Biotechnol. Prog. 1995, 11, 208-213
200
Membrane-Based Cell Affinity Chromatography To Retrieve Viable Cells Evgenia Mandrusov? Abraham Houng? Elias Klein,$and Edward F. Leonard*,+ Artificial Organs Research Laboratory, Department of Chemical Engineering, Material Science and Metallurgy, Columbia University, New York, New York 10027, and University of Louisville, Louisville, Kentucky 40292
A novel scheme for the separation and live recovery of one cell type from a mixture of cells using a cell affinity chromatography (CAC) system is demonstrated. An antimurine IgG was chemically immobilized to a cellophane support via a carbonyldiimidazole (CDI) link. Murine splenocytes flowed over the support, a n d B-cells were allowed to attach at a shear rate of 15 s-l. Once loading was terminated, t h e support was washed at a shear rate of 315 s-l to remove nonspecifically bound cells. Elution of the B-cells was initiated by the transmembrane diffusion of hydrochloric acid (pH 11, supplied to the side of the membrane opposite the cells. At the same time, a shear flow of normal saline was established on the cell side of the membrane, and cells, freed by acid, were retrieved. Results showed that, on average, 250 cells/mm2 attached to antibody immobilized on cellophane surfaces, at a shear rate of 15 s-l, and t h a t attached cells were successfully displaced by acid supplied to the side of the membrane opposite that holding the cells. On average, at least 60% of the B-cells removed by this elution appeared viable, based on a Trypan Blue dye exclusion assay.
1. Introduction This paper demonstrates a novel scheme for the separation of one type of cell from a mixture of cells using a cell affinity chromatography (CAC) system. This scheme allows the processing of a large volume of cell suspension in order to positively select a viable, immunologically distinct clone. Cell separation methods are necessary to implement a number of living-cell therapies, such as bone marrow transplantation (Egland et al., 1993), adoptive immunotherapy for cancer (Rosenberg, 1988) and AIDS (Whiteside et al., 19931, gene therapy (DePalma, 19921, transplantation of pancreatic islet cells for the treatment of diabetes (Jung and Merrell, 19901, and myoblast transplantation for muscular dystrophy (Law et al., 1991). In living-cell therapies, selected cell types are isolated from the body, treated ex vivo, and then reinfused, either to influence other cells to secrete desired substances in correct biological order or to act as cytotoxic agents against tumors. A number of methods already exist (Amici et al., 1992; Boyem, 19911, among which magnetic bead separation technology (Grunn, 1991; Bjerke, 1993) has gained vast popularity. However, none of these methods addresses the need for a method that positively selects a large number of viable cells. Both the adsorption and release of cells from the surface involve an interaction between mechanical forces (those supplied by fluid movement as well as gravity and, when imposed, centrifugal forces) and chemical binding forces, here of both nonspecific (Bongard, 1988) and immunochemical origin (Benjamini and Leskowitz, 1988). The release process may also involve transport of the agent intended to nullify the chemical binding forces. In this study, the selective chemical force depended on the density of antibody on the support (and available for binding) and on the number of receptor molecules on the
* Author t o whom correspondence
' Columbia University.
should be addressed.
