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WO1993008269A1 - Procede pour enrichir des cellules souches f×tales issues du sang maternel - Google Patents

Procede pour enrichir des cellules souches f×tales issues du sang maternel Download PDF

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Publication number
WO1993008269A1
WO1993008269A1 PCT/US1992/009024 US9209024W WO9308269A1 WO 1993008269 A1 WO1993008269 A1 WO 1993008269A1 US 9209024 W US9209024 W US 9209024W WO 9308269 A1 WO9308269 A1 WO 9308269A1
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WIPO (PCT)
Prior art keywords
cells
fetal
ceus
hgand
maternal blood
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PCT/US1992/009024
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English (en)
Inventor
Sheryl Williams
Ronald J. Berenson
Shelly Heimfeld
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Cellpro, Incorporated
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Publication date
Application filed by Cellpro, Incorporated filed Critical Cellpro, Incorporated
Priority to EP92922837A priority Critical patent/EP0610359A1/fr
Priority to JP5507911A priority patent/JPH07500499A/ja
Publication of WO1993008269A1 publication Critical patent/WO1993008269A1/fr

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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0641—Erythrocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/14—Erythropoietin [EPO]

Definitions

  • the invention relates to a method of enriching fetal progenitor cells from maternal blood.
  • the only way of diagnosing fetal disorders is to obtain cells from the amniotic fluid (amniocentesis) or the surface of the fetal sac (chorionicvillus) of the mother. These procedures are expensive and carry a risk of spontaneous abortion of between V2 0 to 1%. Because of the risk of spontaneous abortion, these collection procedures are recommended only for women who are at a high-risk of carrying a child with a genetic defect. For example, only women over age 35 are advised to have the test because the risk of Down's Syndrome is higher in that group. Some of these women refuse the test because of the risk of spontaneous abortion. Even though many of these high-risk women are tested, only a fraction of fetuses with Down's Syndrome are detected.
  • the high-risk women represent such a small portion of the women having children that the low-risk population still delivers most of the afflicted children. Eighty percent of those children born with Down's Syndrome are from the "low-risk,” under 35-year-old group. This situation is also true of many other genetic defects or disorders. It is therefore desirable to provide a test that would resolve this testing dilemma by providing a safe method which could be available to all pregnant women, irrespective of the risk factor and without risk of spontaneous abortion.
  • FACS Fluorescence-Activated Cell Sorter
  • the procedure used is not adaptable to routine clinical testing procedures, in part, due to the great expense and expertise required to run a FACS machine.
  • the method of Hertzenberg et al is also deficient for routine clinical testing because it requires determination of HLA types.
  • Bianchi et al "Direct Hybridization to DNA From Small Numbers of Flow-Sorted Nucleated Newborn Cells," Cytometry 5:197-202 (1987) also used a FACS machine to detect nucleated cells, although the blood sample was not obtained from the mother but from the newborn's umbilical cord.
  • the present invention provides a method for enriching fetal cells that overcomes these disadvantages, and further provides other related advantages.
  • the present invention is also directed toward methods for enriching fetal nucleated, erythroid cells from maternal blood.
  • a method comprises the steps of (a) incubating a sample of maternal blood with an immobilized ligand capable of specifically binding to fetal nucleated, erythroid cells under conditions and for a time sufficient to allow specific binding of the ligand to the cells, (b) removing unbound blood products, and (c) incubating the bound cells in the presence of erythropoietin such that the fetal cells are preferentially enriched.
  • the ⁇ nmobilized ligand is an immobilized antibody.
  • a method for enriching fetal nucleated, erythroid cells from maternal blood comprising the steps of: incubating a sample of maternal blood with a first member chemically linked to a ligand capable of specifically binding to fetal nucleated, erythroid cells under conditions and for a time sufficient to allow specific binding of the ligand to the cells; adsorbing the cells to an immobilized second member, the second member being capable of binding to the first member with an affinity constant of greater than about 10° M "1 ; removing unbound blood products; and incubating the bound cells in the presence of erythropoietin such that the fetal cells are preferentially enriched.
  • Suitable first member-second member binding pairs include biotin-avidin, biotin-streptavidin, biocytin-avidin, biocytin-streptavidin, mefhotrexate-dihydrofolate reductase, 5-fluorouracfl-thimydylate synthetase, and riboflavin-riboflavin binding protein.
  • the first member which is chemically linked to a ligand is a biotinylated antibody and the immobilized second member is immobilized avidin.
  • the method comprises the steps of: incubating a sample of maternal blood with a first ligand capable of specifically binding to fetal nucleated, erythroid cells under conditions and for a time sufficient to allow specific binding of the first ligand to the cells, incubating the sample with a first member chemically linked to a second ligand capable of specifically binding to the first hgand under conditions and for a time sufficient to allow the second Hgand to bind to the first ligand; adsorbing the cells to an immobilized second member, the second member capable of binding to the first member with an affinity constant of greater than about 10° M , removing unbound blood products; and incubating the bound cells in the presence of erythropoietin such that the fetal cells are preferentially enriched.
  • the first hgand is an antibody which specifically binds to fetal nucleated, erythroid cells.
  • the first member which is chemically linked to the second gand is a biotinylated antibody.
  • the ⁇ mnobilized second member is immobilized avidin.
  • another aspect of the present invention comprises the steps of: (a) incubating the bound cells with ammonia and chloride ions and a carbonic anhydrase inhibitor under conditions and for a time sufficient to allow accumulation of ammonium ions within the bound cells, and (b) incubating the bound cells containing accumulated ammonium ions in the presence of ammonia and carbon dioxide such that selective hemolysis of maternal blood cells occurs.
  • the present invention may also be performed using a combination of both erythropoietin enrichment and the method described above wherein the cells are incubated with ammonia and chloride ions and a carbonic anhydrase inhibitor under conditions and for a time sufficient to allow accumulation of ammonium ions within the bound cells, followed by the incubation of bound cells containing accumulated ammonium ions in the presence of ammonia and carbon dioxide such that selective hemolysis of maternal blood cells occurs.
  • These two enrichment methods may be performed sequentially, and in either order.
  • a method for enriching fetal nucleated, erythroid cells from maternal blood comprising the steps of: incubating a sample of maternal blood in the presence of erythropoietin such that the fetal cells are enriched; incubating the enriched cells with an immobilized ligand capable of specifically binding to fetal nucleated, erythroid cells under conditions and for a time sufficient to allow specific binding of the hgand to the cells; and removing unbound blood products.
  • the enriched cells may be immobilized by incubating them with a first member chemically linked to a hgand capable of specifically binding to the cells under conditions and for a time sufficient to allow specific binding of the hgand to the cells, and adsorbing the cells to an immobilized second member, the second member being capable of binding to the first member with an affinity constant of greater than about 10° M "1 .
