MICROSCALE CELL FILTER

Abstract

A microscale cell filter for trapping a sub-portion of cells within a sample has: an inlet flow channel; an outlet flow channel; and a plurality of post elements arranged between inlet and outlet. The plurality of post elements is interspaced, forming a plurality of gaps, each gap being formed in between two adjacent post elements. The plurality of post elements is arranged such that a flow of the sample from the inlet to the outlet passes through the plurality of gaps. The plurality of post elements is arranged such that a width of each gaps is 3 to 6 micrometers. Each of the plurality of post elements has an elongation such that each gap has an aspect ratio between its width and its height being larger than 3.5, thereby trapping the sub-portion of the cells within the sample at an upstream side of the plurality of post elements.

Claims

1. A microscale cell filter for trapping a sub-portion of cells within a sample, the microscale cell filter comprising: an inlet flow channel; an outlet flow channel; and a plurality of post elements arranged between the inlet flow channel and the outlet flow channel; wherein the plurality of post elements is interspaced, thereby forming a plurality of gaps, each gap being formed in between two adjacent post elements; wherein the plurality of post elements is arranged such that a flow of the sample flowing from the inlet flow channel to the outlet flow channel passes through the plurality of gaps; wherein the plurality of post elements (8) is arranged such that a width of each gap is 3 to 6 micrometers; and wherein each of the plurality of post elements has an elongation such that each gap has an aspect ratio between its height and width in the range of 3.5 to 5, thereby trapping the sub-portion of the cells within the sample at an upstream side of the plurality of post elements.

2. The microscale cell filter according to claim 1, wherein each of the plurality of post elements is cylindrically shaped.

3. The microscale cell filter according to claim 2, wherein the cylindrical shape is a circular cylinder.

4. The microscale cell filter according to claim 1, wherein the height of the plurality of post elements is identical.

5. The microscale cell filter according to claim 1, further comprising a substrate, wherein the plurality of post elements is integrally formed with the substrate.

6. The microscale cell filter according to claim 5, wherein the substrate and the plurality of post elements are formed by a PDMS layer.

7. The microscale cell filter according to claim 1, wherein the microscale cell filter further comprises a cover, wherein the plurality of post elements is arranged in between the substrate and the cover.

8. The microscale cell filter according to claim 1, wherein the cover is transparent.

9. The microscale cell filter according to claim 1, wherein the surfaces of the plurality of post elements comprise a surfactant.

10. The microscale cell filter according to claim 9, wherein the surfactant is provided by contacting the plurality of post elements with a pluronic acid.

11. The microscale cell filter according to claim 1, wherein a width of each post element is 15 to 40 micrometers.

12. A method for filtering a biopsy liquid, comprising: filtering a body fluid through the microscale cell filter according to claim 1.

13. The method according to claim 12, wherein the body fluid is whole blood.

14. The method according to claim 12, wherein the body fluid is urine.

15. The microscale cell filter according to claim 11, wherein the width of each post element is 2510% micrometers.

Description

BRIEF DESCRIPTIONS OF THE DRAWINGS

[0031] The above and other aspects of the present invention will now be described in more detail, with reference to appended drawings showing embodiments of the invention. The figures should not be considered limiting the invention to the specific embodiment; instead they are used for explaining and understanding the invention.

[0032] As illustrated in the figures, the sizes of layers and regions are exaggerated for illustrative purposes and, thus, are provided to illustrate the general structures of embodiments of the present invention. Like reference numerals refer to like elements throughout.

[0033] FIGS. 1a and 1 b illustrates schematic top views of a microscale cell filter.

[0034] FIG. 2 illustrates a cross-sectional view of the cell filter, taken along the line A-A in FIG. 1b.

[0035] FIG. 3 illustrates a schematic perspective view of a microscale cell filter.

[0036] FIG. 4 illustrates a schematic top view of an alternative embodiment of a microscale cell filter.

DETAILED DESCRIPTION

[0037] The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which currently preferred embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided for thoroughness and completeness, and to fully convey the scope of the invention to the skilled person.

[0038] A sample comprising biological cells may flow from an inlet to an outlet while passing a plurality of post elements. Depending on the size and compressibility of the passing cells, some cells may be trapped in the gaps between the post elements. As the size and compressibility is a characteristic of the cell type, desired type of cells may be trapped in the cell filter, without any need for chemical targeting or fixation.

[0039] With reference to FIGS. 1a, 1b and 2, a microscale cell filter 1 for trapping a sub-portion of cells within a sample will now be described. The cell filter 1 comprises an inlet flow channel 2 and an outlet flow channel 3. The inlet flow channel 2 is configured to receive a sample. The sample is preferably liquid. The sample may for example be whole blood. However, other samples of body fluid, e.g. urine may also be used with the present microscale cell filter. The sample comprises cells 5, 5, 5. In the in FIG. 1 shown embodiment the sample comprises three different type of cells 5, 5, 5, namely relatively small cells, relatively large deformable cells 5 and circulating tumor cells, CTCs, 5. However, the sample may comprise more or less type of cells.

