WO2019153032A1 - Isolement de cellules prénatales - Google Patents

Isolement de cellules prénatales Download PDF

Info

Publication number
WO2019153032A1
WO2019153032A1 PCT/AU2019/000013 AU2019000013W WO2019153032A1 WO 2019153032 A1 WO2019153032 A1 WO 2019153032A1 AU 2019000013 W AU2019000013 W AU 2019000013W WO 2019153032 A1 WO2019153032 A1 WO 2019153032A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
fetal
trophoblastic
cell population
cell
Prior art date
Application number
PCT/AU2019/000013
Other languages
English (en)
Inventor
Benjamin Jacques THIERRY
Marnie Alyce WINTER
Meysam Rezaei SHAHMIRZADI
Majid Ebrahimi Warkiani
Original Assignee
University Of South Australia
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AU2018900373A external-priority patent/AU2018900373A0/en
Application filed by University Of South Australia filed Critical University Of South Australia
Publication of WO2019153032A1 publication Critical patent/WO2019153032A1/fr

Links

Classifications

    • 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
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • 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
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
    • B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0652—Sorting or classification of particles or molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B81—MICROSTRUCTURAL TECHNOLOGY
    • B81B—MICROSTRUCTURAL DEVICES OR SYSTEMS, e.g. MICROMECHANICAL DEVICES
    • B81B1/00—Devices without movable or flexible elements, e.g. microcapillary devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B81—MICROSTRUCTURAL TECHNOLOGY
    • B81B—MICROSTRUCTURAL DEVICES OR SYSTEMS, e.g. MICROMECHANICAL DEVICES
    • B81B1/00—Devices without movable or flexible elements, e.g. microcapillary devices
    • B81B1/002—Holes characterised by their shape, in either longitudinal or sectional plane
    • B81B1/004—Through-holes, i.e. extending from one face to the other face of the wafer
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B81—MICROSTRUCTURAL TECHNOLOGY
    • B81C—PROCESSES OR APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OR TREATMENT OF MICROSTRUCTURAL DEVICES OR SYSTEMS
    • B81C1/00—Manufacture or treatment of devices or systems in or on a substrate
    • B81C1/00015—Manufacture or treatment of devices or systems in or on a substrate for manufacturing microsystems
    • B81C1/00023—Manufacture or treatment of devices or systems in or on a substrate for manufacturing microsystems without movable or flexible elements
    • B81C1/00055—Grooves
    • B81C1/00071—Channels
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B81—MICROSTRUCTURAL TECHNOLOGY
    • B81C—PROCESSES OR APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OR TREATMENT OF MICROSTRUCTURAL DEVICES OR SYSTEMS
    • B81C1/00—Manufacture or treatment of devices or systems in or on a substrate
    • B81C1/00015—Manufacture or treatment of devices or systems in or on a substrate for manufacturing microsystems
    • B81C1/00023—Manufacture or treatment of devices or systems in or on a substrate for manufacturing microsystems without movable or flexible elements
    • B81C1/00119—Arrangement of basic structures like cavities or channels, e.g. suitable for microfluidic systems
    • 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
    • C12N2503/00—Use of cells in diagnostics
    • 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
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70539—MHC-molecules, e.g. HLA-molecules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G01N2800/368—Pregnancy complicated by disease or abnormalities of pregnancy, e.g. preeclampsia, preterm labour
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/38—Pediatrics
    • G01N2800/385—Congenital anomalies

