Microfluidic device and method for separating cells

Abstract

The invention relates to a microfluidic device. Said device has at least one magnetic marker, which is designed to bind to surface proteins of cells (30, 31), at least one mixing region, which is designed to bring the at least one magnetic marker into contact with the cells (30, 31), and at least one separating region (14), into which the cells (30, 31) can be introduced and which has at least one magnet (15), preferably on an upper side (16). A method for separating cells by means of the microfluidic device comprises bringing cells (30, 31) into contact with at least one magnetic marker in the mixing region, sedimenting the cells (30, 31) in the separating region (14), applying a magnetic field in the separating region (14), and discharging cells (31) marked with the marker from the separating region by means of a fluid flow (61).

Description

[0001] Description

[0002] title

[0003] Microfluidic device and method for cell separation

[0004] The present invention relates to a microfluidic device. Furthermore, the present invention relates to a method for separating cells using the microfluidic device.

[0005] State of the art

[0006] For biological or diagnostic purposes, it is often necessary to separate isolated cells from cells that lack these characteristics based on certain features present on or within the cells. These features can, for example, be surface proteins on cells that are attributed to a tumor origin.

[0007] US 2003 / 0170613 A1 describes a method for detecting cells as targets in a sample, for example, in a microfluidic device. The cells are coupled to magnetic particles. They are then fixed, for example, in wells using a magnetic field, to distribute them evenly across the well bottom for easy analysis.

[0008] Disclosure of the invention

[0009] The microfluidic device, which is intended in particular for separating cells, comprises at least one magnetic marker configured to bind to cell surface proteins. At least one mixing region is configured to bring the at least one magnetic marker into contact with the cells. The cells brought into contact with the magnetic marker can be introduced into at least one separation region of the device, which is separate from the mixing region. Alternatively, the mixing region can simultaneously function as a separation region. This separation region has, preferably on its upper side, at least one magnet. The magnet is configured so that the magnetic field acting on the separation region can be varied. This can be achieved, for example, by using an electromagnet as the magnet.Alternatively, it is also possible that the magnet is a permanent magnet whose distance from the separation area can be changed.

[0010] The magnetic marker can be stored in a reservoir from which it can be introduced into the mixing area. However, it is also possible for the magnetic marker to be stored upstream in the mixing area.

[0011] A magnetic marker is any substance that is attracted to a magnetic field, in particular the magnetic field of the magnet of the microfluidic device. In particular, the magnetic marker is paramagnetic or superparamagnetic.

[0012] In a preferred embodiment of the microfluidic device, the magnetic marker comprises beads with a diameter in the range of 1 nm to 10 pm. The diameter is particularly preferably in the range of 10 nm to 500 nm. Beads with such a diameter can bind to diagnostically interesting cell types, such as bacteria, human cells, spores, and fungi, without significant steric hindrance. Binding can be achieved, in particular, by means of antibodies conjugated to the beads.

[0013] Furthermore, it is preferred that the microfluidic device contains at least two magnetic markers configured to bind to different cell surface proteins. Particularly preferably, it contains exactly two magnetic markers with, for example, different magnetizabilities. This has the advantage of enabling a distinction to be made between cells that have only one of the surface proteins, so that only one of the magnetic markers binds to them, and cells that have multiple surface proteins, so that multiple magnetic markers bind to them. This variant also makes it possible to distinguish between cell populations in which, for example, one population has the first magnetic marker and the second population has the second magnetic marker.Due to the different magnetizabilities of the two materials used, a separation of the two populations can be achieved.

[0014] The separation region preferably has wells in a lower side. The top side and the bottom side can be understood in particular as two partial regions of the separation region which are located opposite one another at a distance and wherein a fluid flow can pass through the separation region between the top side and the bottom. Such wells prevent cells sedimented there from being entrained by a fluid flow. For this purpose, it is particularly preferred that the depth of the wells lies in the range of 5 pm to 500 pm. The depth is very particularly preferably in the range of 8 pm to 50 pm. A cross-section of the wells can, for example, be round or square. Their structural width is particularly preferably in the range of 5 pm to 500 pm and very particularly preferably in the range of 8 pm to 100 pm. The structural width is understood to be the maximum width dimension of the wells in the plane of the separation region.

