WO2019028445A1 - Dispositifs et procédés pour séparer des cellules tumorales circulantes d'échantillons biologiques - Google Patents

Dispositifs et procédés pour séparer des cellules tumorales circulantes d'échantillons biologiques Download PDF

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WO2019028445A1
WO2019028445A1 PCT/US2018/045294 US2018045294W WO2019028445A1 WO 2019028445 A1 WO2019028445 A1 WO 2019028445A1 US 2018045294 W US2018045294 W US 2018045294W WO 2019028445 A1 WO2019028445 A1 WO 2019028445A1
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cells
microfluidic channel
biological sample
cell
circulating tumor
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PCT/US2018/045294
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Leidong Mao
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University Of Georgia Research Foundation, Inc.
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Priority to EP18841411.4A priority Critical patent/EP3669163A4/fr
Priority to CA3071593A priority patent/CA3071593A1/fr
Priority to US16/636,517 priority patent/US11648559B2/en
Publication of WO2019028445A1 publication Critical patent/WO2019028445A1/fr
Priority to US16/406,440 priority patent/US11292000B2/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502769—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 characterised by multiphase flow arrangements
    • B01L3/502776—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 characterised by multiphase flow arrangements specially adapted for focusing or laminating flows
    • 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
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0668—Trapping microscopic beads
    • 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
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0681—Filter
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0883—Serpentine channels
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/043—Moving fluids with specific forces or mechanical means specific forces magnetic forces
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples

Definitions

  • the present disclosure generally relates to microfluidics and uses thereof.
  • Circulating tumor cells are cancer cells that are detached from primary solid tumors and carried through the vasculature to potentially seed distant site metastases in vital organs, representing the main cause of death in cancer patients.
  • Molecular assessments of CTCs not only could benefit basic cancer research, but also might eventually lead to a more effective cancer treatment.
  • one major limitation has been the limited availability of viable CTCs for investigations, due in part to the small patient blood volumes that are allowable for research, which usually yielded less than 100 CTCs from 1 mL of whole blood.
  • microfluidic devices and methods of using microfluidic devices are provided that overcome one or more of the aforementioned deficiencies.
  • the devices and methods can combine filters, sheathing separation, and flow focusing to provide for high throughput cell separation without the need for labeling the circulating tumor cells.
  • a multi-stage microfluidic device having a (i) a first stage including one or more filters along a length of a first microfluidic channel; (ii) a second stage fluidly connected to the first stage and including a second microfluidic channel, a fluid inlet for a sheathing fluid (which may be a ferrofluid), and a fluid outlet to collect waste particles; and (iii) a third stage fluidly connected to the second stage configured with a magnetic focusing force to focus magnetically labeled cells such as white blood cells, resulting in enrichment of the circulating tumor cells in one or more circulating tumor cell outlets.
  • a first stage including one or more filters along a length of a first microfluidic channel
  • a second stage fluidly connected to the first stage and including a second microfluidic channel, a fluid inlet for a sheathing fluid (which may be a ferrofluid), and a fluid outlet to collect waste particles
  • a third stage fluidly connected to the second stage configured with
  • a multistage a multi-stage microfluidic device for enriching circulating tumor cells in a biological sample.
  • the device can include at least three stages, although there may be more in some applications. Therefore, the terms first, second, third and so-on, when used to describe the stages, should not be considered limiting on the total number of stages but is used for simplicity to describe the relative ordering of the stages. Additional stages, not explicitly described, may in some aspects appear before the first stage.
  • Methods are provided for enriching, separating, or isolating unlabeled rare cells such as circulating tumor cells from a sample, e.g. a biological sample such as whole blood.
  • a biological sample such as whole blood.
  • the biological sample is or includes whole blood.
  • the biological sample includes about 50 to about 250 circulating tumor cells per milliliter of the biological sample.
  • the circulating tumor cells can include those selected from the group consisting of a primary cancer cell, a lung cancer cell, a prostate cancer cell, a breast cancer cell, a pancreatic cancer cell, and a combination thereof.
  • FIG. 1A is a schematic illustration of traditional and frequently used label-based magnetophoresis for CTC separation, in which rare cells were targeted via specific biomarkers such as epithelial cell adhesion molecule (EpCA ) through functionalized magnetic particles in order to pull these cells through magnetic force towards magnetic field maxima in a continuous- flow manner.
  • FIG. 1B is a schematic illustration of a label-free ferrohydrodynamic cell separation (FCS) for CTCs.
  • FCS label-free ferrohydrodynamic cell separation
  • RBC-lysed blood and biocompatible ferrofluids colloidal suspensions of magnetic nanoparticles
  • FIG. 1C is a top-view of the FCS device with labels of inlets, debris filters and outlets. A total of 6 outlets were fabricated in order to account for the broad size distributions of cells. The arrow indicates the direction of magnetic field during device operation. Dimensions of the FCS device and magnet are described below.
  • FIGS. 2A-2D show the results of optimization of FCS devices with their device geometry shown in FIG. 1C for high-throughput, high-recovery and biocompatible CTC separation.
  • a 3D analytical model considering magnetic buoyancy force, hydrodynamic drag force, laminar flow profiles and cancer/blood cell physical properties was developed to guide the optimization. The validity of the model was confirmed by comparing its simulated trajectories with experimental ones.
  • Numerical optimization of deflection distance Yc and separation distance ⁇ (corresponding to recovery rate and purity) at the end of the FCS device was conducted with parameters including: (FIG. 2A) & (FIG. 2B) magnetic field gradient, and (FIG. 2C) & (FIG.
  • FIGS. 3A-3D are micrographs of spiked cancer cells of cell culture lines and undiluted WBCs separation process in a FCS device.
  • 1 x 10 5 cells H1299 lung cancer cells were spiked into 1 mL of undiluted WBCs to increase the cancer cell concentration so that their fluorescent signals were visible.
  • the cell mixture was processed at the flow rate of 6 mL rr 1 .
  • a ferrofluid with its concentration of 0.26% (v/v) was used; magnetic field was fixed at 443 mT and its gradient was fixed at 56.2 T ⁇ 1 ⁇
  • FIG. 3A In absence of magnetic fields, cell mixtures exited the channel through outlets 1 and 2.
  • Scale bar 200 ⁇ .
  • FIG. 3B When magnetic fields were present, larger H1299 lung cancer cells and some WBCs were deflected and exited through outlets 5 and 6 (collection outlets), while smaller WBCs exited through lower outlets (outlets 1-4, waste outlets). Scale bar: 200 ⁇ .
  • FIG. 3C Fluorescence image of spiked H1299 lung cancer cell streams during the separation process when magnetic fields were present. H1299 cells were stained by CellTracker Green. Scale ban 200 ⁇ .
  • FIG. 3D Zoomed-in bright-field images of outlets 1-6 when the magnetic fields were present. Scale bars: 100 ⁇ m.
  • FIGS. 4A-4D show verification of FCS devices for high-throughput and high-recovery spiked cancer cells separation.
  • FIG. 4A Recovery rates of spiked H1299 lung cancer cells from undiluted WBCs at flow rates from 1.2 mL lr 1 to 6.0 mL lr 1 . Approximately 100 HI299 cancer cells were spiked into 1 mL of undiluted WBCs. Recovery rates decreased from 98.6 ⁇ 5.0% to 92.3 ⁇ 3.6% when flow rate increased from 1.2 mL rr 1 to 6.0 mL rr 1 .
  • FIG. 4A Recovery rates of spiked H1299 lung cancer cells from undiluted WBCs at flow rates from 1.2 mL lr 1 to 6.0 mL lr 1 .
  • FIGS. 5A-5D show the effect of FCS on cancer cell viability, proliferation and biomarker expressions.
  • FIG. 5B Representative images of Live/Dead cell staining for before (top) and after (bottom) separation groups. Calcein AM (green, live cells) and EhD-1 (red, dead cells) channels were merged. Scale bars: 100 ⁇ .
  • FIG. 5A Short-term cell viability comparison before and after FCS process using a Live/Dead assay. Cell viabilities of H1299 lung cancer cells before and after separation process were determined to be 98.9 ⁇ 0.9% and 96.3 ⁇ 0.9%, respectively. Error bars indicate standard deviation (s
  • FIG. 5C Bright field images of cultured H1299 cells collected after separation from day 1 to day 5. A Live/Dead staining of the cultured cells on day 5 showed excellent cell viability. Scale bars: 50 ⁇ .
