MICROSCALE CELL FILTER

Abstract

A microscale cell filter for separating and isolating cancer cells within a sample having an inlet flow channel; an outlet flow channel; and a single row of a plurality of post elements between the inlet flow channel and the outlet flow channel, the plurality of post elements being interspaced, forming a plurality of gaps, each gap formed in between two adjacent post elements; the plurality of post elements is arranged such that a sample flowing from the inlet flow channel to the outlet flow channel passes through the plurality of gaps; the plurality of post elements is arranged such that a width of each gaps is 3 to 8 micrometers; and each gap has an aspect ratio between its height and width in the range of 3.5 to 5, thereby trapping the cells within the sample at an upstream side of the single row of a plurality of post elements.

Claims

1. A microscale cell filter for separating and isolating cancer cells within a sample, the cell filter comprising: an inlet flow channel; an outlet flow channel; a substrate and a cover; and a single row of a plurality of post elements arranged between said substrate and the cover; wherein the single row of a plurality of post elements are: arranged between the inlet flow channel and the outlet flow channel; arranged perpendicular to the direction of the flow; the post elements are arranged parallel to each other; wherein the width of each post element is 15 to 40 micrometers; 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 single row of a plurality of post elements are 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 at a linear velocity between 0.5 and 1.5 mm/s; wherein the single row of a plurality of post elements are arranged such that a width of each gap is 3 to 8 micrometers; and wherein each gap has an aspect ratio between its height and width in the range of 3.5 to 5; wherein the substrate, the cover and the single row of a plurality of post elements are made of a material suitable to provide wettability and prevent cell adhesion or have a treatment suitable to provide wettability and prevent cell adhesion; thereby trapping the cancer cells of the sample at an upstream side of the single row of a plurality of post elements.

2. The microscale cell filter according to claim 1, wherein each of the plurality of post element are 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 a height of the single row of a plurality of post elements are identical.

5. The microscale cell filter according to claim 1, wherein the single row of a plurality of post elements is integrally formed with the substrate and the cover.

6. The microscale cell filter according to claim 1, wherein the cover, the substrate and the single row of a plurality of post elements are made of a material selected from the group consisting of silicon, glass, paper, polymers such as SU-8, PDMS, PC, PET, PE/PET, polyimide, PMMA, and combinations thereof.

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

8. The microscale cell filter according to claim 1, wherein a surface of the single row of a plurality of post elements comprise a surfactant.

9. The microscale cell filter according to claim 8, wherein the surfactant is a pluronic acid.

Description

BRIEF DESCRIPTIONS OF THE DRAWINGS

[0033] 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.

[0034] 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.

[0035] FIGS. 1a and 1b illustrates schematic top views of a microscale cell filter.

[0036] FIG. 2 illustrates a cross-sectional view of the cell filter, taken along the line A-A in FIG. 1b.

[0037] FIG. 3 illustrates a schematic perspective view of a microscale cell filter.

[0038] FIG. 4 illustrates a schematic top view of an alternative embodiment of a microscale cell filter.

DETAILED DESCRIPTION

[0039] 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.

[0040] A sample comprising biological cells may flow from an inlet to an outlet while passing a filter section formed by a plurality of interspaced 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.

[0041] 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 a 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.

[0042] The microscale cell filter 1 may be arranged on a microfluidic chip (not shown). 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 μm.sup.2. The length of the flow channels 2, 3 may be, for example, 0.1 to 100 cm.

[0043] 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.

[0044] 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.

[0045] The direction of the flow through the cell filter 1 is schematically illustrated by arrows 7.

[0046] 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 and perpendicular to the direction of the flow. 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.

[0047] 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.

[0048] The plurality of post elements 8 are identical to each other.

[0049] 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 8 micrometers. In one embodiment, the width w of each gap is 3 to 6 micrometers. In a preferred embodiment, the width w of the gaps 9 are 5±10% micrometers.

[0050] 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 4±10%. The gaps 9 may have the cross-sectional dimensions of a width W of 5±10% micrometers by a height h 20±10% 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.

[0051] 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 25±10% 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 25±10% micrometer. Hence, the width b of the post elements 8 are preferably larger than the width W of the gaps 9.

[0052] 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.

