Apparatus and process for separating different cell types

Abstract

The invention relates to an apparatus for separating different cell types, comprising a rotatable chamber (10) having a first side (21) which comprises at least one first affinity reagent (31) for at least one first cell type and a second side (22) which is opposite the first side (21) and comprises at least one second affinity reagent (32) for at least one second cell type. A process for separating different cell types using the apparatus comprises introducing a suspension comprising at least one first cell type and at least one second cell type into the chamber (10) and rotating the chamber so that the first side (21) and the second side (22) are each situated at the bottom at least once.

Description

[0001] Description

[0002] title

[0003] Device and method for separating different cell types

[0004] The present invention relates to a device for separating different cell types. Furthermore, the present invention relates to a method for separating different cell types using the device.

[0005] State of the art

[0006] In cancer diagnostics, the quantification of circulating tumor cells (CTCs) is a promising predictor of therapy response or disease progression. These are tumor cells that have detached from the primary tumor and circulate in the bloodstream. These cells can then re-enter the tissue from the blood at another site and potentially form metastases.

[0007] To quantify CTCs, blood is drawn from a patient and examined for their presence. However, this examination is complicated by a large number of cells present in the blood that are undesirable for analysis and are referred to as background cells.

[0008] WO 2019 / 005967 A1 describes a method in which CTCs from a blood sample are bound in a sponge made of carbon nanotubes. By rotating the sponge, unbound CTCs are released into a culture medium.

[0009] Disclosure of the invention: In a first aspect, a device for separating different cell types is provided, comprising a rotatable chamber. On a first side of the chamber, at least one first affinity reagent for at least one first cell type is arranged. On a second side of the chamber opposite the first side, at least one second affinity reagent for at least one second cell type is arranged. The first cell type can, in particular, be a CTC cell type, and the second cell type can, in particular, be a background cell type.

[0010] This device enables the enrichment of CTCs on the affinity reagent of the first side and the binding of background cells to the affinity reagent of the second side, thus depleting the background cells in the chamber. The chamber's rotatability offers the advantage that a cell suspension containing different cell types, introduced into the chamber, can be alternately exposed to the first and second affinity reagents. This utilizes the fact that cells in the chamber sediment at its bottom due to gravity. By rotating the chamber, either the first or the second affinity reagent can be selectively positioned at the bottom.

[0011] Such a device can be designed as a microfluidic apparatus. It can be used for separating both living and fixed cells. The affinity reagents are arranged in cavities on the first and second sides of the chamber, respectively, so that minimal cell loss is expected.

[0012] The affinity reagents are primarily antibodies.

[0013] When separating cell types, it can be advantageous if cells bound to one of the affinity reagents can be selectively released from the respective wall of the chamber at a freely selectable time. For this purpose, one embodiment of the device provides that the first affinity reagent is preferably bound to first magnetic beads, and the first side has at least one first magnet configured to hold the first magnetic beads on the first side. The second affinity reagent is preferably bound to second magnetic beads. The second side has at least one second magnet configured to hold the second magnetic beads on the second side.Preferably, the first and second magnets are both electromagnets, which allows for the simple release of a cell type by switching off the respective electromagnet, thereby releasing the respective magnetic beads, along with the affinity reagents and cells bound to them, from the respective side of the chamber. However, it is also conceivable that the magnets are designed as permanent magnets that can be mechanically removed from their respective sides of the chamber to allow the release of the beads.

[0014] In a preferred embodiment of the device, the chamber has an inlet on a third side and an outlet on a fourth side. The third and fourth sides are, in particular, orthogonal to the first and second sides. The chamber is rotatable about an axis that passes through the third and fourth sides. The inlet allows a cell suspension to be introduced into the chamber in order to separate different cell types contained therein. The outlet allows cells that have not been bound to any of the affinity reagents to be flushed out of the chamber.