  • FIG. 5D Comparison of expressions of two key biomarkers (epithelial cell adhesion molecule-EpCAM and cytokeratin-CK) on HCC1806 breast cancer cells before (top) and after (bottom) separation. They showed qualitatively similar EpCAM and CK fluorescence. Scale bars: 20 ⁇
  • FIGS. 6A-6C show enrichment of CTCs from NSCLC patient blood using FCS devices, and CTC identification with cytopathology and immunofluorescent staining.
  • CTCs FIG. 6A
  • FIGS. 6A-6C show enrichment of CTCs from NSCLC patient blood using FCS devices, and CTC identification with cytopathology and immunofluorescent staining.
  • WBCs (FIG. 6B) from the blood of two NSCLC patients (PA and PB) were enriched by FCS devices and stained with Papanicolaou procedure, then identified by a cytopathologist. (FIG. 6B)
  • FIG. 7A is a schematic illustration of traditional label-based magnetophoresis for CTC separation, in which rare cells were targeted via specific biomarkers such as epithelial cell adhesion molecule (EpCAM) through functionalized magnetic particles in order to pull these cells through magnetic force towards magnetic field maxima in a continuous-flow manner.
  • FIG. 7B is a schematic illustration of label-free ferrohydrodynamic cell separation (FCS) for CTCs. Large rare cells in ferrofluid experience large magnetic buoyance force and will be pushed towards magnetic field minimum
  • FIG. 7C is a schematic of integrated ferrohydrodynamic cell separation (iFCS) for CTC separation. In iFCS, RBC-lysed blood and biocompatible ferrofluid were processed in a single straight channel.
  • FIG. 7D is a photograph of a prototype iFCS device filled with dye and a permanent magnet.
  • FIG. 7E is a top-view of the iFCS device with labels of inlets, outlets.
  • FIG. 7F is a simulation of magnetic flux density and particle trajectories of magnetic particles and nonmagnetic particles in channel (L*WxH, 57.80mmx0.90mmx0.15mm)
  • FIGS. 8A-8I show optimization of iFCS device via simulation and beads calibration.
  • a 3D model was developed to simulate particle trajectories and validated by comparing simulation and experimental results.
  • Numerical optimization of final position Y, separation distance ⁇ , and the expected range (standard deviation) at the end of the channel was conducted with different parameters: (FIG. 8A)&( FIG. 8D)&( FIG. 8G) throughput, (FIG. 8B)&( FIG. 8E)&( FIG. 8H) ferrofluid concentration, and (FIG. 8C)&( FIG. 8F)&( FIG. 81) magnetic field gradient. Ferrofluid concentration was fixed at 0.292% for (FIG. 8A)&( FIG. 8D)&( FIG.
  • FIG. 8G Magnetic field gradient was fixed at 132T rrr 1 for (FIG. 8A)&( FIG. 8D)&( FIG. 8G), and throughput was fixed at 100 ⁇ _ mirv for (FIG. 8B)&( FIG. 8E)&( FIG. 8H)& (FIG. 8C)&( FIG. 8F)&( FIG. 8I).
  • FIG. 8D &( FIG. 8E)&( FIG. 8F) were simulated particles positions and corresponding standard deviations.
  • FIG. 8G)&( FIG. 8H)&( FIG. 8I) were normalized particle distributions in experiment.
  • FIGS. 9A-9F demonstrate iFCS applied to multiple beads separation.
  • FIG. 9A average separation distance between non-magnetic beads and magnetic beads. Non-magnetic beads (20.3 ⁇ m, 8.0 ⁇ m and 5.7 ⁇ m) and magnetic beads (11.8 ⁇ m) were spiked into 0.292% ferrofluid at throughput between 10 and 350 ⁇ mirv 1 . the magnetic field gradient was fixed at 132 T rrr 1 .
  • FIG. 9B simulated and experimental separation distance between 8.0 ⁇ non-magnetic beads and 11.8 ⁇ m magnetic beads at low ferrofluid concentration between 0.01% and 0.1%, and flow rate between 10 and 210 ⁇ min -1 .
  • FIG. 9C Particle trajectories of 8.0 ⁇ m non-magnetic beads at the flow rate of 100 ⁇ _ min -1 . A ferrofluid with its concentration of 0.049% (v/v) was used; the magnetic field gradient was fixed at 132 T rrr 1 . Scale bar: 200 ⁇ m.
  • FIG. 9D In absence of magnetic fields, all of the beads (20.3 and 8.0 ⁇ m non-magnetic beads, and 11.8 ⁇ m magnetic beads) were randomly distributed in the channel. Scale bar: 200 ⁇ m.
  • FIGS. 10A-10C demonstrate size of cancer cell lines and labelling results of WBCs.
  • FIG. 10A the size distribution of different cancer cell lines - Prostate (PC-3), Breast (MCF-7, MDA-MB-231 , HCC1806) and Lung (H1299, H3122, H69, DMS79), and white blood cells from healthy donor.
  • FIG. 10C Percentage of magnetic content in labelled WBCs. The volume fraction of magnetic content in dynabeads is 11.5%, the magnetic content for labelled WBC was calculated based on volume fraction.
  • FIGS. 11A-11F show iFCS applied to cancer cells separation. Certain number of PC-3 prostate cancer cells and 1 ⁇ 10 8 WBCs were spiked into 1 mL ferrofluid with concentration of
  • FIG. 11 A Bright field and fluorescent images of 1 x 10 5 PC-3 cancer cells and WBCs separation process. Green signal comes from PC-3 cancer cells and red signal comes from WBCs Scale bar: 500 ⁇ m.
  • FIG. 11B Collected PC-3 from outlet 1.
  • FIG. 11C Normalized size distributions of spiked and collected cancer cells from outlet 1 for
  • FIG. 11D Magnetic beads enumeration on collected WBCs from outlet 1 with different concentration of ferrofluid.
  • FIG. 11E Recovery rate and purity for different cancer cell lines ( ⁇ 100 cells per mL). Recovery rate of 97.9 ⁇ 1.0%, 97.6 ⁇ 1.0 %, 98.8
  • FIGS. 12A-12C FCSv2 device design and operating principle.
  • FIG. 12A Top-view of the FCSv2 device with labels of inlets, debris filters and outlets. The arrow indicates the direction of magnetic field during device operation.
  • the device integrates 3 stages into one single device for biomarker- and size-independent CTC enrichment. Stage one filters out large cell debris. Stage 2 depletes the unbound magnetic beads and WBCs bound with >3 beads into waste outlet 1 (FIG. 12B ii). Stage 3 continuously deflects unlabeled CTCs into collection outlet, while at the same time focuses WBCs bound with >1 bead into waste outlet 2 (FIG. 12B iii). (FIG.
  • microfluidic device is sandwiched between the device holders, which consist of top and bottom magnet arrays repelling each other and are secured with screws and nuts.
  • the thickness of channel is 250 ⁇ m.
  • the width of stage 2 is 1600 ⁇ and width of stage 3 is 1200 ⁇ .
  • FIG. 13 The microfluidic device is sandwiched between the two device holders, which consist of top and bottom magnet arrays repelling each other and is secured with screws and nuts.
  • the center of the stage 3's microchannel in Figure 1 is aligned exactly with the center of the magnet arrays.
  • FIG. 14 The simulation result shows the magnetic field is stronger in the center of the stage 3 region in Figure 1.
  • the labeled white blood cells and free magnetic beads are focused in the center of the channel and are collected through the waste outlet.
  • any unlabeled cells including circulating tumor cells are deflected into two sides of the channel and are collected through collection outlets.
  • Whole blood is labeled and lysed by RBC lysing buffer before it is processed with the FCSv2.
  • Leukocyte-specific biotinylated antibodies anti-CD45, anti-CD66b and anti-CD16
  • magnetic Dynabeads are added to the blood sample for incubation.
  • FIGS 15A Recovery rate of separated cancer cells for different cancer cell lines at the flow rate of 12 mLJh. recovery rates of 98.46 ⁇ 0.50%, 99.68 ⁇ 0.46%, 99.05 ⁇ 0.75%, 99.35 ⁇ 0.46%, 99.40 ⁇ 0.85%, 99.13 ⁇ 0.49%, 99.11 ⁇ 1.25%, and 99.11 ⁇ 0.74% are achieved for HCC1806 (breast cancer), HCC70 (breast cancer), CF7 (breast cancer), MDA-MB-231 (breast cancer), H1299 (non-small cell lung cancer), H3122 (non-small cell lung cancer), DMS79 (small cell lung cancer), and H69 (small cell lung cancer) cell lines, respectively.