Definitions

  • the present disclosure relates to methods of isolating cells.
  • the present disclosure relates to methods of isolating fetal cells.
  • NIPT non-invasive prenatal testing
  • the current false-positive rate in NIPT for common aneuploidies is approximately 0.1 %-0.2%, even for low risk pregnancies.
  • the American College of Obstetrics and Gynaecology and the International Society of Prenatal Diagnosis advocate the implementation of cffDNA screening in high-risk pregnancies.
  • cffDNA-based NIPT remains mainly a screening technology and invasive testing is still required for a conclusive diagnosis to guide clinical decision-making as current NIPT technologies do not have the capability to provide information about the full range of chromosomal abnormalities and single gene disorders.
  • cffDNA NIPT detects only a small subset of the chromosomal abnormalities that can be diagnosed using standard invasive procedures.
  • the potential for whole genome fetal sequencing using cell-free fetal DNA is limited due to the high proportion of maternal cell- free DNA in maternal plasma (>90%) and the inability of current technologies to determine the origin of cell free DNA fragments using bioinformatic techniques.
  • cffDNA mainly consists of fragmented short DNA which limits the depth of interrogation of the genome.
  • a method of isolating fetal trophoblastic cells from a suitable biological sample comprising the following steps:
  • a method of isolating fetal trophoblastic cells from a suitable biological sample comprising the following steps:
  • a method of isolating fetal trophoblastic cells from a suitable biological sample comprising the following steps:
  • the step of enriching the fetal trophoblastic cell population is performed two, three or more times to further enrich the fetal trophoblast cell population.
  • the suitable biological sample is a maternal biological fluid sample.
  • the suitable biological sample is selected from maternal blood, maternal cervical mucus, maternal endocervical canal, maternal endometrial cavity, from lavage of the maternal endocervical canal or the uterine cavity, and endometrial biopsy.
  • each microwell within the array of microwells collects an average of one or fewer cells. In another embodiment, following step (c), each microwell within the array of microwells collects an average of one or more cells. Where one or more cells are collected in a microwell, serial dilution may be performed.
  • the array of microwells further comprises a microfilter having a porosity that retains fetal trophoblast cells and allows other cell types to pass through.
  • the array of microwells further comprises a micro filter having a porosity that retains fetal trophoblast cells and other cell types to allow removal of excess liquid to concentrate the fetal trophoblast cells and other cell types.
  • the micro filter is coated with an agent to reduce background autofluorescence and improve fluorescent imaging of fetal trophoblast cells.
  • the micro filter is functionalised with a binding partner that specifically binds to cellular receptors of trophoblastic cells.
  • the method further comprises the following step:
  • the isolated fetal trophoblastic cells are used for cytogenetic, genomic, proteomic, transcriptomic and/or metabolomic assays.
  • the isolated fetal trophoblastic cells may be assessed for genetic or chromosomal abnormalities and/or the isolated fetal trophoblastic cells may be used for proteomic, transcriptomic or metabolomic analysis for the detection of pregnancy complications, such as placental dysfunctions.
  • a method of prenatally diagnosing a genetic or chromosomal abnormality or a placental dysfunction in a fetus comprising isolating fetal trophoblast cells from a biological sample according to the method of the present disclosure to obtain isolated fetal trophoblast cells and assessing material from the isolated fetal trophoblast cells.
  • a method for cytogenic analysis of fetal trophoblast cells comprising isolating fetal trophoblast cells from a biological sample according to the method of the present disclosure to obtain isolated fetal trophoblast cells and assessing genetic material from the isolated fetal trophoblast cells.
  • a method for genomic analysis of fetal trophoblast cells comprising isolating fetal trophoblast cells from a biological sample according to the method of the present disclosure to obtain isolated fetal trophoblast cells and assessing genetic material from the isolated fetal trophoblast cells.
  • a method for proteomic analysis of fetal trophoblast cells comprising isolating fetal trophoblast cells from a biological sample according to the method of the present disclosure to obtain isolated fetal trophoblast cells and assessing material from the isolated fetal trophoblast cells.
  • a method for transcriptomic analysis of fetal trophoblast cells comprising isolating fetal trophoblast cells from a biological sample according to the method of the present disclosure to obtain isolated fetal trophoblast cells and assessing genetic material from the isolated fetal trophoblast cells.
  • a method for metabolomic analysis of fetal trophoblast cells comprising isolating fetal trophoblast cells from a biological sample according to the method of the present disclosure to obtain isolated fetal trophoblast cells and assessing material from the isolated fetal trophoblast cells.
  • Figure 1 is a schematic diagram showing an embodiment of the method of the present disclosure
  • Figure 2 is an example of a typical scatter plot of calcien intensity against bright field (BF) cellular area from (A) a pre-sample and (B) a sample obtained from the trophoblast outlet of an inertial microfluidic device.
  • C Image of JEG3 stained cells, and (D) unstained white blood cells. Images taken at 400 c magnification and have been adjusted to enhance visual appearance;
  • Figure 3 shows micrographs of HLA-G and Hoechst positive, CD45 negative fetal trophoblastic cell (green box) from a pregnant maternal blood sample after separation with an inertial microfluidic device.
  • A Hoechst
  • B HLA-G
  • C CD45
  • D brightfield
  • FIG. 4 shows photos of gold coated microwell microfilters with (A) 150mih wells and (B) 500mih wells taken before functionalization with HLA-G antibody to specifically bind trophoblastic cells.
  • SU8 filters were coated with a 25-1 OOnm gold layer then functionalised with 1 1 -Mercaptoundecanoic acid (MUA) or polyethylene glycol (PEG) before attaching the HLA-G antibody;
  • UAA 1 -Mercaptoundecanoic acid
  • PEG polyethylene glycol
  • Figure 5 shows micrographs of single stained JEG-3 (primary HLA-G antibody with secondary Anti -mouse IgG FITC antibody and Hoechst) in 500 mih well (A) and stained JEG-3
  • FIG. 6 shows micrographs of JEG3 cells within a microwell (in a thin layer of media with mineral oil on top to facilitate for pick up) before (A) and after pick up (B) with a 30mih micropipette; (C) shows that 1 cell was picked up and this was deposited into a PCR tube for downstream analysis (lOOxmagnification); (D) cells deposited onto a glass slide (400* magnification). The positive deposition of a single cell visualised with fluorescence microscopy;
  • Figure 7 shows a cytokeratin positive, DAPI positive cytotrophoblast cell isolated from a pregnant woman’s blood sample before (A) and after pick up (B). The sample was stained for
  • Cytokeratin, DAPI and CD45 (APC). Image taken at 400 magnification.
  • Figure 8 is micrograph of a microfilter with 500mih wells (brightfield) and an 80pm single cell manipulation micropipette (red circle) able to pick up cells within the well, and dispense/remove lysis buffer/lysed solution respectively;
  • F igure 9 shows micrographs of a single JEG3 cell (red circle) lysis within the microwell, before (A) and after lysis (B) within a 150 pm microwell; (C) Visual representation demonstrating single well buffer manipulation (in this case red dye), in order to specifically target and lyse 1 microwell;
  • Figure 10 shows a single stained cell (Hoechst nuclear stain and calcein) before (A) and after (B) lysis in a 500pm honeycomb micro well.
  • An example of a droplet digital PCR profile for KRT19 (Keratin 19), in this instance multiple cells were lysed on filter, the mRNA was recovered, converted to cDNA before undergoing PCR (QX200TM droplet digital PCR system BioRad);
  • Figure 11 is a micrograph of multinucleated syncytial nuclear aggregate (SNA) stained with May-Grunwald Giemsa (400x magnification) (A), and SNAs identified as being keratin positive (pan- KRT) and CD45 negative by imaging flow cytometry (400x magnification) (B);
  • SNA single-nucleated syncytial nuclear aggregate
  • Figure 12 shows micrographs of a CD45 negative, Hoechst Positive syncytial nuclear aggregate from a pregnant maternal blood sample (where the pregnancy was complicated with pre eclampsia) after separation with inertial microfluidic device.
  • A Brightfield before picking
  • B Hoechst before picking
  • C CD45 FITC before picking
  • D Hoechst after picking demonstrating efficient pick up of the designated cell ( 100 magnification);
  • Figure 13 shows an example of a computed colour image of a cytotrophoblast with fluorescence in situ hybridisation probing for chromosomes 13 (green) and 21 (red); and (B, D) Raw red imaging of chromosome 21 signals in the same cell as A and C, respectively;
  • Figure 14 shows examples of fluorescence in situ hybridisation of circulating fetal cells from 3 pregnancies with male fetuses enriched with inertial microfluidics (DAPI nuclear stain , Y chromosome: + (DYZ3), X chromosome: * (DXZl)). Images taken at 400 magnification;
  • Figure 15 shows an example of an isolated circulating fetal cell which after enrichment was stained with specific markers and DAPI nuclear staining (A) and placed in a microwell. This cell was then recovered and placed onto a slide for fluorescence in situ hybridisation for X (*) and Y (+) chromosomes confirming that this fetal cell was from a male fetus (B); and
  • Figure 16 shows an example of a water (represented by #) in oil (represented by *) droplet containing a single cell (circle), Brightfield image (A) and nuclear DAPI image (B) of the same droplet attached to a glass surface.
  • the present disclosure relates to a method for the isolation of fetal trophoblastic cells from biological samples such as fluids, including maternal blood and cervical mucus, using a combination of microfluidic technologies.
  • the method provides isolated fetal cells that can potentially be used for non- invasive prenatal diagnosis using cytogenetic, genomic, proteomic or transcriptomic methods.
