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
Acknowledgments

• Cleveland Clinic Foundation
• Lee Moore
• Dr. Steve Williams
• Dr. Liping Sun
• Kara McClosky
• Funding
• NSF
• NCI
• Whitaker Foundation
• NOVAMEDICS

• Chemical Engineering at Ohio State Univ.
• Kristie Melnik
• Julie Chosy
• Kristin Comella
• Keith Decker
• Ningning Ma
• Dr. Yan Zhao
• Bar-Ham Univ. (Israel)
• Dr. Shlomo Margel

3
Immunological Cell Separation

• Rare cell isolation
• Stem cells (CD34, AC133)
• Natural killer cells (CD56)
• Cancer cells circulating in the blood
• 1 in 106 or less?
• Fetal cells in maternal blood
• Harmful Bacteria (O-157 etc)
• Undesired cell depletion
• Removal of cancerous cells from cell population
• Removal of red blood cells from the leukocyte for
  cancer therapy

4
Cell Separation Technologies

• Centrifugation Density
• Filtration Size
• Flow Cytometry (FACS)
• Size
• Granularity
• Fluorescence
• Batch affinity systems antibody, avidin-biotin
• Batch magnetic systems
• Paramagnetic difference

5
Immunomagnetic Labeling of Cells
Two-Step Labeling
One-Step Labeling
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Paramagnetic particles

• Large size
• 1 to 5 um in diameter
• Magnetic susceptibility of a cell High
• Small number of beads per cell
• 1- 10 beads/cell
• Commercially available

7
Paramagnetic colloidal beads

• Colloidal size
• 50 to 200 nm
• Spread in solution (Brownian Motion)
• Magnetic susceptibility of a cell Low
• Large number of beads per cell
• 100 - 100,000 beads/cell

8
Molecular Magnetic Labeling

• Erbium ion (Er3)
• Paramagnetic ion
• High atomic magnetic dipole moment
• Ionic binding to the cell surface
• Ferritin
• Paramagnetic protein
• Hollow protein shell (13 nm)
• Deposition of Fe2O3 the cavity (7 nm)

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Magnetic Labeling
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Cell Separation Based on Antigen Expression Level
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MACS Cell separation system
Plunger
Separation column
Magnet
Remove the magnet
Unlabeled cells
Immunomagnetically labeled cells
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HG4100 Cell Separation system
13
Quadrupole Magnetic Flow Sorter (QMS)
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High Gradient Magnetic Separator (HGMS)
15
Cell Separation by the QMS
16
Lymphocyte cell separation by the QMS
CD8 cells
CD4 cells
Feed
Feed
a
b
a
b
17
CD34 cell isolation by the QMS
Feed
a
b
Total cell load 2 x108 cells CD34 cell
recovery 81 Throughput 1.75 x 105 cells/s
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Dipole Magnetic Flow Sorter (DMS)
Front view of flow channel
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Colony Formation from CD34 Cells
CFU-Mix
CFU-GM
(immature)
BFU-E
20
CD34 Cell Fractionation by the DMS
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Magnetophoretic Mobility

• Magnetophoretic mobility
• A measure of cells magnetism

Magnetic force per bead
Magnetic force on each cell
Drag force on each cell
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Magnetophoretic Mobility

• Applications
• Evaluation of magnetic labeling
• Type of magnetic beads
• Type and Amount of antibody
• Saturation
• Distribution in a cell population
• Magnetophoretic mobility
• Antigen expression level
• Prediction of cell separation performance

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Cell Tracking Velocimetry (CTV)
Light
Inverted Microscope
Sample Inlet
Magnet
CCD Camera
Sample Outlet
VCR
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Magnetic force field strength in CTV system
Monitored Region
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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.
• Measure the velocity
• magnetically induced velocity
• settling velocity
• Track cells up to hundreds

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CTV Analysis of Magnet-doped Microspheres
Trajectory plots of microspheres
Silica
Magnetite
Polystyrene core
2.8 mm
27
Magnetophoretic Mobility Distributions of
Magnet-doped Microspheres
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Application of the CTV system