[0053] 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 5±10% 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 CTCs are around 10-20 micrometers, or more, in diameter and have a relatively rigid body, due to the size of their nucleus. Hence, CTCs do not deform to great extent and are hence trapped in the cell filter 1. This is illustrated in FIGS. 1b and 1n the middle of FIG. 2.

[0054] 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.

[0055] Cells 5 having a smaller diameter than the width w of the gap 9 pass 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 equal or smaller than 5 micrometers, such as red blood cells (5 micrometers) and 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.

[0056] 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 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 along the z-axis 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 white blood cells. Neutrophils typically have a diameter of 14 micrometers. The cross-sectional area of the neutrophil is thus approximately 150 μm.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.

[0057] The filtered sample that have passed through the cell filter 1 may be discarded. Alternatively, the filtered sample may be analyzed further.

[0058] Analysis of the trapped cells 5″ may be performed while the isolated 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 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.

[0059] 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 height 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 not 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.

[0060] 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.

[0061] The cell filter 1 may have any suitable shape or form, for example a flat and thin shape. The cell filter 1, and its elements, may be manufactured from, essentially consist of, or comprise a material selected from the list consisting of silicon, glass, paper, and polymers, such as, but not limited to, PDMS, PC, PET, PE/PET, polyimide, and PMMA, and combinations thereof.

[0062] Again, with reference to FIG. 3, the cell filter 1 comprises 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 further comprises 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.

[0063] 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.

[0064] 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 in parallel to process more sample.

[0065] 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.

[0066] 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.

[0067] Furthermore, many other body fluids may be analyzed.

[0068] 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.

[0069] 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.

[0070] 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 micrometer×100 micrometer. It is however realized that other shapes of the further post elements 14 may as well be used. Moreover, the further post elements 13 are spaced 100±10% 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 intefere with the cell analysis. For symmetry purposes, especially for ease of manufacturing, and to keep the flow homogeneous, the outlet flow channel 3 may also comprise further post elements 14.

[0071] 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.

[0072] Biological samples, such as for example blood samples, often comprise cancer cells with sizes that overlap with non-cancer cells. This overlapping becomes a challenge for the separation and isolation of cancer cells from non-cancer cells for further analysis. In these samples of interest, cancel cells are rare, i.e., 1 to 10 cells in billions of blood cells, in comparison with the amount of non-cancer cells therein, thus making the separation and isolation of cancer cells even more difficult.

[0073] The microscale cell filter of the present invention overcomes the challenge of non-cancer cell contamination and tumor cell heterogeneity, by filtering a whole biological sample by physical characteristics such as size and deformability of the cells contained therein without the need of a pre-treatment to the sample (i.e., filtering, dilution, etc.). This allows to capture, for example, mesenchymal tumor cells that would otherwise be missed by conventional CTC isolation methods. The microscale cell filter is also suitable to process a large volume of sample, up to 10 mL of sample, which is greater than the capacity of other microscale filters known in the prior art. Larger volumes of sample can be processed by fluidically connecting a plurality of microscale cell filters.

[0074] In view of separating and isolating cancer cells in a biological sample, white blood cells are often seen as contaminants because their sizes can overlap with those of cancer cells. Some white blood cells are large cells, but usually polynucleated or with a lobulated nucleus, while cancer cells can have large rigid nucleus with an average size well above 5 μm, which make up for around 70% of the cell size.

[0075] In this scenario, the presently disclosed microscale cell filter allows non-cancer cells, such as white blood cells, to pass through a plurality of gaps between post elements because white blood cells are flexible and deform along the Z axis, while cancer cells with a size above 5 μm are retained by the gaps between the post elements.

[0076] The microscale cell filter is provided with a single row of a plurality of post elements arranged on a substrate between said substrate and a cover, wherein the single row of a plurality of post elements are: [0077] arranged between the inlet flow channel and the outlet flow channel; [0078] arranged perpendicular to the direction of the flow; [0079] the width of each post element is 15 to 40 μm, and [0080] the post elements are arranged parallel to each other.

[0081] The size of the post elements allows to provide a plurality of gaps in between the post elements, these gaps are critical for separating and retaining cells according to their size and deformability, while allowing the sample and unwanted cells to naturally flow through the available gaps that are still free for this purpose.

