Classifications

• • G01N15/1433—
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
  • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
  • B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation

Definitions

• Rare cell isolation is an important example of an application in which particles (the rare cells) are filtered from a medium (blood).
• a medium blood
• filters used for this purpose a plurality of pores is arranged in a layer that extends perpendicular to the flow direction of the filtered medium.
• a disadvantage of such filters is that the retained cells are often damaged due to high pressure gradients.
• a transfer of the cells is needed if they shall be further analyzed, which means additional stress for the cells.
• a filter element according to a first aspect of the invention serves for retaining particles of a medium flowing in a flow direction through at least one opening of the filter element.
• a method that is related to the first aspect of the invention serves for filtering particles out of a medium by retaining them in at least one opening through which said medium passes in a flow direction.
• This method is characterized in that the retained particles are held between opposite areas (or points) where they contact the opening, wherein the medium continues to flow along said particles outside said areas, through the remaining parts of the opening.
• Such a retention of particles can particularly be achieved with the elongate opening of the above filter element, in which a particle gets stuck between the opposite narrow walls of the opening.
• the filter element and the method according to the third aspect of the invention have the advantage that the particles which are retained in the opening can directly be observed. Hence there is no need to transfer these particles from the filter element to another location prior to such an observation. This simplifies the workflow and, most of all, means less stress for the particles.
• a method is preferred in which filtering of particles out of a medium is achieved by retaining them in at least one opening, wherein said particles are retained at different positions that correspond to their size, wherein the retained particles are held between opposite areas while the medium continues to flow along them, and wherein the opening is visually inspected.
• a high throughput through the filter element can be achieved throughout the whole filtering process.
• a plurality of (similar designed) openings can be used through which the medium flows in parallel.
• several (at least two, preferably at least ten, most preferably at least 100) of these openings are spatially arranged in parallel to achieve an optimal exploitation of available area.
• the openings may be arranged with their large dimensions substantially in a common plane.
• the medium is preferably provided from one side of the array and withdrawn from the opposite side and the openings share a common transparent wall.
• the invention further relates to a filter system comprising a filter element according to any of the embodiments described in the application together with additional components for interacting with said filter element and/or the medium inside.
• the filter system may for example comprise a (microscope) objective adjacent to the transparent wall of the opening for visual inspection of the opening, particularly for a visual examination of the retained particles by a human observer. Additionally or
• an image sensor may be provided adjacent to the transparent wall for generating an image of the opening.
• the generated (typically digital) image can then be evaluated by a human observer and/or by automatic image processing routines that are realized by appropriate software. If the opening has an elongated cross section, the objective and/or image sensor is preferably disposed adjacent to its large dimension.
• At least one valve element is provided for selectively forwarding medium that has passed the opening.
• the medium can be led to different destinations.
• the setting of the valve element may be changed to allow for an analysis of the retained particles.
• Retained cells may for example be destroyed and then washed out into particular reaction chambers, where further analysis like PCR can be done. Thermocycling can be done in this stage, too, for example by introducing the filter element into an appropriate thermal controller.
• the reaction chambers are preferably realized on the same carrier as the filter opening(s), thus allowing the execution of filtering and analysis with one and the same device.
• a (micro-) fluidic system may optionally be provided for supplying reagents that shall be mixed with the medium during the filtering process and/or that are needed for a further processing of retained particles after the filtering. If biological cells are retained, the reagents may for example be needed for washing, fixing and/or staining of these cells. It is a particular advantage of the present invention that such a treatment can be done without a need to first transfer retained cells to another location.
• the fluidic system may particularly comprise a storage for the needed media and/or control elements (valves, pumps etc.) for controlling their flow.
• magnetic particles for example as labels for target components of the medium
• magnetic particle shall comprise both permanently magnetic particles as well as magnetizable particles, for example
• a filter system according to the invention may comprise a magnet.
• the aforementioned magnet may particularly be used to retain magnetic particles (and components bound thereto) within the filter element by magnetic forces, independent of the effect of the opening.
• magnetic particles typically with attached cells
• This attraction may be permanent during the filtering procedure or transient, e.g. followed by a release of the magnetic force for further washing purposes.
