Classifications

• • 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
  • B01L3/502761—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 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/06—Fluid handling related problems
  • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
  • B01L2200/0652—Sorting or classification of particles or molecules
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0809—Geometry, shape and general structure rectangular shaped
  • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
  • B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
  • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/08—Regulating or influencing the flow resistance
  • B01L2400/084—Passive control of flow resistance
  • B01L2400/086—Passive control of flow resistance using baffles or other fixed flow obstructions

Definitions

• the present invention relates to a micro-fluidic device operable to isolate target cells from a biological fluid and a method of isolating target cells from a biological fluid.
• Cancer is a leading cause of death globally and early detection is one of the most effective means to combat the disease.
• Recent clinical studies show that the number of cancer cells in cancer patients' blood can predict the disease development and treatment efficacy. Studying these cells may also lead to a better understanding of the disease.
• getting access to blood samples is relatively easy and less invasive and painful than tumour biopsies.
• CTCs circulating tumour cells
• CTCs are found in patients with metastatic carcinomas (W.J. Allard, J. Matera, M.C. Miller, M. Repollet, M.C. Connelly, C. Rao, A.G. Tibbe, J.W. Uhr, L.W. Terstappen, Clin. Cancer Res. 10, 6897 (2004); S. Steen, J. Nemunaitis, T. Fisher, J. Kuhn. Proc 21 , 127 (2008), Bayl Univ Med Cent.) and are associated with the disease progression (M.
• CTCs in blood of cancer patients varies and depends on the conditions of the patients.
• Leading techniques to enumerate CTCs include immuno-magnetic separation followed by immunocytochemistry detection, such as the CellSearch® system sold by Veridex LLC (a Johnson & Johnson company) of Raritan, NJ, U.S.A. (M. Cristofanilli, G.T. Budd, M.J. Ellis, A. Stopeck, J. Matera, M.C. Miller, J.M. Reuben, G.V. Doyle, W.J. Allard, L.W. Terstappen, D.F. Hayes, N. Engl. J. Med. 351 , 781 (2004); H. Yagata, S.
• the isolated cells are likely to be difficult to retrieve due to the binding of the tumor associated antigens to the device. Retrieving these cells may require high mechanical forces or biochemical agents and the integrity of these cells might be affected as a result (S.F. Chang, C.A. Chang, D.Y. Lee, P.L. Lee, Y.M. Yeh, C.R. Yeh, C.K. Cheng, S. Chien, J.J. Chiu, Proc. Natl. Acad. Sci. USA 105, 3927 (2008)).
• most of these cells may require high mechanical forces or biochemical agents and the integrity of these cells might be affected as a result (S.F. Chang, C.A. Chang, D.Y. Lee, P.L. Lee, Y.M. Yeh, C.R. Yeh, C.K. Cheng, S. Chien, J.J. Chiu, Proc. Natl. Acad. Sci. USA 105, 3927 (2008)).
• a micro-fluidic device operable to isolate target cells from a biological fluid
• the micro-fluidic device comprising: an inlet operable to receive the biological fluid, the biological fluid comprising target cells and other components; an waste outlet operable to receive at least the other components of the biological fluid; a plurality of parallel arrays of cell isolation wells coupling the inlet with the waste outlet, each parallel array of cell isolation wells supporting a flow of the biological fluid from the inlet to the waste outlet in response to a pressure differential thereacross, each array of cell isolation wells comprising a plurality isolation wells, each isolation well being dimensioned to mechanically trap the target cells therein whilst permitting flow of other components of the biological fluid; and at least one pressure maintenance structure operable to assist in maintaining a predetermined pressure differential across each of the plurality of parallel arrays of cell isolation wells.
• the first aspect recognises that there are practical difficulties when attempting to isolate target cells from within a test sample. For example, there are difficulties relating to the physical properties of the test sample being processed and difficulties encountered when attempting to maximise the number of target cells being isolated. For example, because the concentration of target cells to be isolated is typically very low, it may be necessary to increase the volume of sample being processed or increase the efficiency of target cell isolation using, for example, cell isolation structures, wells or traps. Processing an increased volume of sample can take more time. Although the rate of target cell isolation can be increased by placing more cell isolation structures in series to increase the likelihood of a target cell being retained, this typically requires operating at an increased pressure due to the increased resistance to flow. Likewise, whilst it may be possible to place an increased number of cell isolation structures in parallel to decrease resistance to flow, this causes difficulty in maintaining a consistent pressure differential across the increased number of cell isolation structures to keep the cells isolated therein.
• a micro-fluidic device operable to isolate or capture target material, such as cells, from a fluid, such as a biological fluid.
• a sample inlet may receive the biological fluid which may contain target cells and other components.
• a waste outlet may be provided which receives at least some of the other components of the biological fluid.
• Coupling the sample inlet with the waste outlet may be a plurality of parallel arrays of isolation wells.
• Each of the parallel arrays may enable fluid to flow from the sample inlet to the waste outlet following the application of a pressure differential across the arrays of isolation wells.
• Each array may comprise a number of isolation wells and each isolation well may be dimensioned to physically trap the target cells.
• the arrangement of the isolation wells may be such that the likelihood of capturing the other components of the fluid may be reduced.
• One or more pressure maintenance structures may be provided which facilitates achieving a pressure differential within the device when the device is operated.
• a device which may provide an increased number of parallel arrays of isolation wells to enable an increased volume of fluid to be processed using an increased number of traps in order to increase the number of target cells which are retained.
• the volume of sample that can be processed per unit time may be increased. This may reduce the processing time of a sample whilst increasing the efficiency of target cell isolation.
• each of the parallel arrays of cell isolation structures may be operated effectively to ensure correct processing of the sample. In particular, the likelihood of the flow of the sample through each of the parallel arrays of traps being maintained in a controllable manner may be increased.
• the at least one pressure maintenance structure comprises: a primary pre-filter array positioned between the inlet and the plurality of parallel arrays of cell isolation wells, the primary pre-filter array comprising a plurality of rows of primary structures dimensioned to mechanically trap bodies within the biological fluid larger than the target cells.
• a problem with some test samples, such as, for example, blood is that the sample may be of poor quality and may be subject to coagulation. Accordingly, the sample may cause local blockages or disrupt the flow within the arrays of cell isolation wells. Such disruption in flow may prevent a pressure differential from being maintained across cell isolation wells. Accordingly, a pre-filter array may be positioned between the sample inlet and the arrays of cell isolation wells.
• the pre-filter array may comprise structures which are configured to trap components within the sample fluid having predetermined dimensions.
• the provision of such a pre-filter array may enable loose material to be held.
• the materials can be confined outside of the main micro-fluidic structure which enables the dimensions of subsequent micro-fluidic structures to be scaled down considerably, thereby facilitating a reduction in size of the micro-fluidic device.
• the area presented by the pre-filter array may determine how much material can be retained.
