US20170121669A1 - Magnetic separation of rare cells - Google Patents

Abstract

A magnetic separation system configured to separate with high qualitative and quantitative yield magnetized cells from cell mixtures, comprising at least one electromagnet structured to generate a magnetic field flux about a plurality of separation zones and sufficient to attract a majority of the magnetized cells in the mixture, and a pump to drive the cell mixture at a controlled flow rate through a tube disposed within the zones thereby separating a majority of the magnetized cells from the mixture. The system is particularly useful to retrieve rare cells from a fluid mixture of cells having low abundance of the rare cells relative to the rest of the cells while sustaining viability of the cells.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS
• This application is a continuation of U.S. patent application Ser. No. 14/976,895, filed Dec. 21, 2015, which is a continuation of U.S. patent application Ser. No. 13/574,557, filed Jul. 20, 2012, now U.S. Pat. No. 9,217,131, which is a national phase of PCT Patent Application No. PCT/IL2011/00068, filed Jan. 20, 2011, which claims the benefit of priority of U.S. Provisional Patent Application No. 61/296,905, filed Jan. 21, 2010. The contents of the above applications are all incorporated by reference as if fully set forth herein in their entirety.
FIELD OF THE INVENTION
• The invention relates to separation of particles including cells. Some embodiments of the invention relate to magnetic separation of rare cells.
BACKGROUND OF THE INVENTION
• A large number of applications involving the magnetic separation of biological cells are described in the literature, for example in U.S. Pat. No. 4,710,472 and the many publications cited therein. Many such applications require not only the separation of one or more specific types of cells (hereinafter called “target cells”), but also the maintenance ofthe quality of the cell membranes in the target cells and/or in the untargeted cells. Thus, in a positive selection process, the target cells are separated from a sample for examination oruse for research, diagnostic or clinical purposes; whereas in a depletion process, the sample is depleted of the target cells for examination or use of the untargeted cells. The separation of target cells from the untargeted cells, and the maintenance of the membranes of both the target cells and untargeted cells, are particularly important in research presently being conducted with lymphocyte populations and their role in the early detection of cancer.
• One technique in present use for the separation of biological cells utilizes the MiniMACS Separation Columns (Miltenyi Biotec GmbH). This technique uses paramagnetic microbeads which are extremely small, about 50 nm in diameter, i.e., aboutone million times smaller in volume than that of eukaryotic cells, compared to the size of avirus. Such magnetic microbeads are produced with selective affinities for certain cells,i.e., the target cells, such that they magnetically label or stain the target cells. The sample is introduced into a magnetic separation column including a liquid-pervious magnetic body, e.g., steel wool or mesh, and a magnetic field is applied across the column such that themagnetically stained cells are retained in the liquid-pervious magnetic body of the column,while the unstained cells pass through the column. In this known process, however, it wasfound that the membranes of the cells are excessively damaged by the liquid-pervious magnetic body, which reduces the effectiveness of the technique for research or clinical purposes.
• In some cases the target cells are rare in a sample, such as cancer cell, stem cells orfetal cells posing a challenge for the separation process. As a non-limiting example fetal cells are referred below.
• The presence of fetal cells in maternal blood has been known for over a century since Schmorl (1) demonstrated trophoblast cells in the pulmonary circulation of women who died of eclampsia. Since then, five different fetal cell types have been demonstrated to circulate in maternal blood, including trophoblast cells (1), lymphocytes (2), nucleated erythrocytes (NRBCs) (3), granulocytes (4), and stem cells (5). The frequency of fetal cells in maternal circulation is very low, with an average of 1.2 cells/ml maternal blood as detected by a quantitative PCR technique (6). Among all of the fetal cell types isolated from maternal blood, NRBCs have emerged as the best candidate cells to be used in noninvasive prenatal diagnosis (7).
• Researchers have attempted separation and enrichment of fetal NRBC using fluorescence activated cell sorting (FACS) and magnetic cell sorting (MACS) methods, including a large scale study funded by the NIH (NIFTY study). This study achieved low sensitivity of fetal cell detection with 13% recovery for FACS and 44% for MACS (the leading magnetic separation technology) (8) and concluded that as the proportion of fetal NRBC in maternal blood is low, and current separation techniques have low recovery rates, the utilization of FNRBC is not feasible for clinical application. As it is extremely difficult to separate these cells, alternative and less effective methods based on different genetic materials are explored, such as DNA fragments (9). These materials must be multiplied using PCR in order to effectively analyze them and cannot provide the same level of conclusive results regarding trisomies as FNRBCs might.
• U.S. Pat. No. 6,482,328 to some of the inventors of the present invention and others discloses a method and apparatus for magnetically separating target particles of a selected type from a sample in order to produce concentration of the target particles in the sample, or depletion of the sample with respect to the target particles, by producing a sample mixture of the sample with magnetic particles having a selective affinity to magnetically stain the target particles.
• There remains an unmet need for rapid and reliable magnetic separation of living rare cells with high yield and high purity.
SUMMARY OF THE INVENTION
• The present invention provides a system for rapid high qualitative and quantitative magnetic separation of rare cells from a fluid sample having a low abundance of the rare cells relative to the rest of the cells, while preserving the vitality the cells.
• Rare cells comprise, for example, nucleated red blood cells (NRBC) in Thalassemia patients, or fetal nucleated red blood cells (FNRBC) in the maternal circulation, cancer cells in peripheral blood, fetal or adult stem cells or cells characterized by CD71, CD8, CD34 or CD133 within the circulation.
• In some embodiments, the separation provides a sufficient concentration and/or quantity of the rare cells to obtain a significant and reliable clinical, genetic, chemical or other analysis of the cells or parts thereof.
• For example, fetal nucleated red blood cells in the circulation of a pregnant woman are very scarce relative to other cells, in the order of 100 ppb or less, preventing reliable genetic analysis of the fetal cells or DNA. According to the methods disclosed herein it is possible to obtain from a routine maternal blood sample an adequate or sufficient quantity of the FNRBC, such as above 95% of the FNRBC present in the sample. Thus the present methods enable efficient separation and the cells obtained may subsequently be used for genetic analysis or diagnosis according to any of the available methods known in the art.
• In typical embodiments, the cells of a primary sample (e.g. blood sample) comprising target cells are processed to bind or couple the cells to magnetic beads having coupling or binding specificity to the target cells in the primary sample to form, at least substantially, a sample of magnetized cells among other non-magnetized cells. In some embodiments, the target cells that are bound to the magnetic beads are rare or scarce cell types within a majority of irrelevant non-magnetic cells. In some embodiments, such as when live cells are to be separated (or depleted), the cells are processed such that their viability is preserved and the cells are separated in an environment that maintains (sustains) the cells' viability. Examples of suitable media or carrier fluids are described hereinbelow.
