Flexible filter device for capturing of particles or cells in a fluid

A filtering device includes a housing and one or more flexible filter arrays. The flexible filter arrays can be coupled to the housing. Each flexible filter array can include a plurality of members and one or more support members. The plurality of members each can have a proximal and a distal end, the plurality of members being spaced apart by a predetermined distance. The one or more support members can be coupled to one or more of the plurality of members. Each of the plurality of members can be configured to deflect relative to the one or more support members.

FIELD

The subject matter herein generally relates to capturing particles in a fluid and specifically a bodily fluid.

BACKGROUND

Filtration is a process by which particles may be separated from a fluid or mixture of particles by taking advantage of differences in their physical properties. It occurs when a fluid is passed along or across a membrane or other structure that can act as a selective barrier. Particles are either retained by the barrier or pass through in the filtrate.

Since their invention in the 1960s track-etched polymer filters have been widely used for biological cell enrichment because of their low cost and fast sample processing speed. The track-etched filters have randomly distributed pores defined by swift heavy ions typically generated by nuclear reactors and then enlarged to a desired diameter in an etching process. These filters mechanically enrich certain cells from body fluids based on their ability to pass through pores of a particular size.

DETAILED DESCRIPTION

The term “porosity” of a filter is defined here as the percentage of the open area on the two dimensional surface to the total surface area of the two dimensional surface.

The term “flexibility” generally refers to the stiffness of elastic materials characterized by their elastic modulus (Young's modulus). When referring to biological particles this definition takes into account their ability to deform in response to applied forces. When referring to device structural flexibility this definition also takes into account the ability of the structure to bend, flex and absorb stress due to applied forces.

The term cell “viability” refers to a cell's maintenance of its integrity, which can be determined by testing for membrane permeability to various chemicals using commercially available cell viability tests.

The term cell “proliferability” refers to the capability of a cell to replicate and form a colony. A viable cell might not be proliferable, as it can be in dormant state and not actively proliferating for the time of observation.

The term “capture efficiency/recovery” of a CTCs enrichment process is defined as the percentage of the number of tumor cells retained on device after enrichment to the number of tumor cells spiked in. To test capture efficiency/recovery of the CTC enrichment system, a model sample needs to be constructed by spiking a known number of tumor cells into healthy donor blood.

The term “enrichment” of CTCs against leukocytes is defined as the ratio of tumor cells to leukocyte ratio after enrichment to that before enrichment.

The term “coupled” is defined as connected, whether directly or indirectly through intervening components and is not necessarily limited to physical connections.

The present technology presents a filtering device. The filtering device as presented herein can include a housing and flexible filter array. The present technology contemplates the flexible filter array could be implemented with a variety of different arrangements that allow a fluid to flow across the flexible filter array. The flexible filter array can be coupled to the housing. The flexible filter array can include a plurality of members. In at least one implementation, each of the plurality of members can include a proximal and a distal end. The one or more members can be spaced apart by a predetermined distance. The distance of this spacing may be constant or it may be tapered in any dimension. The flexible filter array can include one or more support members. The one or more support members can be coupled to a support portion of the one or more of the one or more members. In at least one embodiment, the one or more members can be unsupported at a distal end. Additionally, the plurality of members can be configured to deflect relative to the support portion of the plurality of members. In another embodiment, both the proximal and distal ends of each of plurality of members can be configured to be coupled to a support member. In at least one embodiment, the distal end of the member can still deflect relative to the proximal end even when the distal end is coupled to a support member. Still further, the member itself can deflect relative to one or more of the support members.

A flexible filter array according to the present disclosure can operate on the principle of size based exclusion for the enrichment of circulating tumor cells (CTCs) from whole blood samples. Although, conventional track-etched microfilters and two dimensional (2D) pore shaped microfilters can be used for fixed blood samples, they are unsuccessful in enriching viable CTCs from blood as cells are damaged or lysed and lost in the flow-through. A flexible filter array100according to the present disclosure can use spring-like arrangements as the active structure for microfiltration. The spring-like geometry incorporates several important advantages over the pores in conventional microfiltration devices. The inherent flexibility of the spring structures allows for deformation in response to the application of flow pressure, thus reducing the initial impact between the cell and the device structure and relieving some of the tensile forces experienced by captured cells. The spring-like layout can also be designed to allow for maximal effective opening space, achieving an effective porosity of 20% or greater. The greater porosity increases the sample volume capacity and also eliminates problems with clogging that other microfilters experience. In this way the flexible filter array100can be used in conjunction with a precise pressure regulation system to limit the mechanical stresses experienced by cells during filtration.