[0040] The microscale cell filter 1 may be arranged on a microfluidic chip (not shown). It is realized that the cell filter may be arranged in other systems, such as in a tube. For the purpose of this application however, the cell filter 1 will be described in the context of a microfluidic chip. The flow channels 2, 3 of the microscale cell filter 1 may have a cross sectional area taken perpendicular to a flow direction of the sample through the cell filter 1 of 25 to 100 000 m.sup.2. The length of the flow channels 2, 3 may be, for example, 0.1 to 100 cm.

[0041] The cell filter 1 may be created using standard manufacturing techniques. The cell filter 1 may be made by using soft lithography. The flow channels 2, 3 may have various suitable shapes and geometries. For example, the geometry perpendicular to the flowing of the sample may be rectangular, oval or circular shaped. The flow channels 2, 3 may further have a serpentine or undulating elongation thereby allowing for a compact design of the microfluidic chip.

[0042] The microscale cell filter 1 may comprise a flow generator 6, configured to provide a flow of the sample through the cell filter 1. The flow generator 6 may be directly or indirectly connected to the inlet flow channel 2. For example, the flow generator 6 may be indirectly connected by means of one or more tubings or tubes, channels or capillaries, or combinations thereof. The flow generator 6 may provide a flow such as a peristaltic flow, a continuous or a periodical flow, or combinations thereof. The flow may be provided at different flow rates. The flow may be turned on and off during different time intervals. The flow generator 6 may be a pump, such as a syringe pump, a peristaltic pump or a pressure pump. The flow generator 6 may be operated manually or energized. The flow generator 6 may also be a capillary or any other narrow channel or passage arranged in connection with the flow channels 2, 3 such that liquid is introduced by means of capillary action. The flow generator 6 may further be connected to an outlet opening of the outlet flow channel 3 and use suction force to generate flow through the cell filter 1.

[0043] The direction of the flow through the cell filter 1 is schematically illustrated by arrows 7.

[0044] The cell filter 1 further comprises a plurality of post elements 8, the plurality of post elements 8 being arranged between the inlet flow channel 2 and the outlet flow channel 3. The plurality of post elements 8 may be parallel to each other. The plurality of post elements 8 may also be positioned oblique relative to each other. The plurality of post elements 8 is interspaced, thereby forming a plurality of gaps 9. The gaps 9 are formed between adjacent post elements 8.

[0045] The sample that passes through the cell filter 1 passes the plurality of gaps 9. There might be a different amount of sample flowing through different parts of the cell filter 1, depending on the chosen dimensions. For example, since the flow velocity in a microfluidic channel is different in the middle of the channel compared too close to the walls, a higher amount of sample may flow in the middle of the channel. Furthermore, the frequency of gaps 9 may be different in different parts of the cell filter 1. For example, the frequency of gaps 9 may be higher in the center of the flow channel than close to the walls.

[0046] The plurality of post elements 8 may be identical to each other. Alternatively, the post elements 8 may also differentiate from each other, such as in geometry or height. Since the geometry of the post elements 8 may be different between the post elements 8, the shape and the geometry of the gaps 9 may be different as well.

[0047] With reference to FIG. 2, the plurality of post elements 8 may be arranged such that a width w of each gap is 3 to 6 micrometers. In a preferred embodiment, the width w of the gaps 9 are 510% micrometers.

[0048] Furthermore, the plurality of post elements 8 may comprise an elongation h such that each gap 9 comprises an aspect ratio between the width w and the elongation h of the gap being more than 3.5. In a preferred embodiment the aspect ratio is in the range of 3.5 to 5. In an even more preferred embodiment the aspect ratio is 410%. The gaps 9 may have the cross-sectional dimensions of a width W of 510% micrometers by a height h 2010% micrometers. A gap 9 comprising the dimensions explained above may be arranged to trap a sub-portion of cells 5, 5, 5 carried in the sample.

[0049] The cross section of the post elements 8, illustrated a post element width b, may be in the range of 15 to 40 micrometer, preferably 2510% micrometer. Hence, in case of circular cylindrical post elements 8 the diameter of the post elements 8 may be in the range of 15 to 40 micrometer, preferably 2510% micrometer. Hence, the width b of the post elements 8 are preferably larger than the width W of the gaps 9.

[0050] There are different mechanisms acting in conjunction to trap the sub-portion of cells 5, 5, 5 carried in the sample in the gaps 9. For the facilitation of reading, each mechanism will be explained separately but in practice, a combination of the mechanisms is in play.