  • FIG. 15B A series of spike-in separation experiments in which a certain number (20, 50, 100, and 200) of HCC70 breast cancer cells are spiked into 1 mL of labeled white blood cells. An average recovery
  • FIGS. 16A-16B Bright field and fluorescence image of spiked MDA-MB-231 breast cancer cells (labeled with CellTracker Green) during the separation process confirmed the cancer cell trajectories at the end of stages 2 and 3 in FIGS. 12A-12C. Scale bars: 500 ⁇ m.
  • FIG. 17 Measured magnetic field and its gradient of the center of magnet's surface vs. distance between the magnet's surface and the microfluidic channel wall.
  • FIGS. 18A-18B Schematic and relevant dimensions of a FCS device.
  • FIG. 18A Top- view of the FCS device and relevant dimensions.
  • FIG. 18B Cross-section view of the FCS device. The red arrow indicates the direction of permanent magnet's magnetization.
  • FIG. 19A Cell trajectory simulation of H1299 lung cancer cell (16.9 ⁇ ) and WBCs (11.1 ⁇ m) in a FCS device.
  • FIG. 19B Zoomed-in view of cell trajectories at the end of FCS device. Blue and red trajectories indicate H1299 and WBCs, respectively.
  • Flow rate of cell inlet (Inlet A) was fixed at 6 mL h 1
  • ferrofluid concentration was fixed at 0.26% (v/v)
  • magnetic field was fixed at 443 mT and its gradient was fixed at 56.2 T rrr 1 for this simulation.
  • FIG. 20 FCS device calibration with H1299 cells (replaced with beads of similar size, 15.6 ⁇ m) and WBCs (11.1 ⁇ ).
  • the left-bottom number in each figure indicates the associated flow rate of cell inlet A (mL h ⁇ 1 ).
  • Flow rate of cell inlet (Inlet A) was fixed at 6 mL rr 1
  • ferrofluid concentration was fixed at 0.26% (v/v)
  • magnetic field was fixed at 443 mT and its gradient was fixed at 56.2 T rrr 1 for this calibration.
  • ⁇ 1 *10 4 polystyrene microparticles were mixed with 1 mL of undiluted WBCs. Scale bars: 500 ⁇ m.
  • FIG. 21 Comparison of cell trajectories from calibration experiments and simulations of H1299 cells and WBCs at the end of FCS device. Blue lines are the boundary of the simulated H1299 cell trajectory, and red lines are the boundary of the simulated WBC trajectory.
  • the simulated trajectories considered the initial width of microparticle and cell streams at the entry of the channel, therefore had an up and low bound of trajectories. Overall the simulated trajectories matched well with the experimental calibration trajectories, therefore could be used for subsequent FCS device optimization.
  • Flow rate of cell inlet (Inlet A) was fixed at 6 mL h 1 , ferrofluid concentration was fixed at 0.26% (v/v), and magnetic field was fixed at 443 mT and its gradient was fixed at 56.2 T rrr 1 for simulation and calibration. Scale bar: 500 ⁇ m.
  • FIGS. 22A-22F Characterization of custom-made ferrofluids.
  • FIG. 22A Magnetization of the as-synthesized ferrofluid. Solid red lines are the fitting of the experimental date to the Langevin function. Saturation magnetization of this ferrofluid was 0.96 kA rrr 1 , corresponding to a 0.26 volume fraction or concentration.
  • FIG. 22B Rheological plots of the ferrofluid and blood. The viscosity of ferrofluid was measured to be 2.92 mPa-s.
  • FIG. 22D Size distribution of maghemite nanoparticles was measured by dynamic light scattering (DLS). Hydrodynamic diameter was 40.77 ⁇ 12.71 nm.
  • FIG. 22E Zeta potential of ferrofluid was measured to be - 27.2 ⁇ 11.4 mV, indicating a negative surface charge on the particles.
  • FIG. 22F A transmission electron microscopy (TEM) image of the maghemite nanoparticles. Scale bar: 20 nm.
  • FIGS. 23A-23D FIGS. 23A-23D
  • FIGS. 23A-23D FIGS. 23A-23D
  • FIGS. 23A-23D FIGS. 23A-23D
  • FIGS. 23A-23D FIGS. 23A-23D
  • FIG. 23A Cell viability of H1299 lung cancer cells in different concentrations of ferrofluids was evaluated by a MTT assay. Cell viability was 80.8 ⁇ 2.4% after 12-h incubation with a 0.26% (v/v) concentration ferrofluid.
  • FIG. 23B Colloidal stability of biocompatible ferrofluids. The maghemite nanoparticles remained colloidally stable for at least 10 months in solution and there was no visible precipitation over time.
  • FIG. 23C Blood cells, mixed with a commercial water-based ferrofluid, showed an irreversible flocculation.
  • FIG. 23D No flocculation or aggregation of blood cells was found within the biocompatible ferrofluid. Scale bars
  • FIG. 24 An image of a FCS device and an attached collection chamber.
  • the FCS device was connected to a serpentine collection chamber that was used to accurately enumerate cancer cells for the FCS calibration using cultured cancer cell lines.
  • the depth of collection chamber is 50 ⁇ .
  • the size of the glass slide is 75 * 50 mm. Blue dye was used to visualize the microchannel.
  • FIG. 25 Representative micrographs of lung cancer H1299 cells and WBCs after a separation of spiked cancer cells in a FCS device at a throughput of 6 mL IT 1 .
  • ⁇ 100 CellTracker Green stained H1299 cells were spiked into 1 mL of undiluted WBCs.
  • B and (C) H1299 lung cancer cells and WBCs were identified in the serpentine collection chamber. Scale bars: 50 ⁇ m.
  • FIG. 26 Cell type distribution of cells collected from outlets 1-6 after a separation of ⁇ 100 H1299 cells spiked into 1 mL of undiluted WBCs using a FCS device at a throughput of 6 mL h- 1 .
  • FIG. 27A The average cell size of six cancer cell lines and WBCs measured by a cell counter.
  • FIG. 27B Size distribution of cancer cells and WBCs.
  • FIG. 28 Representative images of CTC identification from patient A and B, with their blood processed by FCS devices. Black arrows indicate the identified CTCs. Scale bars: 50 ⁇ m.
  • microfluidic devices and methods of using microfluidic devices are provided for separating and/or enriching circulating tumor cells in a biological sample such as whole blood.
  • the methods are capable of high throughputs with high levels of retention and separation of the circulating tumor cells.
  • ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.
  • a numerical range of "about 0.1% to about 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within the indicated range.
  • the stated range includes one or both of the limits
  • ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase "x to y" includes the range from 'x' to 'y' as well as the range greater than 'x' and less than 'y'.
  • the range can also be expressed as an upper limit, e.g.
  • 'about x, y, z, or less' should be interpreted to include the specific ranges of 'about x', 'about y', and 'about z' as well as the ranges of 'less than x', less than y', and 'less than z ⁇
  • the phrase 'about x, y, z, or greater' should be interpreted to include the specific ranges of 'about x', 'about y', and 'about z' as well as the ranges of 'greater than x', greater than y', and 'greater than z'.
  • the term "about” can include traditional rounding according to significant figures of the numerical value.
  • the phrase "about 'x' to 'y'", where 'x' and 'y' are numerical values includes "about 'x' to about 'y'".
  • a biocompatible substance or fluid, as described herein, indicates that the substance or fluid does not adversely affect the short-term viability or long-term proliferation of a target cell within a particular time range.
  • microfluidic devices and methods of using microfluidic devices are provided for high throughput sorting, separation, and/or enrichment of circulating tumor cells and other unlabeled rare cells in a biological sample such as blood.
  • the devices are single-stage devices, while in some aspects the devices have multiple stages.
  • a multistage a multi-stage microfluidic device for enriching circulating tumor cells in a biological sample.
  • the device can include at least three stages, although there may be more in some applications. Therefore, the terms first, second, third and so-on, when used to describe the stages, should not be considered limiting on the total number of stages but is used for simplicity to describe the relative ordering of the stages. Additional stages, not explicitly described, may in some aspects appear before the first stage.
  • the first stage in the multi-stage microfluidic device includes a first end, a second end, a first microfluidic channel fluidly connecting the first end and the second end, a first fluid inlet fluidly connected to the first microfluidic channel at the first end, and one or more filters along a length of the first microfluidic channel.
  • the first fluid inlet can be used for introducing, pumping, and/or injecting a biological sample into the device. Where there is a stage prior to the first stage, the inlet may be fluidly connected to the previous stage in such a way as to receive a sample (a biological sample) from the previous stage.
  • the one or more filters can filter large cell debris from the biological sample.
  • a second stage in the multi-stage microfluidic device includes a third end, a fourth end, a second microfluidic channel fluidly connecting the third end and the fourth end.
  • the second stage can be used to separate at least a portion of the white blood cells that are associated with the magnetic beads by flowing the filtered sample through a sheath flow in a nonuniform magnetic field to produce a first enriched sample.
  • the second stage can include, for example, a second fluid inlet fluidly connected to the second microfluidic channel at the third end, and a first fluid outlet fluidly connected to the second microfluidic channel at the fourth end; wherein the second fluid inlet is configured to receive a sheathing fluid; wherein the first fluid outlet is configured to receive a second plurality of waste particles from the biological sample.
  • the third end of the second microfluidic channel can be fluidly connected to the second end of the first microfluidic channel, i.e. the first stage and the second stage can be fluidly connected such that there is a fluid connection from the first microfluidic channel to the second microfluidic channel.
  • a third stage in the multi-stage microfluidic device includes a fifth end, a sixth end, a third microfluidic channel fluidly connecting the fifth end and the sixth end; a second fluid outlet fluidly connected to the third microfluidic channel at the sixth end, and one or more circulating tumor cell outlets fluidly connected to the third microfluidic channel at the sixth end; wherein the second fluid outlet is configured to receive a third plurality of waste particles from the biological sample; and wherein the one or more circulating tumor cell outlets are configured to receive a majority of the circulating tumor cells from the biological sample.
  • the fifth end of the third microfluidic channel can be fluidly connected to the fourth end of the second microfluidic channel, i.e.
  • the seoncd stage and the third stage can be fluidly connected such that there is a fluid connection from the second microfluidic channel to the third microfluidic channel.
  • the third stage can be used for isolating a majority of the unlabeled rare cells by magnetic flow focusing to separate magnetically labeled white blood cells from the unlabeled rare cells.
  • the microfluidic device includes one or more magnetic sources, wherein the one or more magnetic sources cause one or both of: (a) a non-uniform magnetic field along a length of the second microfluidic channel having a component sufficiently perpendicular to the second microfluidic channel to cause magnetic particles in the second microfluidic channel to be deflected into the first fluid outlet; and (b) a focusing magnetic field having a field maximum along a length of the third microfluidic channel sufficient to cause magnetic particles in the third microfluidic channel to be focused toward a center of the third microfluidic channel.
  • the device can include a first magnet array and a second magnet array; wherein the third stage is sandwiched between the first magnet array and the second magnet array; wherein the first magnet array and the second magnet array are oriented to repel each other; and wherein the third stage is oriented such that the length of the third microfluidic channel is centrally aligned between the first magnet array and the second magnet array.
  • one or more of the first microfluidic channel, the second microfluidic channel, and the third microfluidic channel have a thickness of about 10 ⁇ m to about 10000 ⁇ , about 10 m to about 1000 ⁇ m, about 10 ⁇ m to about 500 ⁇ m, about 150 ⁇ m to about 350 ⁇ m, about 220 ⁇ m to about 280 ⁇ m, or about 250 ⁇ m.
  • the second stage has a width of about 50 ⁇ m to about 10000 ⁇ m, about 500 ⁇ m to about 5000 ⁇ m, about 1200 ⁇ m to about 2000 ⁇ m, about 1400 ⁇ m to about 1800 ⁇ m, or about 1600 ⁇ m.
  • the third stage has a width of about 50 ⁇ m to about 10000 ⁇ m, about 500 ⁇ m to about 5000 ⁇ m, about 800 ⁇ m to about 1600 ⁇ m, about 1000 ⁇ m to about 1400 ⁇ m, or about 1200 ⁇ m.
  • the methods are capable of isolating a majority of the unlabeled rare cells.
  • the unlabeled rare cells are circulating tumor cells in a whole blood sample, and the majority of the circulating tumor cells comprises about 90%, about 92%, about 95%, about 97%, or more of the circulating tumor cells as compared to a total number of circulating tumor cells present in the biological sample inserted into the first fluid inlet when in operation.
  • the biological sample includes whole blood, wherein the whole blood includes a plurality of components.
  • the plurality of components comprises magnetically labelled white blood cells, and wherein at least 95%, at least 98%, at least 99%, at least 99.9%, or more of the white blood cells are not collected in the one or more circulating tumor cell outlets as compared to a total number of white blood cells present in the whole blood inserted into the first fluid inlet when in operation.
  • a single-stage microfluidic device for enriching circulating tumor cells in a biological sample, the device comprising a first stage comprising: a first end, a second end, a microfluidic channel fluidly connecting the first end and the second end, a fluid inlet fluidly connected to the microfluidic channel at the first end, three fluid outlets each fluidly connected to the microfluidic channel at the second end, and a magnet along a length of the microfluidic channel to create a non-uniform magnetic field along the microfluidic channel.
  • the microfluidic channel has a length of about 1 cm to about 100 cm, about 2 cm to about 50 cm, about 2 cm to about 10 cm, or about 3 cm to about 5 cm.
  • the microfluidic channel has a width of about 50 ⁇ m to about 10000 ⁇ m, about 100 ⁇ m to about 5000 ⁇ m, about 500 ⁇ m to about 1500 ⁇ m, or about 700 ⁇ m to about 1200 ⁇ m. In some aspects, the microfluidic channel has a thickness of about 10 ⁇ m to about 10000 ⁇ m, about 10 ⁇ m to about 1000 ⁇ m, about 10 ⁇ m to about 100 ⁇ m, or about 30 ⁇ m to about 80 ⁇ m. In some aspects, a gradient of magnetic field flux density of the magnet is about 0.001 T/m to 1000 T/m, about 10 T/m to about 500 T/m, or about 10 T/m to about 90 T/m.
  • Methods are provided for enriching, separating, or isolating unlabeled rare cells such as circulating tumor cells from a sample, e.g. a biological sample such as whole blood.
  • a biological sample such as whole blood.
  • the biological sample is or includes whole blood.
  • the biological sample includes about 50 to about 250 circulating tumor cells per milliliter of the biological sample.
  • the circulating tumor cells can include those selected from the group consisting of a primary cancer cell, a lung cancer cell, a prostate cancer cell, a breast cancer cell, a pancreatic cancer cell, and a combination thereof.
  • the methods can include enriching circulating tumor cells in a sample of whole blood, wherein the whole blood includes unlabeled rare cells and white blood cells, the method including: (i) adding a plurality of magnetic beads to the sample to produce a magnetically labeled sample, wherein at least some of the white blood cells are associated with the magnetic beads; (ii) filtering the magnetically labeled sample in a microfluidic device to produce a filtered sample by removing large cell debris from the magnetically labeled sample; (iii) separating at least a portion of the white blood cells that are associated with the magnetic beads by flowing the filtered sample through a sheath flow in a nonuniform magnetic field to produce a first enriched sample; and (iv) isolating a majority of the unlabeled rare cells by magnetic flow focusing the first enriched sample in a microfluidic channel.
  • the methods can include introducing the biological sample, which may be mixed with a ferrofluid prior to introduction, into the first fluid inlet of a microfluidic device described herein at a flow rate sufficient to cause the biological sample to flow along the microfluidic channels) of the device such that a majority of the circulating tumor cells from the biological sample are collected in the one or more circulating tumor cell outlets.
  • the methods can include introducing a biocompatible ferrofluid, which may include mixing the biological sample with the ferrofluid as well as the use of the ferrofluid as a sheathing fluid flow in the operation of the device.
  • the methods are devices are capable of high throughput.
  • the throughput is about 6 milliliters to about 25 milliliters of the biological sample per hour.
  • the flow rate is about the flow rate is about 10 L to about 600 ⁇ _ per minute.
  • FCS laminar-flow microfluidic ferrohydrodynamic cell separation
  • FCS devices The capability of the FCS devices was verified by successfully separating low-concentration ( ⁇ 100 cells mL 1 ) cancer cells using six cultured cell lines from undiluted white blood cells (WBCs), with an average 92.9% cancer cell recovery rate and an average 11.7% purity of separated cancer cells, at a throughput of 6 mL per hour.
  • the recovery rates of cancer cells were 92.3 ⁇ 3.6% (H1299 lung cancer), 88.3 ⁇ 5.5% (A549 lung cancer), 93.7 ⁇ 5.5% (H3122 lung cancer), 95.3 ⁇ 6.0% (PC-3 prostate cancer), 94.7 ⁇ 4.0% (MCF-7 breast cancer), and 93.0 ⁇ 5.3% (HCC1806 breast cancer), and the corresponding purities of separated cancer cells were 11 1% ⁇ 1.2% (H1299 lung cancer), 10.1 ⁇ 1.7% (A549 lung cancer), 12.1 ⁇ 2.1% (H3122 lung cancer), 12.8 ⁇ 1.6% (PC-3 prostate cancer), 11.9 ⁇ 1.8% (MCF-7 breast cancer), and 12.2 ⁇ 1.6% (HCC1806 breast cancer).
  • FCS Three-dimensional model of ferrohydrodynamic cell separation
  • the magnetic buoyancy force is expressed as, where is the permeability of free space, V c is the volume of a single cell, is its magnetization, s magnetization of the magnetic fluid surrounding the body, and is the magnetic field strength at the center of the body. 4 The magnetization of the ferrofluid
  • ⁇ is the concentration (volume fraction) of the magnetic nanoparticles in the ferrofluid. 4
  • f D hydrodynamic drag force coefficient of a moving cell considering the influence with a solid surface in its vicinity, which is referred to as the "wall effect".
  • the velocity vectors of the fluids were extracted from a 3D velocity
  • Average diameters of WBC and H1299 cells were 11.1 ⁇ m and 16.9 ⁇ .
  • Dimensions of the permanent magnet were 50800 ⁇ m (length) ⁇ 12700 ⁇ m (width) * 12700 ⁇ m (height) and the B field at the polar surface was measured to be 0.5 T.
  • Trajectories of the cells in the device were obtained by (1) calculating the 3D magnetic buoyancy force via an experimentally verified and analytical distribution of magnetic fields as well as their gradients, together with a nonlinear Langevin magnetization model of the ferrofluid, (2) deriving the hydrodynamic viscous drag force with an velocity profile of the channel obtained from COMSOL Multiphasics (Version 3.5, COMSOL Inc., Burlington, MA), (3) solving governing equations of motion using analytical expressions of magnetic buoyancy force and hydrodynamic viscous drag force in MATLAB (Math Works Inc., Natick, MA).
  • the parameters of simulation (device dimension and geometry, fluid and cell properties, and magnetic fields) reflected exact experimental conditions.
  • a water-based ferrofluid with maghemite nanoparticle was synthesized by a chemical co-precipitation method and made biocompatible following a protocol previously described.
  • Ammonium hydroxide solution (28%), iron (II) chloride tetrahydrate (99%), iron (III) chloride hexahydrate (97%), nitric acid (70%), iron (III) nitrate nonahydrate (98%), and sodium hydroxide (98%) were purchased from a commercial vendor (Sigma-Aldrich, St. Louis, MO). All reagents were used as received.
  • Maghemite nanoparticles were synthesized by a chemical co- precipitation method.
  • the maghemite nanoparticle suspension was centrifuged at 3000xg for 3 minutes and finally dispersed in 120 mL of deionized (Dl) water, yielding a stable dispersion with a pH of 1.5- 2.
  • the pH of the dispersion was adjusted to 2.9 by 1 M sodium hydroxide solution.
  • the dispersion was then vigorously stirred at room temperature for 1 hour, and the resulted ferrofluid was dialyzed with a dialysis membrane (Spectrum Labs, Collinso Dominguez, CA) against Dl water for one week.
  • H1299, A549 and H3122 were cultured in RPMI-1640 medium (Mediatech, Inc., Manassas, VA) supplemented with 10% (v/v) fetal bovine serum (FBS; Life Technologies, Carlsbad, CA) and 1% (v/v) penicillin/streptomycin solution (Mediatech, Inc., Manassas, VA).
  • MCF-7 cells were cultured in Dulbecco's modified eagle medium (DMEM; Life Technologies, Carlsbad, CA) supplemented with 10% (v/v) FBS, 1% (v/v) penicillin/streptomycin solution and 0.1 mM MEM non-essential amino acid (NEAA; Life Technologies, Carlsbad, CA). All cell cultures were maintained at 37 °C under a humidified atmosphere of 5% C02. Cell lines were released through incubation with 0.05% Trypsin-EDT A solution (Life Technologies, Carlsbad, CA) at 37 °C for 5-10 minutes before each use.
  • DMEM Dulbecco's modified eagle medium
  • NEAA penicillin/streptomycin solution
  • NEAA non-essential amino acid
  • Cancer cells were fluorescently stained by incubation with 2 ⁇ CellTracker Green (Life Technologies, Carlsbad, CA) for 30 minutes before each use. Probe solution was replaced with culture medium by centrifuging at 2QOxg for 5 minutes. Cells were counted with a hemocytometer (Hausser Scientific, Horsham, PA) and serially diluted in culture medium to achieve a solution with approximately 1 x 104 cells per mL. Cells were then counted with a Nageotte counting chamber (Hausser Scientific, Horsham, PA) to determine the exact number of cells per ML Desired number of cancer cells (50, 100, 200, 500, 1000, or 2000) were spiked into 1 ml. of WBCs (RBC-lysed whole blood).
  • WBCs RBC-lysed whole blood
  • the number of cancer cells spiked was determined by the average of two counts, with an average of 5.2% difference between the counts.
  • H1299 cells from a FCS device were collected into a centrifuge tube and washed three times with culture medium to remove the nanoparticles, and then the cells were suspended in culture medium and seeded into a 24-well plate (Coming Inc., Coming, NY). Cells were then cultured at 37°C under a humidified atmosphere of 5% C02, the medium was refreshed every 24 h during the first 3 days. Cellular morphology was inspected every 24 hours.
  • FCS FCS and seeded on a coverslip. After 24-h incubation, cells were fixed with 4% (w/v) PF A for
  • Nuclei were stained with 4',6-Diamidino-2-Phenylindole (DAPI; Life Technologies, Carlsbad, CA). After immunofluorescence staining, cells were washed with PBS and stored at 4 °C or imaged with a fluorescence microscope.
  • DAPI 4',6-Diamidino-2-Phenylindole
  • Microfluidic devices were made of polydimethylsiloxane (PDMS) using standard soft lithography techniques.
  • the thickness of the microfluidic channel was measured to be 52 ⁇ by a profilometer (Veeco Instmments, Chadds Ford, PA).
  • One NdFeB permanent magnet (K&J Magnetics, Pipersville, PA) was embedded into the PDMS channel with their magnetization direction vertical to the channel during the curing stage.
  • the magnet is 5.08 cm in length, 1.27 cm in both width and thickness.
  • Flux density at the center of magnet's surface was measured to be 0.5 T by a Gauss meter (Sypris, Orlando, FL) and an axial probe with 0.381 mm diameter of circular active area.
  • Fabricated devices were first flushed by 70% ethanol for 10 minutes at the flow rate of 6 mL h and then primed with 1 x PBS supplemented with 0.5% (w/v) BSA and 2 mM EDTA (Thermo Fisher Scientific, Waltham, MA) for 10 minutes at the flow rate of 6 mL lr 1 before each use.
  • a microfluidic device was placed on the stage of an inverted microscope (Carl Zeiss, Germany) for observation and recording. Two fluid inputs were controlled by individual syringe pumps (Chemyx, Stafford, TX) at tunable flow rates. Blood samples were injected into inlet A of a FCS device, sheath flow (ferrofluids) was injected into inlet B. Images and videos of microparticles and cells were recorded with a high-resolution CCD camera (Carl Zeiss, Germany). After separation, cells were collected in a serpentine collection chamber for cell counting.
  • NSCLC Patient blood processing.
  • a stainless-steel sphere (BC Precision, Chattanooga, TN) with a diameter of 1.6 mm was also loaded into a syringe. A magnet was used to gently agitate the sphere to prevent blood cells from settling down every 5-10 minutes. After separation, the FCS device was flushed by PBS or ThinPrep PreservCyt solution (Hologic, Marlborough, MA) at 30 mL h 1 for 20 minutes to remove any cells in outlet reservoir. During the separation, the cells from outlet 6 of a FCS device were directly preserved in ThinPrep PreservCyt solution for further analysis.