[0015] In the method for separating cells using the microfluidic device, the cells are first brought into contact with the at least one magnetic marker in the mixing region. The marker attaches to the surface of cells that have surface proteins complementary to the marker. The cells are then sedimented in the separation region. Sedimentation can be achieved by introducing a suspension of the cells into the separation region and allowing it to stand there without any flow through the separation region during sedimentation. Sedimentation then occurs under the influence of gravity. Sedimentation preferably takes place over a period in the range of 0.01 h to 12.0 h, particularly preferably over a period in the range of 0.2 h to 2.0 h.

[0016] A magnetic field is now applied to the separation area. Cells marked with the magnetic marker are moved by the magnetic field and can thus be separated from unmarked cells, in particular in the direction of the magnet. If the magnet is preferably located in an area above the underside, in particular on the top side, the cells marked with the magnetic marker are lifted from the underside towards the area, in particular towards the top side, while unmarked cells remain on the underside. Cells marked with the marker can then be removed from the separation area by means of a fluid flow. This fluid flow can be introduced into the separation area before, during or after the application of the magnetic field, in particular before the application of the magnetic field.The time between applying the magnetic field and initiating the fluid flow should not be so long that the labeled cells accumulate at the top of the separation area.

[0017] If the microfluidic device has wells on its underside into which the cells are sedimented, the fluid flow can only entrain those cells that have been lifted out of the wells by the magnetic field. If, however, the device does not have wells, it is preferable for the fluid flow to have a parabolic flow profile. Due to the low velocity vectors at the underside of the separation area, the sedimented cells there are not moved, while the cells lifted into the center of the separation area by the magnetic field are carried along by the fluid flow due to the larger velocity vectors there.

[0018] In a preferred embodiment of the method, the cells are brought into contact with at least two magnetic markers, particularly preferably with exactly two magnetic markers, which are designed to bind to different cell surface proteins. First cells have only one type of surface protein, and second cells have multiple types of surface proteins. A first application of a magnetic field in the separation region occurs with a first magnetic field strength. Second cells marked with multiple markers are then carried out of the separation region by means of the fluid flow, since even a low first magnetic field strength is sufficient to lift them from the underside of the separation region. This is followed by a second application of a magnetic field in the separation region with a second magnetic field strength that is greater than the first magnetic field strength.This allows the first cells to be lifted from the bottom. The first cells marked with only one marker are then removed from the separation area by the fluid flow, while the unmarked cells remain in the separation area.

[0019] The device and method thus make it possible to separate cells with a specific surface protein from those that do not. It is also possible to separate cells that have two different specific surface proteins, cells that have only one of the specific surface proteins, and cells that have none of the specific surface proteins into three fractions. The separated cells can be transferred to a further processing level in a subsequent process step to transport only the cells located there to a sampling point of the microfluidic device. Using the method, cell populations can be automatically separated from one another in order to remove a specific cell separation from the microfluidic device and, for example, analyze it externally using molecular diagnostic methods. Brief description of the drawings

[0020] Embodiments of the invention are illustrated in the drawings and are explained in more detail in the following description.

[0021] Figure 1 schematically shows a microfluidic device according to an embodiment of the invention.

[0022] Figure 2 shows schematically a cell which has been marked with a magnetic marker in an embodiment of the method according to the invention.

[0023] Figure 3 shows a flowchart of an embodiment of the method according to the invention.

[0024] Figure 4 shows a schematic sectional view of a separation region of an embodiment of the microfluidic device.

[0025] Figure 5 shows a schematic sectional view of a separation region of a microfluidic device according to another embodiment of the invention.

[0026] Figure 6 shows schematically a cell which was marked with two different magnetic marking substances in an embodiment of the method according to the invention.

[0027] Figure 7 shows a flowchart of another embodiment of the method according to the invention.

