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Magnetic Cell Separation Development of Flowthrough Separators and Cell Tracking Velocimetry

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1 Department of Chemical Engineering, Ohio State University ... Typical elution peaks. QMS. b. Cell Separation by the QMS. Fm. a' b' a. b. Axial Symmetry ... – PowerPoint PPT presentation

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Title: Magnetic Cell Separation Development of Flowthrough Separators and Cell Tracking Velocimetry


1
Magnetic Cell SeparationDevelopment of
Flow-through Separators and Cell Tracking
Velocimetry
  • Masayuki Nakamura1,
  • Jeff Chalmers1 , Maciej Zborowski2

1 Department of Chemical Engineering, Ohio State
University 2 Department of Biomedical
Engineering, Cleveland Clinic Foundation
Research supported by National Science
Foundation National Cancer Institute Whitaker
Foundation Novamedics
2
Contents
  • 1. What is magnetic cell separation?
  • 2. QMS (Quadrupole Magnetic Separator)
  • 3. DMS (Dipole Magnetic Separator)
  • 4. CTV (Cell Tracking Velocimetry)
  • 5. Optimization of magnetic cell separation
  • 6. Future Work

3
Immunological Cell Separation
  • Rare cell detection / enrichment
  • Subsets of leukocytes (T-cells, B-cells, NK
    cells)
  • Hematopoietic stem cells (CD34)
  • Cancer cells circulating in the blood
  • Fetal cells in maternal blood
  • Harmful Bacteria (E. Coli O-157 etc)
  • Undesired cell depletion
  • T-cells to suppress immunological response
  • Cancerous cells from bone marrow
  • Red blood cells from leukocytes for cancer therapy

4
Cell Separation Technologies
  • Centrifugation Density
  • Filtration Size
  • Batch affinity systems Antibody, Avidin-biotin
  • Flow Cytometry (FACS)
  • Size
  • Granularity
  • Fluorescence
  • Magnetic separation systems
  • Paramagnetic

5
Paramagnetic beads
  • Particulate
  • DynabeadTM M450 Diameter 4.5mm
  • Dynal AS, Oslo, Norway

Colloidal MACSTM microbead Diameter 50 nm
Miltenyi Biotec, Germany
6
Molecular Magnetic Labeling
  • Paramagnetic protein
  • Ferritin
  • Hollow protein shell (13 nm)
  • Deposition of Fe2O3 in the cavity (7 nm)
  • Paramagnetic element
  • Erbium ion (Er3)
  • Paramagnetic ion
  • High atomic magnetic dipole moment
  • Ionic binding to the cell surface

7
Immunomagnetic Labeling of Cells
Two-Step Labeling
One-Step Labeling
8
HG4100 Cell Separation system
Immunicon Co. (USA)
  • Batch operation
  • Cell retention on the tube wall

9
MACS Cell separation system
Mintenyi Biotec (Germany)
  • Flow through
  • Cell retention on iron spheres

10
Contents
  • 1. What is magnetic cell separation?
  • 2. QMS (Quadrupole Magnetic Separator)
  • 3. DMS (Dipole Magnetic Separator)
  • 4. CTV (Cell Tracking Velocimetry)
  • 5. Optimization of magnetic cell separation
  • 6. Future Work

11
Quadrupole Magnetic Separator (QMS)
Top view
  • No cell retiontion
  • High throughput (up to 107 cells/s) gtgt FACS (104
    cells/s)

12
Quadrupole Magnetic Separator (QMS)
syringe pump with carrier fluid
Typical elution peaks
particle sample
2
2
1.5
1.5
injection valve
1
1
0.5
0.5
0
0
injection signal
50
100
150
QMS
UV b
UV a
a
b
syringe pump
13
Cell Separation by the QMS
Flow Parameters - Total Flow Rate - Inlet Flow
Ratio - Outlet Flow Ratio
14
Lymphocyte cell separation by the QMS
CD8 cells (Cytotoxic T cells)
CD4 cells (T helper cells)
Feed
Feed
a
b
a
b
15
Contents
  • 1. What is magnetic cell separation?
  • 2. QMS (Quadrupole Magnetic Separator)
  • 3. DMS (Dipole Magnetic Separator)
  • 4. CTV (Cell Tracking Velocimetry)
  • 5. Optimization of magnetic cell separation
  • 6. Future Work