  • Fetal cells including lymphoid and erythroid cells, myeloid precursors, and trophoblastic (epithelial) cells are found in maternal circulation in very low numbers (estimated to be ⁇ 1-2 trophoblasts per mL of blood; Mouawia el al, 2012).
  • Extravillous trophoblast cells isolated using ISET Isolation by Size of Epithelial Tumor Cells" described in Patent US7651838) were consistently larger than 15 mih. Trophoblasts could be detected as early as 5 weeks gestation and notably were also found not to persist post-termination/birth.
  • Syncytial nuclear aggregates (SNAs) have also been consistently observed in maternal blood.
  • SNAs are large fragments of the outer layer of the placenta, the syncytiotrophoblast, which is formed by the fusion of progenitor cytotrophoblasts into a continuous cell layer. Elevated numbers of placental extravillous trophoblastic cells and SNAs are observed in pregnancy complications including preeclampsia, reduced fetal movements, intra-uterine growth restriction and stillbirth (Correa et al, 2008). Trophoblastic cells can also be found in the uterine cavity and a recent study demonstrated the feasibility of using syncytiotrophoblasts isolated from the cervical mucus for prenatal diagnostic
  • the present disclosure relates to an isolation method for fetal trophoblasts from suitable biological samples using a series of efficient separation techniques.
  • fetal trophoblasts are larger than many other cell types, including white blood cells.
  • an extraordinarily efficient isolation procedure is required to isolate fetal trophoblast cells from suitable biological samples containing these cells with the level of purity required for most diagnostic assays.
  • the present disclosure relates to sequential application of microfluidic technologies aimed at isolating fetal trophoblastic cells. The identity of these cells can then optionally be confirmed (eg using trophoblast cell markers), and potentially assessed for diagnostic purposes.
  • SNAs may alternatively or additionally be used in the method disclosed herein in place of fetal trophoblastic cells.
  • the present disclosure provides a method of isolating fetal trophoblastic cells from a suitable biological sample comprising the following steps:
  • the present disclosure provides a method of isolating fetal trophoblastic cells from a suitable biological sample comprising the following steps:
  • the present disclosure provides a method of isolating fetal trophoblastic cells from a suitable biological sample comprising the following steps: (a) providing a suitable biological sample comprising fetal trophoblastic cell population within a population of other cell types;
  • the suitable biological sample comprises a fetal trophoblastic cell population within a population of other cell types.
  • the suitable biological sample is a maternal biological sample, that is, the sample is derived from a pregnant female, in order to isolate fetal trophoblasts derived from the fetus of the current pregnancy.
  • the suitable biological sample is a maternal biological fluid.
  • the suitable biological sample is maternal blood, cervical mucus or lavage fluid of the endometrial cavity obtained during the pregnancy; however, it is envisioned that other types of biological sample may be suitable for use in the method of the present disclosure.
  • the biological sample is derived from cotton swabs or a cytobrush of the endocervical canal and the endometrial cavity, from lavage of the endocervical canal or the uterine cavity, from aspiration of cervical mucus, or from endometrial biopsy using, for example, a Pipelle.
  • the cells derived from the sample may be resuspended in a suitable fluid such as sterile buffers or media well known to those skilled in the art.
  • the biological sample is maternal blood.
  • the biological sample is derived from a human female; however it is anticipated that the method of the present disclosure could easily be adapted for use in pregnant non-human animals of interest or commercial value.
  • the sampling is performed in the first trimester of the pregnancy. However, it is envisioned that the sampling could alternatively occur during the second or third trimester of the pregnancy. In certain embodiments, the sampling occurs before the 16 th week of the pregnancy. In certain embodiments, the sampling occurs before the 14 lh week of the pregnancy. In certain embodiments, the sampling occurs before the l2 fll week of the pregnancy. In certain embodiments, the sampling occurs before the 10 th week of the pregnancy. In certain embodiments, the sampling occurs before the 8th week of the pregnancy. In certain embodiments, the sampling occurs before the 6 lh week of the pregnancy. The week of the pregnancy can be determined using conventional techniques which are well known in obstetrics.
  • the biological sample is treated to fix the cells in the sample prior to application of the sample to the inertial microfluidic device.
  • fixation of cells in the biological sample can be carried out using any of the reagents and methods known for that purpose in the art, such as by treatment with a suitable fixative.
  • suitable chemical fixatives can be selected from one or more of the group consisting of formaldehyde (eg. formalin), glutaraldehyde, methanol and ethanol.
  • the method comprises an initial sample processing step.
  • the initial sample processing step eg an enrichment step
  • the enrichment is through density gradient methods.
  • the enrichment step is a density gradient method that uses dextran using a method described by Lin el al (2017), the disclosure of which is hereby incorporated in its entirety.
  • the initial processing step isolates the nucleated fraction of the sample using standard red blood cell lysis methods.
  • the inertial microfluidic device is a spiral device.
  • a spiral device mediates cell separation based on size and deformability of the cells, relative to other cells and components in the sample, typically with larger cells along the inner wall and the smaller cells away from the inner wall.
  • Suitable inertial microfluidic devices include Clearbridge BioMedics CTChip®FR (Clearbridge BioMedics Pte Ltd, Singapore), and devices described in PCT/US201 1/027276 (WO 2011/109762), PCT/SG2013/000412 (WO 2014/046621) or PCT/US2013/024403 (WO2013116696), the disclosures of which are hereby incorporated in their entirety.
  • the inertial microfluidic device is a vortex device.
  • Suitable vortex devices include devices described in US9133499 B2, the disclosure of which is hereby incorporated in their entirety. However, it is anticipated that other devices may also be suitable for use in the method of the present disclosure to enrich the population of fetal trophoblastic cells.
  • the inertial microfluidic device is modified to optimise separation of fetal trophoblastic cells from other cell types in the biological sample.
  • modifications may include optimising the pressure within the inertial microfluidic device (eg using a pressure compensating device), altering the length of the channels used in the device, altering the shape and/or dimensions of the channels within the device, altering the geometry of cross section (eg trapezoidal), altering the outlet position (which alters resistance), altering the shape of the device, altering the angle of the channels (eg the curvature of the spiral), altering the viscosity of the medium, altering the length and height of tubing (eg relative to a syringe pump) etc, as would be well understood by a person skilled in the art.
  • the biological sample is run through the inertial microfluidic device at a rate of at least 100 microlitres per minute (pL/min), for example, at a rate between approximately 100 pL/min and 5000 pL/min, or 1000 pL/min and 3000 pL/min, such as 1300 pL/min, 1400 pL/min, 1500 pL/min, 1600 pL/min, 1700 pL/min, 1800 pL/min, 1900 pL/min, 2000 pL/min, 2100 pL/min, 2200 pL/min, 2300 pL/min, 2400 pL/min or 2500 pL/min.
  • pL/min microlitres per minute
  • the inertial microfluidic device comprises 1, 2, 3 or more inlets. In certain embodiments, the inertial microfluidic device comprises 1, 2, 3 or more outlets.
  • the channel cross section has a width of between 50 pm and 1000 pm, for example, between 300 pm and 800 pm, such as between 400 pm and 700 pm or 500 pm and 600 pm. In certain embodiments, the channel cross section has a height of between 20 pm and 500 pm, for example, between 30 pm and 300 pm, such as between 50 pm and 200 pm or 80 pm and 160 pm.
  • the channel may have any suitable cross section, for example, circular, elliptical, rectangular, equilateral triangular, non-equilateral triangular, irregular quadrilateral, trapezoidal or teardrop with vertex cut off.
  • the channel may have different inner and outer heights which may increase the efficiency of cell migration and focusing.
  • An inner height to outer height ratio may be between about 19:20 to about 1 :4, for example, between about 10: 11 to about 1 :3 or between about 9: 12 to about 1 :2.
  • an inner height may be, for example, between 70 p and 90 pm and an outer height may be between 120 pm and 140 pm.
  • a path length of the inertial microfluidic device is between 50 mm and 200 mm, for example, between 60 mm and 180 mm, such as between 70 mm and 150 mm or 80 mm and 120 mm.
  • the channel dimensions may be selected such that only the larger cells undergo inertial focusing, while migration of the smaller blood cells is solely affected by the Dean drag.
  • the inertial microfluidic device comprises a spiral of between 5 and 12 loops, for example, between 6 and 10 loops, such as between 7 and 9 loops.
  • a radius of the spiral increases.
  • the radius may increase by any amount between about 1mm to about 100 mm, for example, from about 2 mm to about 60 mm, or from about 3 mm to about 50 mm, or from about 4 mm to about 40 mm, such as about 5 mm to about 30 mm or about 8 mm to about 24 mm.
  • a radius of the spiral decreases.
  • the radius may decrease by any of the above described amounts by which the radius increases.
  • the device can be fabricated using standard photolithographic and etching procedures including soft lithography techniques (e.g. see Shi I, et al, Applied Physics Letters 91, 153114 (2007); Chen Q., et al., Journal of Microelectromechanical Systems, 16, 1 193 (2007); or Duffy et al., Rapid Prototyping of Microfluidic Systems in Poly(dimethylsiloxane), Anal.
  • soft lithography techniques e.g. see Shi I, et al, Applied Physics Letters 91, 153114 (2007); Chen Q., et al., Journal of Microelectromechanical Systems, 16, 1 193 (2007); or Duffy et al., Rapid Prototyping of Microfluidic Systems in Poly(dimethylsiloxane), Anal.