[0028] Figure 8 shows a schematic sectional view of a separation region of the microfluidic device according to an embodiment of the invention in one method step of the method according to the invention. Figure 9 shows a schematic view of a separation region of a microfluidic device according to an embodiment of the invention in another method step of the method according to the invention.

[0029] Embodiments of the invention

[0030] An embodiment of the microfluidic device 10 according to the invention is shown in Figure 1. This device has a filling area 11 that is designed to be filled with a cell suspension. The filling area 11 is connected to a mixing area 13 by means of a microfluidic channel 12. The mixing area 13 opens into a further microfluidic channel that functions as a separation area 14 and in which, in particular on its upper side, a magnet 15 is arranged. In the present embodiment, this is designed, for example, as an electromagnet. A magnetic marker 21 is arranged upstream of the mixing area 13. This can be, for example, antibodies conjugated to superparamagnetic beads with a diameter of, for example, 5 pm.

[0031] When cells 31 are guided through a filling area 11 and the microfluidic channel 12 into the mixing area 13, the magnetic marker 21 binds there to complementary surface proteins 41 of the cells 31. This is shown in Figure 2.

[0032] According to the flow diagram in Figure 3, in a first exemplary embodiment of the method according to the invention, after the start 50 of the method, the cells 31 are first filled 51 into the filling area 11, in order to then be brought into contact 52 with the magnetic marking substance 21 in the mixing area 13. This is followed by a further conveyance 53 of the cells 31 into the separation area 14 by means of a fluid flow. This fluid flow is then interrupted for one hour, for example, so that sedimentation 54 of the cells 31 in the separation area 14 is made possible. A fluid flow is then again introduced 55 into the separation area 14, and a magnetic field is created in the separation area 14 by means of the magnet 15. As shown in Figure 4, the magnet 15 is arranged on an upper side 16 of the separation area 14.In general, it is preferred if the magnet 15 is arranged in a region above the underside 17 in order to lift magnetic or magnetizable substances from the underside 17, in particular towards the top side 16. On the underside 17 of the separation region 14, in addition to the cells 31 marked with the magnetic marker 21, further unmarked cells 30 that do not have the surface protein 41 have sedimented. If the magnetic field is now applied, only the cells 31 marked with the magnetic marker 21 are lifted from the underside 17 towards the top side 16, and thus towards the magnet 15. The introduced fluid stream 61 has a parabolic flow profile. As a result, its velocity vectors near the underside 17 of the separation region 14 are small, so that the unmarked cells 30 sedimented there cannot be entrained by the fluid stream 61.The cells 31, which are lifted by the magnetic field into the center of the separation area 14, are exposed to the higher velocity vectors of the fluid flow 61 there and are discharged 57 from the separation area 14. In this way, the two types of cells 30, 31 are separated from each other. After discharge 57, the process is terminated 58.

[0033] In a second embodiment of the device according to the invention, its separation region 14 differs from the separation region 14 shown in Figure 4 in that it has wells 18 in its underside 17. These wells have, for example, a depth of 20 pm and a structural width of 30 pm. If the wells have a round cross-section, their structural width corresponds to their diameter plus approximately 100%. All cells 30, 31 sediment in wells 18 in step 54 of the method. The unlabeled cells 30 that are not lifted out of the wells 18 by the magnetic field cannot therefore be entrained by the fluid flow, regardless of the velocity of the fluid flow. It is therefore not necessary, as when using the first embodiment of the microfluidic device 10, to use a fluid flow 61 with a parabolic flow profile.Instead, a fluid flow 62 with a uniform flow profile can be used in the method when using the microfluidic device 10 according to the second embodiment.