16
Dipole Magnetic Separator (DMS)
  • No cell retention
  • Fractionate cells depending on traveling distance

Front view of flow channel
17
Fractionation of CD34 Cells by DMS
CD34 Cells (Hematopoietic Stem Cells)
1. Separation of CD34 cells by MACS
2. Fractionation of CD34 cells by DMS
3. Colony formation by culturing fractionated
cells
(immature)
CFU-Mix
CFU-GM
BFU-E
18
CD34 Cell Fractionation by the DMS
90
80
70
60
Percent type of colony per fraction
50
40
30
20
10
0
Fraction 4
Fraction 5
Fraction 6
Fraction 7
Fraction 8
19
Contents
  • 1. What is magnetic cell separation?
  • 2. QMS (Quadrupole Magnetic Separator)
  • 3. DMS (Dipole Magnetic Separator)
  • 4. CTV (Cell Tracking Velocimetry)
  • 5. Optimization of magnetic cell separation
  • 6. Future Work

20
Magnetophoretic Mobility
  • A measure of cells magnetism

Magnetic force per bead
Magnetic force on each cell
Drag force on each cell
21
Cell Tracking Velocimetry (CTV)
  • Measure magnetophoretic mobility experimentally

Light
Inverted Microscope
Sample Inlet
Magnet
CCD Camera
Sample Outlet
VCR
22
CTV system
Monitored Region
CTV system
Magnetic field
23
Cell Tracking Algorithm in CTV system
  • Modification of a 3-D Particle Tracking
    Velocimetry (PTV) (Guezennec et al., OSU)
  • Fully automated algorithm
  • Determine the path of a particular cell
  • Five consecutive images
  • Track cells up to hundreds

24
CTV Analysis Data output
25
CTV Analysis of Magnet-doped Microspheres
Trajectory plots of microspheres
Dr. Shlomo Margel Bar-Ham Univ. (Israel)
Silica
Magnetite
2.8 mm
Polystyrene core
26
Magnetophoretic Mobility Distributions of
Magnet-doped Microspheres
27
Magnetophoretic Mobility of CD34 Cells
Enriched Fraction
Cord Blood Luekocytes
Depleted Fraction
MACS system
28
Magnetophoretic Mobility Distribution at
Different Labeling Conditions
Saturation curve for secondary Ab-MACS beads
complex
Magnetophoretic mobility of cultured breast
cancers (HCC-1954 HER-2/neu)
29
Contents
  • 1. What is magnetic cell separation?
  • 2. QMS (Quadrupole Magnetic Separator)
  • 3. DMS (Dipole Magnetic Separator)
  • 4. CTV (Cell Tracking Velocimetry)
  • 5. Optimization of magnetic cell separation
  • 6. Future Work

30
Factors Controlling Separation Performance
Magnetic Field Strength
Magnetophoretic Mobility Distribution
Column Geometry
Flow Rate Flow Ratio
The theoretical model to predict the performance
of magnetic cell separation by the QMS.
31
Theoretical Model (Part1)
  • 3 possible outcomes
  • Depleted fraction (Fa)
  • Enriched fraction (Fb)
  • Retained on the wall (Fw)
  • Cell trajectory
  • A cell with a particular magnetophoretic
    mobility