• a distance between each primary structure may affect the amount of material which is able to pass through the pre-filter array.
• such a pre-filter array may be used to trap emboli which may also contain CTCs.
• adjacent rows have primary structures which are offset with respect to each other. Offsetting rows of primary structures provides a mechanically robust array which is an effective filter, since large material is likely to have its path obstructed by such offset structures.
• each primary structure presents a width to the flow of the biological fluid and a distance between adjacent primary structures in a row is approximately the width.
• the distance between adjacent primary structures in each row decreases.
• the distance between adjacent primary structures decreases from around ⁇ to 30 ⁇ .
• the width presented by primary structures in each row decreases.
• a distance between adjacent rows is greater than the width
• each primary structure has a generally curved cross-section. Providing a generally curved cross-section helps to reduce any flow impedance through the pre-filter array. In addition, a curved or round cross-section reduces additional stresses on any target cells within the fluid passing through the pre-filter array.
• each primary structure comprises a cylindrical pillar.
• each primary structure has a radius of approximately 20 ⁇ , the distance between adjacent primary structures in a row is approximately 20 ⁇ and the distance between adjacent rows is approximately 40 ⁇ .
• the dimensions of components the pre-filter array will typically be determined based on the size of the target cells and the likely size of large material which needs to be trapped, determined by properties of the fluid being analysed.
• the primary pre-filter array is positioned away from the plurality of parallel arrays of cell isolation wells by a predetermined distance. Positioning the pre-filter array away from the array of cell isolation wells helps to restore uniform flow and may equalize the pressure across the arrays of cell isolation wells. In other words, the effect of any local obstructions within the pre-filter array on the flow of material through each of the arrays of cell isolation wells can be reduced by providing a buffer region between the pre-filter array and the arrays of cell isolation wells to equalize any local pressure variations.
• the primary pre-filter array comprises an inlet flow distributor positioned between the inlet and the plurality of rows of primary structures, the inlet flow distributor comprising a tree of fluidic channels. Accordingly, a flow distributor structure may be provided between the inlet and the primary structures.
• a flow distributor distributes the biological fluid to the plurality of rows of primary structures to provide an even pressure distribution across the rows of primary structures and to obviate the effects of local flow disturbances caused by clogging. The greater the number of levels within the tree, the smoother the transition. It will also be appreciated that providing such an inlet flow distributor may become necessary as the device is scaled up to include many numbers of parallel primary pre-filter arrays and therefore helps to provide for unfettered scalability.
• a root level of the tree presents a number of fluidic channels to the inlet and a leaf level of the tree presents 'n' fluidic channels to the plurality of rows of primary structures. It will be appreciated that the number of fluidic channels presented to the plurality of rows of primary structures need not necessarily exactly match the number of primary structures in the first row of the plurality of rows of primary structures.
• each leaf level away from the root level and towards the plurality of rows of primary structures presents at least an additional fluidic channel.
• the primary pre-filter array comprises a flow combiner positioned between the plurality of rows of primary structures and the plurality of arrays of cell isolation wells, the flow combiner comprising a tree of fluidic channels.
• the micro-fluidic device comprises a collection outlet operable to receive debris trapped by the pre-filter array.
• the at least one pressure maintenance structure comprises: a secondary pre-filter array positioned between the primary pre-filter array and the plurality of parallel arrays of cell isolation wells, the secondary pre-filter array comprising at least one row of secondary structures dimensioned to mechanically trap bodies within the biological fluid larger than the target cells. Accordingly, a secondary pre-filter array may be provided positioned between the arrays of cell isolation wells and the primary pre-filter array. Such a secondary array may enhance the ability to pre-filter the fluid.
• each secondary structure presents a width to the flow of the biological fluid and a distance between adjacent secondary structures in a row is less than the width.
• the secondary pre-filter array comprises a plurality of rows of secondary structures.
• each secondary structure has a generally rectilinear cross-section.
• each secondary structure is has a width of approximately 35 ⁇ and the distance between adjacent secondary structures in a row is approximately 20 ⁇ . It will be appreciated that, once again, the exact dimensions of the secondary structure will need to enable target cells to pass through whilst preventing the passage of any larger bodies into the subsequent structures.
• the row of secondary structures is partitioned by supporting structures positioned to align with each boundary of the plurality of parallel arrays of cell isolation wells.
• Providing supporting structures helps increase the mechanical integrity of the secondary pre-filter array. By aligning the secondary structures with the boundaries of the arrays of cell isolation wells, the flow of material from the pre-filter array may be aligned with the arrays of cell isolation wells, thereby improving flow.
• the secondary pre-filter array is positioned away from the plurality of parallel arrays of cell isolation wells by a predetermined distance. Again, by positioning the secondary structures away from the arrays of cell isolation wells provides a buffer region within which pressure equalization can occur despite local obstructions being present.
• the predetermined distance is approximately 500 ⁇ .
• the distance between the pre-filter array and the array of cell isolation wells will depend on the characteristics of the fluid being processed and the dimensions of the device.
• the at least one pressure maintenance structure comprises: a flow combiner positioned between the plurality of parallel arrays of cell isolation wells and the waste outlet and, the flow combiner comprising a tree of fluidic channels operable to maintain a uniform pressure presented by the outlet to each of the plurality of parallel arrays of cell isolation wells. Accordingly, a flow combination structure may be provided between the arrays of cell isolation wells and the waste outlet. Without such a combiner, the pressure experienced by each of the arrays of cell isolation wells may vary considerably, dependent on the relative location of the waste outlet to that array of cell isolation wells.
• a root level of the tree presents one fluidic channel to the waste outlet and a leaf level of the tree presents 'n' fluidic channels to the plurality of parallel arrays of cell isolation wells. It will be appreciated that the number of fluidic channels presented to the arrays of isolation wells need not necessarily exactly match the number of arrays of cell isolation wells.
• each leaf level away from the root level and towards the plurality of parallel arrays of cell isolation wells presents an additional fluidic channel.
• the fluidic channels have a width of approximately 150 ⁇ . It will be appreciated that the exact dimensions of the flow combiner will depend on the
• the micro-fluidic device comprises: a target cell recovery outlet and in which the at least one pressure maintenance structure comprises: a conduit coupling the plurality of parallel arrays of cell isolation wells with the target cell recovery outlet, the conduit being shaped to restrict flow of fluid therethrough. Accordingly, an outlet may be provided through which target cells may be recovered. It will be appreciated that the target cells may be recovered from the arrays of cell isolation wells and so the target cell recovery outlet will typically need to be coupled with those arrays of cell isolation wells. In order to prevent the presence of the target cell recovery outlet from disrupting the flow through the arrays of cell isolation wells, a conduit which restricts flow from the arrays of cell isolation wells to the target cell recovery outlet may be provided.
• the conduit is shaped to restrict flow of fluid by causing at least one change in direction of flow.
