Autologous Bone Marrow Transplantation - Blog Science Connections

134 Magnetic Separation of Marrow Cells
solid Co 2
B. A second addition of the cobalt-citrate complex results in the
deposition of additional Co 2
B onto these nuclei to form spherical particles
30-60 nm in diameter. Affinity-purified avidin is added, binding essentially
irreversibly to the surface of the colloid. The solution is stabilized with human
serum albumin and to avoid dissolution of the Co 2
B particles passivated with
potassium dichromate. The fluid is then concentrated by tangential filtration
(Amicon, Lexington, MA) and purified by gel permeation chromatography on
Sepharose CL-6B-200 (Sigma, St. Louis, MO). The MAC is adjusted to 20
OD 4 0 0
/ml, filtered through a 0.45-rim Millex-HA filter unit (Millipore, Bedford,
MA), and is ready for use. The entire procedure requires about 3 hours.
Bone marrow mononuclear cells are purified by discontinuous Percoll
gradient centrifugation (5). The cells (5 * 10 7 /ml) are incubated with cocktails
of MAbs at concentrations previously determined to be saturating for 30
minutes at 4° in 10-mM phosphate-buffered saline (PBS) (pH 7.4) containing 2%
fetal bovine serum (FBS). The cells are washed twice by centrifugation in PBS
(2% FBS) and incubated at 5 x 10 7 /ml with a combination of a biotinylated
affinity-purified IgG fraction of goat antimouse immunoglobulins (B-GAM) at
100 uql'ml and biotinylated soybean agglutinin (B-SBA) at 100 uq/rvX in PBS
(2% FBS) for 30 minutes on ice. The cells are again washed twice and incubated
at 5 x 107ml with MAC (at 20 OD 400
/ml) for 30 minutes on ice. The MAC
procedure is shown in Figure 1. The cells are pelleted, resuspended at 5 x 10 7
cells/ml in PBS (2% FBS), and separated on a high-magnetic gradient column.
The column (1.9 x 40 cm) is made by stacking 100 tinned steel screen disks
(3/4-in diameter, .0065-in wire, 60 mesh; Tetko, Elmsford, NY) in a 20-ml
syringe (Becton Dickinson, Rutherford, NJ) and fitting it with an external
samarium cobalt permanent magnet (2.25 * 0.55 x 0.6 cm, grade 25,
magnitude point parallel to a 0.6-cm dimension; Permag, Richardson, TX). The
syringe is fitted with a rubber stopper at the top and a three-way stopcock at the
bottom and is sterilized with ethylene oxide. The cells pass through the column
in PBS (2% FBS) at 1 ml/minute, and the column is washed at 5 ml/minute with
100 ml of PBS (2% FBS). For clinical separations with larger numbers of cells,
two such columns are arranged in tandem. The separation apparatus is
depicted in Figure 2.
The cell suspensions are analyzed before and after MAC separation by flow
cytometry and for clonogenic target cells and normal hematopoietic progenitors.
The cells are stained with MAbs, B-GAM and B-SBA, and fluorescein
isothiocyanate (FITC) avidin, and analyzed on either an Ortho 2150 or a Coulter
Epics C flow cytometer. The control cells are treated similarly, except that the
FITC avidin is preincubated with an excess of biotin. T cells are analyzed in a
limiting dilution clonal assay we have previously described (6). Hematopoietic
progenitors are analyzed by colony formation in methylcellulose for
granulocyte-macrophage colony-forming units (CFUs-GM), erythroid burstforming
unit (BFU-E), and mixed colonies arising from granulocyte-erythrocytemacrophage-megakaryocyte
colony-forming units (CFCls-GEMM) and in agar