[0015] In another preferred embodiment of the device, it has a filter arranged between the first and second sides. The pore diameter of the filter is preferably selected such that blood cells can pass through it, while one of the cell types to be separated, in particular CTCs, is retained. If bead-coupled affinity reagents are used in the chamber that are not held against one side by a magnet, the filter can also be used to prevent unwanted mixing of the different beads. For this purpose, the pore diameter of the filter is smaller than the diameter of the beads.In addition to separation using affinity reagents, the filter also achieves separation based on cell size. Larger cells, such as CTCs not yet bound to affinity reagents, are retained by the filter, while smaller cells, such as leukocytes, can pass through to repeatedly come into close proximity with their corresponding affinity reagents and bind to them. The chamber's rotation prevents the filter from clogging.

[0016] In this embodiment of the device, it has an inlet on a third side, a first outlet on a fourth side between the first side and the filter, and a second outlet on the fourth side between the second side and the filter. If different cell types are separated based on cell size, the separated cells can then be selectively removed from the chamber via the two outlets. If bead-coupled affinity reagents are used in the chamber that are not held in place on one of the sides by a magnet, it is further preferred that the inlet and each of the outlets have an additional filter whose pore diameter is smaller than the diameter of the beads, in order to hold them inside the chamber.

[0017] It is further preferred that the chamber has two separable parts. The first part contains the first side, and the second part contains the second side. This makes it possible, after the separation of the cell types is complete, to separate the two parts of the chamber in order to examine the contents of one of the parts microscopically.

[0018] In another aspect, a method for separating different cell types using the device is provided. The method comprises introducing a suspension containing at least one first cell type and at least one second cell type into the chamber. This suspension is, in particular, a blood sample. To remove erythrocytes from the background before introducing the suspension, it is preferred that the blood sample first undergo selective erythrocyte lysis, be centrifuged, and then resuspended in a physiological buffer before being introduced into the chamber. After introduction, the chamber is rotated so that at least once the first side and at least once the second side are at the bottom. This brings cells sedimented at the bottom of the chamber into contact with the affinity reagent located at the bottom.In principle, this can be achieved by placing either the first or second side of the chamber at the bottom when introducing the suspension and then rotating the chamber once to swap the positions of the first and second sides. Preferably, however, the chamber is rotated multiple times so that the cells are repeatedly exposed to each of the affinity reagents, thus ensuring the most complete possible binding of the first cell type to the first affinity reagent and of the second cell type to the second affinity reagent.

[0019] In this context, the term "below" means that the first side is positioned along a vertical axis below the second side, and vice versa.

[0020] Once the rotation is complete and the cells of the first and second cell types are bound to the first and second affinity reagents, respectively, several further procedural steps can be performed to analyze the cell types. These procedural steps can be carried out individually or in combination.

[0021] In one analytical step, the first affinity reagent can be selectively separated from the first side and / or the second affinity reagent from the second side to release the first cell type and / or the second cell type into the interior of the chamber. If the affinity reagents are magnetically bound to their respective sides, this can be achieved by removing the magnetic field. If this is not possible, affinity reagents can be detached from their respective sides, for example, using UV light, especially if they are bound to the respective sides via an ortho-nitrobenzyl bridge. Separation of affinity reagents from their respective sides could also be achieved by introducing at least one protease into the chamber, which digests the affinity reagents in the form of antibodies at surface receptors.If the affinity reagents are coupled to single-stranded nucleic acids and the respective side of the chamber to which they are bound has a corresponding reverse complementary nucleic acid, they are bound to that side via hybridization. This interaction can be reversed by heating or by adding a nuclease. It is particularly preferred that the first affinity reagent is bound to the first side via a different binding mechanism than the second affinity reagent is bound to the second side. This makes it possible to selectively separate only one of the affinity reagents from its side of the chamber.

[0022] Another analytical step involves fluid exchange within the chamber. This is achieved by introducing a liquid through the chamber inlet, flushing out cells that are not bound to either the first or second affinity reagent. This is particularly advantageous before selectively lysing a cell type or selectively separating an affinity reagent from its chamber side, thus pre-removing interfering background cells.

[0023] If the first side is made of gold, the cells of the corresponding cell type can be examined using surface plasmon resonance spectroscopy (SPR). If the first side is made of glass, the cells of the first cell type can be examined using transmitted light microscopy and / or fluorescence microscopy. This may be preceded by introducing the dye through the inlet into the chamber to stain the cells.