FIG. 1Aillustrates a flexible filter array100according to the present disclosure. The flexible filter array100can be incorporated into a first level array102comprising a plurality of flexible filter arrays100. Furthermore, the first level array102can be incorporated into a second level array104. As illustrated the first level array102includes one hundred flexible filter arrays100. The flexible filter arrays100can include a plurality of members110. As illustrated the flexible filter arrays100have a length (a) and a width (b). In the illustrated example, the length (a) and width (b) are the same, thereby forming a square. The depth (in/out of the paper) of the flexible filter array100can be uniform or it can vary. In the illustrated embodiment, the depth is uniform. In other embodiments, the depth can vary in a uniform or a non-uniform pattern so as to provide different flexibility characteristics. For example, it can be desirable to provide for a stiffer flexible filter array or a more flexible filter array which can be achieved by varying the thickness of the flexible filter array100while maintaining the other dimensions.

The second level array has four first level arrays that each measure 4.742 mm by 4.754 mm. Each first level array has a 10×10 array of flexible filter arrays100. While the second level array is illustrated as having four first level arrays arranged as quadrants, the present technology can be implemented as a single first level array or with a pair of first level arrays. If a larger surface area is required, the second level array can include more than four first level arrays. In at least one embodiment, the number of additional first level arrays are combined in pairs when added. While the first level arrays as illustrated have the same shape and size, the shape and size of the first level arrays can vary.

The flexible filter array100can vary in size depending upon the desired stiffness and/or porosity. In the illustrated example, the length (a) of the flexible filter array can be between 0.5 cm and 1.5 cm. Likewise, the width (b) of the flexible filter array can be between 0.5 cm and 1.5 cm. The whole flexible filter array can be removed from the device housing and used as a culture site for those particles that are captured or located on the flexible filter array100. Alternatively, patches of the flexible filter array can be selectively removed either by mechanical or thermal cutting or use micro laser dissection. In the latter case, a single cell or a single cluster of cells can be isolated before or after immunocytochemical detection with surface marker(s). In at least one embodiment, the flexible filter array100can be made of a material such as parylene-C or polydimethylsiloxane to allow the flexible filter array100to be implanted in a living organism. In other embodiments, the flexible filter array100can be made from a transparent, micromachinable thin film material. In still other embodiments, the flexible filter array100can be polylactic acid, polyglycolide, or any other micromachinable biodegradable material. The plurality of members can be in turn made from parylene-C, polydimethylsiloxane, a transparent, micromachinable thin film material, polylactic acid, polyglycolide, or any other micromachinable biodegradable material, or a combination thereof. The present disclosure contemplates the implementation of other materials that can be implanted as well. In other embodiments, where implantation is not required, the material of the flexible filter array can be selected based upon the desire to control the flexibility and other characteristics of the flexible filter array100as described herein. The material and device structural design can also be chosen to ensure that cells or other particles that impinge upon the flexible filter array100can survive the impact. In embodiments, where the fluid circulating through the flexible filter array100is returned to the organism from which it was obtained, the material can be selected for biocompatibility.

In one example, the flexible filter array100process can begin with the coating of a thin layer of transparent polymer on a silicon wafer through a deposition process. A metal can then be deposited onto the polymer film. The metal layer can be patterned using a photolithography technique and then etched with a suitable etching process. The patterned metal layer can be used as an etching mask for the underlying polymer layer. The desired pattern can be etched into the parylene layer by reactive ion etching using plasma. Finally the polymer layer can be released and cut into individual devices. In other embodiments, different construction methods and assemblies can be implemented in order to provide the filter having the flexible micro spring array according to the present teachings.

The plurality of members110as illustrated inFIG. 1Aare in the form of flexible micro-springs. A flexible micro-spring as used herein refers to the arrangement of a member110in which the member110is arranged to act like a spring. In at least one example, the micro-spring can be arranged such that a one of the plurality of members110can be substantially parallel to another member110. The members110can be joined by a connecting portion116. The members110and connecting portion116can be substantially linear, as illustrated. In other embodiments, the members110and connecting portions116can be slanted or inclined. In yet other examples, the shape of the members110can be selected based upon the structure into which the flexible filter array110is to be installed for filtering. Additionally, the shape of the flexibility filter array110and members110can be chosen based upon whether the flexible filter array will be implanted or further removed for processing. In each row, the flexible micro-spring can be implemented with fourteen and a half turns of a spring-like shape.