  • ThinPrep slides were afterwards evaluated by a cytopathologist using light microscopy to identify and count the number of CTCs. Collected cells were also fixed with 4% (w/v) PFA for 30 minutes and subsequently permeabilized with 0.2% (v/v) Triton X-100 in PBS for 10 minutes. Cells were then blocked by 0.5% (w/v) BSA in PBS for 20 minutes. Afterblocking nonspecific binding sites, cells were immunostained with primary antibodies, anticytokeratin 8/18/19, human EpCAM/TROP-1 , and anti-CD45 (Abeam, Cambridge, MA). Following, the appropriately matched secondary Alexa Fluor-conjugated antibodies (Life Technologies, Carlsbad, CA) were used to identify cells. After immunofluorescence staining, cells were washed with PBS and stored at 4 °C or imaged with a fluorescence microscope. Results and discussion
  • FCS ferrohydrodynamic cell separation
  • the parameters that will affect the above- mentioned criteria include device geometry, magnetic field and its gradient, flow rate of cells, and ferrofluid properties (i.e., magnetic volume fraction or concentration, pH, tonicity, materials and surfactants of nanoparticles, colloidal stability). These parameters are highly coupled with each other and for this reason an effective model was needed for systematic device optimization.
  • ferrofluid properties i.e., magnetic volume fraction or concentration, pH, tonicity, materials and surfactants of nanoparticles, colloidal stability.
  • FCS ferrohydrodynamic cell sorting
  • f D is the hydrodynamic drag force coefficient for a cell moving near a solid surface, often referred to as the "wall effect". Because of the low Reynolds number in FCS devices, inertial effects on the cell were neglected and motion of cells in ferrofluids could be determined by the balance of hydrodynamic viscous drag force and magnetic buoyancy force. From Equations 1-3, it can be seen that cells with different volumes experience different magnitudes of magnetic buoyancy force, which can result in the separation of these cells in ferrofluids in a continuous-flow manner.
  • FIG. 2A shows when the magnetic field gradient increased, the deflection distance of cancer cells Yc increased monotonically for all flow rates. This was because the driving force, magnetic buoyancy force on cells, was proportional to the magnitude of magnetic field gradient. As the cell inlet flow rate increased, Yc decreases due to reduced time in the channel.
  • FIG. 2B shows similar trend of separation distance ⁇ increasing as the field gradient increased when flow rates are 4.8, 6.0 and 7.2 mL rr 1 Interestingly, when cell input flow rates are smaller (e.g., 1.2, 2.4 and 3.6 mL rr 1 ), the separation distance ⁇ between two cell types had different trends.
  • ferrofluid After optimizing flow rate and magnetic field gradient, another critical parameter that still needs to be optimized is the ferrofluid itself.
  • the ferrofluid needs to possess properties that are not only biocompatible to CTCs but also enable its colloidal stability under high flow rates and strong magnetic fields. Therefore, its pH value, tonicity, materials and surfactants of nanoparticles need to be optimized as a biocompatible medium for cells, while at the same time the overall colloidal stability of the ferrofluid will have to be well maintained.
  • the particles had a mean diameter of 11.24 nm with a standard deviation of 2.52 nm.
  • the diameter of the nanoparticles was chosen to preserve the colloidal stability of ferrofluids against agglomeration due to gravitational settling and magnetic dipole-dipole attraction. As a result, our fenofluids remained colloidally stable after at least 10 months' storage.
  • the nanoparticles were functionalized with a graft copolymer as surfactants to prevent them from coming too close to one another when there was a magnetic field.
  • the volume fraction of the magnetic content of the ferrofluid is 0.26%.
  • Fig. 2D shows there was an optimal ferrofluid concentration close to 0.6% (v/v) at 6.0 ml. h-1 flow rate for /; Y. Concentrations higher than 0.6% (v/v) resulted in larger Yc but smaller ⁇ . This again was because both cell types achieved sufficient deflections in a strongly magnetized ferrofluid, resulting in mixing rather than separation of the two. In addition, ferrofluid biocompatibility could be compromised as its nanoparticle concentration increases. Based on these considerations, we chose a 0.26% (v/v) ferrofluid concentration to strike a balance between high-recovery and biocompatible cell separation at a flow rate of 6 mL rr 1 .
  • WBCs The concentration of WBCs was 3-7x106 cells mL 1 ; CTCs were simulated by spiking
  • FIGS. 3B-3D ⁇ 100 Cell Tracker Green stained H1299 cancer cells into 1 mL of WBCs.
  • the cells were loaded into a FCS device at variable flow rates of 1.2-6 mL rr 1 for recovery rate evaluation.
  • Fig. 3 shows a typical cancer cell (Lung cancer H1299) separation process in the FCS device.
  • the magnetic field was not present, all cell types including cancer cells and WBCs were flowing near the bottom sidewall of the channel and exiting through outlets 1 and 2 (Fig. 3A).
  • Fig. 3A When the magnetic field was present, a separation between cancer cells and WBCs was visible. Magnetic buoyancy forces deflected larger H 1299 cancer cells with a mean diameter of 16.9 j..Lm from the cell mixture toward outlets 5 and 6, as shown in FIGS. 3B-3D.
  • Fig. 4A shows the relationship between cancer cell recovery rates and flow rates for HI299 cancer cells. As flow rates increased from 1.2 mL rr to 6 mL lr 1 , recovery rates decreased from 98.6 ⁇ 5.0% to 92.3 ⁇ 3.6%. An average recovery rate of 92.3% was achieved for current FCS devices with a throughput of 6 mL IT 1 when ⁇ 100 HI299 cancer cells were spiked into 1 mL of WBCs. To validate that the device has the potential to process clinically relevant blood samples, a series of spike-in experiments in which a certain number of HI299 cells (50, 100, 200, 500, I000, and 2000) were spiked into 1 mL of WBCs. As shown in Fig.
  • Fig. 4C shows the relationship between removal rates of WBCs and cell input flow rates. As the flow rate increased, more WBCs were removed during the separation process. For example, 99.92 ⁇ 2.2% of WBCs were removed at the flow rate of 6 mL lr 1 when ⁇ IOO HI299 cancer cells were spiked into I mL of WBCs. The corresponding purity of separated cancer cells was 11.1% ⁇ 1.2%.
  • the purities of separated cancer cells in other spike-in experiments were 4.8%-67.4% (4.8 ⁇ 1.6%, 20.3 ⁇ 2.8%, 31.2 ⁇ 4.7%, 41.7 ⁇ 4.9%, and 67.4 ⁇ 3.3% when 50, 200, 500, 1000, and 2000 H1299 cancer cells were spiked into 1 mL of WBCs ).
  • the purity was defined as the number of identified cancer cells over the total number of cells from FCS device's collection outlets. As the number of spiked cells increased, the number of separated cancer cells also increased, which leaded to a higher purity value.
  • FCS device [99] After successfully demonstrating low-concentration cancer cell separation using HI299 lung cancer cell line, we also characterized the FCS device with 5 other types of cancer cells lines. Size distribution of CTCs from clinical samples is unknown, it is therefore important to characterize the performance of FCS devices with cancer cell culture lines with different sizes.
  • lung cancer, prostate cancer, and breast cancer cell culture lines were used to characterize the cancer cell recovery rates at 6 mL lr 1 throughput with ⁇ 100 cell mL 1 spike ratio.
  • the average recovery rates of 88.3 ⁇ 5.5%, 93.7 ⁇ 5.5%, 95.3 ⁇ 6.0%, 94.7 ⁇ 4.0%, and 93.0 ⁇ 5.3% were achieved for A549 (lung cancer), H3122 (lung cancer), PC-3 (prostate cancer), MCF-7 (breast cancer), and HCCI806 (breast cancer) cell lines, respectively.
  • the corresponding purities of separated cancer cells for each cell line were 10.1 ⁇ 1. 7% (A549), 12.1 ⁇ 2.1% (H3122), 12.8 ⁇ 1.6% (PC-3), 11.9 ⁇ 1.8% (MCF-7), and 12.2 ⁇ 1.6% (HCC1806), confirming the robustness of the FCS device for cancer cell separation.
  • FCS The recovery rate increased as the mean cell size of cancer cells increased (Table 1), which was expected as FCS was based on size difference of cell types.