[0051] The cell filter 1 may filter out a sub-portion of cells 5, 5, 5 from the sample 4 based on size. For example, a cell having a diameter that is wider than the width w of the gaps 9, e.g. wider than the preferred width w of 510% micrometers, may not enter through the gaps 9. This since such cells may be physically stopped by the plurality of post elements 8. An example of cells that are normally wider than the width w of the gaps 9 are CTSs 5. CTCs are around 10-20 micrometers in diameter and have a relatively rigid body. Hence, CTCs do not deform to great extent and are hence trapped in the cell filter 1. This is illustrated in FIG. 1b and in the middle of FIG. 2.

[0052] In the case that the trapped cell does not cover the whole gap 9, the flow of the sample may continue to flow through the gap 9.

[0053] Cells 5 having a smaller diameter than the width w of the gap 9 passes through the cell filter 1. Hence, such cells 5 are freely moving from the inlet flow channel 2 to the outer flow channel 3. This is illustrated in FIG. 1 and in the right hand side of FIG. 2. In one embodiment, whole blood is filtered, in which many cells have a diameter smaller than 5 micrometers, such as platelets (2 to 3 micrometers). A cell having a diameter smaller than the width w of the gap 9 passes through the cell filter 1, regardless of its other properties, such as deformability and/or density.

[0054] Biological cells may be deformable. The cytoplasm of the cell may be more deformable than the nucleus of the cell. As illustrated in the left hand side of FIG. 2 a cell 5 having larger diameter than the width w of the gap 9 is hindered by the plurality of post elements 8, the cell 5 in its initial non-deformed state is in FIG. 2 illustrated as having the cross section indicated by the solid line 10. In FIG. 2. a deformable cell 5, in its non-deformed state as illustrated by the solid line 10 cross section, is about to encounter the plurality of post elements 8. However, since the cell 5 is deformable, the deformable cell 5 may deform and pass the cell filter 1 through the gap 9. The cell 5 in its deformed state is in FIG. 2 illustrated as having the cross section indicated by the dotted line 11. An example of a deformable type of cell 5 is, neutrophils constituting 60 to 70 percent of circulating white blood cells. Neutrophils typically have a diameter of 14 micrometers. The cross-sectional area of the neutrophil is thus approximately 150 m.sup.2. if the width w of a given gap 9 in which neutrophils is about to pass through is 5 micrometers, the height h of the gap 9 must at least be around 30 micrometers. However, the neutrophils may also deform in the longitudinal direction letting the deformable neutrophils pass through a gap of 5 times 20 micrometers. Hence, a longitudinal elongation of the deformable cell 5 may lower the required cross sectional area of the gap 9 in order for the deformable cell 5 to pass the cell filter 1. As deformable cells 5 may pass through the cell filter 1 as long as there is enough room for them to squeeze through, the upper limits of the dimensions of the gaps 9 may set by dimension requirements.

[0055] The filtered sample that have passed through the cell filter 1 may be discarded. Alternatively, the filtered sample may be analyzed further. Yet alternatively, the filtered sample may be put back in a patient.

[0056] Analysis of the trapped cells 5 may be performed while the cells are situated in the cell filter 1. As a non-limiting example, the trapped cell may be counted using an optical microscope. In connection with analysis, the trapped cells 5 may also be stained for easier visualization. The trapped cells 5 may for example be stained by targeting the sub-portion of cells with antibodies and subsequently marking the antibodies with fluorescent molecules, such as DAPI, cytokeratin and vimentin. A person skilled in the art realizes that there are many different methods for cellular analysis available. The trapped cells 5 may also be extracted and examined outside of the cell filter 1. For example, as the trapped cells 5 are still viable, the cells 5 may be cultured in growth medium and proliferated for more convenient analysis. The trapped cells 5 may also be lysed to recover their nucleic acids content for molecular analysis The trapped cells 5 may be extracted by reversing the flow such that the flow flows from the outlet flow channel 3 to the inlet flow channel 2 thereby releasing the trapped cells 5 from the post elements 8.

[0057] Now referring to FIG. 3, the post elements 8 may be of cylindrical geometry such that each post element 8 comprise a constant cross-sectional shape along the entire length the post element 8. Furthermore, the cylindrical shape may be a circular cylinder. A cylindrical shape of the post elements 8 may facilitate the guidance of the cells into the gaps 9. Further, by having circular cylindrical post elements 8, the cells that are either deformable enough to pass through, given that the dimensions of the gap 9 is such that the cell is allowed passage, or small enough to not be hindered by the plurality of post elements 8 will no adhere to the walls of the post elements 8. The longitudinal friction of cells against the post elements 8 is thereby reduced. Hence, clogging may be reduced.