  • the enriched cells may be immobilized by incubating the enriched cells with a first hgand capable of specifically binding to the cells under conditions and for a time sufficient to allow specific binding of the first hgand to the cells; incubating the sample with a first member chemically linked to a second hgand capable of specifically binding to the first hgand under conditions and for a time sufficient to allow the second hgand to bind to the first hgand; and adsorbing the cells to an immobilized second member, the second member being capable of binding to the first member with an affinity constant of greater than about 10° M . Suitable combinations of first and second hgand are discussed in detail below.
  • the methods may further comprise (subsequent to removing the unbound blood products) the steps of: incubating the bound cells with ammonia and chloride ions and a carbonic anhydrase inhibitor under conditions and for a time sufficient to allow accumulation of ammonium ions within the bound cells; and incubating the cells containing accumulated ammonium ions in the presence of ammonia and carbon dioxide, such that selective hemolysis of maternal blood cells occurs.
  • the cells are incubated with ammonia .and chloride ions and a carbonic anhydrase inhibitor under conditions and for a time sufficient to allow accumulation of ammonium ions within the bound cells, followed by incubation of the cells containing accumulated ammonium ions in the presence of ammonia and carbon dioxide, such that selective hemolysis of maternal blood cells occurs.
  • ligands may be utilized, including antibodies, eiythropoietin, and transferrin,
  • the ligand may be immobilized on any of a variety of sohd supports, such as hollow fibers, beads, magnetic beads, plates, dishes, flasks, meshes, screens, sohd fibers, membranes, and dipsticks.
  • a method for enriching fetal progenitor cells from maternal blood comprising the steps of (a) incubating a sample of maternal blood with an immobilized hgand capable of specifically binding to fetal progenitor cells under conditions, and for a time sufficient to allow specific binding of the hgand to the cells, and (b) removing unbound blood products such that the fetal progenitor cells are enriched.
  • red blood cells are removed from maternal blood, for example by running the maternal blood over a Ficoll gradient.
  • the bound cells are incubated in the presence of erythropoietin.
  • a method for enriching fetal progenitor cells from maternal blood comprising the steps of (a) incubating a sample of maternal blood with a labeled hgand capable of specifically binding to fetal progenitor cells, under conditions, and for a time sufficient to allow specific binding of the hgand to the cells, (b) detecting the presence of the hgand bound cells, and (c) separating the hgand bound cells from the unbound cells, such that said fetal progenitor cells are enriched.
  • red blood cells are removed from maternal blood by, for example, running the maternal blood over a Ficoll gradient.
  • the bound cells are incubated in the presence of erythropoietin.
  • the label is selected from the group consisting of fluoroscein-isothiocyanate, phycoerythrin, rhodamine isothiocyanate, or other such highly fluorescent molecules.
  • the hgand is an antibody such as 12.8.
  • a method for enriching fetal progenitor cells from maternal blood comprising, the steps of (a) incubating a sample of maternal blood with an immobilized hgand capable of specifically binding to cells other than fetal progenitor cells, under conditions and for a time sufficient to allow specific binding of the hgand to said other cells, and (b) removing the nonbound fetal progenitor cells, such that said fetal progenitor cells are enriched.
  • red blood cells are removed from maternal blood by, for example, running the maternal blood over a Ficoll gradient.
  • the hgand is an antibody.
  • a method for typing chromosomes of fetal nucleated erythroid cells comprising the steps of (a) incubating the fetal nucleated erythroid cells in a media containing erythropoietin under conditions and for a time sufficient to induce metaphase in the cells, (b) fixing the DNA of the cells, (c) staining the fixed DNA such that chromosomes may be observed, and (d) examining the stained DNA thereby allowing the typing of the chromosomes.
  • fetal nucleated, erythroid cells may be enriched from as few as 1 in 10" in maternal blood, to an enriched concentration of about 1 in lC , and preferably, to about 1 in 10 2 .
  • nucleated erythroid cells contain a nucleus and generally include erythroblasts as well as other erythroid precursor cells.
  • Maternal blood may be obtained from a pregnant female using conventional techniques well known in the art.
  • peripheral blood is drawn from an easily obtainable source such as the antecubital vein (the arm vein) with conventional venipuncture techniques.
  • the maternal blood Once the maternal blood has been drawn, it may be frozen using conventional techniques, or stored at 4°C for a maximum of 4 to 7 days.
  • Various anticoagulants may be added to the blood as necessary, including, among others, ACD, CPDA, EDTA, and Heparin.
  • the maternal blood is then subjected to a selection method in accordance with the present invention, wherein preferentially enriched fetal cells may be obtained without the need for further purification, for example, by a Fluorescence-Activated Cell Sorter (FACS).
  • FACS Fluorescence-Activated Cell Sorter
  • the methods of the present invention comprise the steps of: (1) incubating the maternal blood with either an immobilized hgand, or a hgand which will subsequently be immobilized, such that the hgand binds to and hence immobilizes the fetal nucleated, erythroid cells, (2) removing unbound blood products, and (3) preferentially enriching the bound cells for fetal nucleated, erythroid cells.
  • these basic steps may be performed in an alternative order, for example, comprising the steps of:
  • a “cell separator” including a column assembly for separating target cells from a sample fluid, the column assembly including a column, a sample fluid supply bag and a fluid coUection bag wherein the column is provided for receiving the sample fluid from the sample fluid supply bag and for separating the target cells from the sample fluid and retaining the target cells, and wherein the fluid collection bag is provided for receiving the target cells after being released from the column, said cell separator comprising an agitation means for agitating the contents of the column to assist in releasing the sample cells retained in the column, the agitation means being responsive to a drive signal for varying amount of agitation of the contents of the column to vary the rate at which the sample cells are released, column sensor means for providing a column signal indicative of the optical density of fluid flowing out of the column and into the fluid coUection bag, a column valve means response to a column valve control signal for selectively enabling the fluid coming out of the column to flow into the fluid coUection bag
  • the maternal blood is incubated with an immobilized hgand capable of specificaUy binding to fetal nucleated, erythroid cells under conditions, and for a time sufficient to aUow binding of the cells to the hgand.
  • GeneraUy incubation of about 15 to 30 minutes at 4°C to 37°C is preferred. If the incubation step occurs as the cells are passed, over a column, the flow rate should be sufficiently slow to aUow the cells to bind.
  • the ceUs should be aUowed at least 15 minutes in which to traverse the bed of the column.
  • the hgand should be chosen such that it is capable of specificaUy binding fetal nucleated, erythroid ceUs.
  • the hgand is defined to be "specificaUy binding" if it is capable of binding to fetal nucleated, erythroid cells, but not to more than about 10% of the maternal blood cells.