• The sample is mixed with a carrier fluid, constituted to sustain living cells, and is then driven through a tube that passes the mixture at a controlled flow rate in the tube irrespective of gravity. The tube passes through one or more magnetization zones (herein also ‘separation zones’) having a magnetic flux generated and actively controlled by electromagnets. The electromagnets are structured to provide magnetic fluxes formed to attain an effective magnetic separation of rare magnetic target cells. According to preferred embodiments the cells are separated and recovered while the mixture flows at a sufficiently high rate to prevent detrimental effects on the viability of the cells, due to prolonged exposure to suboptimal conditions to sustain cell viability such as lack of aeration.
• According to some embodiments the magnetic target cells are retained or separated with an efficiency of at least 50%, alternatively at least 60%, alternatively at least 70%, alternatively at least 80%, alternatively at least 90% or higher of the rare cells originally present in the sample. In other embodiments, the magnetic target cells comprise a majority of the separated cells collected. In various embodiments the magnetic target cells achieve a proportion above 60%, alternatively above 70%, alternatively above 80%, alternatively above 90%, alternatively above 95%, such as 98%, of the separated cells recovered. Each possibility is a separate embodiment of the invention.
• The separated magnetic target cells gather at and/or adhere to the tube wall in one or more of the magnetic separation zones. They are then washed (rinsed) by the carrier fluid (or other fluid) into a collecting vessel after the magnetic field is stopped to diminish the attraction of the magnetic cells to the tube.
• The present invention also provides mechanisms to assist in efficiently removing and releasing the separated cells from the tube wall, increasing the quantitative yield (recovery) of the separation (in quantity of cells). In some embodiments, the efficiency of washing of the separated magnetic target cells from the tube wall is increased by degaussing the separation zones, removing residual magnetism and decreasing the magnetic attraction of the separated target cells on the tube wall. In other embodiments, the washing is mechanically assisted by at least one of passing one or more air bubbles through the tube, abruptly changing the flow rate inducing shocks in the fluid or vibrating the tube or applying ultrasonic waves to the tube at or in the vicinity of the separation zones.
• The electromagnets are designed to provide and concentrate the magnetic flux about the tube with sufficient power for effective separation while avoiding undue or excessive power consumption that might have detrimental effect on the cells or the apparatus e.g., due to heat dissipation
• Furthermore, in order to achieve rapid yet high quality separation, the system is configured to facilitate flowing of the mixture through a tube having a sufficiently large internal diameter or cross-section at a sufficiently high rate providing rapid separation while avoiding impairing the cells.
• In some embodiments, a typical separation cycle of rare cells to purity above 90% and recovery of about or above 90% is completed within several minutes, such as between about 5 and about 15 minutes.
• In some embodiments, the system is structured such that the tube passes through, or is disposed at, the separation zones substantially horizontally thereby providing a handy apparatus for placing and operating at various locations including but not limited to sterile compartments or fume hoods. The horizontal configuration is preferred for such confined spaces as opposed to a vertically oriented system.
• The separation, retrieval and washing sequence between separation zones, the flow rate, the magnetic field at separation zones, the chemical and physicochemical characteristics of the carrier fluid (e.g. viscosity) and washing fluid, the washing rate, and/or other par ameters and operations are all controlled variables. The variables are adapted to provide desired or sufficient results while sustaining the viability of the cells and substantially avoiding aggregation of non-target cells with the magnetic target cells and/or on the tube wall which can reduce the purity and/or yield of the rare cells separation. In some embodiments, the separation working conditions are controlled to yield substantially fractional separation in separation zones according to the magnetic responsiveness of the magnetic target cells. These embodiments are akin to fractional distillation.
• It should be noted that while the separation technique outlined above are exemplified herein in efficient separation of rare cells, it is nonetheless efficient in separating and/or depletion of non-rare cells.
• It should be noted that the terms ‘separation’, and conversely ‘depletion’, do not necessarily imply total separation and conversely total removal of target cells, but practically or sufficiently high qualitative and quantitative separation, and conversely a corresponding depletion.
BRIEF DESCRIPTION OF THE DRAWINGS
• Some non-limiting exemplary embodiments of the invention are illustrated in the following drawings.
• Identical or duplicate or equivalent or similar structures, elements, or parts that appear in one or more drawings are generally labeled with the same reference numeral, optionally with an additional letter or letters to distinguish between similar objects or variants of objects, and may not be repeatedly labeled and/or described.
• Dimensions of components and features shown in the figures are chosen for convenience or clarity of presentation and are not necessarily shown to scale or true perspective. For convenience or clarity, some elements or structures are not shown or shown only partially and/or with different perspective or from different point of views.
• It should be noted that some figures were converted to black-and-white rendering, thereby degrading the pictorial quality such as by reducing certain details or texture or fineness.
• FIG. 1A schematically illustrates a system for separation of rare cells, according to exemplary embodiments of the invention;
• FIG. 1B schematically illustrates a perspective view of a separation unit of a system such as ofFIG. 1A, according to exemplary embodiments of the invention;
• FIG. 1C schematically illustrates a cross-section view of a separation unit of a system such as ofFIG. 1A, according to exemplary embodiments of the invention;
• FIG. 1D schematically illustrates an enlarged cross-section view of a magnetization zone of a system such as ofFIG. 1A, according to exemplary embodiments of the invention;
• FIG. 1E schematically illustrates another enlarged cross-section view of a magnetization zone of a system such as ofFIG. 1A, according to exemplary embodiments of the invention;
• FIG. 1F schematically illustrates another system for separation of rare cells, according to exemplary embodiments of the invention; and
• FIG. 2A schematically illustrates an external depiction of separation system such as ofFIG. 1A, according to exemplary embodiments of the invention.
• FIG. 2B schematically illustrates an external depiction of separation system such as ofFIG. 1A with a mechanism for convenient handling of the sample, according to exemplary embodiments of the invention.
DESCRIPTION OF EMBODIMENTS OF THE INVENTION
• The following description relates to one or more non-limiting examples of embodiments of the invention. The invention is not limited by the described embodiments or drawings, and may be practiced in various manners or configurations or variations. The terminology used herein should not be understood as limiting unless otherwise specified.
• The non-limiting section headings used herein are intended for convenience only and should not be construed as limiting the scope of the invention.
General Terminology
• The terms ‘about’, ‘close’, ‘approximate’ and ‘practically’ denote a respective relation or measure or amount or quantity or degree that has no adverse consequence or effect relative to the referenced term or embodiment or operation or the scope of the invention.