• the magnetic actuation can also be used to bring magnetic particles (cells) towards a surface to enhance the specific binding to that surface by capture molecules which are immobilized on the surface. Once bound, the magnetic force may be removed and the unbound elements may be washed away.
• the "washing" of magnetic particles may be done hydrodynamically (using a wash fluid) and/or magnetically by another magnetic force that pulls magnetic particles away from the filter element.
• the opening through which the medium passes, a part of this opening, and/or parts within flow channels that are connected to the opening may preferably comprise binding sites for components (e.g. particles) of the medium. Additionally or alternatively, the aforementioned magnetic particles may comprise such binding sites on their surface.
• the binding sites may for example be antibodies that specifically bind to target cells of a biological fluid. Thus it is possible to specifically retain and/or capture components even if they are too small to be retained by the opening.
• the invention further relates to a method for filtering a medium in a filter element of the kind described above, said method comprising the following steps:
• This treatment may for example comprise a staining or lysis of retained cells.
• the treated particles may be imaged (at their position in the opening) and/or they may be moved into a separate compartment for further analysis.
• the invention relates to the use of a filter element of the kind described above for cell-based diagnostics and/or for the isolation of cells of interest from a matrix, for example from blood or another body fluid.
• the isolation of cells may be part of a diagnostic procedure, or it may serve other purposes (e.g. therapy). Most preferably, the isolation is specific (selective), i.e. only cells of a certain type and/or with certain
• Fig. 2 shows a section in x-direction through an opening of the filter element of Figure 1, said filter element being part of a filter system;
• Fig. 4 shows a photograph of a filter element after 15 minutes of filtering a medium with polystyrene beads and THP1 cells.
• CTCs circulating tumor cells
• Circulating tumor cells can differ from leukocytes in their size and stiffness as well as the presence of epithelial cell surface markers (EPCAM). It is possible to isolate CTCs based on specific immune-capture of EPCAM positive cells with antibody- functionalized magnetic beads. CTCs which do not express this marker are thus however not captured.
• EPCAM epithelial cell surface markers
• Filters can also be used to get rid of excess magnetic beads which are used for immune-capture and otherwise interfere with imaging.
• a design for a cell capture cartridge will be described below that is based on in-plane filtration with (optional multistep) size selection and marker-specific capture. Captured cells are kept in separate channels to enable separate molecular analysis of individual cells after identification by imaging. No cell transfer is required.
• FIGS 1 and 2 illustrate an exemplary embodiment of this idea in a top view and a sectional view, respectively.
• the drawings show a filter element 100 (or cartridge) with a plurality of (here seven identical) elongated openings 120 (also called “gaps" in the following) through which a medium can flow in x-direction (horizontal arrows in Figure 1).
• Each opening 120 has an elongate, rectangular cross section extending in the y,z-plane perpendicular to the flow direction.
• the ratio between the maximal diameter (b in Figure 1) and minimal diameter (h in Figure 2) of these cross section, i.e. b:h preferably ranges between about 100: 1 and about 1000: 1.
• Figure 2 shows the filter element 100 of Figure 1 as a part of a filter system 10 comprising additional components 1, 2, 3 needed for the manipulation and/or observation of a sample medium in the filter element.
• the aforementioned cross sections of the openings decrease in size in flow direction x.
• this decrease is caused by a reduction of the height h of the openings, wherein the smallest height hmin is also indicated in Figure 2.
• the decrease in height occurs in the shown example discontinuously in (three) steps, partitioning the opening in three compartments 122, 123, and 124 of different cross sections. Accordingly, larger cells CI will be retained in the first (largest) compartment 122, while smaller cells C2 manage to enter subsequent smaller compartments 123 or 124 until they get stuck, too.
• the length L of the opening 120 (measured in flow direction) is preferably larger than the overall minimal diameter h m i n , for example L > 5-h m i n or even L > 10-h m i n .
• a plurality of openings 120 are connected functionally in parallel. To achieve a compact arrangement, these elongated openings 120 are also spatially arranged in parallel as shown in Figure 1.
• the medium to be filtered is provided to the openings 120 by a common inlet channel 110 that begins at an inlet port 111 where the medium can be introduced into the filter element 100.