[0082] Each gap between the post elements has an aspect ratio between its height and width of 3.5 to 5, this being a static dimension, and even with the post elements being made of different materials, this aspect ratio is maintained. In the context of the present invention, the aspect ratio of the gaps is understood as the ratio between the height that is measured from the substrate to the cover in the region between adjacent post elements and the width of the gap that is measured as the distance between two adjacent post elements.

[0083] Another important feature of the microscale cell filter is that the post elements are arranged between the substrate and the cover, wherein the post elements, substrate and/or cover are made of a material suitable to provide wettability and prevent cell adhesion, particularly reducing protein adsorption on the post elements or substrate. This feature allows the cells in the sample to be separated and isolated based on their physical properties without the risk of being retained by adhesion to the surface itself. The microscale cell filter, post elements and/or substrate are manufactured from, comprise, or essentially consist of a material selected from, but not limited to, silicon, glass, paper, and polymers, such as SU-8, PDMS, PC, PET, PE/PET, polyimide, and PMMA, or any combinations thereof. Alternatively, if the chosen material does not provide wettability and prevent cell adhesion by itself, the material may be subject to physical or chemical treatments in order to provide this effect, including oxygen plasma treatments or functionalization with a surfactant.

[0084] It is critical for the microscale cell filter to have a single row of post elements arranged parallel to each other, as shown in any of the FIGS. 1 to 8. Besides facilitating the separation and isolation of cancer cells in a cell sample, this single row arrangement facilitates downstream analysis of cancer cells, enabling high resolution imaging after separation of cancer cells because the cells retained in the gaps are well distributed along the row of post elements, and in one focal plane for imaging, making this microscale cell filter particularly suitable for high resolution fluorescence imaging. Other prior art cell filters, such as US2016146778, US20050042766 or Morton, K. L, et al., PNAS, 2008, have multiple rows of post elements, which are not suitable for downstream imaging analysis in situ, or for recovering the separated cells. In these documents, the multiple rows increase unwanted retention of cells, such as white blood cells.

[0085] Furthermore, the microscale cell filter of the present invention allows for the flow rate to be adjusted according to the type of cancer cells to separate, substantially reducing the time required to process samples to around an hour. The biological sample must be pumped at sufficient high speed through the microscale cell filter to avoid unwanted blood cells being trapped in the filter. Thus, the flow rate can be adjusted between 80 to 150 μL/min. The selection of this range is not made at random, but it was found to provide the necessary linear velocity of cells against the microscale cell filter. This flow rate range is the most suitable to achieve a linear velocity of the cells that is sufficiently high to allow the large unwanted cells, such as white blood cells, to deform and pass through the gaps between the post elements while still trapping efficiently the cancer cells of interest without compromising their integrity. Linear cell velocities below or above this range in the presented embodiment are not sufficient to achieve the desired effect of separation and isolation of cancer cells, and depletion of non-cancer cells. Consequently, the internal pressure in the microscale cell filter is between 1 and 2 bars. The linear velocity achieved is between 0.5 and 1.5 mm/s.

[0086] Furthermore, by using this microscale cell filter, the separated and isolated cancer cells can be recovered for downstream analysis by reversing the flow of the microscale cell filter.

[0087] It is important to note that the microscale cell filter cannot be operated by capillary flow, as opposed to other devices in the prior art, such as US2016146778, US20050042766 or Morton, K. L, et al., PNAS, 2008. These devices would not be able to process samples at the desired flow rate or to achieve the desired linear velocity of the cells against the filter. Also, the devices would not be able to process samples of the desired volume (between 1-10 mL), as they would be limited by the inner volume of the device (around 50 uL).

[0088] The microscale cell filter has been tested for the successful isolation of viable circulating tumor cells in several cancer types, namely bladder cancer, breast cancer, esophageal cancer, renal cell carcinoma and colorectal cancers, among others, independently on the cell phenotype, and including epithelial, mesenchymal and transitioning cells.

[0089] Thus, unlike other devices in the prior art, the microscale cell filter presently disclosed allows to combine enhanced separation efficiency and purity, given that it achieves a cancer cell isolation efficiency up to 80% and white blood cell depletion above 99%, resulting in a purity of round 10%, which is much higher than the 0.05% purity offered by other prior art devices.

[0090] The features of the microscale cell filter allow enumeration and downstream analysis of the captured cells, that has been successfully correlated with patient prognosis, such as overall survival, progression-free survival, patient monitoring and stratification, evaluation of resistance to treatment, and analysis of druggable targets.