An example of two members110is illustrated inFIG. 1B. As illustrated, the members110are arranged such that the left-hand member114can be substantially parallel to a right-hand member112. The right-hand member112can be joined to the left hand member114by a connecting portion116. The connecting portion116can join the right-hand member112and the left-hand member114. The connecting portion116as illustrated can be substantially perpendicular to the right-hand member112and the left-hand member114. As illustrated each member110has a width (d). In other embodiments, the width (d) of the member can vary for a given flexible filter array. For example, the width (d) can decrease from an outer edge of the flexible filter array100to an inner portion of the flexible filter array100. The connecting portion116can also have a width (d). In at least one embodiment, the connecting portion116can have a length (g) which corresponds to the distance that the inner faces of the right-hand member112and left-hand member114are spaced apart. In at least one embodiment, the right-hand member112and left-hand member114can be between one-half (0.5) and two hundred (200) micrometers (μm). In another embodiment, the right-hand member112and left-hand member114can be between one (1) and fifty (50) micrometers (μm). In yet another embodiment, the right-hand member112and left-hand member114can be between three (3) and nine (9) micrometers (μm). The distance that the right-hand member112and left-hand member114are spaced apart can vary depending upon the size of the particle that is desired to be filtered from passing there through.

While the relationship between a right-hand member112and a left-hand member114has been described above, a similar relationship can apply to multiple members110that are adjacent to one another. As illustrated inFIG. 1A, the micro-spring can be formed in an adjacent pattern, such that two adjacent members110follow a similar pattern. In other embodiments, the shape and spacing can vary.

In the illustrated example ofFIG. 1A, the flexible filter array110can include a plurality of members that are all joined together to form a single structure. In at least one embodiment, the single structure can be formed using a molding process. In another embodiment, the single structure can be formed by fastening or otherwise affixing the plurality of members110together via connecting members116and other components as described above.

The single structure includes a plurality of members110and other components. In the illustrated example ofFIG. 1A, the single structure includes one hundred-sixteen (116) parallel members and one hundred-twelve (112) parallel connecting members. The parallel members110have a length that is greater than the connecting members116. The connecting members116can be substantially orthogonal to the members110. As illustrated, the parallel members110can be arranged in four rows that have a substantially uniform length. The rows can be joined together by row joining portions. In the illustrated example, the row joining portions of together with parallel members110can span across the flexible filter array100. The four parallel members110and three row joining portions can span in a first direction across the flexible filter array100and form a spanning member. The spanning member can be coupled to a support structure via a two support members on each end of the spanning member. The spanning member can provide rigidity to the flexible filter array100.

Another example of a flexible filter array100is presented inFIGS. 1C and 1D. As illustrated in1C, the flexible filter array110includes a plurality of members110. The plurality of members110can be substantially parallel to one another as illustrated. In another embodiment one or more of the plurality of members110can have a different orientation relative to one or more other ones of the plurality of members such that spacing between the different oriented member110and an adjacent member110can allow for a varying spacing between them.

The plurality of members110can be formed as cantilever members110. The plurality of members can be formed such that the each member has a proximal end111and a distal end113. When the plurality of members110can be arranged as cantilever members, the distal end113can be configured to flex relative to the proximal end111. The proximal end111can be coupled to a support member120. In at least one embodiment, such as the one illustrated, the support member120can form an outer perimeter of the flexible filter array100. The support member120is shown as also spanning across the middle of the flexible filter array100in one direction. In another embodiment, another support120can be implemented to space across the middle of the flexible filter array100in another direction. In at least one embodiment, another direction can be substantially orthogonal to the one direction.

As illustrated the plurality of members110can be arranged in two rows. In other embodiments, the flexible filter array can have one row or more than two rows. Additionally, the length of the rows can vary such that the flexible filter array100can have a parallelogram shape or step-like shape. In at least one embodiment, the flexible filter array100can be arranged in a circular arrangement. As illustrated, adjacent members110can extend from a proximal end111, wherein the proximal ends111of adjacent members can be located on support members that are opposed to one another such that the member110extend from one of the support members does not reach the other support member120. The distance separating the distal end113from the opposing support member120is distance (h). In the illustrated example, the distance (h) separating the distal end113from the opposing support member can be substantially the same as the distance (g) separating adjacent members110. In other embodiments, the distance (h) separating the distal end113from the opposing support member can be less than the distance (g) separating adjacent members110. When the distance (h) is less than distance (g), the configuration can allow for a more uniform filtering of particles as it allows for the space under deflection of the member110to be the same. In another embodiment, the members110can have a tapered shape to allow for a more uniform gap space to be formed when the member110deflects. As illustrated, the members have a length (l).