• The terms ‘vertical’, ‘horizontal’, ‘parallel’, ‘opposite’, ‘straight’ and other angular and geometrical relationships denote also approximate yet functional and/or practical, respective relationships.
• The terms ‘preferred’, ‘preferably’, ‘typical’, ‘typically’ or ‘optionally’ do not limit the scope of the invention or embodiments thereof.
• The terms ‘substantial’, ‘appreciable’ (or synonyms thereof) denote with respect to the context a measure or extent or amount or degree which encompass a large part or most of a referenced entity, or an extent at least moderately or much greater or larger or more effective or more important relative to a referenced entity or with respect the referenced subject matter.
• The terms ‘negligible’, and ‘slight’ (or synonyms thereof) denote, a sufficiently small respective relation or measure or amount or quantity or degree to have practical consequences relative to the referenced term and on the scope of the invention.
• The term ‘may’ denotes an option or an effect which is either or not included and/or used and/or implemented and/or occurs, yet the option constitutes at least a part of some embodiments of the invention or consequence thereof, without limiting the scope of the invention.
• The terms ‘comprises’, ‘comprising’, ‘includes’, ‘including’, ‘having’ and their inflections and conjugates denote ‘including but not limited to’.
• When a range of values is recited, it is merely for convenience or brevity and includes all the possible sub-ranges as well as individual numerical values within and about the boundary of that range. Any numeric value, unless otherwise specified, includes also practical close values and integral values do not exclude fractional values. A sub-range values and practical close values should be considered as specifically disclosed values.
• Unless the context indicates otherwise, referring to an object in the singular form (e.g. ‘a thing” or “the thing”) does not preclude the plural form (e.g. “the things”).
Contextual Terminology
• In the specification and claims the following terms and derivatives and inflections thereof imply the respective non-limiting characterizations below, unless otherwise specified or evident from the context.
• Magnetized/magnetic (cells)—coupled with a ferromagnetic material, such as with magnetic beads.
• Target cells—cells typically intended for separation or concentration from other cells (such as for examination or diagnosis), of particular type or having distinct characteristics relative to other cells, such as selective mutual affinity to couple with certain antibodies or other compounds or other particles. In particular embodiments a distinct characteristic is selective affinity to couple or bind with magnetic beads to form magnetic target cells.
• Separation—isolation or collection accumulation of target cells from a surrounding fluid bulk, where the bulk is, for example, a fluidic mixture or suspension of emulsion of cells or a combination thereof, implying also concentration or enrichment of target cells relative to the surrounding bulk or a provided sample of cells (obtaining a precipitate in analogy to precipitation or centrifugation).
• Depletion—with respect to separation, removal of target cells from the bulk (obtaining a supernatant in analogy to precipitation or centrifugation).
• High qualitative (separation, depletion)—high purity, separation of target cells substantially exclusive of other cells, or comprising negligible amount of other cells such as between about 5% and about 1% or less of the separated cells, and conversely a depletion.
• High quantitative (separation, depletion)—high recovery, separation of substantially all the target cells, or very high amount of the target cells from the sample, such as between about 95% to about 99% or more or the separated cells, and conversely a depletion.
• Magnetic beads—paramagnetic sub-micron particles, such as microbeads or microsphere having diameter or cross-section in the nanometer range (e.g. 20-100 nm), and adapted for selective affinity for coupling or binding with target cells.
• Rare cells—cells of particular characteristics which are scarce (“low-abundance”) in a source as an organism or other environment, such as about or less than 10 cells/ml (e.g. about 1 cells/ml) or less than 1 ppm (e.g. 1 ppb) relative to other cells in the respective source or environment. For example, fetal nucleated red blood cells (herein also NRBC, FNRBC) or stem cells.
• Magnetized/magnetic (cells)—coupled with a ferromagnetic material, such as with magnetic beads.
• Cells—biological cells comprising also combination of biological and other particles or compounds optionally of non-biological origin such as magnetic beads, and including for example, blood cells, microorganisms and parts thereof, or other particles of biological origin such as spores or pollen, yet not precluding particles of non-biological origin.
• Primary sample (of cells)—a volume comprising cells taken from a cells' source (e.g. blood sample, polluted water or air sample), possibly or optionally physically or chemically or biologically concentrated such as by centrifuge or reagent or antibody.
• Sample (of cells)—a volume comprising cells taken from a cells' source (e.g. blood sample, polluted water or air sample), possibly or optionally chemically and/or biologically and/or physically processed and/or altered (e.g. cooled, centrifuged, diluted, coupled with magnetic beads).
• Considerable (part or portion)—significant relative amount or quantity such as between about 25% and 80% or more of the referred entity.
• ‘ppm’, ‘ppb’—parts-per-million (10⁻⁶), parts-per-billion (10⁻⁹), respectively.
• It should be noted that whenever a reference is made herein to cells attaching or sticking or adhering to a wall of a tube, or similar terms to that effect, it does not necessarily mean that the cells attach directly to the wall, but rather, that they also connect or link or are attracted indirectly to the wall such as by chains of cells or groups of cells.
Overview
• A general non-limiting overview of practicing the invention is presented below. The overview outlines exemplary practice of embodiments of the invention, providing a constructive basis for variant and/or alternative and/or divergent embodiments, some of which are subsequently described.
• FIG. 1A schematically illustrates asystem100A for separation of rare cells from other cells, according to exemplary embodiments of the invention. Generally,system100A comprises magnetization zones124 formed inseparation units122 through which a fluid comprising a sample mixture of magnetic rare cells and other non-magnetic cells passes (flows) in atube110 from asample mixture source106 and drains to acollection vessel108. In the following discussions, unless otherwise specified or clearly evident from the context, the term ‘target cells’ implies ‘magnetic target cells’.
• Asample container102 contains sample cells comprising magnetic target rare cells in a life maintaining fluid, and a carrier container (or reservoir)104 contains a carrier fluid (hereinafter also ‘carrier’) for carrying the sample inseparation system100A. In some embodiments, typically when live cells are separated, the carrier is a life preserving fluid such as physiological saline, optionally comprising additives such as anti-coagulation ingredients.
• The sample and carrier are independently fed bypumps112 and114, respectively, into anintermediate container106 where they are mixed (referred to also as ‘mixture container’). Having independently controlled pumps allows adjusting the ratio of the sample to the carrier to facilitate sufficient or desired separation of the target cells, typically with relation to other parameters ofsystem100A (e.g. number of magnetization zones and field strength). The fluidic mixture of the sample and carrier (hereinafter ‘mixture’) is fed frommixture container106 throughtube section142 further on intotube110 via a connection schematically illustrated as144.
• Typically, the elements indicated bybracket134 are generally vertical as indicated byarrow154.
• For further reference,FIG. 1B schematically illustrates a perspective view of aseparation unit122 of system10A (shown with indicated cross-section plane A-A and top surface indicated by arrow158), andFIG. 1C schematically illustrates a cross-section A-A view ofseparation unit122, according to exemplary embodiments of the invention, whereFIGS. 1D-E schematically illustrate cross-sections A-A of two exemplary variants of magnetization zones124.
• The mixtureform mixture container106 is supplied into generally horizontally orientedtube110 disposed on the generally horizontal top surface of one ormore separation units122, as indicated byplanar coordinates152.Separation unit122 comprises an electromagnet having internal andexternal cores126aand126b, respectively, and acoil128 winded oninternal core126a, providingmagnetization zones124aand124bwhere the field direction is schematically indicated byarrows160uand160d, as also shown inFIG. 1C byrespective field directions160.
• The mixture fed intotube110 frommixture container106 is driven (impelled) intube110 bypump116 that regulates the flow rate independently of gravity and independently of the flow fromsample container102 andcarrier container104 intomixture container106. As the mixture pass in magnetization zones124, the magnetic field generated by the electromagnet is formed to attract the magnetized target cells toward the walls oftube110 and separate and concentrate the target cells from the rest of the mixture onto the walls, while impeding the non-target cells from attaching on the walls.
• As the mixture flow throughtube110 and target cells stick to the walls oftube110, the depleted mixture (substantially without the rare target cells) drains out into a collectingvessel108 viatube section146 that connects totube110 via a connection schematically illustrated as148. Typically, elements indicated bybracket136 are generally vertically oriented as indicated byarrow156.
• Once the separation is finished and the depleted mixture has drained tovessel108, the electromagnets field generation is stopped, thereby diminishing the attraction of the magnetized target cells to the tube, and the target cells are washed (rinsed) out into another collecting vessel such as anothervessel108. In some embodiments, stopping the magnetic field comprises also degaussing the electromagnets to remove residual magnetism that might still pull and stick the separated target cells to the walls oftube110, for example by applying temporally decaying alternating currents in the electromagnets, thereby assisting in releasing the separated cells off the tube wall.
• In order to enhance the yield of the separated target cells (quantitatively increase of the separation or recovery), in some embodiments in addition or alternatively to degaussing one or more measures are taken to promote releasing the target cells from the wall oftube110. For example, flowing one or more air bubbles that push on the target cells, and/or vibrating the tube such as by one or more piezoelectric elements and/or applying ultrasound on the tube, at least about magnetization zones124 , or abruptly changing the flow rate thereby inducing shocks in the fluid. Concurrent with and/or subsequent to applying the dislodging promoting means the target cells washed out by the carrier, or by another fluid such as from another container, and the constituency (e.g. viscosity) and/or flow rate of the washing fluid are optionally set or adjusted to in order to wash out substantially all the separated target cells.
• It should be noted that in various embodiments any number ofseparation units122 may be used in embodiments ofsystem100A, including a single unit, as shown by breaking/continuation indications138.
Separation System Variants
• FIG. 1F schematically illustrates anothersystem100B for separation of rare cells, according to exemplary embodiments of the invention.
• System100B is similar tosystem100A, apart that instead ofseparation units122 ofsystem100A separation units182 of different design are used insystem100B.
• Separation unit182 comprises an electromagnet comprising acore186 around which coils such as188aand188bare winded generating magnetic field indicated byarrows160pand160qabout magnetization zone184 such as184aand184b, respectively. Any number of separation units182 may be used in embodiments ofsystem100B, including a single unit, as shown by breaking/continuation indications138.
• FIGS. 2A-B schematically illustrate anexternal depiction200 of separation system such as100A, according to exemplary embodiments of the invention. The indicated parts are as or similar to those ofsystem100A as described with respect toFIGS. 1A-C.System200 is typically controlled by one or more processors or computers operated according to one or more programs stored on a computer-readable medium and handled, for example, via atouch panel204 and/or via a mouse and/or keyboard and/or any other human interface device such a dedicated keypad or a remote control unit.
• In some embodiments, collecting vessels such as108 are mounted on a mechanism, such as carousel (indicated as202), that shifts or rotates the appropriate vessel to a position for drainingtube110 according to the system operation procedures. For example, during separation a vessel for collecting the depleted mixture is placed for the drain, and during collection of the target cells another vessel is place at the drain.
• As a separation system such assystem200 is typically used in controlled environments such as sterile compartments or fume hoods, access to mount or removesample container102 or fill the sample or add ingredients to the sample may be in some cases limited or awkward if sample container102 (or mounting elements thereof) is fixedly disposed in a high position (relative to the rest of the system such as level of magnetization zone124). In some embodiments, a mechanism to provide convenient access tosample container102 is implemented.
• FIG. 2B further illustrates amechanism206 for convenient handling ofsample container102, according to exemplary embodiments of the invention.Mechanism206 is structured as a parallelogram where oneside204 is fixedly mounted on the framework ofsystem200 and the other one is moveable between an upper position and a lower position, the latter depicted as an overlay, and upward direction is indicated byarrow208.
• Optionally other mechanisms may be used, including for providing convenient access to other parts of a system such assystem200.
Equipment/Variations
• Generally,systems100A,100B and200 (FIGS. 1A, 1F and 2A-B, respectively), share common characteristics as described above, and will be generally referred to in the following descriptions, without limiting, also as system100 (also ‘the system’).FIGS. 1A-F and2A-B are also referred the following descriptions.
• The construction of the electromagnets and magnetization zones124 and184 insystem100A and100B, respectively, and the shape, directions and intensity of the magnetic field (e.g. spatial distribution) generated by the electromagnet, as well as the number of the magnetization zones, each optionally having different field shape and/or intensity, are configured or determined or tuned to achieve an effective separation of the rare target cells on the tube wall. The field flux at the magnetization zones effect a separation of the magnetized cells while impeding the non-target (and not-magnetized) cells from attaching to the tube wall, resulting in a concentration of the rare cells in a range from about 95% to about 99% or more, such as 98%, practically with no non-target cells or negligible amount thereof.
• In some embodiments, the non-target cells are impeded by hindering or delaying the movements thereof in the fluid mixture towards the tube wall, optionally by taking into account the properties of the carrier or mixture such as viscosity or elasticity. In some embodiments, the composition of the carrier (or the mixture to that matter) are set or selected or adjusted such as by additives to effect the adequate hindrance properties, while still allowing the movements of the magnetized target cells towards the tube wall under the pull of the magnetic field.
• The magnetic flux shape and intensity at the magnetization zones is configured, for example, by one or more of the shape and construction (e.g. structure, materials) of a core (e.g.126 or186), the shape and windings of a coil (e.g.128 or188), the current intensity in a coil (including optionally temporal variation of the current) and the formation (e.g. groove, fixture) where the tube (e.g. tube110) is disposed. In some embodiments, in order to achieve the separation as described above, the electromagnets are configured such that at each magnetization zone a considerable amount of the magnetized rare cells in the flowing mixture (e.g. 50% or more) are attracted onto the tube wall yet with negligible amount of or practically no non-target cells. In some embodiments, the electromagnets are designed and constructed according to design rules and/or expertise and modification are determined by checking the effectiveness of separation, for example comparing the concentration or distribution of the rare cells in the sample and in separated collection by methods of the art (e.g. cytometry or MACS). Subsequently the electromagnets or magnetization zone are adjusted or trimmed or tuned to improve the results towards a goal such as separation concentration of 95% and above.
• It is noted that the path frommixture container106 to collectingvessel108 is essentially a continuous conduit or tube, where tube such as110 and tube sections such as142 or146 andconnections144 and148 are of the same or of different diameter or cross-section. In some embodiments, the cross-section oftube110 may vary along the length thereof thereby varying the flow rate, optionally having diameter or cross-section about magnetization zones adapted to promote adherence of the target cells to the tube wall and/or prevent adherence of non-target cells to the wall oftube110 or the target cells and/or prevent coagulation of the non-target cells which may hinder the mixture flow.
• In some embodiments, the inner diameter oftube110, at least about magnetization zone124 or184 is about or larger than 2 mm to enable sufficient flow rate to achieve rapid separation.
• The connections indicated by144 and148 are tube sections such as curved tube or knee-like tube, and are typically bends in tube110 (see for example,FIGS. 2A-B), and connect the generallyvertical tubes section142 and146 to the generallyhorizontal tube110. Optionally or alternatively, the separation system may be structured such that sections of the tube or conduit frommixture container106 to collectingvessel108 may be in other orientations, between a substantially horizontal general orientation (e.g. sample container102 andmixture container106 are about the same level) to a substantially vertical general orientation or any other variation
• Typically, the tubes such as110 or142 or146 andconnections144 and148 are made of or coated with biocompatible material to preserve the live cells, such as silicone tubes. Typically the tubes are flexible and bendable and are elastically compressible (e.g. resilient to compression).
• Impelling pump116 may be disposed at any practical section on the path betweenmixture container106 to and collectingvessels108, such as before the first separation unit such as122 or182, or after the last separation unit.
• In some embodiments, any number of separation units or magnetization zones or electromagnets may be used, as shown by breaking/continuation indication138, implying also a smaller number of electromagnets or units or zones, optionally only one element thereof, as well as a shorter tube. Optionally a plurality of pumps such aspump116, typically synchronized, may be used to flow the mixture intube110.
• In some embodiments, pumps112,114 and116 are any pumps that maintain the vitality of the cells and/or provide regulated flow, such as peristaltic pumps.
• It should be noted that althoughseparation units122 or182 comprise two magnetization zones124 each, yetunits122 or182 or other units may be designed to have one or more than two magnetization zones, optionally of different construction and generated magnetic field.
• In some embodiments,sample container102 is disposable and has a capacity of about 50 ml and used for only for one separation whereas in someembodiments carrier container104 is a larger bottle or other container for supplying carrier fluid for numerous separations. In some embodiments, tube110 (or in fact the continuoustube comprising tube110 andsections142 and146 andconnections144 and148) is made of disposable and used for one separation only and is made of silicone tubing 1.98*3.18. Typically collection vessels are similar to test-tubes and are disposable.
• In some cases where the vitality of the cells is not mandatory or the sample is of non-animate particles the fluids such as carrier or washing fluids are not necessarily life sustaining, and the tubes are not necessarily biocompatible, allowing larger selection of constituents and components for particular properties such as viscosity, elasticity or flow regime.
Magnetization Zones
• The electromagnets are designed to provide and concentrate the magnetic flux in the magnetization zones for effective separation with power efficiency by avoiding excessive or superfluous power consumption that might have detrimental effect on the cells or the apparatus. For example, excessive heat dissipation that can damage the cells or coagulate the cells, or change the viscosity of the carrier, or deform the electromagnets. Keeping the power consumption to a substantially minimal or nominal value, while providing adequate flux for separation, enables also to avoid cooling mechanisms thereby providing a simple compact and reliable apparatus.
• Furthermore, in order to achieve rapid yet high quality separation the system is configured to facilitate flowing the mixture in a tube having a sufficiently large internal diameter or cross-section, e.g. about 2 mm, at a sufficiently high rate, e.g. about 2 ml/min, providing rapid separation while avoiding impairing the cells which may occur if the rate is inadequately low or the tube is inadequately narrow (e.g. about 1 mm or less) resulting with forces in the flowing fluid, such as shear, that may be detrimental to the cells such as coagulation of cells. Accordingly, the electromagnets are structured and controlled to efficiently separate living rare cells that flow in a high rate and in wide tube as described.
• FIG. 10 schematically illustrates an enlarged cross-section view of amagnetization zone124psuch as zone124 of separation unit such asunit122, where the disposition oftube110 is schematically depicted by a section of the tube.Magnetization zone124pis formed as generally parallel flat surfaces separated by a gap for disposing a section oftube110 in the magnetic field flux indicated by arrow160 (the direction of the field may be opposite as shown inmagnetization zones124aand124binFIG. 1C). In some embodiments, the gap is about 3 mm or more to allow placing a sufficientlywide tube110 to facilitate rapid separation.
• FIG. 1E schematically illustrates an enlarged cross-section view of amagnetization zone124qsuch as zone124 of separation unit such asunit122, where the disposition oftube110 is schematically depicted by a section of the tube.Magnetization zone124qis formed as generally opposing rounded surfaces for further concentration of the magnetic flux (such as relative tomagnetization zone124p) substantially without additional power. The rounded surfaces are separated by a gap for disposing a section oftube110 in the magnetic field flux indicated by arrow160 (the direction of the field may be opposite as shown inmagnetization zones124aand124binFIG. 1C). In some embodiments, the gap allows placing a sufficientlywide tube110, such as of external diameter of about 3 mm, to facilitate rapid separation.
• Table-1 below provides as a function of current an example of the efficiency of producing and concentration magnetic field flux at magnetization zones124, and the better efficiency of the round surfaces ofmagnetization zone124qrelative to the flat surfaces ofmagnetization zone124p. The coil of the electromagnet (e.g. coil128 of separation unit124) is made of 2000 windings of a wire of 1.1 mm thick, resulting in a resistance of 9.1 Ω.
• The described and listed values are close approximations.

• TABLE 1
  	Current (Amp)	Current (Amp)
  Flux (mT)	Flat surfaces	Round surfaces

  100	0.145	0.09
  200	0.28	0.22
  300	0.42	0.31
  400	0.58	0.45
  500	0.77	0.60
  600	1.03	0.80
  700	1.49	1.12
  800	2.12	1.60
  900	3.47	2.71
  930	—	3.16
  956	5.15	—

Operation
• In order to achieve high quantitative and qualitative separation yield of rare cells, system100 is operated, optionally at least partially under a programmed processor, according to certain procedures and parameters, some of which are described below.
• In the process of coupling target rare cells with the magnetic beads (see also below) some cells typically couple with larger amount of beads than others, resulting with magnetic target cells having different magnetic attraction (force under given magnetic field). Assuming, for clarity and simplicity, that only one magnetization zone (e.g. magnetization zone124) is used, than applying a strong magnetic field sufficient to attract substantially all the target cells may, in some cases, pull the cells such that they form chunks or lumps that adhere to the wall of the tube (e.g. tube110, hereinafter ‘the tube’) resulting in low qualitative separation yield. Moreover, possibly and probably, as the target cells drift towards the tube wall they would pull with them non-target cells that may adhere or be concealed in the lumps of cells or coagulate on or with the target cells, resulting in lower qualitative separation. On the other hand, if a weak magnetic field is applied than only the target cells with strong attraction (coupled with relatively large amount of beads) are separated, the rest flowing out with the partially depleted mixture resulting in low quantitative separation.
• Therefore, in some embodiments, different intensities of magnetic field and/or different spatial distributions of the magnetic flux are applied on different magnetization zones. The fields are then adjusted such that the target cells are separated and stick to the tube wall sufficiently to withstand the flow of the mixture, negligibly or without attracting non-target cells, while substantially or entirely (at least practically) washed out and released when the magnetic field is terminated. For example, the first magnetization zone provides moderately strong field, attracting the target rare cells with small amount of magnetic beads so that non-target cells are practically not pulled and not carried to the tube wall or coagulate with the target cells. At a subsequent magnetization zone a lower field is applied attracting the target cells with larger attraction yet practically without pulling non-target cells. Such a scheme with several magnetization zones are may be used, each with different magnetic field intensity and flux distribution.
• In some embodiments, in order to reduce space and costs, the tube may pass through given number of magnetization zones, possibly in one or more loops, and the field intensities adjusted to fractionally separate the rare target cells without, or practically without, pulling and capturing non-target cells. Optionally or alternatively, in some embodiments with a given number of magnetization zones (possibly even one), a moderately weak field is used for target cells with high attraction, and subsequently the field is increased and the mixture is recycled back to separate the target cells with higher attraction. Optionally or alternatively, with a given number of magnetization zones a fraction of the rare target cells are separated with certain field intensity, the field terminated and the separated target cells are washed out and collected, and the cycle is repeated again with different field intensities, thus sequentially separating portions of the rare target cells.
• In some embodiments, the magnetic flux is formed with varying intensities (gradients) to attract a variety of magnetized rare cells with only a negligible, if any, non-target cells or accumulation thereof. Similarly, in some embodiments, the magnetic flux is temporally variable (with a rate respective to the mixture flow) to attract a variety of magnetized rare cells with only a negligible, if any, non-target cells or coagulation thereof. In some embodiments, a combination of spatially varying and temporally varying flux is tuned to achieve high qualitative and quantitative rare cells separation.
• The number of magnetization zones and/or the number of separation cycles may be adjusted allowing tuning the system to a fine differential or fractional separation of rare target cells with a cumulative outstanding qualitative and quantitative separation yield.
• It is noted that in the foregoing description qualifying Willis such as strong, weak moderately, large, low or high merely imply relative amounts or intensities to illustrate different cases.
• Optionally or additionally, in some embodiments, other parameters are adjusted, possibly and optionally with respect to parameters such as magnetic field intensity or spatial distribution or type of cells and/or other parameters such as temperature. For example, the flow rate and/or viscosity and/or elasticity of the mixture or carrier fluid may be adjusted such as to allow separation of target cells yet preventing, at least substantially, coagulation of non-target cells. In some embodiments, the carrier fluid is used to wash out the separated target cells, yet in some embodiments the washing fluid is different from the carrier fluid. The flow regime and rate of the washing fluid is optionally adjusted to promote dislodging (e.g. promote the removal or release) the target cells from the tube wall, such abruptly altering the flow thereby inducing turbulences or shocks that help eroding or destabilize the target cells on the tube wall.
• In some embodiments, a typical cycle for high qualitative and quantitative separation yield of rare cells is completed within several minutes, such as about 15 minutes at a throughput of about 2 ml/min where further tuning of the operation parameters and/or additional equipment (e.g. more separation units or magnetization zone for practically simultaneous fractional separation or simultaneously using a plurality of tubes) may reduce the separation cycle time. In some embodiments, the order of magnitude of the magnetic field flux density generated by the electromagnets in a magnetization zone is of 1T (Tesla), and in some embodiments, a considerable portion such as more than half (e.g. about 80%) of the target cells are separated in the first one or two magnetization zones (first separation unit such as unit122).
• It is noted that in some embodiments, promoting the release of separated cells off the tube (e.g. degaussing, bubbling, vibrations) is carried out prior to and/or concurrent with washing the cells out of the tube.
• It should be noted that when high quality or purity depletion is indented (rather than collection of the target cells) sufficiently strong magnetic fields may be applied such as stronger than used for collection, on the expense of non-target cells adhering to the wall and/or coagulating.
Sample
• The sample is typically obtained from a primary sample comprising rare cells intended for examination (target cells) which were processed to specifically couple with magnetic beads. For example, a blood sample from a pregnant woman where rare fetal nucleated red blood cells (FNRBC) as target cells are expected to be present, or stem cells from blood or amniotic fluid.
• In some exemplary cases the primary sample source is other physiological fluid such as lymph or urine whereas in some cases the source is non-biological and the cells are bacteria or other biological particles such as pollen or spores collected from sources such as water or air or other environments.
• In some embodiments, the target cells are, in fact, not cells but rather other micro-particles (e.g. about or less than 1 μm) of any material or composition where the target particles can selectively couple of bind with magnetic beads.
Outline of an Operational Procedure
• According to the descriptions above in some embodiments the system is used to separate with high qualitative and quantitative yield magnetized cells from a fluid mixture of cells, by (a) providing a sample of cells comprising magnetized rare cells, and (b) flowing a mixture of the sample of cells in a carrier fluid inside a tube disposed about a magnetization zone having a magnetic flux, generated by an electromagnet, sufficient to attract a majority of the magnetized rare cells from the mixture onto the tube wall while impeding other cells from attaching to the tube wall, thereby separating magnetized rare cells on the tube wall.
• In some embodiments, the magnetized cells are rare cells in low abundance relative to there rest of the cells.
• In typical embodiments, once the separation of the magnetized cells rare cells from the mixture is completed (or practically completed), the magnetic flux is terminated (diminishing the attraction of the cells) and the separated magnetized rare cells are washed out of the tube.
• In some embodiments, the washing of the separated cells is preceded or accompanied by activation of mechanism to promote dislodging the separated magnetized rare cells off the tube wall, such as degaussing, bubbling or vibrating the tube.
Exemplary Procedure
• A primary blood sample of a few ml (e.g. 5 ml) is taken from a pregnant woman.
• The sample is prepared and coupled with magnetic beads similar or as defined at the kit protocol in http://tools.invitrogen.com/content/sfs/manuals/11331D-Dynal-CD4-Positive-Isolation-Kit-(rev003).pdf
• The prepared sample is placed in the disposable sample container (e.g. container102).
• About 3 ml of washing buffer (carrier fluid) of PBS+2 mM EDTA+0.5% BSA are placed in the carrier container (e.g.104) and the corresponding pump (e.g. pump114) is switched on flowing the washing fluid (carrier fluid) at about 2 ml/min to fill the tubing (e.g. tube110 and connecting sections) in order to prepare the device, including filling mixture container (e.g. container106) for separation and then the pumps are switched off.
• The magnetic field at the separation units (units122) is set at about 450-800 mT and impelling pump (e.g. pump116) is switched on to flow the mixture at about 1 ml/min. When just a few ml (e.g. 2 ml) remain in the mixture container, the impelling pump is switched off and the system is washed by about 15 ml washing fluid at about 2 ml/min rate.
• The magnetic field is switched off and the electromagnets are degaussed.
• The target cells are washed to collecting vessel (e.g. vessel108) by about 3 ml washing fluid at about 2 ml/min rate.
REFERENCES
• (1) Schmorl G. Pathologisch-anatomische undersuchungen uber puerperal-eklampsie. Leipzig; Vogel, 1893.
• (2) Walknowska J, Conte F A, Grumbach M M. Practical and theoretical implications of fetal/maternal lymphocyte transfer. Lancet 1969; 1: 1119-1122.
• (3) Bianchi D W, Flint A F, Pizzimenti M F, Knoll J H M, Latt S A. Isolation of fetal DNA from nucleated erythrocytes in maternal blood. Proc Natl Acad Sci 1990; 87:3279-3283.
• (4) Wessman M, Ylinen K, Knuutila S. Fetal granulocytes in maternal venous blood detected by in situ hybridization. Prenat Diagn 1992; 12:993-1000.
• (5) Bianchi D W, Zickwolf G W, Weil G J, Sylvester S, DeMaria M A. Male fetal progenitor cells persist in maternal blood for as long as 27 years postpartum. Proc Natl Acad Sci 1996; 93:705-708.
• (6) Bianchi D W, Williams J M, Sullivan L M, Hanson F W, Klinger K W, Shuber A P. PCR quantitation of fetal cells in maternal blood in normal and aneuploid pregnancies. Am J Hum Genet 1997; 61:822-829.
• (7) Lamvu G, Kuller J A. Prenatal diagnosis using fetal cells from the maternal circulation. Obstet Gynecol Survey 1997; 52:433-437.
• (8) Bianchi D W, Simpson J L, Jackson L G, et al. 2002. Fetal gender and aneuploidy detection using fetal cells in maternal blood: analysis of NIFTY I data. National Institute of Child Health and Development Fetal Cell Isolation Study. Prenat Diagn 22: 609-615.
• (9) Chui R W, et al. 2008. Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma. PNAS 105(51):20458-63.