• Cell or particle size determination
• Polystyrene beads
• Magnet-doped microsphere
• Measurement of antibody binding capacity, ABC
• Quantum Simply Cellular calibration beads
• Fibrosarcoma 2C4 model cell line
• Evaluation of separator performance
• QMS and DMS
• Saturation studies
• Breast cancer cell line (HER-2/neu, MCF-7)
• Lymphocyte NK cells

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Magnetophoretic Mobility of CD34 Cells Separated
by MACS Separator
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Magnetophoretic Mobility Distribution at
Different Labeling Conditions
Typical distribution of magnetic mobility of
unlabeled PBL and labeled HER-2/neu cells
measured by CTV
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Factors Controlling Predicted Performance
(1) Magnetophoretic mobility distribution (2)
Annular flow geometry (3) Magnetic field
strength (4) Adjustable flow conditions
The theoretical model considers all the above
factors to predict the performance of magnetic
cell separation by the QMS.
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Theoretical Model (Part1)

• Cell trajectory
• Quadrupole magnetic field
• Magnetophoretic mobility of a cell
• 3 possible outcomes
• Depleted fraction (Fa)
• Enriched fraction (Fb)
• Retained on the wall (Fw)

• Equations

Quadrupole magnetic field strength (Sm)
Laminar flow velocity profile in an annular
channel
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Theoretical Model (Part 2)
Relative positions of the ISC (?ISC) and OSC
(?OSC)
Transport lamina thickness (d)
Trajectory of a cell
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Theoretical Prediction of Cell Recovery
Effect of Total Flow Rate
Effect of Transport Lamina Thickness
Fa
Fa
Fb
Fw
Fb
Fw
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Experimental Results Breast Cancer cells
(HER-2/neu cell line)
Fa
Fb
36
Flexible Magnetic Cell Separation by the QMS
Qt X
Qt Y
Fb
Fb
Qt Z
Fb
37
Future Work

• Rare cell isolation/enrichment by the QMS
• Breast cancer cells in circulating blood
• Other type of cancer cells
• Improvement of the CTV
• Electromagnet
• Laser / Fluorescence
• In-time analysis
• Improvement of separator design
• Continuous feeding (QMS)
• Stable flows (QMS, DMS)

38
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

39
Publications

• QMS related
• Chalmers JJ, Zborowski M, Sun L, Moore L. 1998.
  Flow through, immunomagnetic cell separation.
  Biotechnol Prog 14141-148.
• Sun L, Zborowski M, Moore LR, Chalmers JJ. 1998.
  Continuous, flow-through immunomagnetic cell
  sorting in a quadrupole field. Cytometry
  33469-475.
• Williams PS, Zborowski M, Chalmers JJ. 1999. Flow
  rate optimization for the quadrupole magnetic
  cell sorter. Analytical Chemistry 713799-3807
• Zborowski M, Sun L, Moore LR, Williams PS,
  Chalmers JJ. 1999. Continuous cell separation
  using novel magnetic quadrupole flow sorter. J
  Magnetism Magnetic Materials 194224-230.
• Hoyos M, Moore LR, McCloskey KE, Margel S, Zuberi
  M, Chalmers JJ, Zborowski M. 2000. Study of
  magnetic particles pulse-injected into an annular
  SPLITT-like channel inside a quadrupole magnetic
  field. J Chromatography A and B, submitted.
• CTV related
• Chalmers JJ, Haam S, Zhao Y, McCloskey K, Moore
  L, Zborowski M, Williams PS. 1999a.
  Quantification of cellular properties form
  external fields and resulting induced velocity
  Magnetic susceptibility. Biotechnol Bioeng
  64519-526.
• Chalmers JJ, Zhao Y, Nakamura M, Melnic K, Lasky
  L, Moore L, Zborowski M. 1999b. An instrument to
  determine the magnetophoretic mobility of
  labeled, biological cells and paramagnetic
  particles. J Magnetism Magnetic Materials
  194231-241.
• 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.
• 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.