FIG. 1Dillustrates two adjacent members110of the flexible filter array100ofFIG. 1C. As illustrated, the members110can be substantially parallel and straight with a width (d). In another implementation, the members110can be tapered from a proximal end111to the distal end113, such that the distal end113has a width that is smaller than the width at the proximal end111. When the adjacent members110are configured with a corresponding matching taper, the distance (g) between the adjacent members110can remain substantially the same along the length (l) of the members110.

The following statements can apply to one or more of the above described embodiments/implementations. In at least one implementation, the plurality of members can be substantially rigid. In another implementation, the plurality of members can be substantially flexible. When the plurality of members are substantially rigid, the members can still flex or deflect. When the members are configured to flex or deflect, the members can absorb some of the impact from the particles colliding with the member. When the member is substantially flexible, the member110can be made shorter while retaining the desired flexibility for the flexible filter array. In at least one embodiment, the flexible filter array100can be configured to have a desired overall modulus of elasticity that is selected based upon the type of particles being captured.

The flexible filter array100can be configured to have a desired porosity. In at least one embodiment, porosity of the flexible filter array100is between twenty to sixty percent. In yet another embodiment, the porosity is between forty to sixty percent. In another embodiment, the porosity can be between fifty and sixty percent. Lower porosity is not ideal for this application because it reduces the sample capacity, for example maximal volume of blood that can be processed given the specific device surface area. Higher porosity can be eventually limited mainly by the fragility of the device structure.

The flexible filter array100can be implemented to filter a variety of fluids. In at least one implementation, the fluid can be a bodily fluid that contains cells, particles, other living organisms, and/or molecules. The creation of the filter array100can be designed based upon the cells, particles, other living organisms, and/or molecules that are desired to be filtered. For example, the cells, particles, other living organisms, and/or molecules can have a nominal smallest diameter or other dimension that is associated therewith. In some situations, where very small sizes of cells, particles, other living organisms, and/or molecules are desired to be filtered, two or more flexible filter arrays100can be arranged in series to reduce the chance of each of the flexible filter arrays100from becoming clogged.

The flexible filter array100as presented herein can be designed to operate at a low pressure. In one embodiment, the flexible filter array100can be designed to operate at a pressure less than 1245 Pa. In another embodiment, the flexible filter array100can be designed to operate at a pressure less than 800 Pa. In still another embodiment, the flexible filter array100can be designed to operate at a pressure less than 500 Pa. In yet another embodiment, the flexible filter array100can be designed to operate at a pressure less than 249 Pa. When the flexible filter array100is designed and configured to operate at these low pressures, the ability to capture and allow the molecules or particles to survive is greatly increased compared to other filters which require higher pressures to operate. Further still, when the flexible filter array100is configured to operate at one of the above pressures, it can achieve a flow rate between 0.1 mL/min and 50 mL/min.

As indicated above, the flexible filter array100can be configured to capture or filter a variety of different size molecules and particles.FIGS. 2A and 2Billustrate a filter device202configured to receive the flexible filter array100as described above. In the illustrated example, the filter device202is configured to filter whole blood. The filter device200can be configured to filter other fluids including bodily fluids. An inlet204allows for the fluid to be received into the filter device. The fluid then passes through the flexible filter array110. The fluid, particles, and molecules that are too small to be captured by the flexible filter array are collected in a collection area220of the filter device. In order to provide for a fluid flow, the filter device can include a suction outlet226which is coupled to a valve228, which in turn can be coupled to a vacuum source. The vacuum source230can be configured to provide the desired pressure for the filter device202. The filter device202can also include a pressure sensor outlet222, the pressure sensor outlet222can be coupled to a pressure sensor224. While the pressure sensor outlet222and suction outlet226can be in the form of tubes coupled to filter device202, the pressure sensor outlet222and suction outlet226can take other forms such as threaded connections and other fluid coupling devices. In at least one embodiment, the pressure sensor224can be used to provide information that can be used to adjust the pressure in the filter device202by adjusting the valve228and/or the vacuum source. For example, the vacuum source can be configured to provide a fixed vacuum. By adjusting the position of the valve228, the pressure in the filter device202can be varied. In one embodiment, the pressure sensor224can be coupled to the valve228via a controller so that the valve228can be adjusted automatically to maintain a desired pressure. Additionally, the controller can be configured to allow for a varying of the pressure if desired. When the filter device202includes a controller, the precision and accuracy in which the pressure is maintained inside of the filter device202can be increased. When the precision and accuracy of the pressure can be controlled enhanced ability to reduce the forces that the molecules and particles that are captured by the filter array100experience.