Qb
Qa
Quadrupole magnetic field strength (Sm)
Fm
Magnet
Fw
Laminar flow velocity profile in an annular
channel
Fb
Fa
32
Theoretical Model (Part 2)
Trajectory of a cell
Relative positions of the ISC (?ISC) and OSC
(?OSC)
Transport lamina thickness (d)
33
Recovery prediction in the QMS
Recovery Fi Ni / Nfeed
Fa
Fb
Fw
d (um)
Theoretical Recovery
Fw
Fb
Fa
Qt (ml/min)
34
Breast Cancer Cell Separation by QMS100 HCC1954
Cell Sample
Recovery Fi Ni / Nfeed
35
Breast Cancer Cell Separation by QMS1 HCC1954
99 PBL
Recovery Fi Ni / Nfeed
HCC 1954
PBL
36
1 HCC1954 99 PBLPurity of HCC1954
Throughput 3.33x105 (cells/s)
Pi, HCC Ni, HCC / (Ni, HCC Ni, PBL)
37
Integrated Approach to QMS Optimization
Magnetophoretic Mobility
CTV
Immuno- magnetic Labeling
Theoretical QMS Performance
QMS
38
CD34 cell separation by the QMS
Feed
a
b
Total cell load 2 x108 cells CD34 cell
recovery 81 Throughput 1.75 x 105 cells/s
39
Flexible Magnetic Cell Separation by the QMS
Conventional Magnetic Cell Separation
QMS Magnetic Cell Separation
40
Future Work
  • QMS
  • Scale-up
  • Better column design
  • Disposable (FDA approval)
  • Continuous feeding
  • CTV
  • Electromagnet
  • Laser / Fluorescence detection
  • In-time analysis
  • Magnetic Flow Cytometry

41
Conclusions
  • Development of novel systems
  • Flow-through Magnetic Separators
  • QMS
  • DMS
  • Cell Tracking Velocimetry
  • Mathematical theory
  • Magnetophoretic mobility
  • Cell sorting model
  • Performance prediction of QMS
  • Applications
  • Leukocytes
  • Cancer cells

42
Acknowledgments
  • Chemical Engineering at Ohio State Univ.
  • Julia Chosy
  • Kristin Comella
  • Keith Decker
  • Kristie Melnik
  • Oscar Lara
  • Dr. Yan Zhao
  • Bar-Ham Univ. (Israel)
  • Dr. Shlomo Margel
  • Cleveland Clinic Foundation
  • Dr. Kara McClosky
  • Lee Moore
  • Alexander Rodriguez
  • Dr. Liping Sun
  • Dr. Steve Williams
  • Funding
  • NSF
  • NCI
  • Whitaker Foundation
  • NOVAMEDICS

43
Publications
  • QMS related
  • Nakamura M, Decker K, Chosy J, Comella K, Melnik
    K, Moore L, Zborowski M and Chalmers JJ. The
    separation of a breast cancer cell line from
    human blood using a quadrupole magnetic flow
    sorter. Biotechnology Progress, in press.
  • Hoyos M, McCloskey K, Moore LR, Nakamura M,
    Bolwell B, Chalmers JJ and Zborowski M.
    Pulse-injection studies of blood progenitor cells
    in a quadrupole magnetic flow sorter. Separation
    Science and Technology, in press
  • Moore LR, Rodriguez AR, Williams PS, McClosky K,
    Bolwell BJ, Nakamura M, Chalmers JJ and Zborowski
    M. (2001) Progenitor cell isolation with a
    high-capacity quadruploe magnetic flow sorter.
    J. Magnetism and Magnetic Materials, 225, 277-284
  • CTV related
  • Comella K, Nakamura M, Melnik K, Chosy J,
    Zborowski M, Cooper MA, Fehninger TA, Caliguiri
    MA and Chalmers JJ. The effect of antibody
    concentration on the separation of human natural
    killer cells in a commercial immuno-magnetic
    separation system. Cytometry, in press
  • Melnik K, Nakamura M, Comella K, Zborowski M and
    Chalmers JJ. (2001) Evaluation of elutents from
    separations of CD34 cells from human cord blood
    using a commercial, immunomagnetic cell
    separation. Biotechnology Progress, 17, 907-916.
  • Nakamura M, Lasky L, Zborowski M, Margel S,
    Chalmers JJ. (2000) Theoretical and experimental
    analysis of the accuracy of cell tracking
    velocimetry. Experiments in Fluids, submitted.
  • Moore LR, Zborowski M, Nakamura M, McCloskey K,
    Gura S, Lit G, Margel S, Chalmers JJ. (2000) The
    use of magnetite-doped polymeric microspheres in
    calibrating cell tracking velocimetry. J
    Magnetism Magnetic Materials, In press.
  • Chalmers JJ, Zhao Y, Nakamura M, Melnic K, Lasky
    L, Moore L, Zborowski M. (1999) An instrument to
    determine the magnetophoretic mobility of
    labeled, biological cells and paramagnetic
    particles. J Magnetism Magnetic Materials
    194231-241.
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