or expand the holes in the filter element. Such means allow for quicker and easier adjustment of the hole diameter than when using means to achieve any hole diameter, for example, by applying mechanically adjustable pressure to at least one filter element (e.g., by applying a mechanically adjustable force to at least one filter element by a screw of a clamping device). In this embodiment, the apparatus may include a centrifuge. One advantage is that the centrifugal force of the centrifuge accelerates the passage of the liquid and particles through at least one filter element, allowing for faster separation of particles in a liquid than gravity alone. The voltage source can be configured to apply a voltage in the range of 500 to 4000 V to at least one electrical conduction layer, and optionally, at least two electrical conduction layers.
[0020] In this embodiment, the filter element preferably comprises or consists of an electroactive material and / or piezoelectric material having through-holes. Particularly preferably, the material comprises or consists of an electroactive polymer and / or piezoelectric polymer, in particular a magnetoviscous elastomer and / or piezoelectric elastomer. This may be an elastomer containing embedded magnetic and / or piezoelectric nanoparticles (selected from the group consisting of, for example, silicone elastomers, thermoplastic elastomers, and combinations thereof). According to the present invention, the term "nanoparticles" should be understood to mean particles having a diameter of 1 nm to 100 μm as measured by an electron microscope. Therefore, the present understanding of the term "nanoparticles" also includes "fine particles" when the term "fine particles" is assumed to have a diameter of 1 μm to 100 μm. The magnetic particles and / or piezoelectric particles can be selected from the group consisting of lithium niobate, lithium tantalate, and combinations thereof.
[0021] In a preferred embodiment, the apparatus includes a control unit configured to control means for changing the diameter of the holes in the filter element.
[0022] Preferably, the control occurs in a manner such as changing the centrifugal velocity of a centrifuge, which is a means for changing the pore size of the pores of the filter element, preferably increasing the centrifugal velocity in stages during the separation of particles in a liquid. Here, in particular, the increase occurs automatically over time or manually by user input.
[0023] Furthermore, the control unit can be configured to control the means for changing the diameter of the holes in the filter element by changing the voltage of the voltage source of the means for changing the diameter of the holes in the filter element, preferably by gradually decreasing the voltage during the process of separating particles in the liquid. In particular, this decrease occurs automatically over time or manually by user input.
[0024] Furthermore, the control unit can be configured to control means for changing the pore diameter of the filter element in such a way that the pore diameter of the filter element is changed within the range of 100 nm to 100 μm. The pore diameter within this range is advantageous for the separation of blood particles, that is, the separation of blood cells and exosomes present in the blood.
[0025] Furthermore, the control unit can be configured to control means for changing the pore diameter of the filter element in a method of gradually changing the pore diameter of the filter element over time, automatically or manually by the input of the user of the device, to a larger diameter, preferably from a pore diameter of 200 nm to a pore diameter of 20 μm or more, particularly preferably (advantageously for the retention of exosomes) from a pore diameter of 200 nm to (advantageously for the retention of platelets) a pore diameter of 3 μm, (advantageously for the retention of red blood cells) a pore diameter of 4 - 7 μm, (advantageously for the retention of PBMCs) a pore diameter of 8 - 12 μm, (advantageously for the retention of macrophages, tissue cells, and circulating tumor cells) from a pore diameter of 20 μm to (preferably stepwise so that macrophages, tissue cells, and circulating tumor cells can pass through) a pore diameter of 100 μm.
[0026] In a preferred embodiment, the device includes n additional filter elements, which are arranged on at least one filter element in the direction of the upper compartment of the container and in each case have through-holes with a defined pore diameter. Here, the defined pore diameter of the n filter elements is larger than the defined pore diameter of at least one filter element, and in each of the n filter elements, the closer it is to the upper compartment of the container, the larger it is. Also, n is an integer greater than or equal to 2, particularly preferably an integer greater than or equal to 3, and in particular an integer in the range of 4 to 10. Except for the diameter of the holes, each of the n additional filter elements can have one or more or all of the characteristics of at least one filter element of the device. The n additional filter elements create a so-called "deep bed filter", that is, among the n filter elements, a filter that can retain liquid particles according to its size, that is, a filter that cannot reach the filter elements further arranged in the direction of the lower compartment. The advantage of this "deep bed filter" is that it can avoid clogging of the pores of the filter elements.
[0027] The device can comprise at least one second filter element. The second filter element is arranged on the filter element in the direction of the upper compartment of the container and has through-holes with a second defined pore diameter that is larger than the pore diameter of the filter element. Except for the diameter of the holes, at least one second filter element can have one or more or all of the characteristics of at least one filter element of the device. Before increasing the pore diameters of these two filter elements, for example, a first group of smaller particles can be arranged within at least one filter element (= low), and a second (larger) group of particles can be arranged within the second filter element (= high). Only after the corresponding increase in the pore diameter can the second group of particles pass through at least one filter element so as to finally reach the lower compartment of the device.
[0028] Optionally, the apparatus may include at least one third filter element, which is located opposite the filter element of the second filter element and has through-holes with a third defined pore diameter larger than the pore diameter of the second filter element. Except for the pore diameter, at least one third filter element may have one or more or all of the characteristics of at least one filter element of the apparatus. The additional third filter element enhances the “deep filter”. Before increasing the pore diameters of these three filter elements, a first small particle group may be placed in at least one filter element, a second larger particle group in the second filter element, and a third even larger particle group in the third filter element. Only after the corresponding increase in pore diameter can the second particle group pass through at least one filter element, and optionally, the third particle group may pass through the second filter element (also optionally through at least one filter element) so that it eventually reaches the lower compartment of the apparatus. The advantage of avoiding clogging of the filter element pores is even more pronounced in this embodiment.
[0029] Preferably, at least one filter element of the apparatus, preferably each filter element, contains or consists of fibers having through holes.
[0030] Preferably, at least one filter element of the apparatus, preferably each filter element, comprises or consists of an elastic material, preferably an elastic polymer having through holes.
[0031] Furthermore, it is preferable that at least one filter element of the device, preferably each filter element, contains or is made of an electroactive material having through-holes, preferably an electroactive polymer. The advantage of this is that the pore size of the filter element can be controlled by applying a voltage. This allows for quick and fine adjustment of the pore size.
[0032] Separately, at least one filter element of the device, preferably each filter element, may include or consist of a piezoelectric material having through holes. The advantage of this is that the hole diameter of the filter element can be controlled by applying a voltage. This allows for quick and fine adjustment of the hole diameter.
[0033] At least one filter element of the apparatus, preferably all filter elements, may contain or be made of a material selected from the group consisting of silicone elastomer, thermoplastic elastomer (TPE), magnetoviscous elastomer, piezoelectric elastomer, thermoplastic urethane (TPU), and combinations thereof.
[0034] Furthermore, at least one filter element of the apparatus, preferably all filter elements, preferably comprises or consists of an elastomer and embedded (e.g., magnetic, piezoelectric, and / or gravity-sensitive) nanoparticles, or a composite material thereof. The advantage is that, in the case of magnetic and / or piezoelectric particles (nanoparticles), the pore size of the filter element can be controlled by applying a voltage, and in the case of gravity-sensitive nanoparticles, it can be controlled by applying an accelerating force (e.g., centrifugal force). In these cases, the pore size can be quickly and finely adjusted. According to the present invention, the term "gravity-sensitive" means, in particular, that the particles are 2 g / cm³ 3 It should be understood that this means having a specific density greater than or equal to the above. According to the present invention, the term "nanoparticle" should be understood to mean a particle having a diameter of 1 nm to 100 μm as measured by an electron microscope (i.e., if a diameter of 1 μm to 100 μm is assumed for the term "fine particle", this understanding also includes "fine particle"). Gravity-sensitive nanoparticles can be selected from the group consisting of metal particles, coated metal particles (e.g., metal particles coated with ceramic material), ceramic particles, and combinations thereof.
[0035] Furthermore, at least one filter element of the apparatus, preferably all filter elements, may include or consist of knitted fabric made from woven or fiber material. The woven and / or knitted material does not need to have elastic properties (at the molecular level). The woven and / or knitted material can be selected from the group consisting of PES, PET, PC, PMMA, COC, nylon, glass fiber, PVDF, PP, and combinations thereof.
[0036] Separately, at least one filter element of the apparatus, preferably all filter elements, may include or consist of a material embodied as an (asymmetrical) solid foam. If several filter elements are present, it is preferable that some of these filter elements include or consist of a solid foam. Here, particularly preferably, the different pore sizes of the individual filter elements transition steadily to each other within the solid foam, so that only the theoretical layers of the individual filter elements exist within the solid foam. The material of the solid foam can be selected from the group consisting of silicone elastomers, thermoplastic elastomers (TPEs), magnetoviscous elastomers, piezoelectric elastomers, thermoplastic urethanes (TPUs), and combinations thereof. Alternatively, the material of the solid foam may preferably include or consist of a composite material containing or consisting of elastomers and (e.g., magnetic, piezoelectric, and / or gravity-sensitive) nanoparticles embedded therein. Separately, the material of the solid foam can be selected from the group consisting of PES, PET, PC, PMMA, COC, nylon, glass fiber, PVDF, PP, and combinations thereof.
[0037] Furthermore, at least one filter element of the apparatus, preferably each filter element, can have a width greater than 250 μm, preferably 500 μm or more, particularly preferably 1 mm or more, most preferably 2 mm or more, and especially in the range of 3 mm to 10 mm from the top surface to the bottom surface.
[0038] At least one filter element of the apparatus, preferably each filter element, may include a coating suitable for reversibly binding specific particles of a liquid. Preferably, the coating contains or consists of a material suitable for influencing by means of changing the pore size of the pores of the filter element in a manner that disengages specific particles. Particularly most preferably, the material contains or consists of an electroactive material, in particular an electroactive polymer. Also preferably, the coating is placed on the top, bottom, and / or inner surface of the pores of at least one filter element, and particularly preferably, on the top, bottom, and / or inner surface of the pores of all filter elements of the apparatus. The advantage of the coating is that it can retain specific particles regardless of size and then selectively release such particles (especially particles with very small particle sizes). In this way, selectivity can be further increased, and in particular, particles with very small particle sizes can be separated more reliably.
[0039] The lower compartment of the device's container may be equipped with means for drawing liquid from the lower compartment, preferably a valve, and more preferably an acceleration-sensitive valve and / or a voltage-switching valve. In particular, the control unit of the device can be configured to open and close the means for drawing out the liquid automatically over time or manually by user input. In any case after the pore size of the filter element has changed during the separation process, there is the advantage that the liquid containing particles present in the lower compartment can be isolated, i.e., removed from the lower compartment. The removal of each of these liquids can be done intentionally by applying a certain acceleration and / or voltage to the valve, and / or manually or automatically. Automatic removal is associated with less user effort and is therefore more convenient and less susceptible to errors.
[0040] The apparatus containers can be selected from a group consisting of centrifuge tubes, blood collection syringes, blood donation bags, culture bags for biotechnology production of pharmaceuticals, bioreactors for biotechnology production, sample containers, culture vessels, and combinations thereof.
[0041] Particles suitable for separation by the apparatus can be selected from the group consisting of vesicles, viral particles, and living cells, preferably from the group consisting of vesicles, viral particles, and living cells from blood, and particularly preferably from the group consisting of endosomal vesicles, exosomal vesicles (exosomes), viral particles, liposomes, platelets, red blood cells, white blood cells, and combinations thereof.
[0042] Furthermore, the present invention provides a method for separating particles of different sizes in a liquid. The method is: a) A step of providing an apparatus according to the present invention, b) The step of adjusting the holes in the filter element of the apparatus so that particles do not pass through the filter element, or particles up to a desired particle size pass through the filter element, c) The step of filling the upper compartment of the apparatus container with a liquid containing particles of different sizes, d) Preferably, the step of moving the liquid through a filter element using means selected from the group consisting of a centrifuge, a pump, and a combination thereof, e) A step of isolating the liquid containing the particles that have passed through from the lower compartment of the apparatus container, f) The step of increasing the pore size of the pores in the filter element of the apparatus so that particles up to a desired larger pore size can pass through the filter element, g) Preferably, the step of optionally filling the upper compartment of the apparatus container with a particle-free liquid, h) Preferably, the step of moving the liquid through a filter element using means selected from the group consisting of a centrifuge, a pump, and a combination thereof, i) A step of isolating the liquid containing the particles that have just passed from the lower compartment of the apparatus container, j) Preferably, the step of causing the reversible bonding of particles to the coating of at least one filter element of the apparatus to be released by affecting the means for changing the pore size of the pores of the filter element, k) Optionally, repeat steps g) to j) until all the particles of the liquid are present in multiple liquids separated according to their size. Includes.