  • Suitable materials may include, but are not limited to, polydimethylsiloxane (PDMS), thiol-ene polymers, UV- curable optical adhesives such as NOA81 or UV-curable epoxy resin based photoresists such as SU-8.
  • PDMS polydimethylsiloxane
  • thiol-ene polymers polydimethylsiloxane
  • UV- curable optical adhesives such as NOA81 or UV-curable epoxy resin based photoresists such as SU-8.
  • the inertial microfluidic device comprises 2 inlets and 2 outlets having a channel that has a width of about 500 mih and a height of about 160 mih with a total path length of about 100 mm, and where the device is fabricated with photolithography and comprises of PDMS bonded to a glass slide.
  • the inertial microfluidic device comprises an 8- loop spiral microchannel with one inlet and two outlets with the radius increasing from about 8 mm to about 24 mm, where the width of the channel cross-section is about 600 pm, and the inner/outer heights are about 80 pm and about 130 mih, respectively, for the trapezoid cross-section, and where the device is fabricated through micromilling and soft lithography and comprises PDMS bonded to PDMS.
  • the step of enriching the population of fetal trophoblastic cells utilises an inertial microfluidic device that debulks the biological sample.
  • the enriching step removes > 80% of the other cell types from the biological sample.
  • the enriching step removes > 85% of the other cell types from the biological sample.
  • the enriching step removes > 90% of the other cell types from the biological sample.
  • the enriching step removes > 95% of the other cell types from the biological sample.
  • the enriching step removes > 96% of the other cell types from the biological sample.
  • the enriching step removes > 97% of the other cell types from the biological sample. In certain embodiments, the enriching step removes > 98% of the other cell types from the biological sample. In certain embodiments, the enriching step removes > 99% of the other cell types from the biological sample. In certain embodiments, the other cell types removed from the biological sample include cells selected from red blood cells, platelets, white blood cells, other maternal cells, cellular debris etc. In certain embodiments, the enriching step is performed two, three or more times to further enrich the fetal trophoblast cell population.
  • the inertial microfluidic device is coupled with a pressure compensator device attuned to the fluidics required to bring the enriched fetal trophoblastic cell sample to the following device used in the method, that is, either the DLD device in embodiments where a secondary (purifying) step is performed, or to the microwell array device, as described herein.
  • a pressure compensator device attuned to the fluidics required to bring the enriched fetal trophoblastic cell sample to the following device used in the method, that is, either the DLD device in embodiments where a secondary (purifying) step is performed, or to the microwell array device, as described herein.
  • Examples of the pressure compensator device would be known to those skilled in the art.
  • the enriched fetal trophoblast cell population obtained from inertial microfluidic device is treated to fix the cells in the sample.
  • fixation of cells in the biological sample can be carried out using any of the reagents and methods known for that purpose in the art, such as by treatment with a suitable fixative.
  • suitable chemical fixatives can be selected from one or more of the group consisting of formaldehyde (eg. formalin), glutaraldehyde, methanol and ethanol.
  • the method of the present disclosure optionally comprises a second step that is a purification step.
  • a purification step may be mediated by a deterministic lateral displacement (DLD) device; although it is anticipated that other devices may suitably purify the fetal trophoblastic cells.
  • DLD deterministic lateral displacement
  • examples of suitable deterministic lateral displacement (DLD) device may be selected or adapted from those described in Huang el al, 2004; Davis el al, 2006; PCT/US2010/032150 (W02010124155); the disclosures of which are hereby incorporated in their entirety.
  • the purifying step following the purifying step, more than 90% of the other cell types are removed from the biological sample. In certain embodiments, following the purifying step, more than 95% of the other cell types are removed from the biological sample. In certain embodiments, following the purifying step, more than 98% of the other cell types are removed from the biological sample. In certain embodiments, following the purifying step, more than 99% of the other cell types are removed from the biological sample. In certain embodiments, following the purifying step, more than 99.9% of the other cell types are removed from the biological sample. In certain embodiments, following the purifying step, more than 99.99% of the other cell types are removed from the biological sample.
  • isolated fetal trophoblastic cells are collected following the enriching step and optionally following the secondary purifying step by applying a cell population selected from the enriched fetal trophoblast cell population or the purified fetal trophoblast cell population to a microwell array to collect isolated fetal trophoblast cells.
  • suitable microwell arrays have microwells with a diameter range between about 10 to 1000 mih or about 50 to 1000 pm, although other diameters may be suitable, eg in the range of 100, 200, 300, 400, 500, 600, 700, 800, 900, 1200 1500, 2000 or 5000 pm.
  • the microwell arrays may have any suitable well design, for example, square or honeycomb. Suitable microarrays would be known to persons skilled in the art.
  • the microwell array may be in the form of water droplets in oil wherein cells are deposited into a water droplet within the oil.
  • Water droplets in oil can be formed using a micromanipulator or by manual pipetting.
  • the water droplets behave in the same way as microwells allowing containment of the cell or lysis/amplification within the droplet.
  • use of the term“microwell array” or similar terms herein is intended to include classical microwell arrays and functional equivalents of them, such as water droplets in oil. Also, the above approach using droplets can be used on the filter.
  • each microwell collects an average of one or fewer cells. In practice, this means that each microwell contains one or zero cells following the method of the present disclosure. In certain embodiments, the collection of one or zero cells per well allows for single cell study. In alternative embodiments, each microwell collects on average more than one cell. Contents of the microwell may then be serially diluted until there is one cell, enabling single cell analysis. For example, by picking up one or more cells and depositing them in another well leading to a serial dilution.
  • the array of microwells further comprises a microfilter having a porosity that retains fetal trophoblast cells and allows other smaller cell types to pass through.
  • the microfilter may have a porosity in the range of 3 to 40 pin.
  • the porosity of the microfilter may range between approximately 1, 3, 5, 7, 8, 10, 12, 15, 16, 20, 25, 30, 35, 40, 45 or 50 pm.
  • the pores in the microfilter are round or rod shaped.
  • the biological sample is run through the array of microwells at a rate of at least 0.1 mL/min, for example, in a range between 0.5 mL/min and 5 mL/min, or 0.8 mL/min and 4 mL/min or in a range between approximately 0.2 mL/min, 0.6 mL/min, 1 mL/min, 1.5 mL/min, 2 mL/min, 2.5 mL/min, 3 mL/min, 3.5 mL/min, 4 mL/min, 5 mL/min or 10 mL/min.
  • the microfilter filters out unwanted smaller cells and retains fetal trophoblastic cells and cellular aggregates of characteristic dimensions larger than that of the filter.
  • the microfilter is a microfluidic filter.
  • the microfilter is a microfabricated filter membrane ln certain embodiments, the microfilter is located at the bottom of the microarray wells.
  • the array of microwells further comprises a microfilter having a porosity that retains fetal trophoblast cells and other cell types to allow removal of excess liquid to concentrate the fetal trophoblast cells and other cell types.
  • the microfilter may have a porosity in the range of 1 to 20 pm.
  • the porosity of the microfilter may range between approximately 1 , 3, 5, 7, 8, 10, 12, 14, 16, 18 or 20 pm. Accordingly, a liquid volume is reduced and the cells are concentrated without cellular loss that can occur with centrifugation.
  • the microarray wells do not contain a microfilter, and instead are adapted for the collection of cells (eg using sedimentation).
  • the microfilter is functionalised with a binding partner that specifically binds to a cellular receptor or specific antigen of trophoblastic cells.
  • the cellular receptor or specific antigen may be selected from HLA-G and CD105, although other cellular receptors may be suitable as would be well understood by a person skilled in the art.
  • the binding partner may be a specific antibody or a specific ligand for the cellular receptor or antigen.
  • the functionalised microfilter may assist with the specific retention or binding of the fetal trophoblast with the microfilter.
  • the microfilter Prior to functionalisation, may be coated with an agent to reduce background autofluorescence and improve fluorescent imaging of trophoblastic cells.
  • the agent comprises at least one of gold, silver, aluminium, platinum or the like.
  • the coating of the agent has a thickness ranging between approximately 10 nm and 300 nm, for example, 20 nm, 25 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 150 nm, 170 nm or 190 nm
  • the microfilter following filtration with the microfilter, more than 98% of the other cell types are removed from the biological sample. In certain embodiments, following filtration with the microfilter, more than 99% of the other cell types are removed from the biological sample. In certain embodiments, following filtration with the microfilter, more than 99.9% of the other cell types are removed from the biological sample. In certain embodiments, following filtration with the microfilter, more than 99.99% of the other cell types are removed from the biological sample. In certain
  • each of the devices of the method disclosed herein is in microfluidic connection with one or more other devices of the method. In certain embodiments, each of the devices of the method disclosed herein is in microfluidic connection with the other devices of the method. In certain embodiments, the method utilises a combination of microfluidic devices as described herein.
  • the method further comprises the following step:
  • the staining may be any type of staining known to persons skilled in the art, providing it identifies fetal trophoblast cells.