• distribution channels 121 are sequentially connected to the common inlet channel 110, wherein each distribution channel 121 extends perpendicular to the common inlet channel 110 and distributes the medium to one of the openings 120.
• the minimal diameters of the cross sections (i.e. the clearance) in the flow path from the sample inlet 111 to the openings 120 shall be larger than the clearance within the openings, such that any particles of interest will be retained in the opening and not earlier.
• Each opening 120 ends in an associated collection channel 125, wherein all these collection channels 125 lead into a common outlet channel 131.
• This common outlet channel 131 extends perpendicular to the collection channels 125 and terminates in an outlet port 132 where medium can be withdrawn from the filter element 100. All channels can thus be connected to a single waste chamber. Again, the clearance of the flow path behind the openings 120 shall be larger than within the openings. Accordingly, the openings constitute a bottleneck in the overall flow path.
• the filter element 100 is built from a structured bottom element 102 that is covered by a planar top element 101. At least one of these elements, preferably both, is made from a transparent material like glass or plastic to allow for a visual inspection of the interior of the openings 120.
• the top cover 101 is assumed to be transparent such that the interior of the opening 120 can be visually inspected via an adjacent microscope objective 1.
• the microscope objective 1 may generate an image on an image sensor 2 which can optionally be evaluated by digital image analysis procedures in a computer (not shown).
• the design of the openings is preferably such that all erythrocytes and the vast majority of leukocytes of a blood sample pass through. Therefore only little surface area is required for collecting the target cells.
• By creating a lateral stair of parallel sections with decreasing gap height cells which are smaller than the adjacent gap will move into the next section until they get blocked by a gap they cannot enter based on their size and flexibility. In this way always monolayers of cells are created and cells accumulate at the edge of the proximal gap.
• the blocking of cells does not affect the overall flow until the edge is covered with cells over the full length.
• the length b of the edges can be chosen very large to have a low flow resistance and avoid blocking of the opening. Based on the expected small number of positive cells the number of openings (channels) is chosen such that on average only 1 cell will be captured in each opening 120. In addition areas can be defined in the downstream section which contain e.g. anti-EPCAM capture probes at the (structured) surface to capture also small EPCAM positive cells. Figure 2 shows such a capture coating 126 in the smallest compartment 124 of the opening 120. Even cells that are equal and/or smaller than the leukocytes can thus be captured if they are EPCAM positive.
• the imaging optics 1 can recognize the orientation of the analysis area and use that as control for the stepping and imaging approach during analysis.
• Features can be added to the walls which separate the analysis areas or alternatively features can be present inside the analysis area to provide information about the position of the analysis area with respect to the total filter element 100 (cartridge). This can be a marker like a code or symbol which is interpreted by the image analysis software.
• the walls and steps of the openings 120 are integral part of the substrate 102 which can be produced by replication in plastic from a master with the inverse structure, or alternatively by etching in glass.
• the cover 101 of the cartridge is made from a flat thin sheet of plastic or glass which can be coated on the inner surface with reagents.
• the substrate and cover are joined for instance by laser or thermal bonding.
• the resulting filter element 100 can have microscope slide dimensions.
• Inlet and outlet ports 111, 132 can be integrated for coupling to a system for fluid handling. Additionally or alternatively, reservoirs 112 can be integrated in the device optionally with on-board reagents.
• a magnet 3 can be attached.
• cells with attached beads will be pulled to the channel walls or the bottom of the openings and stay there as long as the magnetic field is on. This allows an additional way of collecting cells which are validated already.
• the result of this capture can be compared in situ in the same cartridge and single scan with the size-based capture in the stair channels and (if included) the capture by antibodies which are immobilized on the channel surface.
• the aforementioned attraction of magnetic particles (and cells attached thereto) may optionally be followed by a release of the magnetic force for further washing purposes. Additionally, magnetic forces may be used to actively remove unbound beads away from the binding surface before the assay is evaluated.
• Figure 3 shows a filter element 200 according to the aforementioned embodiment. Components that are similar or identical to those of the first embodiment ( Figures 1 , 2) need not be explained again.
• the novel feature of the filter element 200 is the design of the outlet structure 230:
• a valve mechanism is provided for controlling the flow through the aforementioned channels.
• a first valve system 237 controls the connection between the collection channels 225 and the reaction chambers 236.
