WO2001007911A2 - Nouveau procede d'isolement perfectionne d'une population cellulaire cible - Google Patents

Nouveau procede d'isolement perfectionne d'une population cellulaire cible Download PDF

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Publication number
WO2001007911A2
WO2001007911A2 PCT/DK2000/000408 DK0000408W WO0107911A2 WO 2001007911 A2 WO2001007911 A2 WO 2001007911A2 DK 0000408 W DK0000408 W DK 0000408W WO 0107911 A2 WO0107911 A2 WO 0107911A2
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Prior art keywords
cells
sample
flow
cell population
antibodies
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WO2001007911A3 (fr
Inventor
Britta Christensen
Steen KØLVRAA
John Philip
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Priority to AU59671/00A priority Critical patent/AU5967100A/en
Publication of WO2001007911A2 publication Critical patent/WO2001007911A2/fr
Publication of WO2001007911A3 publication Critical patent/WO2001007911A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/505—Erythropoietin [EPO]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70582—CD71

Definitions

  • the present invention relates to a novel method for the isolation of a target cell population from a liquid cell sample, in particular a mammalian cell population.
  • Fetal cells may be obtained by amniocentesis, the removal of amniotic fluid from the amniotic cavity within the amniotic sac or by chorion biopsy, where biopsies are taken from the placenta, a so-called invasive sampling.
  • nucleated erythrocyte One particular fetal cell type within maternal blood that has been demonstrated to be useful for detecting fetal DNA is the nucleated erythrocyte.
  • fetal leukocytes have been reported to be present in maternal blood.
  • Leuko- cytes are one subpopulation of white blood cells found in the blood.
  • leukocytes which also are referred to as polymorphnuclear leukocytes
  • neutrophils neutrophils
  • basophils basophils
  • eosinophils All leukocytes have a distinctive morphology characterized by the nucleus and cellular granules.
  • fetal lymphocytes and trophoblasts may be present in the maternal blood. Due to the very limited number of fetal cells in maternal blood concentration or enrichment of the maternal blood sample with respect to the fetal cells have been conducted by negative selection, i.e. removal of maternal cells. Enrichment of fetal cells by density gradient centrifugation or by removing maternal cells with an antibody to a cell surface antigen is described in for example US 5,858,649, US 5,731 ,156,
  • Yet another method of removing maternal cells, in particular maternal erythrocytes, is by lysing, again optionally combined with immunologic methods for removing the maternal cells.
  • Another selection procedure is to mark the fetal cells for example by the use of CD71 antibodies. This is called positive selection as opposed to negative selection mentioned above where maternal cells are removed.
  • Immobilisation of a ligand by linking the ligand to a surface is a well-known preparation for a separation technique.
  • a method of photochemical immobilisation of ligands using quinones is described.
  • US 5,759,793 describes a method for the affinity separation of human stem cells in a fluidised bed system, using a magnetically stabilised fluidised bed. Another approach is described in US 4,710,472 wherein a magnetic separation device suitable for the removal of magnetic bead-coated cells is described.
  • the present invention provides a method for the isolation of at least one target cell population, in particular a mammalian cell population, from a liquid sample of cells comprising: - establishing a sample flow over a surface coated with antibodies specific for said target cell population,
  • sample flow is used in its normal meaning, whereby a sample hav- ing a volume larger than the volume of the chamber wherein the surface is arranged may be flowed over and thereby brought in contact with the surface. Merely stirring or agitating a sample in a chamber without replacing at least part of the sample constantly is not understood as flow.
  • Another object of the present invention relates to a system providing for isolating at least one cell population from a liquid sample of cells, comprising a surface surrounded by at least one wall, said surface being coated with antibodies specific for said target cell population, means for establishing a sample flow over said surface allowing said cell sample and surface to be in physical contact such that the cells from the at least one mammalian target cell population may bind to the antibodies on the surface, means for removing liquid sample comprising non-bound cells, and means for identifying target cells on the surface.
  • Yet another object relates to the use of the target cell population for diagnostic pur- poses.
  • Figure 1 depicts a model of the system for the separation of a cell target population.
  • Figure 2 depicts another embodiment of the system of Figure 1.
  • Figure 3 is a schematic drawing of a system according to the invention, wherein fig. 3a is a closure and fig. 3b shows the system comprising the surface.
  • the present invention reveals a novel method and a system for the isolation of mammalian target cell populations.
  • the present method has improved the separa- tion of target cells from other cells in a liquid sample in particular when the ratio of target cells to the other cells in the liquid sample is small.
  • target cell population is meant the cells desired to identify, which cells are distinguishable from the other cells in the sample by the use of species selective identifiers, such as antibodies.
  • the cell sample of the present invention is passed or flowed over an anti- body-covered surface.
  • the method may be conducted by use of a system as defined below.
  • the invention relates to a system providing for the recovery of the target cells from the surface, for example when the target cell population is in a blood sample.
  • the liquid sample of cells is a blood sample. More particularly the present invention has proven successful in the isola- tion of fetal cells from maternal cells. Fetal cells may occur in the maternal blood stream at a ratio of 1 :10 5 to 1 :10 8 with respect to the maternal cells. In the light of this naturally occurring ratio the present invention presents a method wherein fetal cells are isolated from maternal blood without prior enrichment or concentration of the sample. This provides for an improved method of isolation by which the risk of loosing fetal cells due to enrichment or concentration procedures has been greatly reduced.
  • the cell sample is diluted prior to being passed over the surface.
  • the cell sample is diluted 2-10 times, more preferably 4-6 times by the addition of isotonic buffers, such as saline solutions, phosphat buffered saline solutions, PBS, and/or suitable growth media, such as basal media, and tissues growth media.
  • isotonic buffers such as saline solutions, phosphat buffered saline solutions, PBS, and/or suitable growth media, such as basal media, and tissues growth media.
  • the size of the maternal blood sample is preferably in the range of 5 to 40 ml, such as from 10 to 30 ml.
  • an important aspect of the present invention is the establishment of a sample flow over the surface.
  • the flow may be established by any suitable means, such as by gravity, by use of a pump or by suction. It is however of impor- tance that the flow is controllable and adjustable to be adapted to the specific sample and surface used.
  • the flow is preferably created by a pump that preferably is arranged downstream of the surface in order to avoid any cell damage from the pump prior to binding the cells.
  • the flow of the cell sample may be a continuous flow, so that the contact between the surface and the cell sample is at a constant rate.
  • the flow is discontinuous, by changing the flow rate during the flow, such as by alternating high and low flow rates during the passing of the sample over the surface.
  • the flow is sporadically dis- rupted.
  • One way of creating a disrupted flow according to the invention is by applying a stepped stop flow.
  • the stop flow comprises three steps: a flow step, and a sedimentation step followed by another flow step respectively.
  • the time frame for the length of the individual steps may be of any length.
  • the ratio for the preferred time frames for the individual steps is from 1 :5 to 2:1 , such as from 1 :5 to 1 :60, or from 1 :10 to 2:1 for the flow step to the sedimentation step, more preferred from 1 :20 to 1 :40 or from 1 :5 to 1 :1. It is further preferred that at least two sedimentation steps are provided for each volume during its pass over the surface.
  • the flow rate is held within predetermined range to obtain an optimized surface- sample contact. Accordingly, the flow applied to the cell sample may have a flow rate from 0.2-5 ml/min, such as 0.2-3 ml/min or 1-5 ml/min., more preferably about 1 ml, or about 2.5 ml/min.
  • a turbulence in the flow of the cells may be created.
  • the object of the present invention is to provide for improved isolation of a cell target population, and a movement of cells within the sample may increase the probability of physical contact between the cells and the surface.
  • turbulence may be created in the sample ensuring an optimal contact.
  • Turbulence may be arranged by any means suitable for a turbulent flow over the surface avoiding that the cells are damaged due to the turbulence.
  • the turbulence may be cre- ated by arranging a rotating magnet in the flow, such as adjacent the surface, but preferably not in contact with the surface.
  • the magnet is located in the flow upstream the surface creating the turbulence before the flow over the surface.
  • Another means for creating the turbulence may be mechanical hindrance of a laminar flow over the surface.
  • a magnetic field is effecting cells in the sample having metal particles positioned intracellularly and/or extracel- lularly.
  • the positioning of metal particles on or inside the cells provides for the means of attracting the cells to the surface by applying a magnetic field.
  • the magnetic field is attracting the cells towards the surface.
  • the cells comprising metal particles are preferably only the target cells, however, the method may also be applied in case substantially all cells contain the metal particles, in that only the target cells are bound, whereby removal of the magnetic field will cause unbound cells to pass on.
  • metal particles is meant any type of magnetic particles, such as magnetic beads. In particular magnetic beads comprising iron, such as ferro fluidics are used.
  • the magnetic field may be constant or it may be pulsating.
  • constant is meant a magnetic field, which is applied to the cell sample at the same magnitude throughout the isolation procedure.
  • pulsesating means applying a magnetic field to the cell sample in intervals. Pulsating magnetic field is preferred, so that unbound cells continue flowing.
  • Further means for attracting a target cell population to the surface according to the invention are electrical forces, such as a charge flow wherein the knowledge of the different charges of various cell types are employed as a tool of isolation of a target cell population.
  • the surface according to the invention is any type of surface capable of being coated with antibodies under the flow conditions described above.
  • coated is meant any form of having the antibodies attached to the surface.
  • the surface on which the target cell population will bind to is levelled in the meaning of being non-curved.
  • the surface according to the invention may be positioned in any possible angle provided it is levelled.
  • the surface is levelled and even thereby allowing for the identification of the target cell population in a microscope.
  • the surface is surrounded by at least one wall.
  • Said wall may be divided into several wall pieces, such as four wall pieces around a rectangular or square surface.
  • the wall is constructed by an O-ring or the like situated on or around said surface.
  • Said wall may also be arranged by locating the surface in a chamber, and the flow is then created in the chamber.
  • the surface may constitute at least one wall of the chamber, such as the bottom wall. The surface may be situated in a chamber to ensure the enclosure of the cell sample and to allow for the creation of turbulence in the cell sample.
  • the chamber is a closed chamber apart from at least one inlet and at least one outlet, said closed chamber being provided by arranging a closure of the chamber, preferably the closure provides a fluid tight sealing of the chamber.
  • the closure of the chamber according to the invention may be even or uneven, such as grooved, on the side of the closure facing the sample in the chamber. In case of the latter the created turbulence of the flow of the cell sample may be further potentiated by the shape of the closure.
  • Any one or all of the walls and closure may be made of any type of glass or plastic.
  • at least the surface part and the closure are transparent or at least partly transparent allowing the cells to be microscopically identified when the surface is in the chamber.
  • a part of the surface as well as a part of the closure may be produced from metal providing for electrical conduction, in particular in relation to a charge flow as discussed above.
  • Either the whole surface and/or closure is made from metal or metal wires may be incorporated in glass or plastic surface and/or closure.
  • the dimensions of the chamber provided are not crucial for the method to be carried out.
  • the surface area is often between 10 and 100 cm 2 , such as between 10 and 50 cm 2
  • the height of the chamber is below 1 cm, preferably from 0.1 to 2 mm, such as from 0.2 to 2.0 mm, or from 0.1 to 1.0 mm.
  • the system comprises several individual surfaces or chambers, each separated from each other being supplied individually with a flow.
  • Such system may comprise 5 or 10 adjacent surfaces or chambers or even more.
  • the fluid tight sealing of the chamber may be arranged by providing a surrounding wall made of rubber, synthetic rubber or other flexible material. Also the wall may be made of Teflon, thereby reducing the friction and attachment of cells to the wall.
  • the antibodies on the surface are antibodies specifically directed to the target cells, having no or little affinity towards the other cells of the sample. Accordingly, the antibody is preferably specific for cell-surface situated epitopes expressed by the target cells. However, antibodies directed towards intracellular antigens may be used if the cells are conditioned therefor, i.e. by a permeabilising treatment.
  • the antibodies are preferably antibodies directed to EPO eryth n or CD71 antibodies or CD36 antibodies.
  • the identification of the target cells may be carried out by any suitable method, such as labelling of the target cells, for example fetal cells or cancer cells.
  • Fetal cells may be distinguished from maternal cells by the specific recognition of a fetal cell antigen or they may be distinguished from maternal cells by the specific recognition of RNA encoding a protein selectively produced by fetal cells.
  • the selective labelling is based on an antigen-antibody reaction wherein the antibodies are specific for epitopes expressed by the target cell population, such as a protein selectively produced by fetal cells.
  • a protein may be selected from the group consisting of embryonic he- moglobin, such as ⁇ and zeta hemoglobin, and fetal hemoglobin, such as gamma and alpha hemoglobin.
  • the labelling may be carried out by the use of an antibody selected from antibodies against various types of normal globin chains in human hemoglobin, for example anti epsilon ( ⁇ ) antibodies, anti zeta ( ⁇ ) antibodies, anti gamma ( ⁇ ) antibodies, anti alpha ( ⁇ ) antibodies, and anti beta ( ⁇ ) antibodies.
  • an antibody selected from antibodies against various types of normal globin chains in human hemoglobin for example anti epsilon ( ⁇ ) antibodies, anti zeta ( ⁇ ) antibodies, anti gamma ( ⁇ ) antibodies, anti alpha ( ⁇ ) antibodies, and anti beta ( ⁇ ) antibodies.
  • the antibodies may be monoclonal or polyclonal antibodies, however the monoclonal antibodies are preferred. Furthermore, the antibodies may be labelled or unlabelled antibodies.
  • the labelled antibodies are preferably FITC labelled, biotin labelled or TRITC labelled antibodies, but other labellings may be used as well.
  • the labelling may be carried out using anti epsilon ( ⁇ ) monoclonal antibodies or anti zeta ( ⁇ ) monoclonal antibodies, more preferably anti epsilon ( ⁇ ) unlabelled monoclonal antibodies or anti zeta ( ⁇ ) unlabelled monoclonal antibodies, or anti epsilon ( ⁇ ) monoclonal biotin labelled antibodies or anti zeta ( ⁇ ) monoclonal biotin labelled antibodies.
  • the labelling is performed using anti gamma ( ⁇ ) FITC labelled antibodies.
  • Two or more selective labellings may be performed in order to enhance the prob- ability and selectivity of identifying the fetal cells bound to the surface on the background of maternal cells.
  • the combination of two or more labellings may be a combination of any of the labellings used for single labelling.
  • the enhanced selective labelling may also be performed by the use of two or more antibodies directed against the same protein or different proteins.
  • the labelling with two or more labels may be carried out simultaneously.
  • fetal-cell-specific RNA sequences are used as fetal cell markers.
  • RNA is generally messenger RNA (mRNA).
  • mRNA messenger RNA
  • Fetal cells contain distinct mRNAs or RNA species that do not occur in other cell types. The detection of these RNAs, whether as mRNA can serve to identify cells, or even sub- cellular fractions of cells fetal or embryonic in origin.
  • the m-RNA may be coding for a protein selected from the group consisting of embryonic hemoglobin, such as ⁇ and zeta hemoglobin and fetal hemoglobin, such as gamma and alpha hemoglobin.
  • DNA probes (oligos) for the hybridisation are directed against embryonic cell RNA, such as for ⁇ and zeta hemoglobin, and for fetal hemoglobin, such as for gamma and alpha hemoglobin.
  • a DNA probe may be synthesised as an oligodeoxynucleotide using a commercial synthesiser. Probes may be comprised of the natural nucleotide bases or known analogues of the natural nucleotide bases.
  • a combined labelling may be carried out by the use of two or more different hybridisation probes, such as a combination of a DNA probe and a PNA probe for hybridisation with the same fetal RNA or more preferred with different RNAs.
  • two or more different DNA probes (or PNA probes or LNA probes) may be used for hybridisation with different fetal RNAs.
  • a combination of an immunological labelling and a hybridisation labelling may be employed according to the present invention.
  • the labelling is normally carried out sequentially by a first immunological labelling step, then identification of the labelled cells, and then a second step of hy- bhdisation labelling for verification of the identification of the cells labelled by the first step. It is preferred in the first step to use antibodies against epsilon, gamma and/or zeta globin and in the second step to use hybridisation for epsilon and/or zeta globin mRNA to verify the fetal cells identified.
  • the nucleic acid of the fetal cells can also be amplified prior to detection using a known amplification technique, such as the polymerase chain reaction (in situ RT- PCR).
  • Primers for in situ RT-PCR amplification are chosen to specifically amplify a mRNA of interest in the target cell, as mentioned above for hybridisation.
  • the identification step may further comprise a microscopy step, wherein the cells labelled, independent of the labelling method, are identified by microscopy directly on the surface.
  • the rate of cells identified per unit of time is the rate of cells identified per unit of time.
  • very fast scanning microscopes may be used for the identification.
  • laser scanners could be used.
  • the laser scanner is equipped with at least two lasers emitting light with different wavelength capable to excite the various labels on the cells or in one laser emitting sev- eral wavelengths.
  • the position of detected labelled cells on the surface is recorded. This provides for the later collection of the detected cells from the position which has been recorded.
  • the position of the detected labelled cells on the supporting surface may be recorded by use of a scanner provided with detectors registering the light emitted from the labelled cells, such photo-multipliers, CCDs, or the like detectors. Thereby it is possible to identify and specifically isolate substantially only the selectively labelled fetal cells.
  • the scanner is arranged for detecting selectively labelled fetal cells, and when detecting a fetal cell, carrying out a verification step by switching to another wavelength to verify presence of for example staining of the nucleus.
  • a fast scanning system for example a scanner capable of scanning in the range of from 0.1 /sec to 10 m/sec or faster, such as appr. 1 m/sec.
  • the target cells are recovered from the surface by micro-dissection.
  • micro-dissection is used to mean that each target-cell is identified and removed individually.
  • Micro-dissection may be carried out by means of a laser apparatus, whereby a laser beam may liberate the target cells from the surface preserving the viability of the target cells, and thereby ensuring the further in vivo studies of the living target cells.
  • micro-manipulation is micro-manipulation, optionally by means of minute cannulas.
  • the cells recovered and collected according to any of the procedures may be used for diagnostic purposes, i.e. being subjected to further identification and/or investigation, such as microscopic and/or molecular identification and/or investigation.
  • the cells may be subjected to investigations of analysing the presence of genetic dis- eases.
  • the nucleic acid of fetal cells may be analysed for diagnostic or other purposes.
  • the presence or absence of a particular nucleic acid may indicate the presence or absence of certain genes coding for diseases, such as cystic fibrosis.
  • the nucleic acid may additionally be analysed for X or Y specificity.
  • the presence of a Y chromosome encoded genes or gene products is a qualitative distinguishing feature of the cells of a male fetus.
  • viable cells recovered from the surface may be cultured for further investigations.
  • Verification of the selective identification of fetal cells may be carried out by several methods.
  • the method may be performed on maternal blood samples from pregnant women carrying a male fetus.
  • the cells isolated may then be analysed for the presence of a Y chromosome, indicative of cells being from the male fetus.
  • Another verification method which is independent of the sex of the fetus, is verification by the use of identification of small tandem repeats (STR), or variable number tandem repeats (VNTR) to detect genetic input from the father, thereby verifying fetal cells, as the only cells in the sample comprising input from the father.
  • STR small tandem repeats
  • VNTR variable number tandem repeats
  • the present method may be carried out for the isolation of any kind of rare event target cells, and is particular interesting when used for rare event cells being present in very low concentrations, such as those for the fetal cells in maternal blood or for blood-borne cells in some cancer forms.
  • the system 1 is depicted in Fig. 1 , wherein a chamber 1 is arranged in the flow of the sample.
  • a sample optionally diluted, located in a container 3 provided with an outlet 4.
  • the sample is preferably stirred, such as by means of a rotating magnet 5 in the container to obtain a homogenous slurry before entering into the flow.
  • the sample is flowing from the container 3 through outlet 4 into the line 6 towards inlet 7 of the chamber 1.
  • the sample is passing over the surface 8 arranged in the bottom of the chamber 1.
  • the chamber 1 is defined by the walls 12 made of Teflon in the present case, and transparent closure 2.
  • Unbound cells and liquid is flowing further out of outlet 9 into line 10.
  • the flow is created by means of pump 11 arranged in line 10.
  • the liquid comprising unbound cells may then be discarded or used elsewhere.
  • the liquid may also be reflowed to the system at least once in order to increase the number of target cells bound.
  • a rotating magnet 12 is arranged in the chamber 1 for providing turbulence in the flow in the chamber 1.
  • the magnet 12 is located adjacent the closure 2, not to be brought in contact with the antibody-coated surface 8, in order to avoid damaging the surface 8.
  • Means for establishing a magnetic field in the chamber may be provided outside the chamber.
  • Fig. 3a the closure 2 is shown in greater detail.
  • the inlet 7 and outlet 9 is led through the closure 2.
  • the inlet 7 is divided into 3 inlet streams 13 during the flow through the closure.
  • the division into several inlet streams may be carried out before the flow reaches the closure or during the flow through the closure 2.
  • the outlet 9 may collect flows from one to several outlet streams 14, in the depicted example 3 outlet streams 14 are shown. It is within the scope of the invention that from one to several inlet 13 and outlet 14 streams may be comprised in the flow.
  • Variations in the location of the inlet and outlet streams is foreseen with the present invention, such as inlet streams perpendicular to the face of the closure facing the sample or oblique inlet streams, whereby the inlet is led obliquely through the closure towards the face of the closure facing the sample.
  • FIG. 3b an example of the surface 8 surrounded by four wall pieces 12 is shown.
  • a chamber When covered by the closure of Fig. 3a a chamber is created having the surface 8 as one wall of the chamber.
  • a sample may be flowing in line 6 through inlet 7 into the inlet streams 13 onto the surface 8.
  • the liquid comprising unbound cells is removed from the surface through outlet streams 14 via outlet 9 into line 10.

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Abstract

L'invention concerne un procédé d'isolement d'au moins une population cellulaire cible, à partir d'un échantillon liquide de cellules. Ce procédé consiste à établir un écoulement de l'échantillon sur une surface revêtue d'anticorps spécifiques de la population cellulaire cible, et à permettre à l'échantillon de cellules et à la surface d'être en contact physique, ce qui contribue à la liaison des cellules provenant d'au moins une population cellulaire cible mammifère avec les anticorps présents sur la surface. Le procédé consiste ensuite à enlever de la surface l'échantillon liquide comprenant les cellules non liées, et à identifier les cellules cibles restant sur la surface. L'invention concerne en outre l'utilisation d'un tel procédé, ainsi qu'un système d'isolement de populations cellulaires cibles à partir d'un échantillon liquide de cellules.
PCT/DK2000/000408 1999-07-21 2000-07-18 Nouveau procede d'isolement perfectionne d'une population cellulaire cible Ceased WO2001007911A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU59671/00A AU5967100A (en) 1999-07-21 2000-07-18 A novel method for the improved isolation of a target cell population

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DKPA199901052 1999-07-21
DKPA199901052 1999-07-21

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Publication Number Publication Date
WO2001007911A2 true WO2001007911A2 (fr) 2001-02-01
WO2001007911A3 WO2001007911A3 (fr) 2001-11-08

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111757928A (zh) * 2017-12-01 2020-10-09 环球生命科技咨询美国有限责任公司 用于细胞富集和分离的方法

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2112833T3 (es) * 1988-12-06 1998-04-16 Flinders Technologies Pty Ltd Aislamiento de celulas fetales a partir de sangre materna para permitir el diagnostico prenatal.
CA1340565C (fr) * 1989-06-29 1999-05-25 Thomas B. Okarma Dispositif et methode pour capter et recuperer des cellules
DE4222573C1 (fr) * 1992-05-16 1993-08-12 Dorothee Dr. 4400 Muenster De Ahlert
US5275933A (en) * 1992-09-25 1994-01-04 The Board Of Trustees Of The Leland Stanford Junior University Triple gradient process for recovering nucleated fetal cells from maternal blood

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111757928A (zh) * 2017-12-01 2020-10-09 环球生命科技咨询美国有限责任公司 用于细胞富集和分离的方法

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WO2001007911A3 (fr) 2001-11-08

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