The flexible filter array100can be held in place through a variety of different configurations. As illustrated inFIG. 2A, the flexible filter (not shown) can be held between an upper plate208and a lower plate210. The upper plate208and lower plate210can be held together by two clamps212. The clamps212as illustrated can be configured to contact the bottom surface of the lower plate210and a top surface of a clamp plate206. In the illustrated embodiment ofFIG. 2B, the clamp plate206is eliminated and the clamps212contact the top surface of the upper plate208and the bottom surface of the lower plate210. While the upper plate208and lower plate210are illustrated as having a circular perimeter, the upper plate208and lower plate210can have a perimeter that is of a variety of different shapes. The perimeter of the upper plate208and lower plate210can be configured to match the shape of the filter device202and/or provide for the desired fastening device. For example, the fastening devices as illustrated can include two clamps212. In other embodiments, other fastening devices can be implemented. For example, bolts and screws can be implemented to couple the upper plate208to the lower plate. Another example of a filter device is illustrated inFIG. 11, as will be described below.

FIGS. 3A-10Billustrate various implementations of flexible filter arrays100according to the present disclosure. In the illustrated example, the flexible filter arrays100can be micro-spring filter arrays. These results are provided to provide an enhanced understanding to one of ordinary skill in the art the characteristics of flexible filter array100according to the present disclosure. In one example, a 1 cm2micro-spring filter array, according to the present disclosure, can process 7.5 mL of whole blood in under ten minutes at less than 1 inch water column (inch WC) driving pressure for each of the more than one hundred blood samples without clogging.

FIG. 3Aillustrates a color coded out-of-plane displacement of an exemplarily micro-spring filter array under 0.1 psi pressure. Line1and Line2label positions forFIGS. 3F-G.FIGS. 3B-Eillustrates geometric effects (member thickness t, member width d, member length and number of support member per array) on the maximal out-of-plane deformation of the exemplarily micro-spring filter array ofFIG. 3A, wherein the circle denotes the geometric parameters used in a specific implementation of the micro-spring filter array.FIGS. 3F-Gillustrate exemplarily gap width changes measured from top and bottom of the gaps along Line1and Line2of the micro-spring filter array ofFIG. 3A.

In the example, the micro-spring filter array was designed as a single layer parylene membrane with flexible spring-like structures capable of deformation in response to applied stress. The stiffness of the micro-spring filter array can be controlled by the member length l, the member thickness t, the member width d and the number of anchor pairs n. A model of the system was created to perform finite element analysis. Finite element analysis was carried out to study the effects of these geometric parameters on the micro-spring filter array flexibility. Boundary conditions were applied to the surface of the anchors that connect the members to the frame and all six degrees of freedom were restricted. The Young's modulus and Poisson's ratio of the member material was set to be 2.76 GPa and 0.4, respectively. A uniform pressure of 0.1 pounds per square inch (psi) equivalently 2.77 inch WC was applied to the top surface of the micro-spring filter array. The maximal out-of-plane deformation was calculated in post-processing. Each of the four geometric parameters was studied independently while keeping the other three at the final design values. The design values, as illustrated inFIG. 3A, for these four studied geometric parameters are: member thickness t=10 μm, member width d=8 μm, member length l=80 μm, and number of anchors=2. These values are illustrated as the data points that are circled inFIGS. 3B-3E.

InFIG. 3B, the influence of the member thickness t on the out-of-plane deformation of the micro-spring array was examined. As the member thickness increases from 6 to 20 μm, the deformation decreases dramatically from 10.7 to 0.6 μm. Thus, the member thickness is a parameter that can be used to significantly change the flexibility of the micro-spring array during fabrication for a given mask design.

InFIG. 3C, the influence of member width d was examined, while keeping the periodicity d+g as a constant. The maximal out-of-plane deformation decreases slightly as the member width increases. The member width alone appears to have very small impact on the out-of-plane deformation.

InFIG. 3D, the influence of member length1was examined. The simulation results of changing member length l shows the maximal out-of-plane deformation increases as the member length l increases.

FIG. 3Eillustrates the influence of the number of anchors per array has on the out-of-plane deformation.

Using a load-deflection relationship for a rectangular membrane, the calculated effective Young's modulus of a single micro-spring array (or the structure rigidity) is 44 MPa, which is about two orders of magnitude smaller than that of material itself. Perforation of the material only reduces the effective Young's modulus proportional to the porosity, and this significant reduction of the effective Young's modulus is due to the flexible micro-spring array.

Contrary to the out-of-plane deformation, the in-plane deformation and the change of gap width due to the applied pressure were found to be inconsequential. For the final design parameters, the maximal gap width change under 0.1 psi was less than 60 nm. Since the gap width changes are so small, the micro-spring array is expected to effectively maintain a constant gap width during enrichment.