• a change in direction of flow may be utilised to restrict the flow of fluid, it will be appreciated that other mechanisms for restricting flow could equally be used.
• the conduit is shaped to cause at least two orthogonal changes in direction of flow.
• the conduit is shaped to cause at least four orthogonal changes in direction of flow.
• each section of the conduit has a constant width.
• the conduit has a width of approximately 50 ⁇ .
• the exact dimensions of the conduits will typically depend on the size of the target cells which need to be recovered.
• each array of cell isolation wells comprises a plurality of sub- arrays of cell isolation wells spaced apart in a direction of the flow and a plurality of the conduits are provided, each conduit coupling a sub-array with the target cell recovery outlet. Accordingly, rather than providing just one conduit for recovering the target cells from an array of cell isolation wells, it is possible to split the array of cell isolation wells into sub- arrays and provide a conduit for each of those sub-arrays. It will be appreciated that this considerably facilitates the recovery of target cells from the array of cell isolation wells.
• each conduit is received by a common conduit coupled with the target cell recovery outlet.
• the waste outlet and the target cell recovery outlet are operable to maintain a pressure differential to cause a change in direction of flow within the parallel arrays of cell isolation wells to release trapped cells to be conveyed to the target cell recovery outlet.
• the at least one pressure maintenance structure comprises: a flow distributor coupling the plurality of parallel arrays of cell isolation wells with the inlet, the distributor comprising a tree of fluidic channels.
• Such a flow distributor distributes the biological fluid to the arrays of cell isolation wells to provide an even pressure distribution across the arrays of cell isolation wells and to obviate the effects of local flow disturbances caused by clogging.
• a root level of the tree presents a number of fluidic channels to the inlet and a leaf level of the tree presents 'n' fluidic channels to the arrays of cell isolation wells. It will be appreciated that the number of fluidic channels presented to the arrays of cell isolation wells need not necessarily exactly match the number of arrays of cell isolation wells.
• each leaf level away from the root level and towards the plurality of arrays of cell isolation wells presents at least an additional fluidic channel.
• the micro-fluidic device comprises: a target cell recovery outlet and the flow distributor couples the plurality of parallel arrays of cell isolation wells with the target cell recovery outlet. Accordingly, an outlet may be provided through which target cells may be recovered. It will be appreciated that the target cells may be recovered from the arrays of cell isolation wells and so the target cell recovery outlet will typically need to be coupled with those arrays of cell isolation wells.
• the flow distributor provides a uniform flow field for efficient cell collection. Once again, it will be appreciated that this facilitates the scaling of the device to include many more arrays of cell isolation wells. Such an arrangement, when the flow is reversed, helps to concentrate target cells recovered from the arrays of cell isolation wells when being provided to the target cell recovery outlet.
• the target cell recovery outlet is located on the inlet side of the plurality of parallel arrays of cell isolation wells.
• the micro-fluidic device comprising: a reagent inlet operable to receive a reagent, the reagent inlet comprising a conduit operable to deliver the reagent to between the inlet and the plurality of parallel arrays of cell isolation wells. Accordingly, a reagent can be added to react with the contents of the micro-fluidic device.
• the conduit is operable to deliver the reagent to between primary pre-filter array and the secondary pre-filter array. Accordingly, a reagent can be added between the pre-filter array and the arrays of cell isolation wells in order to avoid any clogging within the pre-filter array and to enable the reagent to reach the target cells much more quickly.
• the reagent comprises at least one fluorescence in situ
• the reagent comprises at least one of a first fluorescent probe and a second fluorescent probe.
• a primary pre-filter array is provided between the reagent inlet and the plurality of parallel arrays of cell isolation wells.
• each array of cell isolation wells is coupled in parallel between the inlet and the outlet.
• the cell isolation structures comprise crescent-shaped structures operable to retain target cells such as, for example, cancer cells, fetal cells or stem cells.
• each cell isolation structure defines at least one gap in the crescent-shaped structure.
• each gap is around 6 ⁇ to 9 ⁇ .
• the crescent-shaped structures are tilted with a tilt axis which is non-orthogonal with respect to a direction of flow through the array of cell isolation wells.
• each cell isolation well within each row of cell isolation wells is tilted with the same tilt axis.
• each cell isolation well within adjacent rows of cell isolation wells is tilted with opposing tilt axes.
• cell isolation wells within each row are spaced apart by gaps and cell isolation wells within adjacent rows are positioned to align with the gaps.
• a distance between adjacent rows of cell isolation wells increases in a direction of flow from the inlet to the outlet.
• a method of isolating target cells from a biological fluid comprising the steps of: receiving the biological fluid at an inlet, the biological fluid comprising target cells and other components; applying a pressure differential across a plurality of parallel arrays of cell isolation wells coupling the inlet with a waste outlet operable to receive at least the other components of the biological fluid, each parallel array of cell isolation wells supporting a flow of the biological fluid from the inlet to the waste outlet in response to a pressure differential thereacross, each array of cell isolation wells comprising a plurality isolation wells, each isolation well being dimensioned to mechanically trap the target cells therein whilst permitting flow of other components of the biological fluid; and maintaining a predetermined pressure differential across each of the plurality of parallel arrays of cell isolation wells.
• the step of maintaining a predetermined pressure differential comprises the step of: positioning a primary pre-filter array between the inlet and the plurality of parallel arrays of cell isolation wells, the primary pre-filter array comprising a plurality of rows of primary structures dimensioned to mechanically trap bodies within the biological fluid larger than the target cells.
• adjacent rows have primary structures which are offset with respect to each other.
• each primary structure presents a width to the flow of the biological fluid and a distance between adjacent primary structures in a row is approximately the width.
• the distance between adjacent primary structures in each row decreases.
• the distance between adjacent primary structures decreases from around ⁇ ⁇ to 30 ⁇ .
• the width presented by primary structures in each row decreases.
• each primary structure has a depth and a distance between adjacent rows is greater than the width.
• each primary structure has a generally curved cross-section.
• each primary structure comprises a cylindrical pillar.
• each primary structure is has a radius of approximately 20 ⁇ , the distance between adjacent primary structures in a row is approximately 20 ⁇ and the a distance between adjacent rows is approximately 40 ⁇ .
• the step of positioning comprises: positioning the primary pre- filter array away from the plurality of parallel arrays of cell isolation wells by a predetermined distance.
• the primary pre-filter array comprises an inlet flow distributor positioned between the inlet and the plurality of rows of primary structures, the inlet flow distributor comprising a tree of fluidic channels.
• a root level of the tree presents a number of fluidic channels to the inlet and a leaf level of the tree presents 'n' fluidic channels to the plurality of rows of primary structures.
• each leaf level away from the root level and towards the plurality of rows of primary structures presents at least an additional fluidic channel.