  • the staining method specifically identifies fetal trophoblasts, for example, the staining may utilise a specific binding partner of a specific cellular receptor or antigen that are understood to be useful in identifying fetal trophoblast cells, such as HLA-G, cytokeratin-7 and/or CD105.
  • the isolated fetal trophoblastic cells are assessed for genetic or chromosomal abnormalities or placental dysfunctions using standard techniques well understood by persons skilled in the art.
  • the present disclosure provides a method of prenatally diagnosing a genetic or chromosomal abnormality or a placental dysfunction in a fetus comprising isolating fetal trophoblast cells from a biological sample according to the method of the present disclosure to obtain isolated fetal trophoblast cells and assessing material from the isolated fetal trophoblast cells.
  • diagnosis can be performed using cytogenetic, genomic, proteomic, transcriptomic or metabolomics methods and performed using single cells or through pooled analyses of the enriched fraction.
  • references herein to‘material’ may be to, for example, genetic material, proteins or metabolites, depending on the method of analysis or diagnosis.
  • the fetal trophoblastic cells are characterised using cytogenetic technology including FISH to identify
  • chromosomal abnormalities Other suitable diagnostic techniques could also be applied to the cells isolated by the disclosed method as would be understood by the person skilled in the art.
  • fetal cell isolation techniques not only have the potential for comprehensive fetal genome analysis, but may allow also simultaneous transcriptome analysis which may enable early biomarker development for pregnancy complications, including preeclampsia, intrauterine growth restriction and stillbirth.
  • efficient enumeration of circulating fetal cells may also allow risk stratification for these placental complications.
  • the placental transcriptome in pregnancies complicated by preeclampsia differs from healthy pregnancies, which may provide insight into the pathophysiology of disease, allowing development of diagnostic biomarkers and possible therapeutic targets.
  • Trophoblasts and SNA from the placenta circulating in the maternal blood therefore provide a unique opportunity to non-invasively profile placental function in normal and complicated pregnancies. Further, the number of trophoblastic cells increases with pre-eclampsia and so just the number identified provides a prognosis for the development of pre-eclampsia.
  • a method for genomic analysis of fetal trophoblast cells comprising isolating fetal trophoblast cells from a biological sample according to the method of the present disclosure to obtain isolated fetal trophoblast cells and assessing genetic material from the isolated fetal trophoblast cells.
  • Genomic analysis technologies may include DNA sequencing or array-type assays as would be well understood by a person skilled in the art.
  • a method for proteomic analysis of fetal trophoblast cells comprising isolating fetal trophoblast cells from a biological sample according to the method of the present disclosure to obtain isolated fetal trophoblast cells and assessing material from the isolated fetal trophoblast cells.
  • Proteomic analysis technologies may include affinity arrays or microfluidic separation, such as microchip capillary electrophoresis (or other chromatography- or electrophoresis-based separation) and detection via chemiluminescence or mass spectrometry as would be well understood by a person skilled in the art.
  • a method for transcriptomic analysis of fetal trophoblast cells comprising isolating fetal trophoblast cells from a biological sample according to the method of the present disclosure to obtain isolated fetal trophoblast cells and assessing genetic material from the isolated fetal trophoblast cells.
  • Transcriptomic analysis technologies may include RNA sequencing or array-type assays as would be well understood by a person skilled in the art.
  • a method for metabolomic analysis of fetal trophoblast cells comprising isolating fetal trophoblast cells from a biological sample according to the method of the present disclosure to obtain isolated fetal trophoblast cells and assessing material from the isolated fetal trophoblast cells.
  • Metabolomic analysis technologies may include direct injection mass spectrometry, liquid chromatography coupled to mass spectrometry, gas chromatography-mass spectrometry (GC-MS), capillary electrophoresis-mass spectrometry (CE-MS), nuclear magnetic resonance spectroscopy, Fourier transform-infrared (FT-IR) spectroscopy and Raman spectroscopy as would be well understood by a person skilled in the art. Also, the person skilled in the art would be understood that mass spectral imaging may use different ionisation methods, for example, matrix assisted laser desorption ionisation (MALD1), desorption electrospray ionisation (DESI) or secondary ion mass spectrometry (SIMS).
  • MALD1 matrix assisted laser desorption ionisation
  • DESI desorption electrospray ionisation
  • SIMS secondary ion mass spectrometry
  • the fetal trophoblastic cells within the microwells are lysed using either a micromanipulator or manual pipetting.
  • a lysis buffer is delivered to individual microwell to lyse the cells of interest. The cell lysate can then be aspirated for genetic analysis.
  • the RNA or DNA from the lysed trophoblastic cells is amplified m situ in the microwells (eg in microwells, in water droplets in oil or on the filter).
  • amplification of the DNA or RNA can occur following removal from the microwells.
  • lysed trophoblastic cells are characterized using genomic
  • transcriptomic, proteomic and/or metabolomic technologies as described herein and as would be well understood by a person skilled in the art.
  • the present disclosure provides an apparatus comprising a combination of microfluidic devices as described herein.
  • the apparatus mediates the isolation of fetal trophoblastic cells and/or SNAs from a suitable biological sample.
  • EXAMPLE 1 DESIGN AND FABRICATION OF INERTIAL MICROFLUIDIC
  • a first inertial microfluidic device (‘device G) comprised 2 inlets and 2 outlets having a channel that had a width of 500 pm and a height of 160 pm with a total path length of about 100 m .
  • the device was fabricated with photolithography and comprised PDMS bonded to a glass slide or PDMS bonded to PDMS.
  • a second inertial microfluidic device (‘device 2’) comprised an 8-loop spiral microchannel with one inlet and two outlets with the radius increasing from 8 mm to 24 mm.
  • the width of the channel cross-section was 600 pm, and the inner/outer heights were 80 and 130 pm, respectively, for the trapezoid cross-section.
  • the device was fabricated through micromilling and soft lithography and comprised PDMS bonded to PDMS. Both devices are effective for enrichment of trophoblastic cells.
  • Calcein stained JEG3 cells (a placental cell line) spiked into whole blood were enriched using an inertial microfluidic device as described herein (device 2) (See, also,‘Enrichment step G in Figure 1) and recovery was enumerated with imaging flow cytometry ( Figure 2).
  • JEG3 cancer cells were spiked into 8-9 mL of whole blood (ie‘pre-sample’) with ⁇ 1000 JEG3 cells per mL of blood and the red blood cells were lysed leaving white blood cells (WBC) and the JEG3 cells intact (ie nucleated cells). Nucleated cells were resuspended in PBS at 3x initial blood volume, but this can vary according to the number of cells recovered.
  • the nucleated cell fraction was pushed through the inertial microfluidic device with a syringe pump at 1700 pL/min, (but this can change with device optimisation) and sample was collected from the trophoblast outlet (ie large cell outlet of the inertial microfluidic device).
  • the number of WBC was reduced to less than 1000 contaminating WBC per mL of blood. That is, approximately 90% depletion from the previously separated sample.
  • Approximately 80% of the JEG3 cells (-6000 cells, from 8000 JEG3 cells spiked into 8 mL of fresh blood) were recovered after a single pass through the inertial microfluidic device. Approximately 54% of JEG3 cells were recovered following two passes through the inertial microfluidic device.
  • Figure 2 shows an example of a typical scatter plot of calcien intensity against brightfield (BF) cellular area from (A) a pre-sample and (B) a sample obtained from the trophoblast outlet of the inertial microfluidic device.
  • C is an image of JEG3 stained cells
  • D is an image of unstained white blood cells. Images taken at 400x magnification and have been adjusted to enhance visual appearance.
  • Fetal trophoblastic cells from a pregnant woman’s blood sample were enriched using an inertial microfluidic device as described herein (device 2).
  • Maternal blood was collected in two lOmL EDTA tubes with 16mL of blood used in the procedure. Maternal blood was kept on ice or at 4°C and processed within 3 hours after collection. Blood was taken off ice and left to come to room temperature for -15 minutes before processing. l6mL of blood was added to 64mL of RBC lysis buffer (gBioscience) and incubated at room temperature on a shaker for 15 minutes. Sample was then centrifuged with soft deceleration at 500g for 10 minutes. Supernatant was removed and discarded.
  • the white blood cell pellet was first resuspended in lmL of PBS (tips pre-coated in Pluronic to reduce binding), an additional 47mL of PBS was then added. Sample was then loaded into a syringe, placed in a syringe pump and connected to the inertial microfluidic device. Sample was run through the inertial microfluidic device at
  • trophoblastic (large cell) outlet was collected and rerun through the spiral (double pass).
  • the trophoblastic outlet was then centrifuged at 500g for 10 minutes, supernatant removed and the cell pellet resuspended in lOOpL PBS with 1% fetal bovine serum.
  • HLA-G APC 0.5ug/100pL
  • CD45 FITC 0.5pL/100pL fluorescent antibodies were then added and incubated for 30 minutes on ice. After 20 minutes Hoechst was added and incubated for 10 minutes. After incubations PBS was added and the sample centrifuged. Supernatant was removed and the sample was washed in PBS.
  • Figure 3 shows micrographs of a HLA-G and Hoechst positive, CD45 negative JEG3 cell (square box) from after separation using an inertial microfluidic device as described herein (device 2), images taken at 100/ magnification.
  • EXAMPLE 2 OPTIMISATION OF FLOW RATES AND PORE SIZES FOR
  • Table 1 Recovery of Fetal trophoblastic cells (FTCs) using micro fabricated filters AT different flow rates and round pore sizes.