[0058] To reduce friction against the post elements 8 of the cells 5, 5 passing through the cell filter, a surfactant may be placed on the post elements 8. A non-limiting example of surfactant is pluronic acid. Further examples of surfactant are tween, sodium desoxycholate, SDS. The surfactant may further facilitate the releasing of the trapped cells 5 upon extraction. The cell filter 1 may have any suitable shape or form, for example a flat and thin shape. The cell filter 1 may be manufactured from, essentially consist of, or comprise a material selected from the list consisting of silicon, glass, paper, and polymers, such as PDMS, PC, PET, PE/PET, polyimide, and PMMA, and combinations thereof.

[0059] Again with reference to FIG. 3, the cell filter 1 may comprise a substrate 12. The post elements 8 may be arranged on the substrate 12. The plurality of post elements 8 may be integrally formed with the substrate 12. The substrate 12 may be made of a polymer material such as polydimethylsiloxane, PDMS. The post elements 8 may be made of a polymer material such as polydimethylsiloxane, PDMS. It is however, realized that the substrate 12 and/or the post elements 8 may be manufactured from, essentially consist of, or comprise a material selected from the list consisting of silicon, glass, paper, and polymers, such as PDMS, PC, PET, PE/PET, polyimide, and PMMA, and combinations thereof. The cell filter 1 may further comprise a cover 13. The cover 13 may be transparent. As a non-limiting example, the cover 13 may be made of glass. A transparent cover 13 may facilitate on-chip analysis.

[0060] An example of manufacturing method for the microscale cell filter 1 may be to have the plurality of post elements 8 be integrally formed with the substrate 12. The substrate 12 may form part of a flow channel for the sample. The substrate 12 may furthermore be molded in PDMS at the same time as the molding of the plurality of post elements 8.

[0061] It is realized that other modules may be connected to the cell filter 1 such as other microfluidic components. For example, any of the following: DNA extraction with reagent kits, sample preparation modules, CTC isolation and lysis modules, ctDNA separation, mixing module and biosensors enabling fast and cost-effective in-situ tumor cell phenotypic and molecular profiling. Multiple cell filters 1 may also be connected to process more sample.

[0062] Below a non-limiting example of a manufacturing process of a microscale cell filter 1 will be discussed. Deep Reactive Ion Etching (DRIE) may be used to etch a microstructure into a silicon wafer. Each cycle of etching may be followed by a deposition of Teflon-like polymer on the walls to ensure unidirectional etching. The fabrication of a polymeric replica of the microstructure may be done by a molding procedure, in which the microstructures are placed on a silicon wafer and PDMS is poured on top. The silicon wafer, including the silicon microstructures may be removed and a PDMS with a negative of the microstructures may be provided. Glass may be bonded to the PDMS using oxygen plasma treatment.

[0063] The person skilled in the art realizes that the present invention by no means is limited to the preferred embodiments described above. On the contrary, many modifications and variations are possible within the scope of the appended claims.

[0064] For example, there might be numerous rows of post elements 8. There are many ways to place the post elements 8 in the cell filter 1, such as in a v-formation or any other formation. Moreover, the cell filter may comprise one or more additional rows of post elements 8. The gap between post elements 8 of one specific row may be different than the gap between post elements 8 of another row. The different rows may display decreasing gap weights. For example, for urine samples, several rows of post elements 8 may be used. The different rows may display decreasing gap weights, from 5010% micrometer to 510% micrometer.

[0065] Furthermore, many other body fluids may be analyzed.

[0066] Further, the number of inlet flow channels 2 may vary. The microscale cell filter 1 may comprise one single inlet flow channel 2. The microscale cell filter 1 may comprise a plurality of inlet flow channels 2.

[0067] Moreover, the number of outlet flow channels 3 may vary. The microscale cell filter 1 may comprise one single outlet flow channel 3. The microscale cell filter 1 may comprise a plurality of outlet flow channels 3.

[0068] Further, as illustrated in FIG. 4 the inlet flow channel 2 may comprise a further filtering stage comprising a plurality of further post elements 14. The further post elements 14 may be formed by rectangular-shaped posts of 200 micrometer100 micrometer. It is however realized that other shapes of the further post elements 14 may as well be used. Moreover, the the further post elements 13 are spaced 10010% micrometer apart. The further filtering stage is configured to trap any possible large cell debris from entering the core of the cell filter 1. Such large cell debris may interfere with the cell analysis. For symmetry purposes, especially for ease of manufacturing, the outlet flow channel 3 may also comprise further post elements 14.

[0069] Additionally, variations to the disclosed embodiments can be understood and effected by the skilled person in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims.