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Isolation and enrichment of rare cells with an immunomagnetic cell selection system

K. Wagner, C. Jörke, T. Kroll, K. Höffken, J.H. Clement
Clinic for Internal Medicine II, Dept. of Haematology, Oncology, Bone marrow transplantation
University Clinic Jena

Introduction
Cells of biomedical interest are, despite their functional significance, often present in very small numbers. Therefore the analysis and isolation of previously inaccessible rare cells, e.g. peripheral
haematopoietic stem cells or circulating tumor cells, require efficient, sensitive and specific procedures that work under physiological conditions.
Furthermore the automation of laboratory processes is an important requirement for streamlining and standardizing technical procedures.
Methods RoboSep®

Allows positive or negative selection of
RoboSep® (StemCell Technologies Inc.) Schematic of Easy Sep® TACs
Magnetic labeling of cells
Schematic of a positive selection procedure:
Incubation and separation regiment may be varied
virtually any cell type from any single
cell suspension or species. Isolates cells
automates an immunomagnetic cell for either optimizing purity or recovery.
Robotic arm: uses 1 ml tips to
directly from any sample source
including whole blood. Isolated cells are
selection procedure (Easy Sep®) that uses A Labeling of cells
add reagents and 5 ml tips to add
buffer and transfer or mix cell
immediately
cytometry,
available for
functional studies,
flow
or Fits into a standard
specific antibodies and tiny magnetic sample. downstream analysis. biocontainment hood for
sterile operation.
nanoparticles in a column-free magnetic incubation
(usually about 15 min)
incubation
(usually about 10 min)

system. Cells can be targeted either for
Tetrameric Antibody magnetic

selection or depletion using monoclonal TACs are comprised of two mouse IgG1 monoclonal
Complex (TAC) nanoparticles
Technical specifications :
cell suspension at a concentration of 1-2 x 108 nucleated cells per ml

antibodies directed against cell surface antibodies held in tetrameric array by two rat anti-
mouse IgG1 monoclonal antibody molecules. One
Dimensions:
• Height with lid: 56 cm
antigens. These labeled cells are then mouse antibody recognizes the specific cell surface
antigen while the other recognizes dextran on the
B Separation of labeled cells Carousel: each quadrant
• Width: 71 cm
• Depth: 39 cm
contains a magnet, sample, tip
crosslinked to magnetic dextran iron magnetic particles.
(http://www.stemcell.com/technical/28960_RoboSep_v4.pdf)
rack and reagents. This enables
• Weight: 26 kg
Power Requirements:
separation of up to 4 samples in
nanoparticles and separated in a specially + fraction - fraction
parallel.
• 50/60 Hz, AC 100-240V
• Connections: RJ-45 10/100 Ethernet port,
designed ring magnet. Unlike larger 2 USB ports
Conditions for Operation:
microparticles these nanoparticles do not • Temperature: 10-30°C (50-86°F). RoboSep®
is not specified for use in a cold room (4°C, 39°F)
interfere with subsequent analysis (e.g. • Humidity 20-85% (non-condensing)

FACS). We used the RoboSep® System to usually 3 x 5-minute
separations in the ring Intuitive touch screen operation: after
magnet
separate rare cells from the vast majority It is also possible to use a biotin-, PE-, APC- or FITC-
selecting the relevant protocol, a list of
required reagents and resources is displayed. Tip racks: each disposable rack
Capacity:
of peripheral blood cells. Selected cell conjugated, or any mouse IgG1 antibody directed
against a cell surface antigen on your cell of interest.
The instrument calculates the reagent
addition volume based on sample volume. Positive selection: up to 2x109 each sample
contains sterile tips to process a
single sample.
Negative selection: 1x109 each sample
subpopulations were analyzed by (http://www.stemcell.com/technical/28960_RoboSep_v4.pdf)
Sample volume: from 250 µl to 8,5 ml per sample

immunohistochemistry, immunofluores-
cence and flow cytometry.
http://www.stemcell.com/product_catalog/robosepinstrument.aspx

cells in +fraction
Results: Immunofluorescence Flow cytometry
Immunohistochemistry (modified) ABC-method

Initially CD90-positive cells were
enriched from the leucocyte- apoptotic

fraction of peripheral blood. unstained control

These cell population was further CD90 -fraction

CD90 +fraction

characterized. non-apoptotic
CD29 CD90

It is possible to isolate high yields
CD90-positivity is detected with a fluorescence- The use of labeled antibodies (e.g. CD90- CD90-positivity is detected by flow cytometry,
of vital CD90-targeted cells with CD90-positivity is detectable by a
biotinylated secondary antibody which
CD29 is detected using a biotinylated
primary antibody to avoid a parallel
The cell vitality is detected by staining cells
with 7AAD, which labels the nucleus of scanning-cytometer (ScanR) by labeling FITC) for separation allows the analysis of using a fluorescence (AF488, green)-labeled
targeted cells with a fluorescence (AF488, targeted cells in comparison to other secondary antibody which recognizes the
the RoboSep® Sytem. recognizes the CD90-antibody bound
during separation.
recognition of the antibody used for
separation (CD90).
apoptotic cells (red).
green)-labeled secondary antibody which markers, e.g. CD45. CD90-antibody bound during separation.
recognizes the CD90-antibody bound during
separation. The nucleus of apoptotic cells is
Conclusions: stained with 7AAD (red).

The magnet-assisted cell sorting system RoboSep® is a suitable device for reliable enrichment of rare cell populations.