Introduction - Chemical and Biomolecular Engineering - The Ohio by wuyunyi

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									 An Integrated Approach to Optimizing
    Immunomagnetic Cell Separation
using a Quadrupole Magnetic Flow Sorter
         Masa Nakamura1*,, Keith Decker1,
    Kristin Comella1, Julia Chosy1, Lee Moore2,
       Maciej Zborowski2 and Jeff Chalmers1


1 Department of Chemical Engineering, Ohio State University
2 Department of Biomedical Engineering, Cleveland Clinic Foundation


                                Research supported by:
                Cleveland                 National Science Foundation
                  Clinic                  National Cancer Institute
                Foundation                Whitaker Foundation
                                          Novamedics
Magnetic Cell Separation            Magnetic
                                    Bead

    Labeling Cells with        Marker         Antibody
     Magnetic Beads             Protein

       Micron (2-5 um)           Target
                                   Cell     135 nm
       Submicron (50-150 nm)
                                             Bead

    Separation Based on:
       Retention in a column

       Trajectory Difference



    Quadrupole Magnetic Flow
     Sorter (QMS)                          2 µm
Quadrupole Magnetic Flow Sorter (QMS)
     b’ a’      Core rod
                 Inlet flow splitter           Top view
                           Magnetic cell
                           Non-magnetic cell
                                                 M


                v
                                          M               M
                                                  Fm



                                                 M
                    Outlet flow
    b   a           splitter

            Continuous operation
            High throughput (up to 107 cells/s)
             >> Flow Cytometry (104 cells/s)
Circulating Cancer Cells
                         An early step in tumor-cell
                          invasion
       Tumor             Quantification of circulating

Blood
                          cancer cells
Stream       Circulating    Diagnostic
             Cancer Cell
                            Prognostic

                         1-1000 cancer cells / 106
                          mononuclear cells in breast
                          cancer pacients (Kraeft et al.,
      Metastatic          2000)
        Tumor
                         Enrichment step of cancer
                          cells is required
Objective of Research
Research Goals
 Optimization of QMS operation based on the
  theoretical model
 Using the QMS for circulating cancer cell
  detection
Approach
 Construction of a model of QMS sorting

 Calculation of theoretical cell recovery

 QMS sorting using cancer cells spiked in
  blood cells
 Comparison of prediction and experiment
Cell Type and Labeling
    Human breast cancer        Peripheral Blood
     cell line: HER-2/neu        Lymphocytes (PBL)
       Over-expression of         Density gradient

        HER-2/neu proteins         Lysis of red cells

       Large cell size




                    2º mAb + 50nm
1º mAb+FITC         Magnetic bead             1º mAb+PE

               HER-2/neu                         CD45
                Protein                         Protein

        HER-2/neu                       PBL
          Cell
Cell Diameter Distribution
by a Particle Size Analyzer                         Coulter Multisizer II®
                      0.14
                      0.14 5.92     12.57                                           30.07
                      0.12 5.92     12.57                                           30.07
                      0.12        PBL        HER-2/neu
Fraction of of cell




                      0.10        PBL
 population (-) (-)



                                             HER-2/neu
                                                    100




                                                    Particle Analyzer
  Fraction cell




                      0.10
   population




                                                     Purity (%) by
                      0.08                                              10
                      0.08
                      0.06                                               1
                      0.06
                      0.04                                              0.1
                                                                           0.1      1     10       100
                      0.04                                                         Purity (%) by
                      0.02                                                       Flow Cytometry
                      0.02
                      0.00
                      0.00 5       10    15     20     25                               30          35
                            5            15     20     25
                                   10 Cell diameter (m)                                30          35
                                        Cell diameter (m)
Cell Tracking Velocimetry (CTV)
   Measure magnetophoretic mobilities of cells
   Constant magnetic energy gradient (Sm)
                      Magnet
                                                   Sm=198 (T-A/mm2)

              Light                                    mobility
  Inverted
 Microscope                                              Dc
                                                  Fm              Fd

Sample                  CCD        Sample
                                                       Fm = Fd
 Outlet                Camera       Inlet

VCR                   PC (CTV Programs)
                                                       Vm
                      - Image acquisition
                                                 m             Nb
                      - Image processing
                      - Cell trackingargorithm      3  Dc 
                      - Velocity determination
Magnetophoretic Mobility Distribution by CTV




                                                                                     Mean m x 104 (mm3/T-A-s)
Unlabeled PBL Labeled HER-2/neu                                                                                 4.5
                     (8L)                                                                                      4.0
                   0.8                                   0.06                                                            n=7




                                                                Fraction for HER-2
                                                                                                                3.5
Fraction for PBL