  • the relative percentage of bound fetal nucleated, erythroid ceUs to other ceUs may be reach y determined by analysis with a specific marker such as Alpha Fetal Protein (AFP).
  • AFP Alpha Fetal Protein
  • the relative percentage of fetal cells to maternal ceUs may be determined with either glucose oxidase or fluorescein conjugated anti- AFP antibody.
  • Ligands which specificaUy bind to fetal nucleated, erythroid cells are known in the art, including erythropoietin (Amgen, Thousand Oaks, Calif.), transferrin (Sigma Chemical Co., St. Louis, Mo.) and selected antibodies. Monoclonal antibodies which specificaUy recognize nucleated erythroid cells are particularly preferred.
  • Monoclonal antibodies to nucleated erythroid ceUs such as anti-transferrin receptor antibodies may be obtained from conventional supphers (Becton Dickinson Immunocytometry Systems, Mountain View, Calif.).
  • monoclonal anti-erythroid antibodies, such as EP-1 may be produced using techniques weU known in the art. See Yokochi etal, "Monoclonal Antibodies Detecting Antigenic Determinants With Restricted Expression On Erythroid CeUs: From the Erythroid Committed Progenitor Level to the Mature Erythroblast," Blood 63:1376 (1984); see also Heddy Zola (ed.), Monoclonal Antibodies: A Manual of Techniques, CRC Press, Boca Raton, Fla.
  • ceUs may be generated for immunization from fetal liver clonal erythroid cultures, and enriched for progenitor ceUs.
  • the population of cells used for antigens and for primary screening may contain immature erythroblasts, erythroblasts of an intermediate degree of maturity, and presumably, progenitor ceUs of BFU-E and CFU-E types.
  • These cells may be used for intravenous immunization, foUowed by removal of the spleen and fusion of the spleen cells with a myeloma line such as NSI, using standard techniques.
  • the resulting fused ceUs, or hybridomas may then be screened against the above-described ceUs using conventional techniques.
  • the entire specificaUy binding antibody need not be used as the hgand. More specificaUy, only the binding region of the antibody is necessary to specificaUy bind fetal nucleated, erythroid ceUs. Thus, antibody fragments such as Fab or F(ab')2 fragments may be used within the present invention. Additionally, the binding regions of the specificaUy binding antibody may be incorporated into a new protein, which may be used as the hgand.
  • the hgand is immobilized in order to aUow separation of bound cells from other blood products.
  • suitable supports include, among others, hoUow fibers (Amicon Corporation, Danvers, Mass.), beads (Polysciences, Warrington, Penn.), magnetic beads (Robbin Scientific, Mountain View, Cahf.), plates, dishes and flasks (Corning Glass Works, Corning, N.Y.), meshes (Becton Dickinson, Mountain View, Calif.), screens and solid fibers (see Edelman et al, U.S. Patent No. 3,843,324; see also Kuroda et at, U.S. Patent No.
  • a support such as Biogel P-60TM (BIORAD, Richmond, Calif.).
  • Biogel P-60TM is a porous polyacrylamide hydrogel bead.
  • the beads are generally spherical, on average about 250 microns in size, and have an average pore size which excludes molecules larger than approximately 60,000 daltons.
  • a variety of methods may be used to immobUize the hgand onto a support.
  • a hgand such as an antibody, may be directly coupled to the support by various methods wiU known in the art. See J. K.
  • the maternal blood may be incubated with the immobilized hgand under conditions, and for a time sufficient to aUow binding of the hgand to the ceUs.
  • suitable conditions for binding to occur include incubation in a physiological buffer at about 4°C to about 37°C. Particularly preferred temperatures range from about 4°C to room temperature.
  • the time of incubation depends on the affinity and avidity of the hgand for the ceU, and may be readUy determined. GeneraUy, incubation for about 15 minutes to one hour is preferred. FoUowing incubation, unbound blood products may be removed, and fetal ceUs enriched using the methods described herein.
  • a sample of maternal blood is incubated under suitable conditions with a hgand which is chemicaUy linked to a first member, and then adsorbed to a second member which is immobilized on a sohd support.
  • the first member shduld be capable of binding to the second member with an affinity of greater than about 10° M .
  • Many suitable first member-second member binding pairs are weU known in the art.
  • biotin-avidin biotin-streptavidin
  • biocytin- avidin biocytm-streptavidin
  • methotrexate-dihydrofolate reductase 5-fluorouracU-thimydylate synthetase
  • riboflavin-riboflavin binding protein antibody-protein A
  • antibody-protein G antibody-protein G.
  • the first member is biotin and the second member is avidin.
  • Either member of the above described binding pairs may function as the second member, with the complementary member functioning as the first member.
  • combinations of the first member-second member binding pair may be employed.
  • biotin may be linked to the hgand, as weU as adsorbed to the support.
  • the ceU, hgand, biotin-complex and biotin, support- complex may then be bound together through an incubation step with avidin.
  • Avidin is multivalent, permitting the formation of a ceU, hgand, biotin, avidin, biotin, support-complex which immobilizes the ceU.
  • a sample of maternal blood is incubated with a biotiny ted antibody under conditions and for a time sufficient to aUow binding to occur.
  • the sample is then incubated with, or passed over, a support which contains immobilized avidin.
  • CeUs which are coupled to the biotinylated antibody are adsorbed to the immobilized avidin, thus aUowing separation of ceUs from unbound blood products.
  • unbound blood products may be removed, and fetal ceUs enriched using methods described below.
  • a two-step method is used to immobilize the fetal nucleated, erythroid cells.
  • a first hgand is incubated with a sample of the maternal blood under suitable conditions as described above. Subsequently, a second hgand which has been chemically linked to a first member is added. The second hgand is capable of binding to the first hgand. The cell, first hgand, second ligand, first member-complex may then be adsorbed onto an immobilized second member, thus allowing the separation of cells from unbound blood products. Representative examples of the first member- second member binding pair have been discussed above. Representative examples of the first hgand include erythropoietin, transferrin and selected antibodies.
  • the second ligand is chosen such that it specifically recognizes and binds to the first ligand.
  • the second hgand is an antibody, for example, an anti-erythropoietin (Terry Fox Laboratory, Vancouver, B.C., Canada), anti-transferrin (Chemicon Intl., Inc., Temecula, Cahf.), or anti-immunoglobulin antibody.
  • Anti- immunoglobulin antibodies may be prepared using techniques weU known in the art, or may be obtained from conventional sources, including, among others, Sigma Chemical Co., St. Louis, Mo., and Becton Dickinson Immunocytometry Systems, Mountain View, Calif.
  • the first hgand is an antibody which specifically recognizes fetal nucleated, erythroid cells such as an anti- transferrin receptor antibody (Becton Dickinson Immunocytometry Systems, Mountain View, Cahf.).