  • EXAMPLE 3 RECOVERY OF TROPHOBLASTIC CELLS USING DIFFERENT MICROFILTERS
  • the recovery of trophoblastic cells from a trophoblastic cell line was analysed using different microfilters in a microarray, as shown in Table 2. Specifically, sample from a single pass through the inertial microfluidic device as described herein (device 2) was passed through the SU8 microfilter using a syringe pump. Four different SU8 filters were used with 6, 8, 12 and 16 pm rod shaped pores. The flow rate was varied from 250pL/min to 5mL/min. The results show that the highest recovery was obtained using a filter with 6 p rod pores.
  • EXAMPLE 4 RECOVERY OF TROPHOBLAST1C CELLS US1NG FUNCT10NAL1SED MICROFILTERS
  • microfilters functionalised with 1 1-Mercaptoundecanoic acid (MU A) or polyethylene glycol (PEG) were prepared by coating the standard SU8 filters in a 25-100nm gold layer (See Figure 4 for a photo of gold 150pm wells (A) and 500pm wells (B) taken before functionalisation). Filters were washed with 100% ethanol and surface cleaned with oxygen plasma.
  • MU A 1-Mercaptoundecanoic acid
  • PEG polyethylene glycol
  • the filters were soaked in MUA (alkane acid and alkane thiol) solution overnight, washed with ethanol, then MQ water and then activated with N- (3-dimethyl amino propyl)-N-ethylcarbodiimide hydrochloride (EDC) for 10 minutes.
  • EDC N- (3-dimethyl amino propyl)-N-ethylcarbodiimide hydrochloride
  • PEG functionalisation the filters were soaked in PEG (HS-PEG-COOT1 and HS-PEG-OME) solution overnight, washed with MQ water and then activated with N-hydroxysuccinimide (NHS) for 10 minutes. Filters were washed with MQ water, then PBS, and then conjugated with HLA-G antibodies for 2 hours.
  • Table 3 Recovery of Fetal trophoblastic cells (FTCs) using gold coated micro fabricated filters (Square microwells, lml/min flow rate).
  • EXAMPLE 5 STAINING OF TROPHOBLASTIC CELLS ON MICROFILTER
  • cells were stained on the filter with specific antibodies to distinguish WBC from fetal trophoblastic cells. This was done by passing, for example, antibodies/fixative/ solutions through the filter as per sample from the inertial microfluidic device. Cells were stained with HLA-G (either fresh or fixed) and Hoechst (fresh), or Cytokeratin (fixed) and DAPI (fixed) to specially stain trophoblastic cells. Cells were fixed with formalin, glutaraldehyde or ice cold methanol (and permeabilized with Triton-x 100).
  • Cells were processed (stained/fixed permeabilized) while still in a holder on the syringe pump at a flow rate of 100 m ⁇ ./min and then the flow was stopped to reduce the amount of antibody used.
  • the cells were visualised using fluorescence and brightfield microscopy, with trophoblastic cells distinguished based on staining and their morphology (ie trophoblastic cells were larger than the surrounding WBC).
  • FIG. 5 shows a single stained JEG3 (primary HLA-G antibody with secondary Anti mouse IgG FITC antibody and Hoechst) in 500 pm well (A) and stained JEG3 (Cytokeratin FITC +DAPI) in 220 pm honeycomb well (B). Detection of HLA-G or Cytokeratin and a nuclear signal indicates the presence of a fetal cell, the cell can then be picked up or lysed with a manual pipette or single cell manipulator system.
  • EXAMPLE 6 SINGLE CELL PICK UP FOR DOWNSTREAM ANALYSIS
  • Fetal cells were isolated from maternal blood by enrichment with an inertial microfluidic device as described herein (device 2), collected into a microarray as described herein and single cells were picked-up as described below.
  • the microwell was either manually or automatically scanned with a fluorescence microscope detected by setting fluorescent thresholds i.e. pick up CD45 negative cytokeratin/HLA-G positive.
  • Cells of interest were identified based on their brightfield, nuclear staining and/or specific antibody stain (i.e. HLA-G or Cytokeratin) and either manually or automatically chosen on the computer, with detection based upon size and also presence of positive/negative staining.
  • Cellsorter software https://www.singlecellpicker.com/
  • the Cellsorter software provided for automated detection, based on user defined parameters, cell minimum area, cell maximum area, detection sensitivity, cell minimum intensity, cell maximum intensity, noise threshold and comparison of these values with other channels.
  • Single cells were either manually or automatically picked up using a programmable motorised arm attached to a micropipette (normally 30-70pm inner diameters) and deposited into a PCR tube, glass cover slip or into another micro well (for serial dilution) or water in oil droplet. To achieve this, a small vacuum was applied to the micropipette (commercially available single cell manipulation technology). Once single cells were isolated they were used for various downstream applications, such as Karyomapping, Next Generation Sequencing, fluorescence in situ hybridisation and droplet digital PCR.
  • FIG. 6 shows JEG3 cells within a microwell (in a thin layer of media with mineral oil on top to facilitate for pick up) before (A) and after pick up with a 30pm needle (B).
  • 1 cell was picked up and deposited into a PCR tube as shown this can easily enable downstream analysis techniques (C); images taken at 100 c magnification.
  • Cells can also be deposited onto a glass slide (D); image taken at 400 c magnification.
  • the positive deposition of a single cell can be visualised with fluorescence microscopy.
  • Figure 7 shows a cytokeratin positive (green) DAPI positive cytotrophoblast cell isolated from a pregnant woman’s blood sample before (A) and after pick up (B). Sample was stained for Cytokeratin, DAPI and CD45. Image taken at 400 magnification.
  • Figure 8 shows a micrograph of a microfilter with 500mih wells (brightfield) and an 80pm single cell manipulation micropipette (red circle) able to pick up cells within the well, and
  • EXAMPLE 7 SINGLE CELL LYSIS FOR DOWNSTREAM ANALYSIS
  • a standard cell lysis buffer can be used (eg l OmM Tris-CL, l OOmM NaCL, ImM EDTA, 1 % TritonX-100, 10% glycerol, 1 %SDS and 0.5% sodium deoxycholate or 400mM KOH, lOmM EDTA, lOOmM DTT) or propriety buffers as used on laser capture microdissection or for example Singleshot cell lysis kit - BioRad or Single Cell Lysis Buffer for Whole Genome Amplification DOPliiyTM- Reproductive Health Science).
  • a manual or automated pipette was used to dispense the lysis solution (volume dependent on the size of the microwell), which was left on top of the cell for -15 minutes. The solution containing the lysed cell was then picked up in the same way it was deposited and then deposited into a PCR tube for further downstream analysis.
  • Figure 9 shows a single JEG3 cell (circle) lysis within the microwell, before (A) and after lysis (B) of a single cell within a 500 pm microwell that is removed.
  • C Visual representation demonstrating single well buffer manipulation (in this case red dye), in order to specifically target and lyse 1 microwell.
  • Figure 10 shows a single stained cell (Square) before (A) and after (B) lysis in a 500pm honeycomb microwell.
  • An example of a droplet digital PCR profile for KRT19 (Keratin 19), in this instance multiple cells were lysed on filter, the mRNA was recovered, converted to cDNA before undergoing PCR (QX200TM droplet digital PCR system BioRad).
  • the droplet digital PCR plot shows positive droplets i.e. the presence of KRT19 mRNA after lysis of multiple cells. This demonstrates that the genetic material can be recovered after lysing the cells.
  • EXAMPLE 8 ENRICHMENT AND COLLECTION OF FETAL CELLS FROM
  • Fetal cells were isolated from maternal blood by enrichment with an inertial microfluidics device as described herein (device 2) and collected into a microarray as described herein. Enrichment and collection were performed as per Example 1B with the following variations. Maternal blood was collected in two 1 OmL EDTA tubes (or Streck BCT® cell free DNA tubes, kept at room temperature and processed within 24 hours).
  • fetal cells were isolated from maternal blood as follows: Chemical red blood cell lysis was performed at room temperature with a 1 : 10-1 :25 blood to lysis solution (145mM NH 4 C1, lOmM KHC0 3 0. lmM EDTA in MilliQ water; Sigma Aldrich) dilution (proprietary lysis buffer or dextran could also be used). After 10 minutes of vigorous shaking, samples were centrifuged at 500g for 10 minutes, the cell pellet was then washed and resuspended in PBS (eg. up to c 6 dilution) and the cellular suspension was loaded in a syringe.
  • PBS eg. up to c 6 dilution
  • the sample may be fixed before red blood cell lysis and before running through the inertial microfluidic device.
  • Samples were fixed with 4% formaldehyde for 10 minutes at room temperature, red blood cell were then lysed using 0.1% Triton X 100 for 10 minutes at room temperature in PBS. Sample is then centrifuged for 10 minutes at 500g to recover the nucleated cell fraction, supernatant is removed and sample is resuspended in PBS.
  • an inertial microfluidics device as described herein (device 2) was used to further enrich the fetal cells obtained from the maternal sample.
  • the suspension was injected through tygon tubing with a syringe pump into the inertial microfluidic device (1700 pL/min) using a standard syringe pump.
  • Eluted suspension, enriched in trophoblastic cells were then collected and processed for downstream analyses.
  • sample from the trophoblast outlet was plated into well plates, the samples were left to adhere for approximately 24 hours and then stained with May-Grtinwald Giemsa. Briefly, May-Grtinwald dye was placed onto the plated cells and incubated for 5 minutes.
  • Giemsa dye was diluted 1:20 with autoclaved MQ water, added to the cells and incubated for 20 minutes. The sample was washed with water and left to air dry. Microscopy was carried out using a Nikon Ti Eclipse inverted Microscope fitted with a colour camera. In another instance, sample from the trophoblastic outlet was analysed using an imaging flow cytometer (IFC). The cells were stained with the nuclear stain DAPI (4',6-diamidino-2-phenylindole) and with the fluorescently labelled fluorochromes Anti-Human CD45 PE-Cy5.5 and Anti-Pan-Cytokeratin (AE1/AE3) Alexa Fluor® 488 (eBioscience, San Diego, CA, USA).
  • IFC imaging flow cytometer
  • IFC was performed using the ImageStreanP Mark II (AMNIS, Seattle, WA, USA) and analysis of the cellular population was performed with IDEAS software Version 6.1 (AMNIS, Seattle, WA, USA).
  • Potential trophoblast cells were identified as DAPI positive, CD45 negative with cytokeratin staining. Events per sample were acquired at 400* magnification and 10,000 - 500,000 events were collected depending on sample volume.
  • FIG. 11 shows (A) a micrograph of multinucleated syncytial nuclear aggregate (SNA) stained with May- Grunwald Giemsa (400x magnification), and (B) SNAs identified as being keratin positive (pan-KRT) and CD45 negative by imaging flow cytometry (400x magnification).
  • SNA multinucleated syncytial nuclear aggregate
  • a syncytial nuclear aggregate was isolated from the blood of a pregnant woman with pre eclampsia using the enrichment step as described herein, with an inertial microfluidic device as described herein (device 2). Enrichment and collection were performed as per Example 1 B with the following variations. Maternal blood was collected in two lOinL EDTA tubes (but could also be collected in Streck BCT® cell free DNA tubes, kept at room temperature and processed within 24 hours).
  • FIG. 12 shows micrographs of a CD45 negative, Hoechst Positive syncytial nuclear aggregate from a pregnant women’s blood sample (pregnancy complicated with pre eclampsia) after separation with images A, B, C taken at 400 c magnification.
  • EXAMPLE 10 ISOLATION OF FETAL CELLS FROM MATERNAL BLOOD WITH CONFIRMED TRISOMY 21
  • Fetal cells with confirmed trisomy 21 were isolated from maternal blood using the enrichment step as described herein, with an inertial microfluidic device as described herein (device 2).
  • Chemical red blood cell lysis was performed at room temperature with a 1 : 10-1 : 25 blood to lysis solution (145mM NH 4 C1, lOmM KHCCR 0. ImM EDTA in MilliQ water; Sigma Aldrich) dilution. After 10 minutes of vigorous shaking, samples were centrifuged at 500g for 10 minutes, the cell pellet was then washed and resuspended in PBS (x2 dilution) and the cellular suspension was loaded in a syringe.
  • an inertial microfluidic device was used to further enrich the fetal cells obtained from the maternal sample.
  • the suspension was injected through tygon tubing with a syringe pump into the inertial microfluidic chips (1700 pL/min) using a standard syringe pump.
  • Eluted suspension, enriched in trophoblastic cells, were then collected and processed for downstream analyses for FISH and IFC. Sample from the trophoblastic outlet analysed using an IFC.
  • the cells were stained with the nuclear stain DAPI (4',6-diamidino-2-phenylindole) and with the fluorescently labelled fluorochromes Anti-Human CD45 PE-Cy5.5 (0.5pg/100pL) and Anti-Pan-Cytokeratin (AE1/AE3) Alexa Fluor® 488 (0.25pg/100pL) (eBioscience, San Diego, CA, USA).
  • IFC was performed using the ImageStreanP Mark II (AMNIS, Seattle, WA, USA) and analysis of the cellular population was performed with IDEAS software Version 6.1 (AMNIS, Seattle, WA, USA).
  • Potential trophoblast cells were identified as DAPI positive, CD45 negative with cytokeratin.
  • DAPI anti-fade Vector Shield
  • cover slip added for imaging.
  • Microscopy was carried out using a Nikon Ti Eclipse inverted fluorescence microscope and the CompuCyte iCysTM Laser Scanning Cytometer using DAPI , FITC (*) and Cy3/PE (+) channels for detection of nuclei, Chromosome X (or 13 in Trisomy 21 (T21) sample) and chromosome Y (or 21 in T21 sample), respectively.
  • FIG. 13 shows (A, C) an example of a computed colour image of a cytotrophoblast with fluorescence in situ hybridisation probing for chromosomes 13 (*) and 21 (+); and (B, D) Raw red imaging of chromosome 21 signals in the same cell as A and C, respectively.
  • a cell from a trisomy 21 pregnancy has also been isolated, amplified and sequenced demonstrating the applicability of these samples for more complex downstream analysis.
  • EXAMPLE 11 ISOLATION OF FETAL CELLS FROM MATERNAL BLOOD WITH MALE FETUSES
  • Example 10 The protocol described in Example 10 was repeated for pregnancies with a male fetus.
  • Fetal cells from maternal blood were enriched using the enrichment step as described herein, with an inertial microfluidic device as described herein (device 2).
  • Blood samples were collected in Streck BCT® cell free DNA tubes, kept at room temperature and processed within 24 hours.
  • Whole blood was fixed with 4% formaldehyde for 10 minutes at room temperature.
  • Red blood cells were then lysed with 0.1% Triton X 100 in PBS after 10 minutes of vigorous shaking, samples were centrifuged at 500g for 10 minutes, the cell pellet was then washed and resuspended in PBS (*6 dilution) and the cellular suspension was loaded in a syringe. From this point an inertial microfluidic device was used to further enrich the fetal cells obtained from the maternal sample.
  • the suspension was injected through tygon tubing with a syringe pump into the inertial microfluidic chips (1700 pL/min) using a standard syringe pump.
  • Eluted suspension, enriched in trophoblastic cells, were then collected and processed for downstream analyses for FISH and/or immunofluorescence (IF) in a microwell before then performing FISH.
  • the cells were stained with the nuclear stain DAPI (4',6-diamidino-2-phenylindole) and with the fluorescently labelled fluorochromes Anti -Human CD45 APC and Anti-Cytokeratin FITC (Miltenyi Biotec). After staining cells were placed in a microwell and scanned as previously described.
  • Cytokeratin positive cells were collected using the single cell manipulator before being deposited onto a glass slide for FISH (in one instance).
  • FISH FISH
  • maternal blood samples were processed and the large cells were isolated with an inertial microfluidics as described above.
  • the trophoblast outlet sample was deposited onto a glass slide and then treated with hypotonic solution (0.05mol/L potassium chloride) and left at room temperature for 10 minutes before removal.
  • hypotonic solution 0.05mol/L potassium chloride
  • Carnoy’s fixative 3: 1 methanol/acetic acid
  • Slides were left to air dry before probes (either probe set X spectrum green, Y spectrum orange, AneuVysion Multicolor DNA probe kit) were applied to the slide and a coverslip placed on top and sealed with rubber cement to avoid drying out. Samples were hybridised at 75°C for 5 minutes, before being incubated at 30°C overnight. Slides were washed with 0.4 Saline-sodium citrate buffer (SSC) with NP-40 (pH 7) at 75°C for 2 minutes and then with 2xSSC with NP-40 (pH 7) for 1 minute. Slides were mounted with DAPI anti-fade (Vector Shield) and a cover slip added for imaging.
  • SSC Saline-sodium citrate buffer
  • NP-40 pH 7
  • Slides were mounted with DAPI anti-fade (Vector Shield) and a cover slip added for imaging.
  • Microscopy was carried out using a Nikon Ti Eclipse inverted fluorescence microscope using DAPI,
  • FITC and Cy3 channels for detection of nuclei, Chromosome X and chromosome Y, respectively. In this instance we recover 0.6 circulating fetal cells per mL of mother’s blood.
  • Figure 14 shows examples of fluorescence in situ hybridisation of circulating fetal cells from 3 pregnancies with male fetuses enriched with inertial microfluidics (DAPI nuclear stain. Y chromosome: + (DYZ3), X chromosome: * (DXZl)). Images taken at 400 c magnification.
  • Figure 15 shows an example of an isolated circulating fetal cell which after enrichment was stained with specific markers and DAPI nuclear staining (A) and placed in a microwell. This cell was then recovered and placed onto a slide for fluorescence in situ hybridisation for X (*) and Y (+) chromosomes confirming that this fetal cell was from a male fetus (B).
  • EXAMPLE 12 USE OF WATER IN OIL DROPLETS AS MICROWELLS
  • the step of collecting isolated fetal trophoblast cells was carried out by applying the enriched fetal trophoblast cell population or the purified fetal trophoblast cell population to water droplets in oil.
  • the water droplets behave in the same way as microwells allowing containment of the cell or lysis/amplification within the droplet.
  • Figure 16 shows an example of a water (represented by #) in oil (represented by *) droplet containing a single cell (circle), Brightfield image (A) and nuclear DAPI image (B) of the same droplet attached to a glass surface.
  • Droplet size can vary from microlitres to nanolitres and the water droplet acts as a reaction vessel to contain cells for further analysis including lysis and genomic amplification.
  • Each oil droplet may contain 1 or more water droplets.
  • Droplets may contain 1 or more cells and may be free floating, or attached to the surface.
  • the surface chemistry of the glass may be altered (to change contact angle) so that the water droplet forms a near perfect sphere while also being attached to the surface.
  • DNA/RNA amplification may occur within the droplet, which may be located, for example, on a filter or on chip (ie in a microwell).
  • the methods of the present disclosure provide isolated fetal cells that can potentially be used for non-invasive prenatal diagnosis using cytogenetic, genomic, proteomic or transcriptomic methods.
  • Mantzaris D. Cram D.S., Potential of syncytiotrophoblasts isolated from the cervical mucus for early non-invasive prenatal diagnosis: evidence of a vanishing twin, Clinicazia acta;
  • Mouawia H., Saker A., Jais J.P., Benachi A., Bussieres L., Lacour B., Bonnefont J.P., Frydman R., Simpson J.L., Paterlini-Brechot P., Circulating trophoblastic cells provide genetic diagnosis in 63 fetuses at risk for cystic fibrosis or spinal muscular atrophy, Reprod. Biomed. Online, 25 (2012) 508-520.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Zoology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Gynecology & Obstetrics (AREA)
  • Reproductive Health (AREA)
  • Developmental Biology & Embryology (AREA)
  • Pregnancy & Childbirth (AREA)
  • General Engineering & Computer Science (AREA)
  • Virology (AREA)
  • Biophysics (AREA)
  • Toxicology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé d'isolement de cellules trophoblastiques fœtales présentes dans un échantillon biologique approprié. Le procédé comprend : la fourniture d'un échantillon biologique approprié comprenant une population de cellules trophoblastiques fœtales dans une population d'autres types de cellules ; l'enrichissement de la population de cellules trophoblastiques fœtales par application de l'échantillon biologique à un dispositif microfluidique inertiel pour séparer les cellules trophoblastiques fœtales de la population d'autres types de cellules pour obtenir une population de cellules trophoblastiques fœtales enrichie ; et la collecte de cellules trophoblastiques fœtales isolées par application de la population de cellules trophoblastiques fœtales enrichie à un réseau de micropuits pour collecter des cellules trophoblastiques fœtales isolées.
PCT/AU2019/000013 2018-02-07 2019-02-04 Isolement de cellules prénatales WO2019153032A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AU2018900373 2018-02-07
AU2018900373A AU2018900373A0 (en) 2018-02-07 Pre-natal cell isolation