[0024] A further analytical step involves separating the chamber into two parts, with the first part containing the first side and the second part containing the second side. This allows the cells fixed to the first side to be examined microscopically or using surface resonance plasmonography after separation. For this purpose, the first side is typically made of gold or silicon.

[0025] Brief description of the drawings: Exemplary embodiments of the invention are shown in the drawings and are explained in more detail in the following description.

[0026] Figure 1 shows a schematic cross-sectional view of a first embodiment of the device according to the invention.

[0027] Figure 2 schematically shows the sequence of an embodiment of the method according to the invention using the device according to the first embodiment of the invention.

[0028] Figure 3 shows a schematic cross-sectional view of a device according to a second embodiment of the invention.

[0029] Figure 4 schematically shows the sequence of an embodiment of the method according to the invention using the second embodiment of the device according to the invention.

[0030] Exemplary embodiments of the invention

[0031] In a first embodiment of the invention, it is designed as a microfluidic device comprising a chamber 10. This is illustrated in Figure 1. The chamber 10 consists of two separable parts 11 and 12. It has a first side 21, which is made, for example, of gold. A second side 22 is located opposite the first side 21. A third side 23 connects the first side 21 to the second side 22. A fourth side 24 is located opposite the third side 23. An inlet 25 is arranged in the third side 23. An outlet 26 is arranged in the fourth side 24. The first side 21 is a component of the first part 11, and the second side 22 is a component of the second part 12. The chamber 10 is rotatably mounted in the microfluidic device about an axis which passes through the third side 23 and the fourth side 24 and which runs parallel to the first side 21 and the second side 22.The first page 21 has a first affinity reagent 31 in the form of antibodies on its inner surface. These exhibit, for example, an affinity for the epithelial cell adhesion molecule (EpCAM) and are thus able to bind CTCs. The second page 22 has a second affinity reagent 32 in the form of antibodies, which exhibit an affinity for the receptor type tyrosine protein phosphatase C (CD45). They are thus able to bind leukocytes.

[0032] In one embodiment of the method according to the invention, a blood sample of, for example, 7.5 ml is taken from a patient. This sample is subjected to selective erythrocyte lysis using the ammonium chloride method, centrifuged, and resuspended in phosphate-buffered saline (PBS). As shown in Figure 2, the cell suspension thus obtained is introduced into chamber 10 through inlet 25. Chamber 10 is arranged such that its first side 21 is at the bottom. The cell suspension contains, as the first cell type 41, EpCAM-positive CTCs. As the second cell type 42, it contains leukocytes, which are CD45-positive cells. As the third cell type 43, it contains EpCAM-negative CTCs. As the fourth cell type 44, it contains CD45-negative cells. After a short incubation period, the cells of the first cell type bound to the first affinity reagent 31 on the first side 21.Chamber 10 is then rotated 180° (53). The cells of cell types 42 to 44, which were initially sedimented on the first side 21 and are not bound to the first affinity reagent 31, now reach the second side 22, which is now at the bottom. There, the cells of the second cell type 42 bind to the second affinity reagent 32. Subsequently, chamber 10 is rotated another 180° (54), so that the cells of the third and fourth cell types 43 to 44 return to the first side 21.

[0033] To prepare for an analysis of the CTCs of the first cell type 41, various procedure steps 55 to 57 can now be carried out. By selectively separating 55 the first affinity reagent 31 from the first side 21, the cells of the first cell type 41 can be detached from the first side 21 in order to subsequently rinse them out of the outlet 26 from the chamber 10 and analyze them outside the chamber 10.

[0034] Through a fluid exchange 56, the cells of the third and fourth cell type 43 to 44 are flushed out of the outlet 26 at a high flow velocity of a fluid introduced into the chamber 10, while the cells of the first cell type 41 and the second cell type 42 are held in place by their respective affinity reagents 31, 32 on the first side 21 and the second side 22.

[0035] Separating 57 of the second part 12 from the first part 11 of the chamber makes it possible to examine the cells of the first cell type 41 in the first part 11 of the chamber 10 microscopically.