  • the antibody is incubated with a sample of maternal blood.
  • a biotinylated anti-immunoglobulin antibody such as biotinylated goat anti-mouse IgG (the second hgand which is chemically hnked to a first member) is then added and incubated with the sample.
  • the sample is then incubated with, or passed over a bed of material which contains the immobilized second member, in this case, immobilized avidin.
  • the ceU, antibody, anti-immunoglobulin antibody, and biotin-complex will be adsorbed to the immobilized avidin, thus allowing the subsequent removal of unbound blood products.
  • the immobilized ceUs are rinsed with a physiological buffer, thereby removing the unbound blood products.
  • a physiological buffer Various methods maybe used to rinse the immobilized ceUs, depending upon the type of support chosen. These methods include, among others: washing or flushing the support; magnetically attracting the support out of solution, foUowed by resuspension in a physiological buffer; and centrifugation foUowed by resuspension.
  • physiological buffers are also weU known in the art, including PBS, PBS plus albumin, such as Bovine Serum Albumin (BSA), normal saline and ceU culture media.
  • BSA Bovine Serum Albumin
  • bound ceUs may be preferentiaUy enriched for fetal nucleated, erythroid cells.
  • at least two alternative methods may be used either separately, or together. If the two methods are performed together, either method may be performed first.
  • the bound ceUs are cultured under selected culture conditions in the presence of erythropoietin (Amgen, Thousand Oaks, Calif.).
  • Emerson et al "Developmental Regulation of Erythropoiesis by Hematopoietic Growth Factors: Analysis on Populations of BFU-E From Bone Marrow, Peripheral Blood and Fetal Liver," Blood 74(l):49-55 (1989); see also Linch et al, "Studies of Circulating Hemopoietic Progenitor CeUs in Human Fetal Blood,” Blood 59(5):976-979 (1982).
  • Selected culture conditions generaUy include growth in standard ceU culture media, without any other cytokines other than erythropoietin. This preferentiaUy aUows fetal, but not maternal, nucleated erythroid ceUs to grow.
  • Particularly preferred media includes Iscoves' Modified Dulbecco's Medium (Gibco, Grand Island, N.Y.) containing a final concentration of 20% fetal bovine serum, and 2 U/ml purified urinary human EPO.
  • fetal nucleated, erythroid ceUs are preferentiaUy enriched based upon their uptake of ammonium ions and the selective hemolysis of maternal blood cells, or more specificaUy, of maternal erythroid cells. See generally Jacobs and Stewart, "The Role of Carbonic Anhydrase in Certain Ionic Exchanges Involving the Erythrocyte," /. Gen.
  • suitable sources of ammonia include, among others, ammonia and the ammonium salts.
  • suitable sources of chloride ions include, among others, NaCl, KC1, MgCi2 and CaC -
  • Suitable sources for carbon dioxide include, among others, carbon dioxide in solution, carbonate and bicarbonate.
  • carbonic anhydrase inhibitors are known in the art, including, among others, most monovalent anions such as cyanide and cyanate, monovalent sulfides, sulfonamides, and acetazolamide. See Lindskog et al, The Enzymes 5:587 (1971); Ward and Cull, Arch. Biochem. Biophys. 750:436 (1972); and Pocker and Watamori, Biochem. 12:2475 (1973).
  • the carbonic anhydrase inhibitors should be selected so as to function under physiological conditions.
  • Preferred carbonic anhydrase inhibitors include sulfanUamide and acetazolamide.
  • Bound ceUs may be released by various methods either subsequent to, or prior to enrichment. If bound cells are released prior to enrichment, they may be subsequently enriched using the above-described methods.
  • Various methods are known in the art for releasing ceUs.
  • ceUs may be cultured with or without cytokines. Cytokines, such as EL-2, can cause proliferation of cells, or changes in the surface characteristics of the cells, such that the ceUs or their progeny are released from a support.
  • cleavage of the hgand or of the ceU-hgand bond may release the cell.
  • Various cleavable ligands and cleaving enzymes are known in the art, including among others, papain and trypsin.
  • the ceUs may be released by mechanical, gravitational or electromagnetic forces. A particularly preferred method is mechanical agitation, for example, by agitation of the beads through pipetting, stirring, shaking, vibration, or sonication.
  • fetal ceUs may be enriched by first incubating maternal blood with ammonia and chloride ions, and a carbonic anhydrase inhibitor. After the ceUs have been incubated under conditions and for a time sufficient to allow accumulation of ammonium ions, the ceUs are treated with ammonia and carbon dioxide such that selective hemolysis of maternal blood ceUs occurs.
  • Fetal cells may also be enriched by incubation with erythropoietin as discussed above, or by a combined treatment using both erythropoietin and the method discussed above wherein the ceUs are treated with ammonia and carbon dioxide. Furthermore, as discussed above, the two methods may be performed in either order.
  • the enriched cells remaining after the method(s) described above may then be immobilized using any of the above discussed methods, including: (1) an immobilized hgand which specificaUy binds to fetal nucleated, erythroid cells remaining after enrichment, (2) incubating ceUs remaining after enrichment with a first member linked to a Hgand, the Hgand being capable of specificaUy binding to fetal nucleated, erythroid cells, foUowed by adsorption of the cells to an.
  • the second member being capable of binding to the first member with an affinity constant of greater than about 10° M
  • unbound blood products may be removed using methods described above. If the ceUs were initiaUy enriched using erythropoietin, subsequent to removing unbound blood products, the cells may be treated using the method wherein ammonia and carbon dioxide are used. Si ⁇ iilarly, if these cells were initially treated with the method wherein ammonia and carbon dioxide are used, subsequent to removing unbound blood products, the cells may be treated with erythropoietin.
  • Fetal ceUs which have been enriched from maternal blood may be characterized by incubating the preferentiaUy enriched ceUs with a marker capable of selectively binding to fetal ceUs.
  • a marker selectively binds to fetal cells when its presence on the target cells is greater than 10-fold more than the quantity on maternal cells.
  • markers are known in the art, including, for example, antibodies to Alpha Fetal Protein ("AFP"). See Kulozik et al, “Fetal CeUs in the Maternal Circulation: Detection by Direct AFP- Immunofluorescence," Human Genet. 62:221-224 (1982), or antibodies to antigen "i”. See Y.W. Kan et al, "Concentration of Fetal Red Blood CeUs From a Mixture of Maternal and Fetal Blood by Anti-i Serum," Blood 43(3):411-415 (1974).
  • Antibodies to the fetal ceU marker may be labeled using techniques weU known in the art and used to detect the presence of fetal ceUs.