FIGS. 3F and 3Gillustrate the in-plane filter structure deformation, i.e. the change of gap width, at different positions of the filter patched being simulated. The simulation illustrates that the change of the in-plane gap width is orders of magnitude smaller that the out-of-plane deflection of the flexible structure.

FIG. 4Aillustrates a bright field optical microscopic image of an area of an exemplarily micro-spring filter array.FIG. 4Billustrates an SEM image of an exemplarily micro-spring filter array with shorter spring width than the micro-spring filter array illustrated inFIG. 4A.FIG. 4Cillustrates a SEM image of an exemplarily micro-spring filter array after refill.FIG. 4Dillustrates a SEM image showing tumor cell growth on an exemplarily micro-spring filter array. Scale bars forFIGS. 4A, 4B and 4Dare 30 μm. Scale bar forFIG. 4Cis 10 μm.

FIG. 5Aillustrates capture efficiency under exemplarily gap widths and driving pressures. In the illustrated example, four carcinoma cell lines (MCF-7, MDA-MB231, C8161, WM35) were used separately using model systems. A known number of pre-labeled cells was spiked in healthy donor blood and processed to determine recovery rates. Each data point shows the mean value and its standard deviation (n≧3).FIG. 5Billustrates enrichment of whole blood under exemplarily gap widths and driving pressures. The number of leukocytes that remained after enrichment of whole blood was quantified through microscopy and image analysis then compared to the known initial leukocyte count to determine an enrichment ratio. Each data point shows the mean value and its standard deviation (n=3).

FIG. 6illustrates the effects of the freshness of blood samples and the types of anticoagulant on the blood volume that can be processed through an exemplarily 1 cm×1 cm micro-spring filter array. The filtered volumes were the blood volumes that were tested. For the cases of sodium citrate as the anticoagulant and fresh blood in EDTA (labeled with upward arrows), the driving pressures were so low and the tested blood volumes are expected to be lower than the maximal volumes that can be processed. The rest of the data points represent the maximal blood volumes that can be processed under the specified pressure for the specified anticoagulants and blood storage times.

InFIG. 7, an exemplarily effect of pressure on cell viability is shown for a 5 μm gap micro-spring filter array. Calcein AM dye and Ethidium Homodimer-1 were used to assay MDA-MB 231 cell membrane integrity after enrichment from 1 mL of whole blood at various driving pressures. Viable cell recovery was determined as the percentage of viable cells retained out of the total number of cells spiked into the blood. Calculated viability is presented as the viable cell recovery divided by the known capture efficiency for MDA-MB 231 cells at each pressure condition. Limiting the driving pressure to 1 inch water column allowed a viable cell recovery of >80%, which corresponds to a calculated viability of >90%. 5 inch water column driving pressure lowered viable cell recovery to below 50% and 10 inch water column pressure effectively prohibited any viable cell capture

FIG. 9Aillustrates an exemplarily positive control of a spiked sample on glass slides.FIG. 9Billustrates an example positive control of a spiked sample after exemplarily enrichment by the micro-spring filter array.FIG. 9Cillustrates an example of a single CTC enriched from a clinical sample.FIG. 9Dillustrates a cluster of CTCs enriched from another clinical sample. Scale bars are 20 μm.

Immunofluorescent detection was established to identify CTCs from clinical samples after enrichment with the micro-spring filter array. As positive controls, MDA-MB 231 cells were spiked into 1 mL of peripheral blood and either deposited on glass slides (FIG. 9A) or filtered through the device (FIG. 9B). The protocol described herein was used to stain the cells with monoclonal antibodies for cytokeratins and CD45. The MDA-MB 231 cells were observed as large nucleated cells that stained positively for cytokeratins and negatively for CD45. The contaminant leukocytes were noticeably smaller in size, and stained positively for CD45 and negatively for cytokeratins.

Successful immunofluorescent detection was then demonstrated with clinical samples. An example of a CTC detected from a patient diagnosed with Stage IV Non-Small Cell Lung Cancer is shown inFIG. 9C. 7.5 mL of blood was filtered through the device and stained using the previously described immunofluorescent detection process.FIG. 9Dshows an aggregate cluster of 3 CTCs and 3 leukocytes obtained using the same process from a Stage IV Breast Cancer patient. Aggregate clusters of CTCs have been previously reported in Lung Cancer patients and Breast Cancer patients.