Publications (1)

Publication Number Publication Date
WO2019153032A1 true WO2019153032A1 (fr) 2019-08-15

Family

ID=67547779

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU2019/000013 WO2019153032A1 (fr) 2018-02-07 2019-02-04 Isolement de cellules prénatales

Country Status (1)

Country Link
WO (1) WO2019153032A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190344273A1 (en) * 2016-01-28 2019-11-14 Clearbridge Biomedics Pte Ltd Multi-stage target cell enrichment using a microfluidic device
JP2023527246A (ja) * 2021-05-20 2023-06-27 広州凱普医薬科技有限公司 妊婦の子宮頸部剥離細胞から胎盤栄養膜細胞を分離する方法

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006108101A2 (fr) * 2005-04-05 2006-10-12 Living Microsystems Dispositifs et procedes d'enrichissement et de modification de cellules et autres particules
WO2007035498A2 (fr) * 2005-09-15 2007-03-29 Artemis Health, Inc. Dispositifs et methodes d'enrichissement magnetique de cellules et d'autres particules
WO2008014516A2 (fr) * 2006-07-28 2008-01-31 Living Microsystems, Inc. Sélection de cellules au moyen de biomarqueurs
WO2008130977A2 (fr) * 2007-04-16 2008-10-30 The General Hospital Corporation D/B/A Massachusetts General Hospital Systèmes et procédés de focalisation de particules dans des micro-canaux
US20100021984A1 (en) * 2008-05-23 2010-01-28 Edd Jon F Microfluidic Droplet Encapsulation
WO2011109762A1 (fr) * 2010-03-04 2011-09-09 National University Of Singapore Dispositif de tri microfluidique pour la détection et l'isolement de cellules
WO2014070776A1 (fr) * 2012-10-29 2014-05-08 The Regents Of The University Of Michigan Dispositif microfluidique et procédé permettant de détecter des cellules rares
WO2015057159A1 (fr) * 2013-10-16 2015-04-23 Clearbridge Biomedics Pte Ltd Trieur microfluidique pour détection et isolation de cellules
WO2017131580A1 (fr) * 2016-01-28 2017-08-03 Clearbridge Biomedics Pte Ltd Enrichissement en cellules cibles en plusieurs étages utilisant un dispositif microfluidique

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006108101A2 (fr) * 2005-04-05 2006-10-12 Living Microsystems Dispositifs et procedes d'enrichissement et de modification de cellules et autres particules
WO2007035498A2 (fr) * 2005-09-15 2007-03-29 Artemis Health, Inc. Dispositifs et methodes d'enrichissement magnetique de cellules et d'autres particules
WO2008014516A2 (fr) * 2006-07-28 2008-01-31 Living Microsystems, Inc. Sélection de cellules au moyen de biomarqueurs
WO2008130977A2 (fr) * 2007-04-16 2008-10-30 The General Hospital Corporation D/B/A Massachusetts General Hospital Systèmes et procédés de focalisation de particules dans des micro-canaux
US20100021984A1 (en) * 2008-05-23 2010-01-28 Edd Jon F Microfluidic Droplet Encapsulation
WO2011109762A1 (fr) * 2010-03-04 2011-09-09 National University Of Singapore Dispositif de tri microfluidique pour la détection et l'isolement de cellules
WO2014070776A1 (fr) * 2012-10-29 2014-05-08 The Regents Of The University Of Michigan Dispositif microfluidique et procédé permettant de détecter des cellules rares
WO2015057159A1 (fr) * 2013-10-16 2015-04-23 Clearbridge Biomedics Pte Ltd Trieur microfluidique pour détection et isolation de cellules
WO2017131580A1 (fr) * 2016-01-28 2017-08-03 Clearbridge Biomedics Pte Ltd Enrichissement en cellules cibles en plusieurs étages utilisant un dispositif microfluidique

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
CHOOLANI, M. ET AL.: "The promise of fetal cells in maternal blood", BEST PRACTICE & RESEARCH CLINICAL OBSTETRICS AND GYNAECOLOGY, vol. 26, 2012, pages 655 - 667, XP055074842, doi:10.1016/j.bpobgyn.2012.06.008 *
HAHN, S. ET AL.: "Noninvasive prenatal diagnosis of fetal aneuploidies and Mendelian disorders: new innovative strategies", EXPERT REVIEW OF MOLECULAR DIAGNOSTICS, vol. 9, 2009, pages 613 - 621 *
KARABACAK, N.M. ET AL.: "Microfluidic, marker-free isolation of circulating tumor cells from blood samples", NATURE PROTOCOLS, vol. 9, no. 3, 2014, pages 694 - 710, XP055237737, doi:10.1038/nprot.2014.044 *
REZAEI, M. ET AL.: "A Reappraisal of Circulating Fetal Cell Noninvasive Prenatal Testing", TRENDS IN BIOTECHNOLOGY, vol. S 0167-7, no. 18, November 2018 (2018-11-01), pages 30311 - 1 *
VONA, G. ET AL.: "Enrichment, Immunomorphological, and Genetic Characterization of Fetal Cells Circulating in Maternal Blood", AMERICAN JOURNAL OF PATHOLOGY, vol. 160, 2002, pages 51 - 58, XP002236986 *
WARKIANI, M.E. ET AL.: "Slanted spiral microfluidics for the ultra-fast, label-free isolation of circulating tumor cells", LAB ON A CHIP., vol. 14, 2014, pages 128 - 137, XP055425605, doi:10.1039/C3LC50617G *
WINTER, M. ET AL.: "Isolation of Circulating Fetal Trophoblasts Using Inertial Microfluidics for Noninvasive Prenatal Testing", ADVANCED MATERIAL TECHNOLOGIES, vol. 3, no. 7, July 2018 (2018-07-01) *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190344273A1 (en) * 2016-01-28 2019-11-14 Clearbridge Biomedics Pte Ltd Multi-stage target cell enrichment using a microfluidic device
US10898898B2 (en) * 2016-01-28 2021-01-26 Clearbridge Biomedics Pte Ltd Multi-stage target cell enrichment using a microfluidic device
JP2023527246A (ja) * 2021-05-20 2023-06-27 広州凱普医薬科技有限公司 妊婦の子宮頸部剥離細胞から胎盤栄養膜細胞を分離する方法
JP7368642B2 (ja) 2021-05-20 2023-10-24 広州凱普医薬科技有限公司 妊婦の子宮頸部剥離細胞から胎盤栄養膜細胞を分離する方法

Similar Documents

Publication Publication Date Title
JP6524082B2 (ja) 精子を選別するシステム及び方法
US10613015B2 (en) Methods for classification and sorting of cancer cells
US8969021B2 (en) Methods and compositions for detecting non-hematopoietic cells from a blood sample
WO2021170009A1 (fr) Module de capture de cellules foetales et puce microfluidique pour la capture de cellules foetales et leurs procédés d'utilisation
Talebjedi et al. Exploiting microfluidics for extracellular vesicle isolation and characterization: potential use for standardized embryo quality assessment
US20070059774A1 (en) Kits for Prenatal Testing
US20070059680A1 (en) System for cell enrichment
US20070059716A1 (en) Methods for detecting fetal abnormality
US20070059719A1 (en) Business methods for prenatal Diagnosis
US20070059718A1 (en) Systems and methods for enrichment of analytes
US20070059683A1 (en) Veterinary diagnostic system
CA2888746C (fr) Identification et analyse des cellules trophoblastiques foetales dans la glaire cervicale pour diagnostic prenatal
EP1613743B1 (fr) Isolement et enrichissement de cellules foetales
WO2011063416A2 (fr) Dispositifs microfluidiques destinés à capturer des composants d'un échantillon biologique
CN104797340A (zh) 微流体装置及其用途
US20140315295A1 (en) Polymer microfilters, devices comprising the same, methods of manufacturing the same, and uses thereof
WO2007035585A2 (fr) Systemes et methodes d'enrichissement d'analytes
JP2009511001A (ja) 細胞及びその他の粒子を磁気濃縮するためのデバイス並びに方法
JP2012520687A (ja) 循環細胞の捕獲用デバイス
KR101794218B1 (ko) 비침습적 산전 진단을 위한 산모 혈액 내 유핵 적혈구의 농축 분리 방법
WO2013126774A2 (fr) Dispositifs microfluidiques pour la capture d'espèces cibles
WO2013158044A1 (fr) Appareil et procédé pour séparer une entité biologique d'un volume d'échantillon
Kantak et al. Lab-on-a-chip technology: impacting non-invasive prenatal diagnostics (NIPD) through miniaturisation
WO2019153032A1 (fr) Isolement de cellules prénatales
US10564077B2 (en) Device for and method of isolating and analyzing a fraction in a biological sample

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19750539

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19750539

Country of ref document: EP

Kind code of ref document: A1