  • Hgand is removed from the ceU surface through the method described in a related apphcation (U.S. Serial No. 07/513,056) entitled “Methods for Removing Ligands from a Particle Surface", which is incorporated herein by reference. Such removal may be particularly advantageous prior to characterization, as discussed above.
  • a method for enriching fetal progenitor cells from maternal blood comprising the steps of (a) incubating a sample of maternal blood with an immobilized hgand capable of binding to fetal progenitor ceUs under conditions, and for a time sufficient to aUow specific binding of the hgand to the ceUs, and (b) removing unbound blood products such that the fetal progenitor cells are enriched.
  • fetal progenitor cells includes totipotent hematopoietic stem ceUs as weU as early progemtor cells such as colony forming cells (CFCs).
  • CFCS include CFU-E, CFU-G, CFU-M, CFU-GM, CFU-GEMM and BFU-E ceUs.
  • CD 34 positive ceUs may be stem ceUs or colony-forming ceUs, it should be understood that when CD 34 cells are concentrated or purified, fetal progenitor ceUs are likewise understood to be concentrated or purified.
  • a Hgand capable of binding to fetal progenitor ceUs.
  • a Hgand "binds to" fetal progenitor cells if it recognizes an antigen associated with either both adult and fetal cells or only fetal progenitor ceUs.
  • a representative antigen in this regard is the CD-34 antigen.
  • Representative examples of antibodies which specifically recognize the CD 34 antigen include MY-10 and HPCA2, (Becton-Dickinson, Mountain View, Calif.), QBEND-10 (Quantum Biosystems, Cambridge, U.K.) and 12.8 (CeUpro ® , Bothell, Wash.).
  • This Hgand may be utilized in the above-described devices and methods in order to immobhize the fetal progenitor ceUs.
  • the unbound blood products are removed as described above, such that the fetal progenitor ceUs are enriched.
  • cells are "enriched" if greater than 0.001% of the ceUs are fetal progenitor cells.
  • the fetal progenitor ceUs are enriched to greater than .1% and particularly, to greater than 1% of the ceUs present.
  • a method for enriching fetal progenitor ceUs from maternal blood comprising the steps of (a) incubating a sample of maternal blood with a labeled Hgand capable of specificaUy binding to fetal progenitor cells under conditions and for a time sufficient to aUow specific binding of the Hgand to the ceUs, (b) detecting the presence of the hgand bound cells, and (c) separating the Hgand bound cells from the unbound cells, such that the fetal progenitor cells are enriched.
  • ceUs are incubated with a Hgand such as that described above which is capable of specificaUy binding to fetal progenitor cells, except that rather than being immobilized, this Hgand is labeled.
  • a Hgand such as that described above which is capable of specificaUy binding to fetal progenitor cells, except that rather than being immobilized, this Hgand is labeled.
  • Various labels may be utilized within the context of the present invention, although fluoroscein- isothocyanate, phycoerythrin, rhodamine isothiocyanate, or other such highly fluorescent molecules are particularly preferred.
  • FACS flow cytometry
  • red blood ceUs may be removed from maternal blood, for example, by running the maternal blood over a FicoU gradient.
  • the bound ceUs are incubated in the presence of erythropoietin utilizing culture methods described above.
  • a method for enriching fetal progenitor ceUs from maternal blood comprising the steps of
  • CD 34 is a ceU-surface antigen that may be found on fetal progenitor cells. Many ceU-surface antigens however, can not be found on progenitor cells, and thus maybe utilized to deplete a sample of maternal blood from cells other than fetal progenitor ceUs. Representative examples of such antigens include la, Glycophorin, CD 3, CD 19, CD 11, CD 14, CD 33, and CD 45. Ligands, such as antibodies, which specificaUy bind to such antigens may be purchased from commercial supphers such as Becton-Dickinson, Mountain View, Calif.
  • red blood cells are removed from maternal blood by, for example, running the maternal blood over a FicoU gradient.
  • enriched fetal ceUs have a variety of uses. For example, through in situ hybridization the presence of a selected genetic material may be detected in a fetal ceU. SimUarly, DNA or RNA amplification may also be used to detect a selected genetic sequence in fetal ceUs.
  • the methods of the present invention are particularly useful for fetal ceUs which are enriched to such an extent that they are amenable to chromosomal typing by conventional cytogenetics techniques wherein spreads of the ceUs' chromosomes are examined under a microscope.
  • In situ hybridization may be used as a method for detecting the presence of a selected genetic material within cells. See Pinkel et al, Proc. Nail Acad. Set USA 55:9138-42 (1988); see also Hopman et al, "Detection of Numerical Chromosome Aberrations in Bladder Cancer by In Situ Hybridization," Am. J. of Path. i35(6):1105-1117 (1989). Briefly, the genetic material associated with the preferentially enriched ceUs is first exposed using techniques well known in the art. The genetic material is then incubated with a labeled probe capable of specificaUy hybridizing to the genetic material under conditions and for a time sufficient to aUow hybridization to occur. See, for example, K. E.
  • the genetic material is denatured after the step of exposing.
  • genetic material includes whole chromosomes, DNA and RNA
  • a selected genetic sequence may be amplified using techniques well known in the art, and then probed for presence of that sequence. See Kogan et al, "An Improved Method For Prenatal Diagnosis of Genetic Diseases by Analysis of Amplified DNA Sequences," The New Eng. J. ofMed. 377(16):985-990 (1987); see also Witt and Erickson, "A Rapid Method for Detection of Y-Chromosomal DNA from Dried Blood Specimens by the Polymerase Chain Reaction," Human Genet. 52:271-274 (1989).
  • Methods for amplification include Polymerase Chain Reaction ("PCR") (see MulHs et al, U.S. Patent No. 4,683,195; MulHs et al, U.S. Patent No. 4,683,202; and Mullis et al, U.S. Patent No. 4,800,159, which are incorporated herein by reference), and RNA- based amplification techniques.
  • PCR Polymerase Chain Reaction
  • RNA- based amplification techniques See Lizardi et al, Bio/Technology 6:1197-1202 (1988); Kramer et al, Nature 339:401-402 (1989); and Lomeli et al, Clinical Chemistry 35(9): 1826-1831 (1989); see also Kramer et al, U.S. Patent No.4,786,600, which is incorporated herein by reference.
  • PCR is the most commonly used method for amplifying DNA sequences. Briefly, amplification entails adding the appropriate primer(s), enzymes and nucleotides into a reaction mixture, foUowed by several (20-80) cycles of denaturation and annealing in order to amplify the smaU amount of target DNA The DNA mixture is then separated by electrophoresis and hybridized with a labeled probe to detect the presence of the target sequence of DNA
  • the preferentiaUy enriched fetal ceUs may also be chromosomaUy typed. See Human Cytogenetics, D.E. Rooney and B.H. Czepulkouski (eds.), IRL Press, Oxford, U.K. (1986).