• the primary pre-filter array comprises a flow combiner positioned between the plurality of rows of primary structures and the plurality of arrays of cell isolation wells, the flow combiner comprising a tree of fluidic channels.
• the method comprises the step of applying a pressure differential to receive debris trapped by the pre-filter array at a collection outlet.
• the step of maintaining a predetermined pressure differential comprises the step of: positioning a secondary pre-filter array between the primary pre-filter array and the plurality of parallel arrays of cell isolation wells, the secondary pre-filter array comprising at least one row of secondary structures dimensioned to mechanically trap bodies within the biological fluid larger than the target cells.
• each secondary structure presents a width to the flow of the biological fluid and a distance between adjacent secondary structures in a row is less than the width.
• the secondary pre-filter array comprises a plurality of rows of secondary structures.
• each secondary structure has a generally rectilinear cross-section.
• each secondary structure is has a width of approximately 35 ⁇ and the distance between adjacent secondary structures in a row is approximately 20 ⁇ .
• the method comprises the step of: partitioning the row of secondary structures by supporting structures positioned to align each boundary of the plurality of parallel arrays of cell isolation wells.
• the step of positioning comprises: positioning the secondary pre- filter array away from the plurality of parallel arrays of cell isolation wells by a predetermined distance.
• the predetermined distance is approximately 500 ⁇ .
• the step of maintaining a predetermined pressure differential comprises the step of: positioning a flow combiner between the plurality of parallel arrays of cell isolation wells and the waste outlet and, the flow combiner comprising a tree of fluidic channels operable to maintain a uniform pressure presented by the outlet to each of the plurality of parallel arrays of cell isolation wells.
• a root level of the tree presents one fluidic channel to the waste outlet and a leaf level of the tree presents 'n' fluidic channels to the plurality of parallel arrays of cell isolation wells.
• each leaf level away from the root level and towards the plurality of parallel arrays of cell isolation wells presents an additional fluidic channel.
• the fluidic channels have a width of approximately 150 ⁇ .
• the step of maintaining a predetermined pressure differential comprises the step of: providing a conduit coupling the plurality of parallel arrays of cell isolation wells with a target cell recovery outlet, the conduit being shaped to restrict flow of fluid therethrough.
• the method comprises the step of recovering target cells from the target cell recovery outlet.
• the step of recovering target cells from the target cell recovery outlet comprises reversing a flow through the cell isolation wells by applying a differential pressure between the waste outlet and the target cell recovery outlet.
• the conduit is shaped to restrict flow of fluid by causing at least one change in direction of flow.
• the conduit is shaped to cause at least two orthogonal changes in direction of flow.
• the conduit is shaped to cause at least four orthogonal changes in direction of flow.
• each section of the conduit has a constant width.
• the conduit has a width of approximately 50 ⁇ .
• each array of cell isolation wells comprises a plurality of sub- array of cell isolation wells spaced apart in a direction of the flow and a plurality of the conduits are provided, each conduit coupling a sub-array with the target cell recovery outlet. In one embodiment, each conduit is received by a common conduit coupled with the target cell recovery outlet.
• the step of recovering target cells from the target cell recovery outlet comprises reversing a flow through the cell isolation wells by maintaining a pressure differential between the waste outlet and the target cell recovery outlet to cause a change in direction of flow within the parallel arrays of cell isolation wells to release trapped cells to be conveyed to the target cell recovery outlet.
• the step of maintaining a predetermined pressure differential comprises the step of: providing a flow distributor coupling the plurality of parallel arrays of cell isolation wells with the inlet, the flow distributor comprising a tree of fluidic channels.
• a root level of the tree presents a number of fluidic channels to the inlet and a leaf level of the tree presents 'n' fluidic channels to the arrays of cell isolation wells.
• each leaf level away from the root level and towards the plurality of arrays of cell isolation wells presents at least an additional fluidic channel.
• the method comprises the step of providing a target cell recovery outlet, the flow distributor coupling the plurality of parallel arrays of cell isolation wells with the target cell recovery outlet.
• the method comprises the step of recovering target cells from the target cell recovery outlet.
• the step of recovering target cells from the target cell recovery outlet comprises reversing a flow through the cell isolation wells be applying a differential pressure between one of a buffer port and the waste outlet and the target cell recovery outlet.
• the method comprises locating the target cell recovery outlet on the inlet side of the plurality of parallel arrays of cell isolation wells.
• the method comprises the step of: receiving a reagent at a reagent inlet, the reagent inlet comprising a conduit operable to deliver the reagent to between the inlet and the plurality of parallel arrays of cell isolation wells.
• the conduit is operable to deliver the reagent to between primary pre-filter array and the secondary pre-filter array.
• the reagent comprises at least one fluorescence in situ
• the reagent comprises at least one of a first fluorescent probe and a second fluorescent probe.
• a primary pre-filter array is provided between the reagent inlet and the plurality of parallel arrays of cell isolation wells.
• each array of cell isolation wells is coupled in parallel between the inlet and the outlet.
• the cell isolation structures comprise crescent-shaped structures operable to retain target cells such as, for example, cancer cells, fetal cells or stem cells.
• the cell isolation structures comprise 'U' or 'V'-shaped structures.
• each cell isolation structure defines at least one gap in the crescent-shaped structure. In one embodiment, each gap is around 6 ⁇ to 9 ⁇ .
• the crescent-shaped structures are tilted with a tilt axis which is non-orthogonal with respect to a direction of flow through the array of cell isolation wells.
• each cell isolation well within a row of cell isolation wells is tilted with the same tilt axis. In one embodiment, each cell isolation well within . adjacent rows of cell isolation wells is tilted with opposing tilt axes. In one embodiment, cell isolation wells within each row are spaced apart by gaps and cell isolation wells within adjacent rows are positioned to align with the gaps.
• a distance between adjacent rows of cell isolation wells increases in a direction of flow from the inlet to the outlet.
• the method comprises the step of performing fluorescence in situ hybridization (FISH).
• FISH fluorescence in situ hybridization
• the method comprises the step of performing a secondary fluorescence in situ hybridization (FISH).
• FISH secondary fluorescence in situ hybridization
• Figure 1 illustrates a diagnostic system incorporating a micro-fiuidic device according to one embodiment
• Figure 2 shows a laboratory prototype of a fabricated micro-fluidic device
• Figure 3 illustrates an arrangement of the micro-fluidic device according to one embodiment in more detail
• Figure 4A illustrates a configuration of a first pre-filter array in more detail
• Figure 4B illustrates a configuration of a secondary pre-filter in more detail
• Figure 4C illustrates an arrangement of a plurality of arrays of cell isolation wells in more detail
• Figure 4D illustrates a configuration of a gradient generator in more detail
• Figure 4E illustrates a configuration of flow restrictors in more detail
• Figure 5 illustrates cell isolation wells in more detail
• Figure 6 illustrates an arrangement of the micro-fluidic device according to another embodiment in more detail
• FIG. 7A, Figure 7B and Figure 7C illustrate a flow combiner in more detail
• Figure 8 illustrates a pre-filter array in more detail
• FIG. 9 illustrates cell isolation wells in more detail.