  • FCS device was capable of separating cancer cells from WBCs with a flow rate of 6 ml_ rr ⁇ with a cancer cell recovery rate of 92.9% and a separated cancer cell purity of 11.7% averaged from all 6 cancer cell lines at ⁇ 100 cell mL 1 spike ratio, which allowed us to use the devices to process the clinical samples.
  • Fig. 5C shows the images of the cultured H1299 cells over a 5-day period. These cells were able to proliferate to confluence and maintain their morphologies after the ferrohydrodynamic separation process. Fluorescence image in Fig. 5C also confirms that cells were viable after the 5-day culture.
  • Fig. 6A shows a few Pap-stained CTCs and WBCs separated from two NSCLC patients.
  • CTCs were identified from 6.5 and 5.6 mL of blood samples, respectively. Purity of CTCs (defined as the number of identified CTCs over the total number of cells from FCS device's collection outlets) from these two patients was 17.0 ⁇ 7.8%. Additionally, Immunofluorescent staining of CKS/18/19, EpCAM, and leukocyte marker CD45 was also used to confirm the presence CTCs separated from patient B's blood. Cells were identified as CTCs if the staining pattern is CK +/CD45- or EpCAM+/CD45- or CK +!EpCAM+/CD45-, otherwise, cells were identified as WBCs. Typical fluorescent images are shown in Fig. 6B based on this immunostaining detection criteria.
  • FCS ferrohydrodynamic cell separation
  • FCS devices We have developed and validated such an analytical model that considered magnetic buoyancy force, hydrodynamic drag force, laminar flow profiles and cancer/blood cell physical properties to guide the optimization and design of a high- throughput, high recovery rate FCS devices.
  • FCS devices We also considered the chemical makeup of the ferrofluids, including its nanoparticle concentration, pH value, nanoparticle size and surfactant, tonicity to optimize a colloidially stable and biocompatible ferrofluid suitable for cancer cell separation.
  • FCS devices were capable of separating various types of low-concentration cancer cells of cultured cell lines 0 100 cell ml_-i> from WBCs under a flow rate of 6 ml.
  • FCS devices demonstrated a high-recovery and biocompatible separation of rare cancer cells at a clinically relevant throughput, and was validated with NSCLC patient blood, it was still at its early stage of development and could benefit from further system optimization or integration with other methods in order to achieve high-throughput, high- recovery, high-purity separation of intact CTCs.
  • rate of recovery of cancer cells was higher than the current average reported value of 82%, including methods based on standing surface acoustic wave (>83%), dean flow(>85%), vortex technology (up to 83%), and deterministic lateral displacement (>85%).
  • FCS device (6 mL h 1 ) was sufficiently high to process clinically relevant amount of blood, it was slower than a few hydrodynamics-based methods that had extremely high flow rates, including the dean flow (56.25 mL rr 1 ) the vortex technology (48 mL rr 1 ) and DLD (10 mL mhr 1 ). Further system optimization, scale-up or multiplexing of FCS devices should be conducted in order to process more blood quickly.
  • the average purity of separated cancer cells in current FCS devices was 11.7%. Reported purity values varied dramatically from 0.1% to 90% in label-free methods, ⁇ 5 as most of them focused on improving recovery instead of purification of rare cells.
  • FCS currently distinguished cells primarily based on their size difference. For cancer cells that have similar size as WBC's, this method will result in lower separated cancer cell purity than label-based method. Additional cell characteristics or methods could be integrated with FCS to further improve the purity of separated cancer cells.
  • One possible strategy is for future FCS devices to exploit both size and magnetic labels of cells for ere separation.22 For example, WBC's in blood can be labeled with sufficient number of anti-CD45 magnetic beads so that the overall magnetization of the WBC-bead complex is larger than its surrounding ferrofluids. The direction of magnetic force on the complex is then pointing towards magnetic field maxima.
  • Example 2 iFCS technology and device (medium magnetic field gradient, one- stage, sheath-free)
  • Circulating tumor cells contains abundant information regarding the location, type and stage of cancer and have significant implications in both diagnostic target and guiding personalized treatment.
  • the traditional isolation technologies rely on the properties of CTCs such as antigens (e.g., epithelial cell adhesion molecule or EpCA ) or size to separate them from blood.
  • Integrated - ferrohydrodynamic cell separation(iFCS) a size-independent and marker-independent method, can isolate cancer cells with large size distribution in biocompatible ferrofluids with a throughput 6ml_ rr 1 , an average recovery rate of 97.9%, and an 99.95% WBC depletion.
  • iFCS Integrated ferrohvdrodvnamic cell separation
  • the whole blood was firstly labeled with antibodies for 30min and lysed by RBC lysis buffer (eBioscience, San Diego, CA) for 7 min at room temperature. Cell mixtures were centrifuged for 5min at 800*g and the pellet were suspended in PBS with Dynabeads. Incubate the tube for 25 min on the rocker. Added ferrofluid and 0.1% (v/v) Pluronic F-68 non-ionic surfactant (Thermo Fisher Scientific, Waltham, MA) to achieve the same volume with whole blood and keep mixing for 5 more minutes. The number of dynabeads on each WBC were counted with microscope and the average was 34 ⁇ 11 dynabeads per WBC ( Figure 10B).
  • the corresponding purities of separated cancer cells for each cell line were 22.0 ⁇ 1.3 % (MCF-7), 23.5 ⁇ 0.7 % (MDA-MB-231), 25.2 ⁇ 1.5 % (HCC1806), 23.1 ⁇ 0.9 % (H1299), 22.2 ⁇ 0.9 (H3122), and 23.3 ⁇ 0.4 (PC-3), confirming the robustness of the iFCS device for cancer cell separation.
  • MCF-7 MCF-7
  • MDA-MB-231 MDA-MB-231
  • HCC1806 23.1 ⁇ 0.9 %
  • H1299 22.2 ⁇ 0.9
  • PC-3 23.3 ⁇ 0.4
  • Example 3 iFCS technology and device (high magnetic field gradient, multiple- stage, sheath-flow)
  • the magnet holder consists of top and bottom parts and is secured with screws and nuts. Two magnet arrays repelling each other are placed in the top and bottom holders, respectively.
  • the microfluidic device is sandwiched between the magnet holders and the center of the stage 3's channel is exactly aligned with the center of the magnet arrays (FIGS. 12A-12C and FIG. 13).
  • CTCs enrichment is achieved by inputting red blood cell (RBC)-lysed blood that is pre-labeled with magnetic beads (Dynabeads MyOne Streptavidin T1) targeting WBCs via CD45, CD16 and CD66B surface antigens (FIG. 14).
  • RBC red blood cell
  • magnetic beads Dynabeads MyOne Streptavidin T1
  • the recovery rates of cancer cells were 98.46 ⁇ 0.50% (HCC1806 breast cancer), 99.68 ⁇ 0.46% (HCC70 breast cancer), 99.05 ⁇ 0.75% (MCF7 breast cancer), 99.35 ⁇ 0.46% (MDA-MB-231 breast cancer), 99.40 ⁇ 0.85% (H1299 non-small cell lung cancer), 99.13 ⁇ 0.49% (H3122 non-small cell lung cancer), 99.11 ⁇ 1.25% (DMS79 small cell lung cancer), and 99.11 ⁇ 0.74% (H69 small cell lung cancer).
  • FIGS. 15A-15B show the separation of spiked cancer cells (stained with CellTrack Green) at the end of stages 2 and 3.
  • a multi-stage microfluidic device for enriching circulating tumor cells in a biological sample, the device comprising: (i) a first stage comprising a first end, a second end, a first microfluidic channel fluidly connecting the first end and the second end, a first fluid inlet fluidly connected to the first microfluidic channel at the first end, and one or more filters along a length of the first microfluidic channel; wherein the first fluid inlet is configured to receive the biological sample; and wherein the one or more filters are configured to remove a first plurality of waste particles from the biological sample; (ii) a second stage comprising a third end, a fourth end, a second microfluidic channel fluidly connecting the third end and the fourth end, a second fluid inlet fluidly connected to the second microfluidic channel at the third end, and a first fluid outlet fluidly connected to the second microfluidic channel at the fourth end; wherein the second fluid inlet is configured to receive a sheathing fluid; wherein the
  • Feature 2 The microfluidic device according to Feature 1 , further comprising one or more magnetic sources, wherein the one or more magnetic sources cause one or both of: (a) a non-uniform magnetic field along a length of the second microfluidic channel having a component sufficiently perpendicular to the second microfluidic channel to cause magnetic particles in the second microfluidic channel to be deflected into the first fluid outlet; and (b) a focusing magnetic field having a field maximum along a length of the third microfluidic channel sufficient to cause magnetic particles in the third microfluidic channel to be focused toward a center of the third microfluidic channel.