  • a sample containing at least 1 fetal ceU in 10 ⁇ other cells is cultured for 2-6 days in Iscoves' Modified Dulbecco's Medium (IMDM) (Gibco, Grand Island, N.Y.) containing a final concentration of 20% fetal bovine serum (FBS) (HYCLONETM, Logan, Utah) and 2 U/ml of highly purified recombinant erythropoietin (Terry Fox Laboratory, Vancouver, B.C., Canada) in order to increase the proportion of ceUs in metaphase.
  • IMDM Iscoves' Modified Dulbecco's Medium
  • FBS fetal bovine serum
  • HYCLONETM highly purified recombinant erythropoietin
  • the cells are then incubated in colcemid, fixed, attached to microscope shdes, trypsin-treated and stained with Wright's stain.
  • the slides may then be scanned microscopicaUy for abnormal chromosomes
  • PreferentiaUy enriched fetal ceUs may also be utilized therapeuticaUy.
  • a gene may be inserted into a retrovirus, and the retrovirus utilized to infect fetal cells which are deficient in this gene.
  • the infected fetal ceU may then be administered to the fetus (or another individual) in order to remedy the genetic defect.
  • Diseases which may be treated in this manner are numerous, including for example ADA, sickle ceU anemia, Thalassemia, and SCLD.
  • Fetal cells may also be utilized as universal donor cells.
  • fetal progenitor ceUs do not create as strong an immune reaction in recipients as do progenitor cells from adults, they can be transplanted with fewer side effects. These fetal progenitor cells can thus be used for transplant to correct inherited metabohc diseases in recipients. By expanding their numbers in culture, these fetal progenitor cells may even be used in place of a bone marrow transplant foUowing myeloblative therapy for cancer.
  • PreferentiaUy, enriched fetal ceUs may also be made oncogenic in order to study the progress of such oncogenic cells, or in order to simplify expansion and maintenance of fetal cells for research or diagnostic purposes.
  • transforming genes that may be inserted into fetal ceUs (for example, by retroviruses as discussed above) include SV40, Ti, myc, ras and src.
  • enriched fetal cells may also be cryopreserved for future research, or for future therapeutic use. Briefly, methods for freezing stem ceUs are described in a pending apphcation entitled “Method for freezing engrafting ceUs" (Attorney's Docket No. 200072.409). These methods may also be utilized to freeze fetal progenitor cells.
  • the beads are washed several times with distiUed or deionized water, foUowed by several washings of PBS using a coarse glass filter connected to a vacuum.
  • the carboxylated gel may be stored in PBS at 4°C, and is stable for up to one year if sterilized or stored with a preservative.
  • EXAMPLE 2 Avidin Conjugating the Carboxylated Biogel
  • PBS is first removed from a measured amount of carboxylated Biogel by filtering with a coarse glass filter connected to a vacuum.
  • the gel is then equihbrated in distiUed or deionized water for 15-30 minutes. EquiUbration in water causes an expansion of the gel to a volume of about 4 times its previously measured amount.
  • the gel is resuspended in 10 ml of distiUed or deionized water for each ml of gel (as originaUy measured in PBS). Thirty mg of l-ethyl-3-(3-dimethylaminopropyl) carbodiimide
  • EEC-HC1 (Sigma Chemical Co., Catalog No. E7750, St Louis, Mo.) is added for each ml of gel as originaUy measured.
  • the pH is rapidly adjusted to 5.5 by dropwise addition of Hd Care is taken to maintain the pH at 5.5; pHs of less than 5.0 or greater than 6.0 result in significantly less activation of the Biogel.
  • the mixture is stirred for five minutes.
  • Avidin International Enzymes, Inc., FaUbrook, Calif.
  • a concentration of between 10 and 100 mg/ml in deionized water 1000 ⁇ g of avidin is rapidly added for each ml of gel (as originaUy measured in PBS). The mixture is stirred for 1.5 hours.
  • 2 M glycine is added to give a final concentration of 0.2 M glycine in the mixture and stirred for an additional 1 hour.
  • the gel is washed with several volumes of PBS using a coarse glass filter and vacuum, and stored in PBS at 4°C. The gel is stable for approximately one year.
  • the nuclei of ceUs which are almost aU lysed by the acetic acid, are counted, in the four ruled fields per chamber of a hemacytometer with improved Neubauer ruHngs (VWR Scientific, San Francisco, Calif.), each field representing 0.1 x 10 " ⁇ ⁇ l sample volume.
  • the average number of nuclei per field is multiplied by a dUution factor of 61 x 10 4 to calculate the number of nucleated ceUs per miUihter of whole blood. If the average is less than 10 nuclei per field, the procedure is repeated, except 50 ⁇ l blood is dUuted into only 1 ml acetic acid, with a resulting new dUution factor of 21 x 10 .
  • Each tube of diluted blood is underlayered with 5 ml of Histopaque 1077 (Sigma Chemical Co.) and centrifuged at 700 x g for 15 minutes at room temperature. CeUs at the interface are coUected and washed once in PBS plus 1% BSA The pellet is resuspended in 100 ⁇ l in PBS plus 1% BSA
  • ceUs are then gently resuspended to 1 ml and 1 ⁇ g/ml of biotinylated goat anti-mouse IgG (Southern Biotech, Birmingham, Ala.) is added. The mixture is incubated for 15 minutes on ice and washed twice as described above with PBS plus 1% BSA
  • Carboxylated Biogel P-30TM (prepared as described above) is allowed to equihbrate to room temperature and placed in a K9/15 column (Pharmacia,
  • the column is washed with PBS, followed by washes with PBS plus 5% BSA. This column functions as a "pre- column.”
  • the avidin column contains avidin-conjugated Biogel P-60TM, which is prepared as described above. The avidin-conjugated Biogel is allowed to equUibrate to room temperature and placed in a K9/15 column to a total bed height of 4 cm. The column is then washed with several volumes of PBS, followed by washes with PBS plus 5% BSA
  • CeUs which have been prepared as described above are resuspended in PBS plus 5% BSA to a volume of 1 ml.
  • the ceUs are then gently transferred onto the top of the gel bed of the pre-column filter.
  • the ceUs are allowed to filter through the pre-column and are washed with 1 ml PBS plus 5% BSA
  • a peristaltic pump (Cole-Parmer, Rockford, HI.) controls flow from the avidin column to a rate of about 1 ml/minute.
  • 1-2 ml of PBS plus 5% BSA is added to the top of the avidin column in order to wash out remaining cells.
  • the column is washed out with 4-6 ml of PBS plus 5% BSA, foUowed by 4-6 ml of PBS.
  • the avidin column is placed on top of a 15 ml centrifuge tube.