• a second valve system 238 controls the connections between the reaction chambers 236 and the common waste collection channel 234.
• a third valve 239 controls the connection between the common outlet channel 231 and the outlet port 232.
• the valve systems 237, 238, and 239 can for instance be realized by pneumatic control lines which act as valves for the associated fluidic channels.
• the medium leaving the openings 220 can selectively be forwarded either directly to the outlet port 232 (third valve 239 open and first valves 237 closed) or to the individual reaction chambers 236 (third valve 239 closed and first valves 237 open). While the first alternative is preferably used during the filtering process, the latter is used after filtering for an analysis of the retained particles in the reaction chambers 236.
• the reaction chambers 236 can contain microarrays for specific hybridization of R A, or DNA. Amplification reactions can be carried out after introducing PCR mix to the crude lysate of the cells. It is clear that the design of the downstream part can be modified to accommodate the necessary reaction steps. Thermocycling can be done for amplification by introducing the cartridge in the appropriate thermal controller.
• the substrate is compatible with fluorescence detection for reading microarrays and monitoring qPCR.
• the dimensions of the filter element can be adjusted for different diagnostic applications. For instance for small cell lung cancer it is known that the CTCs are very large. For other applications where the cell sizes are closer a closer spacing of the gap heights is required.
• the ideal gap heights can be determined experimentally before use in diagnostic application. In the minimum scenario the filter would only be used to separate free magnetic beads from captured cells after immune-capture without further specific separation of the cells and in addition for the benefit of creating a monolayer for identification with staining and pathological investigations.
• a first parameter to be considered is the flow resistance in relation to the flow rate.
• the flow rate is derived from the sample volume and desired analysis time.
• the pressure drop ⁇ ⁇ scales as follows (with ⁇ being the viscosity and V being the velocity of the medium):
• a captured cell will seal off a pore completely so that the pressure inside the pore will be lower than on top of it (the pressure difference is due to the pressure drop due to flow in the neighboring, still open pores). Once all pores are filled the pressure drop will be equal to the applied pressure at the entrance. This pressure difference will occur in a very small region where the cell membrane makes contact to the pore of the filter. This local gradient can rupture the cell membrane.
• the elongated opening is not sealed off by the cell so that the pressure drop remains moderate and continuous over the cell membrane. It is equal to the pressure drop in the wider channel due to flow.
• the structured substrates 102 can be manufactured by injection molding or other replication technology.
• the master structure which is required as a mold can be produced with the aid of photolithography and/or etching.
• One possible approach requires two lithographic masks. The first one contains the analysis areas (i.e. the openings 120, 220).
• a mask representing the channel structure is used in order to create a resist pattern which is used for the selective etching of the substrate (e.g. silica). After etching the etch resist is stripped and the photopolymer on the analysis areas remains on the substrate. In this way three levels are created on the substrate.
• This substrate can then be copied into a Ni shim by electroplating in a way which is well established for instance in optical disc manufacturing. The Ni shim is then used as an insert in the mold for replication in plastic by molding.
• etching in glass can be used to create the stepwise channels with precisely defined gaps.
• reservoirs can be attached or integrated which allow interconnecting to fiuidic tubing or syringes or fluid handling stations, as well as for storing reagents and waste.
• Pumping of the reagents can be done by external pumps, pneumatic interfaces, or other integrated pumps.
• a test medium was filtered in a staircase filter element 300 (similar in design to that shown in Figures 1 and 2, but with only two steps 322, 323).
• 15 ⁇ polystyrene beads (Phosphorex) at a concentration of 1.6- 10 5 beads/ml PBS and THP 1 cells at a concentration of 1.2 ⁇ 10 6 cells/ml PBS were mixed.
• the resulting medium contained 8- 10 4 beads and 6- 10 5 THPl cells per ml PBS. This medium was led through the staircase filter at 0.2 ml/min.
• Figure 4 shows a photograph of the filter element 300. It can be seen that some cells are stuck in the narrowest slit 323 or are caught by the beads B which effectively form a filter with a small pore size. Pretreatment of the device may reduce the adhesion of cells.

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• 2012
  • 2012-10-24 WO PCT/IB2012/055844 patent/WO2013061257A1/en active Application Filing
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