[0034] In a second embodiment of the method according to the invention, a cell suspension is introduced into the microfluidic device in step 51. This cell suspension contains, in addition to the cells 30 that do not have surface proteins 41 complementary to the magnetic marker 21 and the cells 30 that do have these surface proteins 41, further cells 32 that, in addition to the surface proteins 41, have further diagnostically interesting surface proteins 42. In addition to the magnetic marker 21, which is complementary to the first surface proteins 41, in this embodiment of the method, a second magnetic marker 22, which is complementary to the second surface protein 42, is disposed upstream in the mixing region 13. Figure 6 shows the attachment of both magnetic markers 21, 22 to this type of cell 32.In order to separate all three types of cells 30, 31, 32 from one another, a modification of the method in steps 56 and 57 according to the first exemplary embodiment of the method is provided in Figure 7. Initially, only a magnetic field with a low first magnetic field strength is applied 56a. As shown in Figure 8, this is only sufficient to lift the cells 32 marked with both magnetic marking substances 21, 22 from the underside of the separation region 14, so that a removal 57a of these cells 32 from the separation region 14 is possible. The cells 31 marked with only one magnetic marking substance 21 interact so weakly with the magnetic field at the first magnetic field strength that they remain on the underside of the separation region 14. Subsequently, a magnetic field with a greater second magnetic field strength is applied 56b.As shown in Figure 9, this is now sufficient to lift even the cells 31 which are only marked with a magnetic marker 21 into the middle of the separation region 14 so that they can be carried out of the separation region 14 by the fluid stream 61 57b. The second embodiment of the method thus enables the separation of three different types of cells 30, 31, 32. Even if it was explained in Figures 8 and 9 by way of example using the first embodiment of the microfluidic device 10, the second embodiment of the method according to the invention can also be carried out with the second embodiment of the device 10, which has wells 18 in the underside 17 of the separation region 14.

Claims

Claims 1. A microfluidic device (10) comprising at least one magnetic marker (21, 22) configured to bind to surface proteins (41, 42) of cells (30, 31, 32), at least one mixing region (13) configured to bring the at least one magnetic marker (21, 22) into contact with the cells (30, 31, 32), and at least one separation region (14) into which the cells (30, 31, 32) can be introduced and which, preferably on a top side (16), has at least one magnet (15).

2. Microfluidic device (10) according to claim 1, characterized in that the magnetic marker (21, 22) is paramagnetic or superparamagnetic.

3. Microfluidic device (10) according to claim 1 or 2, characterized in that the magnetic marker (21, 22) comprises beads with a diameter in the range of 1 nm to 10 pm.

4. Microfluidic device (10) according to one of claims 1 to 3, characterized in that it contains at least two magnetic markers (21, 22) which are designed to bind to different surface proteins (41, 42) of cells (30, 31, 32).

5. Microfluidic device (10) according to one of claims 1 to 4, characterized in that the separation region (14) has wells (18) in a bottom side (17).

6. Microfluidic device (10) according to claim 5, characterized in that a depth of the wells (18) is in the range of 5 pm to 500 pm.

7. A method for separating cells by means of a microfluidic device (10) according to one of claims 1 to 6, comprising the following steps: bringing cells (30, 31, 32) into contact (52) with at least one magnetic marker (21, 22) in the mixing region (13), sedimenting (54) the cells (30, 31, 32) in the separation region (14), applying (56, 56a, 56b) a magnetic field in the separation region (14), and discharging (57, 57a, 57b) cells (31, 32) marked with the marker (21, 22) from the separation region by means of a fluid stream (61, 62).

8. Method according to claim 7, characterized in that the sedimentation (54) takes place within a period in the range of 0.01 h to 12.0 h.

9. Method according to claim 7 or 8, characterized in that the fluid flow (61) has a parabolic flow profile.

10. The method according to any one of claims 7 to 9, characterized in that the cells (30, 31, 32) are brought into contact with at least two magnetic markers (21, 22) which are designed to bind to different surface proteins (41, 42) of cells (31, 32), wherein first cells (31) have only one of the surface proteins (41) and second cells (32) have several of the surface proteins (41, 42), and wherein the method comprises the following steps: first application (56a) of a magnetic field in the separation region (14) with a first magnetic field strength, Discharging (57a) second cells (32) marked with a plurality of marking substances (41, 42) from the separation region (14) by means of the fluid flow (61, 62), second application (56b) of a magnetic field in the separation region (14) with a second magnetic field strength which is greater than the first magnetic field strength, Discharging (57b) first cells (31) marked with a marking substance (41) from the separation region (14) by means of the fluid flow (61, 62).

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