• a label-free micro-fluidic device capable of isolating target cells, such as cancer cells or other cells, from whole blood via their distinctively different physical properties such as
• the isolation efficiency is on average at least 80% for tests performed on breast cancer, colon cancer and other types of cancer cell lines. Viable isolated cells are also obtained which may give further insights to enhance the understanding of the metastatic process.
• a micro-fluidic device provides a mechanistic and efficient means of isolating viable cancer cells in blood.
• the micro-fluidic device has the potential to be used for routine monitoring of cancer development and cancer therapy in a clinical setting.
• a device that presents a means to enumerate and isolate viable CTCs or other target cells from peripheral blood with a high throughput and high efficiency. Isolation and quantification of CTCs presents an alternate disease marker that assists in monitoring cancer progression and determining overall survival.
• retrieving viable CTCs assists in the further study of key biological
• Micro-fluidic devices provide an alternative technique compared to conventional biochemical separations- Devices utilizing dielectrophoretic forces to separate and manipulate cells are advantageous as they do not require functionalization of the sample or the micro- fluidic device (P.Y. Chiou, A.T. Ohta, M.C. Wu, Nature 436, 370-372 (2005); D.S. Gray, J.L. Tan, J. Voldman, C.S. Chen, Biosens. Bioelectron. 19, 771-780 (2004); A. Rosenthal, J. Voldman, Biophys. J. 88, 2193-2205 (2005); J. Voldman, Curr Opin Biotechnol 17, (2006)).
• efficient cancer cell separation may be difficult due to the low concentration of CTCs or other target cells in blood and the relative similar dimensions of leukocytes with cancer cells.
• the retrieval of CTCs or other target cells is made relatively easy to achieve by controlling the input/output conditions.
• no functional biochemical modification of the device or CTCs or other target cells is necessary to maintain the integrity of CTCs or other target cells.
• the microsystem of embodiments has a high throughput and is able to process the blood samples within minutes.
• embodiments permit real time visualization of the isolation process.
• a system may provide a simple means of enumerating target cells (such as CTCs) using a uniform array.
• a device may permit real time enumeration of isolated cells.
• embodiments enable fluorescence in situ hybridization (FISH).
• FISH fluorescence in situ hybridization
• Such a technique can be used to detect and localise the presence or absence of specific DNA sequences on chromosomes of targets cells.
• Fluorescent probes are introduced via the reagent inlet 150 that bind to only those parts of the chromosome with which they show a high degree of sequence similarity. Fluorescence microscopy can be used to find out where the fluorescent probe is bound to the chromosomes.
• FISH can also be used to detect and localize specific mRNAs within samples and can help define the spatial-temporal patterns of gene expression within cells and tissues.
• FISH involves fixing the cells (i.e., killing the cells and chemically cross-linking proteins, nucleic acids), exposing the fixed cells to a staining agent and visualising it.
• the staining agent typically is a nucleic acid probe which binds to portions of the DNA and has a fluorescent tag so the chromosomes can be visualised.
• the micro-fluidic device 10 enables target cells to be sieved, immobilised in the isolation wells 190 and then visualised or stained to identify target cells in a first round.
• a stain that picks out tumour cells may be used (e.g., an antibody that binds to a tumour specific antigen expressed on the cell surface) to see which cells that have been sieved are tumour cells.
• a second round of staining using FISH can then be used to look at the chromosomes of those particular cells, (e.g., to see if there are chromosomal breaks that might be associated with the tumour phenotype).
• double staining is possible. Because the target cells are immobilised in the isolation wells 190 in which they were caught during the sieving, increased - certainty is possible that whatever cells were identified using the first stain are the cells which are being examined after the second stain.
• the FISH signals from those positions in the matrix which are positive from the first signal can simply be examined. Without the immobilisation, it is not possible to do this.
• FISH may be performed either on the target cells within the isolation wells 190 or may be performed within a FISH structure (not shown) on the microfluidic device 10 or outside the micro fluidic device 10.
• a separate channel may be provided on the side of the microfluidic device 10 (having appropriate couplings for introduction of the reagents) within which FISH may be performed or alternatively or additionally a structure (not shown) may be provided which enables small amounts of reagents to be located near the isolation wells and piped thereto so that minimal reagents are used.
• additional inlet ports and tubing may be required.
• a device in accordance embodiments has potential application to aid clinicians in cancer disease monitoring and retrieval of viable CTCs or other target cells for further analysis.
• the ease of use and portability of the device presents an attractive replacement to current methodologies.
• a high throughput ensures fast recovery time of diagnosis results which can aid in timely drug administering to better the chances of survival.
• a device in accordance with embodiments is fabricated in accordance with the biorheology of cells and depends on the deformability and dimensions of the cells. Studies of various different types of cells' biorheology may be used to determine the dimensions of the device. In such studies, fresh cell samples may be used to ensure consistent biorheological properties of the cells.
• a micro-fluidic device to isolate target cells such as viable cancer cells of breast and colon origins from whole blood using solely the biorheological property differences of cancer cells and blood constituents. No functional modifications of the micro-fluidic device are required as isolation is solely dependent on the biorheological property differences of the target cells such as cancer cells and blood constituents.
• the setup of the device consists of two main components to process samples such as blood.
• a pressure control component maintains the desired pressure setting through software control on a computer which in turn drives the blood sample into the device or draws the blood sample through the device.
• the device works on the physical characteristics of cells such as cancer cells from blood cells which are impeded in a cell isolation region. Pre-filters are added to account for coagulations that might be present in blood and a change of pressure settings will allow the recovery of target cells into the collection point.
• immunofluorescence staining in the device is also possible for enumeration and purity characterization.
• the micro-fluidic device may be used to capture and isolate target cells such as circulating tumor cells from peripheral blood of cancer patients for diagnostic and prognostic purposes.
• the micro-fluidic device is attractive for applications in oncology research, particularly prognostication and prediction of drug response.
• Embodiments overcome the technical challenges posed by the low cancer cell count in blood combined with large sample volumes. By utilizing the biomechanical property differences of cancer cells from blood, embodiments achieve an effective isolation of cancer cells.
• biomechanical means to isolate the target cells need to be provided. This presents a problem in the practical application of biomechanical isolation, since in order to trap sufficient numbers of target cells, large volumes of blood need to be processed. To be able to process large volumes of blood, it is desirable to increase the size of the micro-fluidic device, but this presents practical difficulties in controlling the flow through parts of the device and, in particular, maintaining a required pressure differential across any structures which are used to mechanically isolate the target cells. Hence, a number of arrangements are provided which facilitate the desired flow and pressure differentials within parts of the micro-fluidic device to enable the device to be scaled up to process larger volumes of a sample, such as blood.
• Figure 1 illustrates a diagnostic system, generally 100, incorporating a micro-fluidic device 10 according to one embodiment. It will be appreciated that the diagnostic system 100 may also be utilized with an alternative micro-fluidic device 10' illustrated in Figure 6 below.
• the diagnostic system 100 comprises a control system 20 coupled with a micro-fluidic device assembly 30 incorporating the micro-fluidic device 10.
• the micro-fluidic device assembly 30 comprises the micro-fluidic device 10 coupled with a sample syringe 40 containing the sample to be analysed.
• the sample syringe 40 supplies, under the control of the control system 20, the sample contained therein to a sample inlet 50 of the micro-fluidic device 10.
• a waste syringe 70 Also coupled with the micro-fluidic device 10 via a waste outlet 60 is a waste syringe 70.
• the waste syringe 70 is also controlled by the control system 20 and receives fluids emitted from the micro- fluidic device 10.
• a cell collection syringe 80 Also coupled with the micro-fluidic device 10 is a cell collection syringe 80 which receives target cells trapped by the micro-fluidic device 10 provided via a target cell outlet 90.
• one or more reagent syringes (not shown) which provide reagents into the micro-fluidic device 10 in order to interact with fluid and/or target cells within the micro-fluidic device to assist subsequent analysis.
• micro-fluidic device assembly 30 consists of a custom made holder which is mountable onto a microscope, sealed tubes and the micro-fluidic device 10.
• the control system 20 maintains a desired pressure setting through software control via the computer 1 10 which drives the programmable syringe pump 120 to apply a pressure differential to the syringes to control fluid flow through the micro-fluidic device 10.
• the programmable syringe pump 120 applies a pressure differential to the sample syringe 40 and the waste syringe 70 to control flow from the inlet 50 to the waste outlet 60.
• the programmable syringe pump 120 may also control the application of reagents into the micro-fluidic.
• a pressure transducer 130 is coupled with the syringes which, via a multi meter 140 acting as an interfacing device, provides pressure values to the computer 1 10.
• a pressure transducer 130 is coupled with the syringes which, via a multi meter 140 acting as an interfacing device, provides pressure values to the computer 1 10.
• the pressure settings may be monitored on a regular basis and the programmable syringe pump 120 controlled using software (coded in LabVIEW) to make adjustments, for example every 100 milliseconds.
• a differential pressure is created in the tubes connected to the micro-fluidic device 10 by compressing or expanding the syringes attached to the syringe pump 120.
• the programmable syringe pump applies a differential pressure to force the sample into the sample inlet 50.
• the sample passes through the micro-fluidic device 10 which traps target cells therein, as will be explained in more detail below. Any untrapped target cells and other components of the sample emerge from the waste outlet 60 and are received in the waste syringe 70.
• the sample syringe 40 may be held static whilst one or more reagent syringes are activated to deliver one of more reagents into the micro-fluidic device 10.
• Surplus fluid is continued to be collected within the waste syringe 70 which may be expanded to assist in maintaining a required pressure differential and flow direction.
• the target cells trapped within the micro-fluidic device 10 may then be directly analysed in situ. Additionally or alternatively, the trapped target cells may be recovered from the micro-fluidic device 10.
• this is achieved by changing the direction of flow through the micro-fluidic device 10 towards the target cell outlet 90 for storage in the cell collection syringe 80.
• This may be achieved by maintaining the sample syringe 40 in a static configuration whilst expanding the cell collection syringe 80 and either contracting the waste syringe 70 or contracting a syringe containing a benign fluid coupled to a point at or near the waste outlet 60.
• the reverse directed of flow caused by a reverse pressure differential enables the trapped target cells to disengage from structures within the micro-fluidic device 10 and travel to the cell collection syringe 80.
• Figure 2 shows a laboratory prototype of the fabricated micro-fluidic device 10.
• Fabrication of the device is achieved with soft lithography using a biocompatible polymer (polydimethylsiloxane) bonded onto a glass slide.
• a biocompatible polymer polydimethylsiloxane
• FIG. 3 illustrates an arrangement of the micro-fluidic device 10 in more detail.
• the micro-fluidic device 10 has a sample inlet 50, a waste outlet 60 and a target cell outlet 90.
• the device has the reagent inlet 150 and a debris removal outlet 160.
• the debris removal outlet 160 is typically coupled with a debris syringe (not shown) to enable large items of debris which are unable to pass through the micro-fluidic device 10 to be removed, as will be explained in more detail below.
• trapped emboli which may also contain CTC may be stained and retrieved via the debris removal outlet 160.
• the micro-fluidic device comprises a first pre-filter array 170, which performs initial pre-filtering of the sample to capture large debris such as blood clots or other large bodies, a secondary pre-filter 180, a plurality of arrays of cell isolation structures 190, which capture target cells, and a pressure gradient generator 200.
• flow restrictors 210 are provided between the array of cell isolation wells and the target cell outlet 90.
• pre-filter arrays 170, 180 together with the arrangement of the gradient generator 200 and the flow restrictors 210, enable a large-scale device having many parallel arrays of cell isolation wells to be provided since a. uniform flow and pressure differential across each of those arrays of cell isolation wells can be achieved.
• the operation of the micro-fluidic device 10 is as follows. Initially, a sample is introduced through the sample inlet 50 and a pressure differential is generated between sample inlet 50 and waste port 60 to cause a flow through the device generally in direction A. This causes the sample being tested to pass through the primary pre-filters 170 and the secondary pre-fi Iters 180. The pre-filters pre-filter the sample, thereby removing the need for the sample to be pre-processed prior to being provided to the micro-fluidic device 10. Any large debris within the sample is likely to be trapped within the pre-filter arrays.
• a pressure differential may be created to cause a flow out of the debris removal outlet 160, for example by contracting syringes coupled with the sample inlet 50, the reagent port 150 or the waste outlet-60 and by expanding a syringe coupled with the debris removal outlet 160 to dislodge any large bodies which may be blocking flow to remove these from the micro-fluidic device 10. It will be appreciated that such a blockage will be detected by the pressure transducer 130.
• the pre-filtered sample then passes into the plurality of arrays of cell isolation wells 190.
• the cell isolation wells utilise the characteristic that target cells may have different mechanical properties to other cells within the sample. For example, cancer cells may be stiffer and generally larger than most blood cells, which will impede their flow as they pass through the cell isolation structures. The target cells will then be retained within the cell isolation structures whilst other constituents flow past. By maintaining a pressure differential which causes the flow generally in direction A, the target cells remain retained within the cell isolation wells.
• reagents may then be introduced via the reagent inlet 150.
• the micro-fluidic device 10 enables in situ immunofluorescence staining of the target cells to quantify and enumerate them or to direct them to the target cell outlet 90 for retrieval.
• the benefit of on-chip staining simplifies operations by avoiding any intermediate preparatory steps and minimises cell losses due to sample transfer to maximise yield.
• the reagent use is minimal, as the volume inside the micro-fluidic device 10 is small.
• the sample inlet 50 is closed and the reagent inlet 150 opened.
• on-chip staining can be completed by flowing in each reagent sequentially.
• a readout can be achieved manually or using commercial image processing software to identify cells that carry the tumour markers using a normal fluorescence microscope or any compatible array scanner.
• the target cells may be removed from the device for external processing or analysis by creating a pressure differential which causes a flow within the arrays of cell isolation wells generally against direction A and towards target cell outlet 90. This may be achieved by expanding the syringe coupled with the target cell outlet 90 and contracting a syringe coupled with waste outlet 60. This dislodges any target cells trapped within the isolation wells and they flow to the target cell outlet 90.
• FIG. 4A illustrates the configuration of the first pre-filter 170 array in more detail. Due to considerable clogging in cancer patient blood and also due to the presence of micro- emboli (tumours fragments that have detached), pre-filtering can be of significant importance.
• the first pre-filter array 170 comprises an array of structures 220. These structures are formed into an array of rows and columns. Each row is offset from its adjacent column.
• the first pre-filter array 170 comprises multiple 20 ⁇ circular structures with gaps of 20 ⁇ between structures in each row. Each adjacent row has a 40 ⁇ gap therebetween. Each adjacent row is offset by another 20 ⁇ to ensure that clumps of large material are held in place.
• the gaps are sufficiently larger than cells to allow them to pass through the pre-filter array 170 with ease.
• the distance between each row is set at 60 ⁇ to allow the sample to flow smoothly. Clumps are likely to occur with samples which have been subject to prolonged storage or samples that are improperly stored.
• the first pre-filter array 170 should effectively remove such clumps before the blood reaches the isolation arrays 190 so that they do not hinder the sample processing.
• this embodiment has a uniform spacing of structures 220, it will be appreciated that the spacing could be arranged to reduce in the direction of flow (i.e. the structures 220 could be cascading, having smaller and smaller gaps therebetween). Likewise, a series of differing structures 220 of different diameters and flow gaps may be provided; these may be correspondingly smaller (as in water filter systems) to trap the micro- emboli.
• FIG. 4B illustrates the configuration of the secondary pre-filter 180 in more detail.
• the secondary pre-filter 180 is provided to help to prevent any large debris that may have passed through the first pre-filter 170 from entering the array of cell isolation wells 190.
• the secondary pre-filter 180 is spaced away from the primary pre-filter array 170 to normalise flow and to equalise any local variation in the pressure caused as a result of any blockages in the first pre-filter array 170.
• the secondary pre-filter array 180 is spaced apart from the arrays of cell isolation wells 190 to provide for such flow and pressure normalisation.
• the secondary pre-filter array 180 comprises a row of structures.
• a row of rectangular blocks 230 having dimensions of 35 ⁇ by 20 ⁇ ⁇ and spaced to have 20 ⁇ gaps therebetween.
• the blocks 230 are separated by supporting structures 240 which assist in the mechanical integrity of the micro-fluidic device 10 during fabrication.
• the arrangement of the supporting structures aligns with similar supporting structures which compartmentalise the isolation region into the plurality of arrays of isolation wells 190. This helps to align the flow from the secondary pre-filter 180 to the arrays of isolation wells 190.
• this embodiment has a single row of blocks 230, it will be appreciated that more than one row could be provided either with uniform spacing and/or the spacing could be arranged to reduce in the direction of flow (i.e. the blocks 230 could be cascading, having smaller and smaller gaps therebetween).
• Figure 4C illustrates an arrangement of the plurality of arrays of cell isolation wells or structures 190 in more detail.
• Each array is compartmentalised by a supporting structure 250.
• Each array is approximately 560 ⁇ by 850 ⁇ .
• Each array comprises a number of rows and columns of isolation structures 260 illustrated in more detail in Figure 5.
• compartmentalisation of the arrays of isolation wells facilitates counting, recovery of target cells and provides uniform flow characteristics.
• the compartmentalised arrays also facilitate the fabrication of the micro-fluidic device by providing large supporting structures to prevent the device from collapsing.
• using compartmentalised arrays facilitates further expansion for future scaling up of the device.
• Each compartmentalised array is approximately 560 ⁇ wide and 850 ⁇ in length and holds 200 cell isolation structures 260. Including more isolation structures 260 within each array will affect the recovery process and thus is not favoured.
• the cell isolation structures 260 in each row are staggered to increase the hydrodynamic efficiency.
• the dimension and placement of the isolation structures 260 are optimized for fabrication ease and isolation efficiency.
• Target cells, such as CTCs, are generally less deformable and larger than most blood cells and so will be passively retained by the isolation wells 260 and the multiple rows of isolation wells 260 increases the overall cell retention efficiency.
• the hydrodynamic profile of the structure also prevents build up of cells in any particular region to facilitate the smooth processing of the samples.
• the placement of the cell isolation wells 260 is staggered to improve the isolation yield and the offset assists in capturing cells that miss an earlier row of cell isolation wells 260.
• the gaps of around 5 ⁇ and preferably 6 ⁇ to 9 ⁇ within each isolation structure allow constituents to leave the cell isolation wells 260 whilst retaining the target cells in place. In order to trap fetal cells, these gaps may be reduced to 2 or 3 ⁇ .
• the radius of each cell isolation well 260 is approximately 10 ⁇ to enable entrapment of cells of approximately 6 to 28 ⁇ .
• the parts of the cell isolation wells 260 facing the direction of flow A are rounded to ensure a smoother transition of the cells into the cell isolation wells 260.
• the selection mechanism is based on target cells being stiffer and generally larger than most blood cells which will impede them in the flow as they pass through the cell isolation wells 260. Due to the gaps in between structures, blood constituents are able to deform through which will prevent clogging in the device when processing large volume of blood. Occupied cell isolation wells 260 tend to hold only single or duplet cells due to the design which directs incoming cells downwards when cell isolation wells 260 are filled and this will facilitate counting.
• FIG. 4D illustrates in more detail the configuration of the gradient generator 200.
• the gradient generator 200 is positioned between the plurality of arrays of cell isolation wells 190 and the waste outlet 60.
• the gradient generator 200 consists of a coupling structure which reduces the number of conduits from a maximum amount at a position closest to the plurality of arrays of cell isolation wells 190 to a single conduit coupled with the waste outlet 60.
• This tree structure reduces the number of conduits by at least one at each level.
• This arrangement provides a serial gradient generator that aides the maintenance of uniform conditions in each column of the array of cell isolation wells 190.
• the gradient generator 200 provides for a generally constant pressure presented to each column of the array of cell isolation wells irrespective of its relative location with respect to the waste outlet 60.