[0043] The method can be characterized in that an increase in the pore diameter of the filter element occurs by increasing the centrifugal speed of the centrifuge used to change the pore diameter of the filter element.
[0044] Furthermore, the method can be characterized in that an increase in the diameter of the holes in the filter element occurs by decreasing the voltage of the voltage source of the means for changing the diameter of the holes in the filter element.
[0045] The present invention further describes the use of the apparatus for separating particles of different sizes present in a liquid. The use of the apparatus preferably includes isolating one or more hematological fractions from blood to provide hematological fractions for diagnostic purposes and / or to manufacture blood products, in particular to create cell therapeutics. The use of the apparatus may also include isolating bacterial cells from blood. Furthermore, the use of the apparatus preferably includes isolating exosomes from blood, serum, or biosuspensions to provide exosomes for diagnostic purposes and / or to manufacture vaccines (e.g., liposomal vaccines). Separately, the use of the apparatus may include isolating tissue cells from mixed tissue cell fractions. Furthermore, the use of the apparatus may include isolating cells from a mixed cell suspension derived from a bioreactor. Here, preferably, the cells are selected from the group consisting of plant cells, animal cells, human cells, bacterial cells, yeast cells, and combinations thereof. [Brief explanation of the drawing]
[0046] [Figure 1] Embodiment 1 of the apparatus according to the present invention is shown. [Figure 2] Embodiment 2 of the apparatus according to the present invention is shown. [Figure 3] Embodiment 3 of the apparatus according to the present invention is shown. [Figure 4] The separation of particles of different sizes in a liquid using the apparatus according to the present invention is schematically shown. [Modes for carrying out the invention]
[0047] The subject matter of the present invention will be described in more detail based on the following figures and examples, without limiting it to the specific embodiments shown in this disclosure.
[0048] Figure 1 shows Embodiment 1 of the apparatus according to the present invention. The apparatus comprises a container 1 for receiving liquid and at least one filter element 2 including a top surface 3, a bottom surface 4, and at least one side surface 5 connecting the top surface 3 and the bottom surface 4. The filter element 2 includes through holes 6 having a defined pore diameter. The filter element 2 divides the container 1 into an upper section 7 in the direction of the top surface 3 of the filter element 2 and a lower section 8 in the direction of the bottom surface 4 of the filter element 2, and is arranged in the container 1 such that if liquid particles in the upper section 7 pass through the filter element 2, only those particles can reach the lower section 8. The upper section 7 of the container 1 has an opening 9 for receiving liquid containing particles. The apparatus is characterized by comprising means for changing the pore diameter of the holes 6 of the filter element 2. In this embodiment, these means comprises a centrifuge (not shown) and a body 11 for applying compressive or tensile force to the side wall 5 of the filter element 2. Here, the main body 11 is connected via a cord using a force-fitting method to the outside of at least one side 5 of the filter element 2, which is deflected by the deflection roller 12 provided on the attachment 13. As the centrifugal force of the centrifuge increases, the force acting on the main body 11 increases, and the tensile force acting on the side wall 5 of the filter element 2 increases. As a result, the holes 6 of the filter element 2 widen.