                   0.7
                                                         0.05                                                   3.0
                   0.6
                                                                                                                2.5
                                                         0.04
                   0.5
                                                                                                                2.0
                   0.4                                   0.03                                                   1.5    n=5
                   0.3                                                                                          1.0
                                                         0.02
                   0.2                                                                                          0.5
                                                         0.01
                   0.1                                                                                          0.0
                   0.0                                   0.00                                                   -0.5
                         -2   0    2   4   6    8   10
                                                                                                BL R-2 0uL) 4uL 8uL
                                                                                               P E 1
                                    m x 104                                                                2
                                                                                           led d H ly ( ER- ER-
                                                                                                                2
                                                                                          e e
                                  (mm3/T-A-s)                                          lab bel S* on H H
                                                                                     Un nla -2
                                                                                       U ER
                                                                                        H
Quadrupole Magnetic Flow Sorter (QMS)
               Cell                   Magnetic cell
   Carrier     feed Carrier
                                      Non-magnetic cell
                              b’ a’       Core rod
                                          Inlet flow splitter



               a’   b’
       Waste
                                      v                  76
 QMS                                                     mm

               a    b
                                                Outlet flow
                                                splitter

                              b a
      Flow rate in each pump is adjustable
QMS Theoretical Model (1):
Trajectory for a particular cell
     Qa’       Qb’               Magnetic field strength
                                 Annular flow geometry
                                 Magnetophoretic mobility
           d
                                 Adjustable flow conditions
                                    Total flow rate: Qt

                                    Inlet flow ratio: Qa’/Qt

                                    Outlet flow ratio: Qa/Qt


                     Trajectory of a cell
                      z          v(  )         2  v  r0       1     2
                                                                                     
                                                                              A2 ln 
                     0 dz  
                              1 u (  )
                                         r0 d 
                                                     A1       
                                                               1            mSm
                                                                                       d
      Qa       Qb                 m



Qt = Qa’+Qb’ = Qa+Qb
     QMS Theoretical Model (2):
     Recovery prediction of cell population
                                                                                   Recovery
                               0.06
                                                                                   Fi = Ni / Nfeed
                               0.05
                                      Qa’ Qb’                              1.0
                               0.04
                                                                           0.8
                               0.03                                                                       Fa




                                                    Theoretical Recovery
                                                                           0.6
                               0.02

                               0.01        d                               0.4
                                                                                                     Fb
                                                                           0.2

-2    0   2   4   6   8   10
                               0.00
                                                                           0.0                        Fw
Magnetophoretic                            Fw
                                                                                 -0.2   0.0    0.2    0.4      0.6


                                                                                              d (um)
   Mobility                                                                 1.0

                                           Fb                               0.8          Fw
                                      Fa                                                                    Fb
                                                                            0.6

                                                                            0.4

                                                                            0.2
                                                                                                            Fa
                                      Qa       Qb                           0.0
                                                                                   0    5     10     15   20    25   30


                                                                                         Qt (ml/min)
100% HER-2/neu Cell Sample

Cell Carrier                                 Recovery Fi = Ni / Nfeed
Feed                               1.0
                                              FbHER2

               Recovery, Fa , Fb
                                   0.8
    d
                                   0.6


                                   0.4


                                   0.2
                                                       FaHER2
                                   0.0
  Fa    Fb                               0     100     200    300     400   500   600
                                                             d (m)
                                                                Recovery
1 % HER-2/neu + 99% PBL                                         Fi = Ni / Nfeed
                                             1.0




                        Recovery, Fa , Fb
 Cell Carrier                                0.8                FbHER2
 Feed
                                             0.6

                HER-2                        0.4

                                             0.2
     d                                                              FaHER2
                                             0.0
                                                    0   100                0 :Theory)
                                                              200FbPBL ( =400 500 600
                                                                   300
                                                                d (m)
                                               1


                        Recovery, Fa , Fb
                                                                     FaPBL
                                              0.1                ( = 1: Theory)
                 PBL                                                     FbPBL
                                             0.01                   ( = 0 : Theory)
   Fa    Fb
                                            0.001
                                                    0   100   200    300   400   500   600
                                                                d (m)
1 % HER-2/neu + 99% PBL:                                            Throughput:
Purity of HER-2/neu                                                 3.33x105 (cells/s)
 Cell Carrier
 Feed                                        Pi, HER2 = Ni, HER2 / (Ni, HER2 + Ni, PBL)
                                            100


     d          Purity: Pb, HER ; Pa, HER   10-1                   Pb, HER2

                                            10-2
                                                             Initial Purity (0.01)
                                            10-3
                                                                   Pa, HER2
                                            10-4


   Pa    Pb                                 10-5
                                                   0   100   200     300      400    500   600
                                                                   d (m)
Integrated Approach to QMS Optimization
                                                                                                                  0.06

                                                                                                                  0.05

                                                                                                                  0.04

                                                                                                                  0.03

                                                                                                                  0.02

                                                                                                                  0.01

                                                                                                                  0.00
                              -2                               0       2          4            6         8   10

                              Magnetophoretic
                                 Mobility
               CTV
                                                     1.0




Immuno-                                              0.8




                              Theoretical Recovery
magnetic                                             0.6




Labeling           v
                                                     0.4




                                                     0.2




                                                     0.0
                                                           0       5   10       15        20       25   30

                                                                            Qt (ml/min)



                              Theoretical QMS
                QMS            Performance
Conclusions
   QMS performance was predicted theoretically.

   Reasonable agreement between prediction and
    experimental results were obtained with spiked
    breast cancer cells.

   Integrated approach facilitates rational
    development of optimized QMS separation
    technique.

   Higher throughput is possible, and currently,
    human CD34+ cells are separated in a larger
    system at 107 cells/s (at CCF).

								
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