Claims (11)

What is claimed is:

1. A magnetic separation system comprising:

a mixture container;

a plurality of electromagnets;

a tube extending from said mixture container, passing about said plurality of electromagnets, and terminating at a plurality of collection vessels;

a pump associated with said tube;

a computer that is configured to:

(i) operate said plurality of electromagnets to generate magnetic flux, and simultaneously operate said pump to cause a mixture of (a) magnetized cells, (b) non-magnetized cells, and (c) carrier fluid, to flow inside said tube about said plurality of electromagnets, such that:

a majority of the magnetized cells are attracted to a wall of said tube, about said plurality of electromagnets, and

a majority of the non-magnetized cells are drained to a first collection vessel of said plurality of collection vessels,

(ii) operate said plurality of electromagnets to terminate the magnetic flux, and simultaneously operate said pump to feed a washing fluid from a carrier container, through said mixture container and into said tube, such that attracted magnetized cells are dislodged from the wall of said tube and are washed, with the washing fluid, to a second collection vessel of said plurality of collections vessels.

2. The system according toclaim 1, wherein the tube is disposed generally horizontally about the plurality of electromagnets.

3. The system according toclaim 1, wherein the plurality of electromagnets is arranged about a generally horizontal plane for disposing the tube generally horizontally about the plurality of electromagnets.

4. The system according toclaim 1, wherein the tube is disposed generally vertically about the plurality of electromagnets.

5. The system according toclaim 1, wherein the plurality of electromagnets is arranged about a generally vertical plane for disposing the tube generally horizontally about the plurality of electromagnets.

6. The system according toclaim 1, wherein an electromagnet is configured to generate a magnetic flux about two zones.

7. The system according toclaim 1, wherein the tube is disposed about the plurality of electromagnets in a successive manner, such that each successive electromagnet of the plurality of electromagnets attracts onto the wall of the tube a majority of the magnetized cells remaining in the flowing mixture following attraction by a preceding electromagnet of the plurality of electromagnets.

8. The system according toclaim 1, further comprising at least one mechanism configured to promote the dislodging of attracted magnetized cells from the wall of said tube,

wherein said computer is further configured to operate said at least one mechanism to promote the dislodging of attracted magnetized cells from the wall of said tube.

9. The system according toclaim 8, wherein said computer is further configured to operate said at least one mechanism to degauss the plurality of electromagnets.

10. The system according toclaim 8, wherein the mechanism comprises an apparatus configured to flow an air bubble in the washing fluid inside the tube, and

said computer is further configured to operate said apparatus and said third pump to cause the air bubble to flow in the washing fluid inside the tube.

11. The system according toclaim 8, wherein the mechanism comprises an apparatus to vibrate the tube,

and wherein said computer is further configured to operate said apparatus to vibrate the tube.

Images