  • the valve of the column is opened and 15 ml of RPMI 1 is added to the column with a wide bore, 9-inch transfer pipette.
  • the RPMI is added to the column while the pipette is used for mechanicaUy agitating and resuspending the ceU bed, thus aUowing ceUs to become detached from the gel matrix, and to filter into the centrifuge tube.
  • the tube is then centrifuged at 400 x g for 5 minutes and resuspended in ceU culture media as described below.
  • IMDM Iscoves' Modified Dulbecco's Medium
  • FBS fetal bovine serum
  • CeUs are dUuted to 5 x 10" nucleated cells/ml and 200 ⁇ l is plated into each weU of a 96 weU tissue culture plate with round bottoms (Co ⁇ iing Glass Works, Corning,
  • CeUs which are separated from the Avidin column above are adjusted to a concentration of less than 2 x 10 ' nucleated ceUs/ml.
  • One volume of the ceU mixture is chUled, and a solution at 29°C containing 18 volumes of 0.1844 M NH4CI and 2 volumes of 10 ⁇ M acetazolamide is added. After 2 minutes, 2 volumes of 3mM NH4HCO3 is rapidly added, and the whole mixture gently stirred for 3 minutes.
  • CeUs are washed several times with PBS by centrifugation in order to remove ceUular debris.
  • Enriched ceUs are exposed to a hypotonic solution (.075 M KC1) for 12 minutes at 37°C.
  • the tubes are inverted once during the incubation to keep the ceUs suspended.
  • Twenty drops of freshly prepared fixative (3:1 methanolracetic acid) is added to the ceUs, vortexed, and then centrifuged for 8 minutes at approximately 250 x g.
  • Fresh fixative is added to the ceUs, foUowed by incubation for one hour at room temperature.
  • the cells are centrifuged for 8 minutes at approximately 250 x g.
  • Fresh fixative is added and the process is repeated one more time. FinaUy, the ceUs are resuspended in a small amount of fixative and placed at 4°C overnight.
  • ceUs are vortexed and placed onto microscope shdes (Baxter, McGaw Park, 111.) which have been cleaned with ethanol and dipped in distilled water.
  • the shdes are aUowed to dry for two days at room temperature.
  • a probe is prepared from plasmid DNA according to the method of Page et al ("Single copy sequence hybridizes to polymorphic and homologous loci on human X and Y chromosomes," PNAS 79:5352-5356 (1982), from American Type Culture Collection (ATCC) No. 57261; except that biotin-dATP is incorporated into the probe. Twenty five microhters of the biotinylated probe (5 ⁇ g/ml) per slide is placed in a microfuge tube. The probe is heated to 70°C for 5 minutes then immediately placed on ice. Twenty microhters of the probe solution is placed onto each slide and covered with a 22 x 40 mm covershp. The shdes are placed into a box with a wet paper toweling liner and incubated at 37°C for 12-18 hours.
  • a 50% solution of formamide in 1 x SCC is warmed to 37°C. Covershps are removed from the shdes and immersed into the 50% formamide solution for 30 minutes. The shdes are then placed in 2 x SCC solution for 30 minutes with gentle rocking, then in 1 x SCC for 30 minutes with gentle rocking. Fluoresceinated avidin (Vector, Burlingame, Calif.) is dUuted 1:1000 (1 ⁇ g/ml). The back of the slide and around cell area are wiped. Two hundred microhters of the avidin-fluorescein is added to each slide and incubated in the box for 30 minutes at room temperature.
  • the sHdes are rinsed sequentially in (1) 4 x SCC for 10 minutes with rocking, (2) 4 x SCC, 0.1% Tween-20 for 10 rninutes without rocking, and (3) 4 x SSC for 10 minutes with rocking.
  • the back of the slide and around ceU area is wiped off.
  • Ten microhters of anti-fade plus propidium iodine (10 ml PBS, 100 mg p-Phenylene diamine, 90 ml glycerol, pH 8.0, 10 ⁇ g/ml propidium iodide) is added.
  • the shdes are covered with coversHps and stored at 4°C. The shdes may be stored for several days if necessary.
  • Target DNA may be. observed under a microscope by the presence of fluorescence.
  • the enriched ceUs are exposed to 1 ⁇ g of colcemid (Sigma, St. Louis, Mo.) for one hour at 37°C.
  • a hypotonic solution (0.075 M KC1) is added to the ceUs and incubated for 12 minutes at 37°C.
  • the tubes are inverted once during the incubation to keep the cells suspended.
  • Twenty drops of freshly prepared fixative (3:1 methano acetic acid) is added to the cells, vortexed, and then centrifuged for 8 minutes at approximately 250 x g.
  • Fresh fixative is added to the cells, foUowed by incubation for 1 hour at room temperature.
  • the cells are centrifuged for 8 rninutes at approximately 250 x g and fresh fixative is added again. This process is repeated one more time.
  • ceUs were resuspended in 20 ml of IMDM culture media containing 20% Fetal Bovine Serum (HYCLONE, Logan, Utah), 2mM glutamine, ImM sodium pyruvate, 0.1 mM NEAA (non-essential amino acids; Whittaker Bioproducts, WalkersvUle, Md.), and 1 U/ml EPO (Terry Fox Laboratory, Vancouver, B.C., Canada.
  • Ten miUihters of the ceU suspension was placed into each of two T75 Costar flasks, and incubated overnight at 37°C in a 5% CO2 incubator. Adherent ceUs such as fibroblasts are thus removed prior to separation.
  • Example 7 After incubation overnight, the cell culture was counted and centrifuges 10 minutes at 1200 rpm. The supernatant was removed, and the ceUs were resuspended in 10 ml of PBS containing 1% BSA (Bovine Serum Albumin) and centrifuged once more for 10 minutes at 1200 ⁇ m (250 x g). The supernatant was removed and the ceUs were resuspended in 1 ml of PBS containing 0.1% BSA
  • BSA Bovine Serum Albumin
  • Antibody 12.8 (CellPro, BotheU, Wash.) (an anti-CD 34 antibody) was added to the ceU suspension to a final concentration of 40 ug/ml. The ceUs were then incubated for 30 minutes on ice. After 30 rninutes, the cells were washed twice by centrifugation and resuspension in PBS/1% BSA Next, biotinylated rabbit anti-mouse IgM antisera
  • Adsorbed cells which were separated by the above procedure were resuspended in IMDM containing 20% FBS and 2 U/ml EPO. The cells were- then placed in 96 weU Costar plates in a volume of 200 ⁇ l per weU (approximately one million cells per weU), and incubated for 5 days at 37°C in a 5% CO2 incubator.