[0049] Figure 2 shows Embodiment 2 of the apparatus according to the present invention. The apparatus comprises a container 1 for receiving liquid and at least one filter element 2 including a top surface 3, a bottom surface 4, and at least one side surface 5 connecting the top surface 3 and the bottom surface 4. The filter element 2 has through holes 6 with a defined pore diameter. The filter element 2 divides the container 1 into an upper section 7 in the direction of the top surface 3 of the filter element 2 and a lower section 8 in the direction of the bottom surface 4 of the filter element 2, and is positioned in the container 1 such that only liquid particles in the upper section 7 can reach the lower section 8 if they pass through the filter element 2. The upper section 7 of the container 1 has an opening 9 for receiving liquid containing particles. The apparatus is characterized by comprising means for changing the pore diameter of the holes 6 of the filter element 2. In this embodiment, these means comprises a centrifuge (not shown) and at least two bodies 10 for applying compressive or tensile forces to the holes of the filter element. Here, at least two bodies 10 are embodied as nanoparticles positioned on the opposite side of the holes of the filter element. As the centrifugal force of the centrifuge increases, the force acting on at least two of the main bodies 10 increases, and the tensile force acting on the side edges of the holes 6 of the filter element 2 increases. As a result, the holes 6 of the filter element 2 widen.
[0050] Figure 3 shows Embodiment 3 of the apparatus according to the present invention. The apparatus comprises a container 1 for receiving liquid and at least one filter element 2 including a top surface 3, a bottom surface 4, and at least one side surface 5 connecting the top surface 3 and the bottom surface 4. The filter element 2 has through holes 6 with a defined hole diameter. The filter element 2 divides the container 1 into an upper section 7 in the direction of the top surface 3 of the filter element 2 and a lower section 8 in the direction of the bottom surface 4 of the filter element 2, and is positioned in the container 1 such that only liquid particles in the upper section 7 can reach the lower section 8 if they pass through the filter element 2. The upper section 7 of the container 1 has an opening 9 for receiving liquid containing particles. The apparatus is characterized by comprising means for changing the hole diameter of the holes 6 of the filter element 2. In this embodiment, this means comprises a voltage source 14 and at least one electrically conductive layer on the side wall 5 of the filter element 2. When the voltage of the voltage source is reduced, the compressive force on the side wall 5 of the filter element 2 is reduced. This widens the holes 6 of the filter element 2.
[0051] Figure 4 schematically illustrates the separation of particles of different sizes in a liquid using the apparatus according to the present invention. Here, the means for changing the pore width of the filter element's holes is provided with a voltage source. The voltage source is connected to at least one electrically conductive layer on the side of the filter element. No voltage is applied to the electrically conductive layer, or only a low voltage is applied, to keep the average pore diameter of the holes 6 of the first filter element 2 and the holes 16 of the second filter element 15 as small as possible. A liquid (suspension) containing a first group of particles 18 having a first size and a second group of particles 19 having a second size larger than the first size is added to the upper compartment 7 of the apparatus's container (Figure 4A). By gravity or by the action of a centrifuge and / or pump, the first group of particles 18 passes through the second filter element 15 and reaches the first filter element 2, where it is held. Here, the second group of particles 19 cannot reach the first filter element 2 and is already held by the second filter element 15 (Figure 4B). When the voltage applied by the voltage source is increased, the holes 6 of the first filter element 2 and the holes 16 of the second filter element 15 are widened so that the first group of particles 18 can pass through the first filter element 2 and be collected in the lower compartment 8 of the device's container, and the second group of particles 19 can reach the first filter element 2 and be held there (Figure 4C). After removing the liquid containing the first group of particles 18, the holes 6 of the first filter element 2 are widened further, and the voltage applied by the voltage source is increased further so that the second group of particles 19 can also pass through the first filter element 2 and be collected in the lower compartment 8 of the device's container (Figure 4D). As a result, the first group of particles 18 can be separated from the second group of particles 19 in stages.
[0052] Example 1 - Apparatus comprising a centrifuge and at least one body for changing the pore size In the first selection step, which is characterized by a first centrifugal acceleration, liquid containing particles that can pass through the pores of the filter element during the first centrifugal acceleration flows from the upper compartment to the lower compartment of the container, while liquid containing particles that cannot pass through the pores of the filter element under these conditions remains in the upper compartment of the container. Subsequently, liquid containing particles located in the lower compartment is removed from the lower compartment.
[0053] In the second selection step, a second centrifugal acceleration greater than the first is applied, which results in the action of at least one body increasing the pore size of the filter element. As a result, liquids containing larger particles can pass through the filter element and be collected in the lower compartment of the container, where they can be isolated.