[0036] In a second embodiment of the device according to the invention, the chamber 10 differs from the first embodiment in that a filter 60 divides the chamber 10 into two separate areas parallel to the first side 21 and the second side 22. Instead of a single outlet 26, the chamber 10 in this embodiment has two outlets 27, 28. The first outlet 27 is located in the fourth side 24 between the first side 21 and the filter 60, and the second outlet 28 is located in the fourth side 24 between the second side 22 and the filter 60.

[0037] Figure 4 shows the sequence of the method according to the invention using the device according to the second embodiment of the invention. The same process steps 51 to 57 are carried out as are used when using the device according to the first embodiment of the invention. The pore diameter of the filter 60 is selected such that the cells of the third cell type 43 are retained by it, while the cells of the fourth cell type 44 can pass through it. After the first rotation 53 of the chamber 10, only the cells of the second cell type 42 and the fourth cell type 44 reach the second side 22 of the chamber 10. The cells of the third cell type 43, on the other hand, are retained by the filter 60. When the chamber 10 is rotated again 54, the cells of the fourth cell type 44 pass through the filter 60 again.If a selective separation 55 of the first affinity reagent 31 from the first side 21 is performed, the cells of the first cell type 41 are flushed out of chamber 10 through the first outlet 27. If a fluid exchange 56 takes place, the cells of the third cell type 43 are flushed out of chamber 10 exclusively through the first outlet 27, while the cells of the fourth cell type 44 can also leave chamber 10 via the second outlet 28. The filter 60 is a component of the second part 12 of chamber 10. When parts 11 and 12 of chamber 10 are separated 57, it is removed together with the second side 22 from the first part 11, so that it does not obstruct a microscopic examination of the cells of the first cell type 41.

Claims

Claims 1. Device for separating different cell types (41-44), comprising a rotatable chamber (10) with a first side (21) which has at least one first affinity reagent (31) for at least one first cell type (41), and a second side (22) opposite the first side (21) which has at least one second affinity reagent (32) for at least one second cell type (42).

2. Device according to claim 1, characterized in that the first cell type (41) is a CTC cell type and the second cell type (42) is a background cell type.

3. Device according to claim 1 or 2, characterized in that the first affinity reagent (31) is bound to first magnetic beads and the first side (21) has at least one first magnet which is arranged to hold the first magnetic beads on the first side (21).

4. Device according to one of claims 1 to 3, characterized in that the second affinity reagent (42) is bound to second magnetic beads and the second side (22) has at least one second magnet which is arranged to hold the second magnetic beads on the second side (22).

5. Device according to one of claims 1 to 4, characterized in that the chamber (11) has an inlet (25) in a third side (23) and an outlet (26) in a fourth side (24).

6. Device according to one of claims 1 to 4, characterized in that a filter (60) is arranged between the first side (21) and the second side (22), wherein the chamber (10) has an inlet (25) in a third side (23), a first outlet (27) in a fourth side (24) between the first side (21) and the filter (60), and a second outlet (28) in the fourth page (24) between the second page (21) and the filter (60).

7. Device according to one of claims 1 to 6, characterized in that the chamber (10) has two separable parts (11 , 12) wherein the first part (11) has the first side (21) and the second part (12) has the second side (22).

8. Method for separating different cell types (41-44) using a device according to one of the preceding claims, comprising the following steps: Introducing (51) a suspension comprising at least one first cell type (41) and at least one second cell type (42) into the chamber (10), and rotating (53, 54) the chamber (10) so that at least once the first side (21) and at least once the second side (22) is arranged at the bottom.

9. Method according to claim 8, characterized in that after rotation (53, 54) a selective separation (56) of the first affinity reagent (31) from the first side (21) and / or a selective separation (56) of the second affinity reagent (32) from the second side (22) takes place.

10. Method according to claim 8 or 9, characterized in that after rotation (53, 54) a fluid exchange (57) takes place in the chamber (10).

11. Method according to one of claims 8 to 10, characterized in that the chamber (10) is separated into two parts (11, 12) after rotation (53, 54), wherein the first part (11) has the first side (21) and the second part (12) has the second side (22).

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