  • Feature 3 The microfluidic device according to any one of Features 1-2, wherein the device comprises a first magnet array and a second magnet array; wherein the third stage is sandwiched between the first magnet array and the second magnet array; wherein the first magnet array and the second magnet array are oriented to repel each other; and wherein the third stage is oriented such that the length of the third microfluidic channel is centrally aligned between the first magnet array and the second magnet array.
  • Feature 4 The microfluidic device according to any one of Features 1-3, wherein one or more of the first microfluidic channel, the second microfluidic channel, and the third microfluidic channel have a thickness of about 10 ⁇ m to about 10000 ⁇ m, about 10 ⁇ m to about 1000 ⁇ m, about 10 ⁇ m to about 500 ⁇ m, about 150 ⁇ m to about 350 ⁇ m, about 220 ⁇ m to about 280 ⁇ m, or about 250 ⁇ m.
  • Feature 5 The microfluidic device according to any one of Features 1-4, wherein the second stage has a width of about 50 ⁇ m to about 10000 ⁇ m, about 500 ⁇ m to about 5000 ⁇ m, about 1200 ⁇ m to about 2000 ⁇ m, about 1400 ⁇ m to about 1800 ⁇ m, or about 1600 ⁇ m.
  • Feature 6 The microfluidic device according to any one of Features 1-5, wherein the third stage has a width of about 50 ⁇ m to about 10000 ⁇ m, about 500 ⁇ m to about 5000 ⁇ m, about 800 ⁇ m to about 1600 ⁇ m, about 1000 ⁇ m to about 1400 ⁇ m, or about 1200 ⁇ m.
  • Feature 7 The microfluidic device according to any one of Features 1-6, wherein the majority of the circulating tumor cells comprises about 90%, about 92%, about 95%, about 97%, or more of the circulating tumor cells as compared to a total number of circulating tumor cells present in the biological sample inserted into the first fluid inlet when in operation.
  • Feature 8 The microfluidic device according to any one of Features 1-7, wherein the biological sample comprises whole blood, wherein the whole blood comprises a plurality of components.
  • Feature 9 The microfluidic device according to any one of Features 1-8, wherein the plurality of components comprises magnetically labelled white blood cells, and wherein at least 95%, at least 98%, at least 99%, at least 99.9%, or more of the white blood cells are not collected in the one or more circulating tumor cell outlets as compared to a total number of white blood cells present in the whole blood inserted into the first fluid inlet when in operation.
  • Feature 10 The microfluidic device according to any one of Features 1-9, wherein the plurality of components comprises magnetically labelled white blood cells, and wherein at least 95%, at least 98%, at least 99%, at least 99.9%, or more of the white blood cells are collected in one or more of the filters, the first fluid outlet, and the second fluid outlet as compared to a total number of white blood cells present in the whole blood inserted into the first fluid inlet when in operation.
  • Feature 11 The microfluidic device according to any one of Features 1-10, wherein the plurality of components comprise unlabeled rare cells and at least 90%, 92%, 95%, or more of the unlabeled rare cells are collected in the one or more circulating tumor cell outlets as compared to a total number of unlabeled rare cells present in the whole blood inserted into the first fluid inlet when in operation.
  • a method of enriching circulating tumor cells in a biological sample comprising a plurality of components, the method comprising introducing the biological sample into the first fluid inlet of a microfluidic device according to any one of claims 1-11 at a flow rate sufficient to cause the biological sample to flow along the first microfluidic channel, the second microfluidic channel, and the third microfluidic channel such that a majority of the circulating tumor cells from the biological sample are collected in the one or more circulating tumor cell outlets.
  • Feature 13 The method according to Feature 12, wherein the biological sample is whole blood.
  • Feature 14 The method according to Feature 12 or Feature 13, wherein the biological sample comprises about 50 to about 250 circulating tumor cells per milliliter of the biological sample.
  • Feature 15 The method according to any one of Features 12-14, wherein the flow rate is about 6 milliliters to about 25 milliliters of the biological sample per hour.
  • Feature 16 The method according to any one of Features 12-15, wherein the circulating tumor cells are selected from the group consisting of a primary cancer cell, a lung cancer cell, a prostate cancer cell, a breast cancer cell, a pancreatic cancer cell, and a combination thereof.
  • a single-stage microfiuidic device for enriching circulating tumor cells in a biological sample comprising a first stage comprising: a first end, a second end, a microfiuidic channel fluidly connecting the first end and the second end, a fluid inlet fluidly connected to the microfiuidic channel at the first end, three fluid outlets each fluidly connected to the microfiuidic channel at the second end, and a magnet along a length of the microfiuidic channel to create a non-uniform magnetic field along the microfiuidic channel; wherein the microfiuidic channel has a length of about 1 cm to about 100 cm; wherein the microfiuidic channel has a width of about 50 ⁇ to about 10000 ⁇ m; wherein the microfiuidic channel has a thickness of about 10 ⁇ m to about 10000 ⁇ ; and wherein a gradient of magnetic field flux density of the magnet is about 0.001 T/m to 1000 T/
  • a method of enriching circulating tumor cells in a biological sample comprising a plurality of components, the method comprising introducing the biological sample and a biocompatible ferrofluid into the fluid inlet of a microfiuidic device according to claim 17 at a flow rate sufficient to cause the biological sample to flow along the microfiuidic channel, wherein a majority of the circulating tumor cells from the biological sample are collected in one of the outlets.
  • Feature 19 The method according to Feature 18, wherein the flow rate is about 10 ⁇ . to about 600 ⁇ _ per minute.
  • Feature 20 The method according to Feature 18 or Feature 19, wherein the biological sample is whole blood.
  • Feature 21 The method according to any one of Features 18-20, wherein the biological sample comprises about 50 to about 250 circulating tumor cells per milliliter of the biological sample.
  • Feature 22 The method according to any one of Features 18-21 , wherein the flow rate is about 6 milliliters to about 25 milliliters of the biological sample per hour.
  • Feature 23 The method according to any one of Features 18-22, wherein the circulating tumor cells are selected from the group consisting of a primary cancer cell, a lung cancer cell, a prostate cancer cell, a breast cancer cell, a pancreatic cancer cell, and a combination thereof.
  • a method of enriching circulating tumor cells in a sample of whole blood, wherein the whole blood comprises unlabeled rare cells and white blood cells comprising: (i) adding a plurality of magnetic beads to the sample to produce a magnetically labeled sample, wherein at least some of the white blood cells are associated with the magnetic beads; (ii) filtering the magnetically labeled sample in a microfluidic device to produce a filtered sample by removing large cell debris from the magnetically labeled sample; (iii) separating at least a portion of the white blood cells that are associated with the magnetic beads by flowing the filtered sample through a sheath flow in a nonuniform magnetic field to produce a first enriched sample; and (iv) isolating a majority of the unlabeled rare cells by magnetic flow focusing the first enriched sample in a microfluidic channel.

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Abstract

La présente invention concerne différents dispositifs et procédés pour séparer ou enrichir des cellules tumorales circulantes dans un échantillon biologique tel que du sang total. Dans certains aspects, les dispositifs sont des dispositifs à étages multiples comprenant au moins (i) un étage de filtrage, (ii) un étage d'écoulement de gaine pour la séparation ferrohydrodynamique de globules blancs magnétiquement marqués, et (iii) une étape de focalisation pour la focalisation indépendante du marqueur et indépendante de la taille de particules magnétiquement marquées de façon à séparer ou enrichir des cellules rares non marquées dans l'échantillon biologique. Les dispositifs et les procédés sont, dans certains aspects, capables d'atteindre un rendement élevé supérieur à 6 millilitres par heure tout en obtenant une séparation élevée (> 95 %) des cellules rares non marquées.
PCT/US2018/045294 2017-08-04 2018-08-04 Dispositifs et procédés pour séparer des cellules tumorales circulantes d'échantillons biologiques WO2019028445A1 (fr)

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CA3071593A CA3071593A1 (fr) 2017-08-04 2018-08-04 Dispositifs et procedes pour separer des cellules tumorales circulantes d'echantillons biologiques
US16/636,517 US11648559B2 (en) 2017-08-04 2018-08-04 Devices and methods for separating circulating tumor cells from biological samples
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