[0054] Example 2 - Apparatus equipped with a voltage source for changing the hole diameter In a first selection step, characterized by a first voltage applied by a voltage source, liquid containing particles that can pass through the pores of the filter element while the voltage is present flows from the upper compartment to the lower compartment of the container, while liquid containing particles that cannot pass through the pores of the filter element under these conditions remains in the upper compartment of the container. Subsequently, liquid containing particles located in the lower compartment is removed from the lower compartment.
[0055] In the second selection step, a voltage lower than the first voltage is applied, which results in an increase in the pore size of the filter element. As a result, liquids containing larger particles pass through the filter element and are collected in the lower compartment of the container, where they are isolated. [Explanation of symbols]
[0056] 1: Container 2: Filter element 3: Top of the filter element 4: Bottom of the filter element 5: Aspects of filter elements 6: Through-holes of filter elements having defined pore diameters 7: Upper compartment of the container 8: Lower compartment of the container 9: Opening of the upper compartment of the container 10: Body for applying compressive or tensile force to the holes of the filter element 11: Body for applying compressive or tensile force to the sidewall of the filter element 12: Deflection roller 13: Deflection roller attachment 14: Voltage source 15: Second filter element 16: Through-hole of the second filter element having a defined pore diameter 17: Means for drawing liquid from the lower compartment (e.g., a valve) 18: The first group of particles 19: A second group of particles larger than the first group of particles. Z: Center of the surface of the filter element R: The edge of the surface of the filter element.
Claims
1. A device for separating particles of different sizes in a liquid, a) A container for receiving the liquid, b) At least one filter element having a top surface, a bottom surface, and at least one side surface connecting the top surface and the bottom surface, Equipped with, The filter element includes a through hole having a defined hole diameter, The container is divided into an upper section in the direction of the top surface of the filter element and a lower section in the direction of the bottom surface of the filter element, and the filter element is arranged in the container such that if liquid particles in the upper section pass through the filter element, only those particles can reach the lower section. The upper section of the container has an opening for receiving a liquid containing particles, The apparatus includes means for changing the diameter of the through-hole of the filter element, The means for changing the hole width of the through hole of the filter element is, It is equipped with a centrifugal separator, and further, i) At least one member, which is connected to the outside of at least one side surface of the filter element by force fitting, and which is suitable for compressing or expanding the through hole of the filter element by applying a compressive or tensile force to at least one side surface of the filter element by changing the rotational speed of the centrifuge, and / or, ii) A plurality of members disposed on the upper surface of the filter element and / or within the filter element, which are suitable for compressing or expanding the through-holes of the filter element by applying compressive or tensile force to the through-holes of the filter element by changing the rotational speed of the centrifuge, Device.
2. The means for changing the diameter of the through-hole of the filter element is suitable for applying a force to the filter element from the center of the surface of the filter element toward the edge of the surface of the filter element, or in the opposite direction. The apparatus according to claim 1.
3. The apparatus according to claim 1 or 2, wherein the plurality of members have a higher specific density and / or a higher charge than the particles to be separated.
4. The apparatus according to claim 3, wherein the plurality of members are embodied as a plurality of nanoparticles.
5. The apparatus according to claim 4, wherein the nanoparticles are arranged around the through-holes of the filter element.
6. The device comprises a control unit, The control unit, i) The means for changing the diameter of the through-holes of the filter element, by changing the centrifugal speed of the centrifuge such that the centrifugal speed of the centrifuge increases in stages during the separation of particles in the liquid, and / or ii) Changing the diameter of the through-hole of the filter element to a range of 100 nm to 100 μm, and / or iii) The diameter of the through-hole of the filter element is changed in steps to a larger diameter, either automatically over time or manually by input from the user of the device. The means for changing the diameter of the through-hole of the filter element is configured to control such a method. The apparatus according to any one of claims 1 to 5.