FIG. 10Aillustrates an example of colony expansion of GFP labeled C8161 melanoma cells on the micro-spring array. Cells are spiked in blood, enriched with the micro-spring filter array and cultured on-chip. Pictures are fluorescent images of the same micro-spring filter array area over time. Scale bars are 50 μm.FIG. 10Billustrates an example of cell number over time for cell proliferation on the micro-spring filter array and in Petri dishes (as positive controls). Positive controls of cell proliferation are at various initial seeding levels (6×, 12×, and 96× dilutions). Log phase growth curves are linearly fitted in a semi-log plot as solid lines to extract the population doubling time.

FIG. 11illustrates another example of the filter device1100that incorporates a plurality of the flexible filter arrays100in series to form the tandem flexible filtration device. As illustrated, the filter device1100includes four different flexible filter arrays100arranged in series. In one embodiment, the size of the gap widths g of each of the flexible filter array100is different from the other flexible filter array100of the filter device1100. When different gap spaces g are arranged in series as shown, it is possible to capture different materials, cells, blood cells, subtype of blood cells, stem cells circulating tumor cells, microbes, metabolic aggregates on each of the flexible filter arrays100. For example, the flexible filter array1101with the largest gap space g is placed first in the series of the flexible filter arrays100allowing the first flexible filter array1101to capture the largest materials, particles, cells, and circulating tumor cells. The next flexible filter array1102can be sized smaller than the first flexible filter array1101to capture the materials, particles, cells, and circulating tumor cells. Likewise, each successive flexible filter array100can be sized such that the respective gap space g is smaller than the previous flexible filter array100. In at least one embodiment, two or more of the flexible filter arrays100can have the same gap space. For instance the last two flexible filter arrays (1103,1104) can have the same gap size g. This can be done to increase the capture rate of the most desired materials, particles, cells, and circulating tumor cells. Alternatively, the first two flexible filter arrays (1101,1102) can have the same gap size g so as to prevent the larger materials, particles, cells, and circulating tumor cells from clogging the next size flexible filter array100.

The filter device1100as shown is constructed using a top layer1110that provides for ingress and egress of the fluid to be filtered. For example, the fluid can be blood or water. Other fluids are considered within the scope of this disclosure. The top layer has two ports (1112,1114) that can be configured to accommodate various connectors. One of the ports is an inlet port1112, and the other port is an outlet port1114. The top layer1110can also include one or more perimeter attachment devices1116. The perimeter attachment devices1116are pin receivers that receive a pin1170. Additionally, the top layer can include a center attachment device1118. As illustrated the center attachment device is in the form of a pin receiver that is configured to receive a center pin1172. In at least one embodiment, the top layer1110can be a plastic layer. In other embodiments, the top layer1110can be a glass or metal layer.

The next layer is a top chamber layer1120. In at least one embodiment, the top chamber layer can be made of Polydimethylsiloxane (PDMS). In other embodiments, the top chamber layer can be made of rubber, plastic or other type of polymer. The top chamber layer1120accommodates the fluid upon entrance or exit from the respective flexible filter array100. As illustrated there are four top chambers formed in the top chamber layer1120. A first top chamber1122receives the fluid from the inlet port1112. A second top chamber1124receives fluid after it has passed through the first flexible filter array1101and a second flexible filter array1102. The third top chamber1126receives fluid from the second top chamber1124via a upper coupling port1125that is formed in the second layer1120between the second top chamber1124and the third top chamber1126. The fourth top chamber1128receives the fluid after it has passed through all four flexible filter arrays100and prior to exiting the filter device100through the exit port1114. The top chamber layer also has perimeter coupling devices1121and a center coupling device1123. The perimeter coupling devices1121and the center coupling device1123are the in form of through holes that are configured to receive pins1170and a center pin1172, respectively. Other coupling devices can be implemented as well.

The third layer1130can be a flexible filter layer. The third layer in at least one embodiment can be constructed as a single sheet. In another embodiment, the flexible filter arrays100are formed separately and then bonded to the third layer1130. The third layer1130can also include perimeter coupling devices1131and a center coupling device1133. In the illustrated embodiment, the perimeter coupling devices1131and a center coupling device1133are the in form of through holes that are configured to receive pins1170and a center pin1172, respectively. Other coupling devices can be implemented as well.

The fourth layer is a bottom chamber layer1140. The bottom chamber layer can be formed of PDMS like the top chamber layer1120. The bottom chamber layer1140includes a first bottom chamber1142that receives the fluid that has passed through the first flexible filter array1101. The first bottom chamber1142can be coupled to the second bottom chamber1144by a coupling pathway1162. The fluid in the second bottom chamber1144then passes through second flexible filter array1102. After the fluid has passed through the third flexible filter array1103, the fluid enters the third bottom chamber1146. The third bottom chamber1146can be coupled to the fourth bottom chamber1148by a coupling pathway1164. Fluid in the fourth bottom chamber pass through the fourth flexible filter array1104before entering the fourth top fluid chamber1128.