• the generator is defined by a plurality of a reducing number of supporting structures dimensioned to generally 190 ⁇ by 150 ⁇ defining channels which are generally 150 ⁇ wide.
• FIG. 4E illustrates in more detail the arrangement of flow restrictors 210 between the plurality of arrays of cell isolation wells 190 and the target cell outlet 90.
• a flow restrictor 210 is coupled with each row of arrays of cell isolation wells.
• Each flow restrictor comprises a looped or kinked conduit which changes the direction of flow to increase the fluid resistance and provide a slight back-pressure during, for example, sample processing and reagent application to minimise the presence of any contaminants present in this region during the retrieval of the target cells.
• Providing multiple couplings with the cell isolation region helps to facilitate the recovery of target cells.
• the device is similar to the device described above but has a reconfiguration of parts, changes in the primary isolation structures and a branching network.
• the operation of the micro-fluidic device 10' is as follows. Initially, a sample is introduced through the sample inlet 50' and a pressure differential is generated between the sample inlet 50' and the waste port 60' to cause a flow through the micro-fluidic device 10' generally in direction A. This causes the sample being tested to pass through the primary filters 170', thereby removing the need for the sample to be pre-processed prior to being provided to the micro-fluidic device 10'. The filtered sample then passes into the plurality of arrays of cell isolation wells 190'.
• the cell isolation wells utilise the characteristic that target cells may have different mechanical properties to other cells within the sample, in the manner described above.
• the target cells will then be retained within the cell isolation wells whilst other constituents within the sample flow past.
• By maintaining a pressure differential which causes the flow generally in the direction A the target cells remain retained within the cell isolation wells.
• reagents may then be introduced via the reagent inlet 150'.
• the micro-fluidic device 10' enables in-situ immunofluorescence staining of the target cells to quantify and enumerate them in a similar manner to that described above prior to directing the stained cells to the target cell outlet 90' for retrieval.
• Figure 8 illustrates the arrangement of the primary filters 170'.
• a dual path is provided from the inlet 50' to a parallel pair of primary filters 170'.
• Each primary filter 170' has a branching tree structure 172' which distributes the sample within the primary filter 170'.
• the branching tree structure 172' takes a single conduit and splits into a dual conduit at each level within the tree. This helps to distribute the sample with uniform flow across the width of the primary filter 170'.
• the sample then encounters a series of rows of cylindrical pillars 175', 177' which retain debris and cell clumps.
• the gap between the pillars 175', 177' decreases progressively from row to row in the direction A from a starting gap of around 100 ⁇ down to a gap of around 30 ⁇ .
• the diameter of the cylindrical pillars 175', 177' may reduce from row to row in the direction A.
• the filtered sample is then received by a branching network 174' of conduits which combine to a single conduit 55' which then couples with the parallel arrays of cell isolation structures 190'. It will be appreciated that the branching network may instead reduce to a plurality of conduits.
• the primary filters 170' are placed away from the parallel arrays of cell isolation structures 190' in order to prevent any local flow disturbances caused by debris or cell clumps trapped by the filter 170'.
• a parallel set of primary filters 170' is provided in order that should one primary filter 170' become clogged during operation, the sample can still continue to flow through another primary filter 170'.
• additional primary filters 170' are included to prevent debris or other foreign material from entering the micro-fiuidic device 10' from the reagent port 150' of from the buffer port 195'.
• a primary filter 170' is present in the inlet port 50', the reagent port 150' and the buffer port 195'. These avoid debris entering the chambers. Debris can include dust which makes flow non-uniform in cell isolation chambers.
• Figure 7A shows a portion of the plurality of arrays of cell isolation wells 190' in more detail.
• Figure 7B shows the pressure within a branching network 210' when fluid is flowing in direction A.
• Such an arrangement of parallel arrays of cell isolation wells 190' achieves a processing speed of around 2-6 mL per hour.
• the filtered sample is received at the conduit 55' and is distributed through the branching network 210' to achieve uniform flow through each array of cell isolation wells.
• the branching network 210' forms a tree structure where each conduit splits to two conduits (although each conduit could split to more than two conduits) at each level in the tree. Accordingly, in this example, to expand the single conduit 55' to feed all 16 parallel arrays of cell isolation wells requires four levels of the tree.
• This branching network of inlet channels affects throughput and helps to distribute flow evenly. The flow profile is generally non-uniform as it enters the device and this is ameliorated by the branching network.
• the branching network increases the number of conduits at each level from 1 ->2->4->8-> 16. It is possible to use 16 conduits or more but generally a minimum 8 conduits are required to give an even flow. 16 conduits give at least 2.5 ml sample per hour.
• a reverse arrangement branching network 220' is provided which combines the waste material received from the array of cell isolation wells 190'. This waste is then received at the conduit 65' and flows under pressure into the waste outlet 60'.
• the arrays of cell isolation wells 190' consist of crescent-shaped cell traps 262', 264' to isolate and capture cells.
• the cell traps 262', 264' define two or more gaps in the crescent shape with a size ranging from around 6 ⁇ to around 9 ⁇ .
• the cell traps 262', 264' are tilted left and right handedly to increase capture efficiency.
• the distance between individual cell traps 262', 264' is optimized to increase flow rate through the micro-fluidic device 10'.
• the rows of cell traps 262', 264' are spaced to increase the probability of cell capture. The distance between successive rows of cell traps 262', 264' increases to increase the processing throughput.
• the direction of flow within the micro-fluidic device 10' is reversed' within the arrays of cell isolation wells 190'. This is achieved by preventing any flow in or out of the inlet 50', the reagent port 150' or the waste outlet 60'. Fluid is introduced through the buffer port 195', which is filtered by a filter array 170'.
• the fluid received through the buffer ports 195' is received within the conduit 65' and is distributed by the reverse arrangement branching network 220' and into the parallel arrays of cell isolation structures 190'. This reverses the flow direction (to generate a flow in a direction which is opposite to the direction A) and dislodges any cells trapped within the cell traps 262', 264'.
• Figure 7C shows the pressure within a branching network 210' when fluid is flowing in a direction which is opposite to direction A. The dislodged cells then travel into the branching network 210' and are received at the conduit 55'. A flow is then established toward the target outlet 90' and the cells are retrieved via the target cell outlet 90'.
• the buffer port 195' at the bottom of the parallel arrays of cell isolation structures 190' assists in evenly distributing the flow through the parallel arrays of cell isolation structures 190' during cell retrieval.
• the pressure differential is maintained between the buffer for 195' and the target cell outlet 90'.
• no filters are included between the parallel arrays of cell isolation structures 190' and the target cell outlet 90'.
• the target cell outlet 90' is instead placed the top of the device (i.e., on the same side as the inlet ports).
• the buffer port 195' is placed at the bottom of device. After trapping, buffer solution is pushed from buffer port 195' at the bottom to flush cells out to target cell outlet 90' at top.

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