7. The apparatus includes n further filter elements, the n further filter elements being positioned on at least one filter element in the direction of the upper compartment of the container, and in each case having through holes of a defined diameter, The defined pore diameters of the n filter elements are greater than the defined pore diameter of at least one filter element, and in each of the n filter elements, the pore diameters are greater the closer they are to the direction of the upper compartment of the container, where n is an integer of 2 or more. The apparatus according to any one of claims 1 to 6.
8. The at least one filter element of the apparatus is i) containing or consisting of fibers having through holes, ii) comprising or consisting of an elastic material having through holes, iii) comprising or consisting of an electroactive material having through holes, iv) A piezoelectric material having through holes, comprising or consisting of such material, v) A material selected from the group consisting of silicone elastomers, thermoplastic elastomers, magnetoviscous elastomers, piezoelectric elastomers, thermoplastic urethanes, and combinations thereof, comprising, and / or, vi) Composite materials comprising or consisting of elastomers and nanoparticles embedded therein, and / or vii) A material selected from the group consisting of PES, PET, PC, PMMA, COC, nylon, glass fiber, PVDF, PP, and combinations thereof, or a woven or knitted fabric made of such material, and / or viiii) A solid foam comprising, and / or consisting of, a material selected from the group consisting of silicone elastomers, thermoplastic elastomers, magnetoviscous elastomers, piezoelectric elastomers, thermoplastic urethanes, composite materials made of elastomers embedded with nanoparticles, PES, PET, PC, PMMA, COC, nylon, glass fiber, PVDF, PP, and combinations thereof, or a solid foam made of such materials. ix) Having a width greater than 250 μm from the top surface to the bottom surface, The apparatus according to any one of claims 1 to 7.
9. The at least one filter element of the apparatus includes a coating suitable for reversibly binding specific particles of a liquid, The aforementioned coating is i) A material comprising, and / or comprising, a means suitable for influencing by a means of changing the diameter of the through-holes of the filter element in a manner that breaks the bond with the specific particles. ii) Displaced on the top surface, bottom surface, and / or inner surface of the hole of the at least one filter element: The apparatus according to any one of claims 1 to 8.
10. The apparatus according to any one of claims 1 to 9, wherein the lower compartment of the container of the apparatus is provided with means for drawing liquid from the lower compartment.
11. The container is selected from the group consisting of centrifuge tubes, blood collection syringes, blood donation bags, culture bags for biotechnology production of pharmaceuticals, bioreactors for biotechnology production, sample containers, culture vessels, and combinations thereof. The apparatus according to any one of claims 1 to 10.
12. The apparatus according to any one of claims 1 to 11, wherein the particles are selected from the group consisting of vesicles, virus particles, and living cells.
13. A method for separating particles of different sizes in a liquid, a) the step of providing the apparatus according to any one of claims 1 to 12, b) The step of adjusting the holes in the filter element of the apparatus so that no particles pass through the filter element, or particles up to a desired particle size pass through the filter element, c) The step of filling the upper compartment of the container of the apparatus with a liquid containing particles of different sizes, d) A step of moving the liquid through the filter element using means selected from the group consisting of a centrifuge, a pump, and combinations thereof, e) The step of isolating the liquid from the lower compartment of the container of the apparatus, f) A step of increasing the pore diameter of the through-holes of the filter element of the apparatus so that particles up to a desired pore diameter can pass through the filter element, g) A step of moving the liquid through the filter element using means selected from the group consisting of a centrifuge, a pump, and a combination thereof, h) A step of isolating the liquid containing the particles that have just passed through from the lower compartment of the container of the apparatus, Methods that include...
14. i) The means for changing the diameter of the through-holes of the filter element increases the diameter of the through-holes of the filter element by increasing the centrifugal speed of the centrifuge, The method according to claim 13.
15. Use of the apparatus according to any one of claims 1 to 12 for separating particles of different sizes present in a liquid, i) Isolate one or more blood cell fractions from the blood, and / or ii) Isolate bacterial cells from the blood, and / or iii) Isolate exosomes from blood, serum, or biosuspension, and / or iv) Isolate tissue cells from the mixed tissue cell fraction, and / or v) Isolate cells selected from the group consisting of plant cells, animal cells, human cells, bacterial cells, yeast cells, and combinations thereof from the bioreactor-derived mixed cell suspension. For use.