A fifth layer can be a base layer1150. As illustrated the bottom acrylic layer serves as a base for the filter device. The pins1170and center pin1172can be coupled or affixed to the base layer1150. In other embodiments, other bases or materials for the base and top portion can be implemented. Furthermore, while four chambers and flexible filter arrays100are illustrated, the filter device can implement a greater or lesser number according to the desired filtration scheme.

While the filter device is shown arranged with a series of chambers, the filter device could instead be constructed using a series of stacked layers of flexible filter arrays separated from one another. For example, the flexible filter array layers can be separated by spacers. Alternatively, a series of stacked chambers could be implemented. When the flexible filter array layers are stacked, it can be advantageous as the amount of pressure is reduced.

FIG. 12illustrates one example of using the tandem filtration device, to separate different subtypes of the leukocytes. Gap widths of 5.9, 4.3, and 1.9 μm were chosen for the three filter patches in the tandem device. Granulocyte-to-lymphocyte-ratio was measured to be 3.3 on the 5.9 μm gap-width patch, while the lymphocyte-to-granulocyte-ratio was 2.5 on the 1.9 μm gap-width patch. The non-ideal separation is due to the size overlap (˜30% lymphocytes are “large” lymphocytes and comparable to small granulocytes) and complication of monocytes (largest leukocytes but identified as mononuclear cells in acridine orange staining).

FIG. 13A-Fillustrates another example of using the tandem filtration device, to separate viable cells and cells died from different mechanisms. Serum starvation can result cell death in tumor cells.FIG. 13Aillustrates a measured cell size distribution.FIG. 13Billustrates fluorescence and differential interference contrast (DIC) microscopic images for cell death detection.FIG. 13Cillustrates separation of viable, apoptotic and necrotic cells by a tandem filter array as described herein. Viable, apoptotic and necrotic cells can be detected with fluorescent vital stains. Three distinct populations of cells are found to have different cell size distribution, with necrotic cells the largest, apoptotic cells the smallest and viable cells in between (FIG. 13AandFIG. 13B).FIGS. 13D-Fillustrate composite fluorescence images of separate cells on each flexible filter array. Tandem filtration device with gap widths of 1.5 μm (FIG. 13D), 3.5 μm (FIG. 13E), and 5.5 μm (FIG. 13F) were used to fractionate tumor cells after serum starvation. Approximately 90% of the necrotic cells are found on 5.5 μm gap width filter patch and none on 1.5 μm GW patch, while over 50% of the apoptotic cells are found on 1.5 μm gap width filter patch and none on 5.5 μm gap width filter patch (FIG. 13D-F).

FIG. 14A-Fillustrates another example of using the tandem filtration device, to separate cells of the same type but at different cell cycles.FIG. 14Aillustrated measured size distribution at different stages of the cell cycle.FIG. 14Billustrates capture efficiency of cells at different stages of the cell cycle by a single flexible filter array of a varying gap widths.FIG. 14Cillustrates a separation of early S (red fluorescence labeled) and G2/M (green fluorescence labeled) stage cells by a tandem flexible filter array.FIGS. 14D-Fillustrate composite fluorescence microscopic images of polylactic acid, polyglycolide, or any other micromachinable biodegradable material with gap widths of 2.5 μm (FIG. 14D), 4.3 μm (FIG. 14E), and 6.5 μm (FIG. 14F). Mammalian cell's volume changes during the cell cycle, with the smallest of newly born cells in G1 phase and largest before mitosis in the G2 phase. As a heterogeneous population, cells might be in different phases of cell cycle and this may contribute to cell size variation. In fact, size based separation is an effective physical approach for cell synchronization. MDA-MB-231 cells are synchronized at different cell cycle stages: G0/G1 stage by serum starvation, early S stage by double thymidine (dT) block, late S stage by lease after dT block and G2/M stage by thymindine-nocodazale block. Cell size distributions of individual cell population (FIG. 14A) and the capture efficiency of single FMSA device with different gap widths for each cell population (FIG. 14B) were measured. Based on this information, gap widths of the filters inside the tandem device were chosen. 99% of the cells on 6.5 μm gap width FMSA patch are at G2/M stage, while the ratio of cells in Early S to those in G2/M stage is ˜10 on the 2.5 μm gap width FMSA patch.

While the above embodiments and examples are provided, it is appreciated that this disclosure is for a flexible filter array according to the teachings as supplied herein. Specifically, other configurations and arrays of flexible filter arrays can be made and remain within the scope of this disclosure.