  • the ceUs were transferred into n ⁇ crocentrifuge tubes and exposed to Colcemid (1 ⁇ g in 300 ⁇ l) for one hour at 37°C. The cells were then centrifuged for 5 minutes at 1000 rpm, and 1 ml of 0.075M KQ was added. Twenty drops of freshly prepared fixative (3:1 MethanofcAcetic Acid) was added to the ceU suspension, and the ceUs were centrifuged again for 5 minutes at 1000 ⁇ m. One ml of fixative was added and the cells were left at room temperature for 1 hour. The cells were then centrifuged for 5 minutes at 1000 ⁇ m, and washed three times in 1 ml of fixative, and left in 500 ⁇ l overnight at 4°C. The next day metaphase spreads were prepared.
  • fixative 3:1 MethanofcAcetic Acid
  • the spreads were aUowed to dry several days at room temperature.
  • the shdes were placed in Difco Bactotrypsin for one minute (6 drops/45 ml PBS).
  • the shdes were rinsed in PBS containing 1% FBS, and then in PBS only.
  • the shdes were placed in fresh Wright's stain for 35 seconds, rinsed twice, and scanned under the microscope.
  • CeUs were plated in triplicate at 3-fold dilutions to improve the accuracy of the assay. The highest number of cells plated was 10 5 /plate except for column-purified ceUs which were plated at 3 x 10 3 and less. The ceUs were spread evenly over the surface of each plate and then incubated in a humidified incubator at 37°C with 5% CO2 in air for 10 to 14 days. Colonies were counted if they contained more than 50 cells and scored as CFU-GM, BFU-E, or other (e.g., CFU-GEMM). The number of various types of colonies were summed to give the total number of colony-forming ceUs (CFC).
  • CFC colony-forming ceUs
  • Example 8 Fetal ceUs which had been enriched as described above in Example 8 were subjected to in situ hybridization utilizing a commerciaUy avaUable kit (Chromosome in situ kit S1370, Oncor, Gaithersburg, MD). CFCs number was also determined as described above in Example 10. The results are briefly set forth below in Table 2.
  • Fetal cells which were enriched as described above in Example 8 were submitted to analysis by FACS. Briefly, approximately 125,000 purified ceUs were divided into two tubes. One tube received an IgG control, and the second received QBend-10 (an anti-CD 34 antibody) at a final concentration of 20 ⁇ g/ml. The tubes were incubated for 30 minutes on ice, then washed twice with 4 ml of 1% BSA in PBS.
  • Clonal fetal cells are identical, and thus may be utilized for assays wherein the effect of multiple samples are to be tested on ceUs (ie., to establish whether a compound is carcinogenic). Some of the ceUs may function as a control, whUe other ceUs may be subjected to the compound. Similarly, multiple assays may be performed in order to determine the sensitivity of a ceU to a certain drug. In addition, the colonies may be screened for a desired response, and clones subjected to more detailed analyses.

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Abstract

Le procédé décrit consiste (a) à incuber un échantillon de sang maternel avec un ligand immobilisé susceptible de se fixer à des cellules souches f÷tales dans des conditions et pendant une durée suffisantes pour permettre la fixation spécifique du ligand sur les cellules, et (b) à éliminer les produits sanguins non fixés, de telle manière que les cellules souches f÷tales sont enrichies.
PCT/US1992/009024 1991-10-23 1992-10-22 Procede pour enrichir des cellules souches f×tales issues du sang maternel WO1993008269A1 (fr)

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WO1994025873A1 (fr) * 1993-04-23 1994-11-10 Cellpro, Incorporated Procedes d'enrichissement de cellules souches f×tales provenant du sang maternel
WO1995026417A1 (fr) * 1994-03-29 1995-10-05 Genzyme Corporation Culture et isolation de cellules f×tales a partir de sang peripherique maternel
US5580724A (en) * 1994-03-25 1996-12-03 Board Of Regents, The University Of Texas System Differential expansion of fetal stem cells in maternal circulation for use in prenatal genetic analysis
US5646004A (en) * 1994-08-31 1997-07-08 Activated Cell Therapy, Inc. Methods for enriching fetal cells from maternal body fluids
US5648223A (en) * 1994-08-31 1997-07-15 Activated Cell Therapy, Inc. Methods for enriching breast tumor cells
US5663051A (en) * 1994-08-31 1997-09-02 Activated Cell Therapy, Inc. Separation apparatus and method
US5840502A (en) * 1994-08-31 1998-11-24 Activated Cell Therapy, Inc. Methods for enriching specific cell-types by density gradient centrifugation
WO2001079851A1 (fr) * 2000-04-13 2001-10-25 Imperial College Innovations Limited Diagnostique prenatal non invasif utilisant des cellules foetales cd45-negatives
WO2018010632A1 (fr) * 2016-07-11 2018-01-18 山东亚大药业有限公司 Procédé de séparation et de purification de globules rouges nucléés fœtaux, et kit de réactifs

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JP2010029178A (ja) * 2008-07-01 2010-02-12 Osaka Univ 細胞ピッキングシステム、スクリーニング方法および哺乳類細胞を取得する方法

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THE LANCET, Volume 336, July 1990, U.W. Mueller et al., "Isolation of fetal trophoblast cells from peripheral blood of pregnant women" *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994025873A1 (fr) * 1993-04-23 1994-11-10 Cellpro, Incorporated Procedes d'enrichissement de cellules souches f×tales provenant du sang maternel
US5580724A (en) * 1994-03-25 1996-12-03 Board Of Regents, The University Of Texas System Differential expansion of fetal stem cells in maternal circulation for use in prenatal genetic analysis
WO1995026417A1 (fr) * 1994-03-29 1995-10-05 Genzyme Corporation Culture et isolation de cellules f×tales a partir de sang peripherique maternel
US5646004A (en) * 1994-08-31 1997-07-08 Activated Cell Therapy, Inc. Methods for enriching fetal cells from maternal body fluids
US5648223A (en) * 1994-08-31 1997-07-15 Activated Cell Therapy, Inc. Methods for enriching breast tumor cells
US5663051A (en) * 1994-08-31 1997-09-02 Activated Cell Therapy, Inc. Separation apparatus and method
US5840502A (en) * 1994-08-31 1998-11-24 Activated Cell Therapy, Inc. Methods for enriching specific cell-types by density gradient centrifugation
WO2001079851A1 (fr) * 2000-04-13 2001-10-25 Imperial College Innovations Limited Diagnostique prenatal non invasif utilisant des cellules foetales cd45-negatives
WO2018010632A1 (fr) * 2016-07-11 2018-01-18 山东亚大药业有限公司 Procédé de séparation et de purification de globules rouges nucléés fœtaux, et kit de réactifs

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EP0610359A1 (fr) 1994-08-17
JPH07500499A (ja) 1995-01-19

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