University of Louisville. 8756-7938/95/3011-0208$09.00/0
cell surface. The principal mechanical force was usually the surface shear rate, regulated by the flow rate of the cell suspension. Measurements of shear rate and antibody density are important in defining and regulating any such cell separation operation. CAC can be resolved into three consecutive steps: loading, washing, and elution. During loading, a flowing suspension of mixed cells is exposed to a ligand-bearing surface. The concentration of cells in the effluent from the region containing the immobilized ligand can be plotted as a function of time to produce a breakthrough curve, with the breakthrough point defined as the point where the feed concentration drops to one-half of its original value. Loading is terminated a t a prescribed concentration near the breakthrough point. The membrane is then washed to remove nonspecifically adhered cells. Following washing, elution is accomplished by a releasing agent supplied to the immobilized cells to dissociate the antigen-antibody bond. The scheme proposed in this paper employs a cellophane membrane with a n appropriate ligand on the cell-facing surface; the membrane support is loaded and washed as a bed of particles would be in conventional CAC, but elution is initiated by a releasing agent introduced on the side of the membrane opposite the adhered cells. The agent is thus positioned to reach the cell-support interface first, and it need never surround the bound cells. As diffusion of agent occurs, the shear flow of medium is established on the cell side of the membrane. The force of this flow facilitates the removal of cells from the surface, while its volume dilutes the releasing agent. This paper demonstrates the proposed system using murine splenocytes and a membrane whose surface bears anti-mouse IgG, which specifically binds B-lymphocytes. The effects of flow, antibody density, and the novel, as well as conventional, releasing methods are studied. 2. Materials and Methods 2.1. Cellophane Activation. Cellophane membrane (SpectrdPor 4), standard for laboratory dialysis, was cut
0 1995 American Chemical Society and American Institute of Chemical Engineers
Biotechnol. Prog., 1995,Vol. 11, No. 2
L
209
I
Figure 1. Experimental apparatus showing pumps, reservoir, flow chamber, and fraction collector.
into 9 cm2sheets and washed in distilled water to remove glycerol. The cellophane was dehydrated in acetonitrile (Sigma A-6914) for 30 min and reacted with 0.1 M carbonyldiimidazole (Sigma (2-7625) for 10 min. Sheets were then briefly washed in acetonitrile to remove unreacted CDJ and immediately incubated with 1.33 mg/ mL anti-mouse IgG whole molecule (raised in goat) (Sigma M-8642) in PBS (pH 9) overnight a t room temperature. Control membranes were incubated with 1%BSA (Sigma A-8022) in place of IgG. To remove unbound proteins, antibody- and albumin-bearing sheets were washed in 0.2 M glycine buffer (pH 2.5) for 10 min and blocked with 1% BSA for 15 min. 2.2. Enzyme-Linked Immunoadsorbent Assay (ELISA). To estimate the membrane’s protein-bearing capacity, activated membranes were exposed to 5 pg of horseradish peroxidase-conjugated anti-goat IgG (Sigma A-3540) for 10 min in 5 mL of 50 mM sodium bicarbonate buffer (pH 8.3). The membranes were then washed in PBS for 30 min and exposed to 5 mL of a substratecofactor mixture for 20 min. The mixture consisted of 42 mM 3,3’,5,5’-tetramethylbenzidine (TMB) in DMSO (Sigma T-2885), diluted 1:lOO into 100 mM sodium citrate (pH 6.0) containing 1.3mM HzOz. The absorbance of the solution was measured a t 410 nm and compared to a standard curve. The standard curve was generated using 0-10 pg of the anti-mouse IgG, physisorbed by the method of Wysocki and Sat0 (1978) onto 60 x 15 mm untreated polystyrene Petri dishes (Fisher 8-757-13A). 2.3. Cell Preparation. Fresh lymphocytes were obtained daily from murine spleens. Cells were removed from the spleen by injecting PBS into the excised but intact organ, thus inflating and disrupting the spleen pouch. Cells were filtered, washed twice, and resuspended in 2 mL of fresh Dulbecco’s Modified Eagle’s medium (DMEM) (Sigma D-5648). Cells were labeled with the fluorescent 2’,7’-bis(2-~arboxyethyl)-5(and6)carboxyfluorescein acetoxymethyl ester dye (BCECF)
(Molecular Probes, Inc.): the entire cell harvest, generally containing lo8cells, was incubated with 20 pL of the dye and 5 mL of the DMEM for 30 min in the dark, at room temperature. The cells were washed twice and resuspended in the DMEM medium containing 1%BSA by weight. All experiments were conducted using fluorescent cells, unless specified otherwise. 2.4. Experimental Apparatus. Flow experiments were conducted with the system shown in Figure 1. The activated cellophane membrane was sandwiched between the faces of two methyl methacrylate cylinders. The face of each cylinder was machined to define a flow channel that matched that in the other cylinder. One set of cylinders defined two contiguous Z-shaped paths with a rectangular cross section; another defined two spiralshaped paths with a semicircular cross section. One rectangular cross section was 1.0 mm in height ( a d , and the other (also referred to as the slit channel) was 0.2 mm in height (az).The semicircular channel had a radius of 0.55 mm. The flow properties of all channels were characterized by a friction factor:
f = k/Re
(1)
The A values for the semicircular cross section, the rectangular cross section of height al, and the slit cross section were 63.07. 59.55, and 90, respectively (Blevins, 1984). The hydraulic diameter was calculated to be 0.0672 cm for the semicircular channel, 0.1240 cm for the rectangular channel of height al, and 0.04 cm for the slit channel. The wall shear rate (the rate of change in velocity with respect to distance measured from and near the wall) was given by the following equation:
y =spflsp
(2)
Equations 1 and 2 allowed the calculation of the wall
Biotechnol. Prog., 1995, Vol. 11, No. 2
210
shear rate in each cross section as a function of the flow rate (Q = U x area of the channel cross section). Vegetable oil (Krasdale pure vegetable oil), immiscible with the cell suspension, was pumped using the first head of a dual-head syringe pump to displace an equivalent volume of cell suspension into the upper flow channel. The cells within the stirred reservoir were kept cold by UTEK refrigerant packs. A single-head syringe pump was used to supply releasing agent to the lower flow channel. The second head of the dual-head syringe pump was used to flush the upper flow chamber with saline. 2.5. Stationary Experiments = 0). Fluorescently labeled cells, a t known concentrations, were pipeted onto circles of membrane (1cm2 area), each held in one well of a 24-well culture plate (Corning 25820-241, and incubated for a specified time a t room temperature. &r incubation, the circles of membrane were removed and washed with PBS to remove nonspecifically adhered cells (Wysoki and Sato, 1978). By using an upright Olympus BH-2 microscope with a Mercury-100 light source, coverage was observed and recorded a t four locations on the surface of the membrane. 2.6. Flow Experiments (p > 0). Flow experiments were conducted in three consecutive steps: (i) loading, where cells were deposited on the activated cellophane, (ii) washing, where nonspecifically bound cells were washed off the membrane, and (iii) elution, where specifically bound cells were released into a flowing stream. During loading, oil was pumped into the stirred reservoir a t a controlled flow rate, causing the cell suspension to be supplied to the activated membrane. During elution, hydrochloric acid (pH 1)was circulated through the lower channel a t a flow rate of 0.1 mumin. At the same time, unbuffered saline flowed through the upper part of the chamber, countercurrent to the acid, a t 0.2 mumin. Effluents from the loading, washing, and elution stages were collected with a fraction collector. Concentrations of the eMuents were determined wih a Nageotte bright line 0.5 mm deep hemacytometer (Hausser Scientific) suited to measuring low cell concentrations. To observe membrane coverage, the flow chamber could be disassembled after any of these three steps. The cells that adhered to the part of the membrane in contact with the flow path were counted at a distance of 0.02 mm inward from the edges of the channel, using the Olympus microscope. Four readings per membrane were taken a t randomly chosen locations. Cell coverage was reported as the number of cells per square millimeter and was compared to the cell density required to form a monolayer (about lo4 cells/mm2). 2.7. Viability Assay. The viability of the nonfluorescing cells was determined by dye exclusion, usiong Trypan Blue dye (0.4%, w/v) (Sigma T-8154).
3. Results 3.1. Membranes were prepared as described in section 2.1, except that the concentration of antibody in which membranes were incubated was 2.0 mg/mL. The amount of immobilized IgG was determined as described in section 2.2, showing that 0.13 ,ug/cm2functionally active antibody attached to the cellophane support. Simple settling (stationary) experiments were performed to establish the membrane's ability to bind target cells. One million cells in 1cm3 of medium were placed over the membranes for times ranging from 5 to 60 min. The settling velocity of a cell, calculated using Stokes' law (Bird et al., 1960), was 0.016 c d m i n . Thus, all cells supplied to the membrane should have settled after 30 min of incubation. Figure 2 shows a linear relationship
1200
1
c.l
"
0
I
0
10
20
30
40
50
60
70
time (min) Figure 2. Stationary experiment. Influence on surface coverage of the exposure time of the cells to the membrane. The arrow indicates the time when all cells have settled. The upper curve corresponds to the active membrane, and the lower curve (almost touching the abscissa) corresponds to the control membrane.
between coverage and time, even after a time sufficient for all cells to settle onto the surface. After 60 min of incubation, the surface coverage of the activated membrane was about 1350 cells/mm2. Coverage of the control membranes remained below 100 cells/mm2. A group of membranes activated on the same day was also tested after varying storage times for the loss of IgG activity. Results indicate that membranes could be used for up t o 24 days from the time of activation. 3.2. Two experiments were conducted to optimize membrane preparation. In the first, CDI-activated cellophane membrane was incubated with increasing concentrations of IgG. By using the loading and washing procedures described earlier, 5 x lo6 cells were supplied to the membrane a t a shear rate of 5.4 s-l, in a rectangular channel of height al. Slight evidence was found for a finite maximum a t a concentration of about 3 pg/mL IgG in PBS (data not shown). In the second experiment, membranes were incubated with this IgG concentration for different periods of time. The number of adhered cells increased linearly over the first hour and then more slowly, approaching a plateau (data not shown), a surprising result since only superficial ligands should have aided cell attachment. In all subsequent experiments, CDI-immobilized membranes were incubated overnight in a solution containing 1.33pg/mL IgG. 3.3. A quantity of cells more than sufficient to form a monolayer were centrifuged for 10 min onto membrane samples in order to ascertain maximum possible coverage. Four g-levels were studied: lg, 4g, log, and 28g. Coverage reached a plateau of about 3000 cells/mm2 when a centrifugal force of 28g was applied (data not shown). 3.4. The influence of shear rate on cell attachment was investigated by using the loading and washing procedures described earlier. Cells a t a concentration of 5 x lo6cells/mL were passed over the membrane. For shear rates above 50 s-l, the semicircular channel was used, and for shear rates between 0 and 50 s-l, the rectangular channel of height a1 was used. Figure 3 shows that membrane surface coverage increases sharply with decreasing shear rate. The strength of cell adherence was also examined. In four separate experiments, the membrane was loaded and then washed a t a shear rate of either 5.4, 28, 62.5, or 315 s-l. For the three lower shear rates, the rectangular channel of height a1 was used; for 315 s-l, the slit
211
Biotechnol. Prog., 1995, Vol. 11, No. 2 1500
r
14 L
l
l
1
i
t \ .
0
1 1
9,
,
,
,
,
,
,
,
,
shear ;&e (Us)
,
i
t
0
+ :,;
100
Figure 3. Influence of shear rate on membrane surface coverage. Data represented by open circles were obtained with the rectangular channel; those represented by solid circles were
,.
i
-.,s-
' - " ' ' '
5
0
1s
10
20
25
time (min) Figure 4. Washing curve. Washing was conducted at a shear rate of 315 s-l.
obtained with the semicircular channel.
channel was used. A typical washing curve, conducted at a shear rate of 315 s-l, is shown in Figure 4. The washing was considered to be complete 'when the effluent concentration dropped below 1000 celldml. At a shear rate of 5.4 s-l, the washing process was completed in 90 min, whereas at a shear rate of 315 s-l, the process was completed in 25 min. The asymptotic surface coverage averaged 250 cells/mm2, irrespective of the shear rate a t which the surface was washed. Loading was studied using the slit flow channel. The membrane was supplied with a suspension of 1.1x lo6 cells/mL for 65 min at a shear rate of 15 s-l. Loading curves are plotted in Figure 5 for both active and control membranes. The shape of these curves is typical of all the loading experiments. No clear breakthrough point was observed. Each data set was fit with a second-order polynomial, and the difference between the fitted functions was used to estimate the number of cells bound to the membrane. The calculated coverage corresponded to 1600 celldmm2. To examine elution, cells were supplied to the slit flow channel a t a feed concentration of 1 x lo6 cells/mL and a shear rate of 15 s-l. The chamber was washed a t a shear rate of 315 s-l. Releasing agent a t pH 1 was supplied to the side of the membrane opposite the adhered cells, while saline was supplied to the cell side. After elution the chamber was disassembled. No cells were observed on the surface of the membrane. Thus, within 15min all cells were removed from the membrane. Figure 6 shows a typical elution curve. On average, the calculated number of eluted cells corresponded to 500 cells/mm2. The viability of eluted cells was assessed. The first two steps of the flow experiments were accomplished as previously described, using nonfluorescent cells in the slit flow channel at a feed concentration of 1 x l o 6 cells/mL and a shear rate of 15 s-l. The membrane was washed at a shear rate of 315 s-l. Then, in three separate runs, elution was induced with pH 5 and pH 2 acids supplied directly to the cell side of the membrane and pH 1 acid supplied to the side of the membrane opposite the cells. In each case, all cells were removed from the surface of the membrane. Figure 7 shows that, on average, the percentage of live cells in the effluent was 30%, lo%, and 60% for pH 5, pH 2, and pH 1, respectively. These elutions were carried out on the same day with the same cell preparation. A subsequent diffusive elution showed even higher viability, shown in Figure 7.
*
t
i
12
[
I
0
10
I
I
I
40
50
I T
10 v)
5:
a
30 6
4
2
0 20
30
60
70
time (min) Figure 5. Loading curves. The solid circles correspond to the active membrane and open diamonds correspond to the control
membrane.
t
n
1
0
" " " ' " ' ~ " " ' ' '
0
5
10
d
15
20
25
,
i
30
35
time (min) Figure 6. Elution curve when eluant was diffised through the membrane.
4. Discussion Cellophane membrane, chosen as the solid phase because of its permeability to the acid eluant, has not been used previously for CAC. Results reported in sections 3.1 and 3.2 showed that cellophane membranes with stable and effective cell selectivity can be prepared, but with less capacity than was expected even after the extensive effort to optimize the procedure that is described here.
Biotechnol. Prog., 1995, Vol. 11, No. 2
212 100
'
'
'
V
v
t 80
60
1
$
' J 1
V
v
1
V
V
v
v
v
v
v
v
1
v-
40
$
0
[
+
20
.,
+ ,
0 0
2
4
I
li_
6
time
8
-
LA
10
assumed assumed fiber flowrate calcd total required size required number of radius Q fiber (cells) area (cm2) fibers N R Cum) (mumin) length (cm) clone
lo6 107 lo8 lo9
40
400 4000 (0.4 m2) 40000 (4.0 m2)
2600 2600 2600 7200 33000 67000 20000
1
~-
Table 1. Envisioned Hollow Fiber CAC Devices
12
14
(min)
Figure 7. Percentage of viable cells in effluent. Cells were eluted by two different methods: (i) triangles correspond to elution via transmembrane difision with pH 1 acid; (ii) diamonds correspond to elution with acid at pH 2, and circles correspond to elution with acid at pH 5. The solid points refer to runs completed on the same day.
Less cell coverage than expected was obtained during stationary experiments. It was speculated that there were barriers, possibly charge repulsion between the cellophane surface (Benavente, 1991) and the cells (Witkowski and Mysliwski, 1991) or that adhesion was inhibited by the roughness of the cellophane surface. Another speculation was that the low coverage was due to the formation of multiple bonds between IgG and CDI (Hearn, 1987). Such bonds could hold antibody molecules flat on the membrane surface, allowing them to be active in the ELISA assay but preventing them from contacting cell surface antigens. Centrifugation experiments (section 3.3) were conducted to maximize the proximity of the cells and the immobilized antibodies. This maneuver produced a coverage of 3000 cells/mm2. This value, although about 2 times higher than that reached during simple settling (with an incubation time of 60 min, was still a small fraction of the monolayer coverage. However, since the main goal of the study was to show that the proposed CAC system was able to separate and recover live cells, efforts to raise membrane capacity were not carried further. The influence of shear rate on coverage was studied (section 3.4, preliminary experiments). It was shown that coverage increased sharply with decreasing shear rate. However, after washing, 250 cells/mm2 remained on the surface, indifferent to shear over the range tested. Thus, the number of adhered cells was extremely sensitive to the shear rate during deposition and relatively insensitive to the shear rate during washing. These observations were used in the design of subsequent experiments: washing time was shortened by using a shear rate of 315 s-l, and membrane coverage was optimized by using a shear rate of 15 s-l. The breakthrough curve for the CAC system was not sharp (section 3.4). A n exact material balance was not obtained: the actual number of cells found on the membrane after loading and washing was about one-half of the number calculated by integrating the elution curve. However, elution by transmembrane diffusion of acid, compared to elution by supplying acid directly to the immobilized cells, proved to be significantly more gentle. The CAC system proposed in this paper was successful, in that cells could be immunospecifically immobilized on the cellophane support and subsequently eluted with a t least 60% viability.
175 175 175 125 75 75 125
10
10 10 10 10
20 30
0.15 1.5 14.0 7.0 25.3 12.7 23.0
Systems of this type may be easy to realize since cellophane hollow fiber devices are produced commercially as artificial kidneys. Standard designs might easily be converted to a CAC system by immobilizing antibodies specific to the cells of interest onto the luminal surface of cellophane fibers. A typical artificial kidney consists of 12 000 cellophane fibers, 20 cm in length and 250 pm in luminal diameter, providing a surface area of about 2 m2, This already developed unit could be used to immobilize antibodies and adhere about 5 x lo8 immunospecific cells, with a large fraction recoverable in a viable state and presumably useful for living-cell therapies. Clearly, smaller (and perhaps larger) units could accommodate different clone sizes. Table 1 envisions units capable of isolating clones between 1 and 1000 million cells. A membrane capacity of 250 cells/mm2 (attached a t a shear rate of 15 s-l and subsequently washed a t a shear rate of 315 s-l) was used to calculate the total surface area needed for each unit. The number of fibers (Min each unit was calculated by using the following formula:
N = 4Q/7cR3p
(3)
which guarantees that the required shear rate will be achieved. Under normal clinical conditions, Q (the blood flow rate available to the unit) might range from 10 to 30 mL/min. Fiber radius R was chosen as 75, 125, or 175 pm. These sizes are common for cellophane fibers. The total fiber length required to accommodate the clone (the required surface area divided by the circumference of the fiber) was then divided by N to produce the length of each fiber. For the small clone size of lo6 cells, a fiber length of 0.15 cm clearly is not feasible. However, increasing the fiber length by decreasing N is not possible because N is fixed by the shear rate, blood flow rate, and fiber radius (eq 3). Therefore, a larger unit with excess surface area would be used. For a clone size of lo9 cells, surface area of 4 m2 is indicated. While such a surface is feasible from a device perspective, it could require a large quantity of antibody for activation. This problem can be solved by cyclic use of a smaller unit, since the CAC column appears capable of being regenerated and reused indefinitely. The device area will decrease in proportion to the number of cycles. CAC operation with multiple cycles may be accomplished by using either two devices alternately or one device that is on line only part of the time. If two devices are used to isolate 80% of a clone of lo9 cells (fiber length, 25.3 cm), then the flow-limited operation time, calculated by the following equation,
t = V/Q l n ( l / ( l - 0.8))
(4)
will be 13.4 h a t 10 cm3/min and 4.5 h a t 30 cm3/min. However, if one device is used, then the operation time
Biotechnol. Prog., 1995, Vol. 11, No. 2
will be increased by the time needed to wash and elute the device between each cycle. If 10 cycles are used and washing and elution take 30 min, the total cell isolation procedure will be increased by 5 h. In either case, increasing the number of cycles corresponds to decreasing the area of the device and reduces the antibody requirement. The purpose of this work was to model and evaluate a proposed CAC system in which cells are immunospecifically adhered to IgG immobilized onto a cellophane support, from which they can be eluted by acid supplied to the side of the membrane opposite the cells. Even though the system did not demonstrate a sharp breakthrough curve and the cell material balance was not closed, the model system indicates that a clinically useful system is feasible and attractive. It should be possible to obtain high yields of viable cells. Calculations for prospective CAC units demonstrate that a lower than expected membrane capacity for cells is not a n impediment to a successful system.
Notation a C
co f k N
Q R Re U t V Y P
r
height of rectangular duct, cm concentration, cells/mL initial concentration, celldml dimensionless friction factor dimensionless friction coefficient number of fibers flow rate, mumin radius of cellophane fiber, pm Reynolds number velocity of cell suspension, c d m i n time, min blood volume, mL shear rate, s-l viscosity, g4cm.s) fluid density, g/cm3
Acknowledgment The authors acknowledge financial support from National Institutes of Health Grants HL-38306 and HL44535, from contributions to the Artificial Organs Research Fund at Columbia University, and from the International Exposition Company's Tomkowit scholarship, awarded to E.M.
Literature Cited Amici, C. Human Peripheral Blood Mononuclear Cell Subfractionation Using Counterflow Centrifugation. Haematologia 1991, 70 (21, 89-93.
213 Benavente, J . A Study on the Variation with Temperature of Fixed Charge and Membrane Structure of Cellophane Membrane. Sep. Sei. Technol. 1991,26(2), 189-198 Benjamini, E.; Leskowitz, S. Immunology: A Short Course; Alan R. Liss, Inc.: New York, 1988. Bird, R. B.; Stewart, W. E.; Lightfoot, E. N. Transport Phenomena; John Wiley and Sons: New York, 1960; pp 519-553. Bjerke, T. Purification of Human Blood Basophiles by Negative Selection Using Immunomagnetic Beads. J . Zmmunol. Methods 1993,157 (1-81, 49-51. Blevins, R. D. Applied Fluid Dynamics Handbook; Van Nostrand Reinhold Company: New York, 1984; pp 38-47. Bongrand, P. Physical Basis of Cell-Cell Adhesion; CRC Press: Boca Raton, FL, 1988. Boyum, A. Separation of Leukocytes; Improved Cell Purity by Fine Adjustment of Gradient Medium Density and Osmolality. Scand. J . Zmmunol. 1991,34 (61, 697-712. DePalma, A.; CellPro, Inc. Tests its Stem Cell Therapy in Clinic Trials. Genet. Eng. News 1992 (May). Egeland, T.; Tjonnfijor, G.; Steen, R.; Gaudernack, G.; Thorsby, E. Positive Selection of Bone Marrow-Derived CD43 Positive Cells for Possible Stem Cell Transplantation. Transplant. Proc. 1993,1261-1263. Grunn, B. Model Experiments for Immunomagnetic Elimination of Leukemic Cells from Human Bone Marrow. Zmmunobiology 1991,83 (51, 374-85. Hearn, M. T. W. CDI-mediated Immobilization of Enzymes and M i n i t y Ligands. Methods Enzymol. 1987,135(B), 102-17. Jung, P. J.; Merrell, R. C. Update on Pancreatic Islet Cell Separation. Semin. Surg. Oncol. 1990,6 (21, 122-5. Law, P. K.; Goodwin, T. G.; Fang, 0. W.; Chen, M.; Li, H. J.; Florendo, J. A.; Kirby, D. S. Myoblast Transfer Therapy of Duchenne Muscular Dystrophy. Acta Paediatr. Jpn. 1991, 33 (2), 206-15. Rosenberg, S. A. The Development of New Immunotherapies for the Treatment of Cancer Using Interleukin-2. Ann. Surg. 1988,208 (2), 121-135. Teshigawara, K.; Maeda, M.; Nishino, K.; Nikaido, T.; Uchiyama, T.; Tsudo, M.; Wano, Y.; Yodoi, J. Adult T Leukemia Cells Produce A Lymphokine that Augments Interleukin-2 Receptor Expression. J . Mol. Cell. Zmmunol. 1985,2 (l), 17-26. Witkowski, J. M.; Mysliwski, A. Influence of Age on Transmembrane Potential and Cell Surface Charge of Human Peripheral Blood Lymphocytes Estimated with Fluorescent Probes. Folia Histochem. Cytobiol. 1991,29(31, 115-20. Wysocki, L. J.; Sato, V. L. "Panning" for Lymphocytes: A Method for Cell Selection. Proc. Natl. Acad. Sci. U S A . 1978, 75 (61, 2844-2848. Accepted October 5, 1994.@ BP940077P Abstract published in Advance ACS Abstracts, December 15, 1994. @
