Separation of Lectin-Binding Cells Using Polystyrene Culture Devices with Covalently Immobilized Soybean Agglutinin

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<ul><li><p>JOURNAL OF HEMATOTHERAPY 3:37-46 (1994)Mary Ann Liebert, Inc., Publishers</p><p>Separation of Lectin-Binding Cells Using Polystyrene CultureDevices with Covalently Immobilized Soybean Agglutinin</p><p>LISA R. SCHAIN, DAVID OKRONGLY, THOMAS B. OKARMA, andJANE S. LEBKOWSKI</p><p>ABSTRACT</p><p>The plant lectin, soybean agglutinin (SBA), has been widely used to separate heterogeneouspopulations of cells. In the field of bone marrow transplantation, SBA has been used for partialdepletion of T cells from bone marrow allografts to reduce graft-vs.-host disease. SBA's high affinityfor many different tumor cells has also indicated its use as a tumor purging agent for autologous bonemarrow transplants. We have compared two methods of cell separation using either soluble SBAagglutination, or SBA covalently attached to an activated polystyrene surface. The nonbinding SBA-cell populations generated by these two procedures were very similar in terms of cell recovery, lightscatter properties, and phenotypic profile. Notably, both SBA- fractions were enriched in cells withthe known progenitor markers, CD34, CD33, and HLA-DR, and were relatively depleted of SBAbinding cells. In addition, the activity of each SBA- cell population was measured in vitro inshort-term progenitor assays. Here, both SBA- populations were significantly enriched for CFU-GM. When device-separated SBA- cell populations were seeded into long-term bone marrow culture,they produced both increased progenitor activity and cell proliferation compared to unseparatedBMMCs. The polystyrene technology described here could reduce or eliminate many of thedrawbacks of soluble SBA agglutination, making SBA cell separation a viable and convenienttechnique for clinical application.</p><p>INTRODUCTION</p><p>THE PLANT LECTIN, SOYBEAN AGGLUTININ (SBA) is atetrameric glycoprotein that, in its native form, existsprimarily as -pleated sheets ( 1 ). Each of its four subunitsis 30 kDa, with an abundance of acidic and hydroxylicamino acid residues. One SBA molecule contains fourcarbohydrate binding sites, which have the highest affin-ity for N-acetylgalactosamine and its derivatives (1).</p><p>Historically SBA has been used for a variety of pur-poses, for example, to purify glycoproteins and to sepa-rate heterogeneous cell populations. In mice, SBA can be</p><p>used to fractionate splenocytes into B cells (SBA+) and Tcells (SBA-) (2). Moreover, murine hematopoietic stemcells bind both SBA and peanut agglutinin (PNA), and,when isolated, will engraft allogeneic recipients withoutoccurrence of graft-vs.-host disease (3,4).</p><p>In humans, SBA has been used to fractionate a varietyof cell types. SBA agglutinates both B and T cells and hasbeen used to crudely separate T helper (SBA+) and Tsuppressor (SBA-) cells (5). In addition, SBA binds tocells transformed by viral or chemical agents, and, as aresult, has been suggested as a tumor-purging agent inautologous bone marrow transplantation (6-8).</p><p>Applied Immune Sciences, Inc., Santa Clara, CA 95054.</p><p>37</p></li><li><p>SCHAIN ET AL.</p><p>Lastly, soybean agglutinin has been used extensively toprocess allogeneic bone marrow grafts (9,10). SBA bindsapproximately 60-90% of all bone marrow mononuclearcells, including fibroblasts, red blood cells, stromal cells,and mature cells of both the myeloid and lymphoidlineages, yet depletes only 10-25% of hematopoieticprogenitor activity (11,12). Over 400 bone marrow trans-plants that have been T cell depleted using SBA have beenused to engraft patients (9,10) clearly demonstrating thatSBA has minimal toxic effects on hematopoietic stemcells.</p><p>Soluble soybean agglutination is a process commonlyused in the laboratory to separate heterogeneous mixturesof cells, and, though it is effective in removing the desiredSBA+ population of cells, nonspecific trapping of SBA-cells is problematic. Soluble agglutination has otherdrawbacks, especially in the clinical setting. A standardbone marrow graft contains approximately 2-10 x 109bone marrow mononuclear cells. Using the standardagglutination process with a sample of this size is bothtime-consuming and cumbersome. The potential expo-sure to outside contaminants during this procedure isappreciable.</p><p>In order to take advantage of the binding properties ofSBA, yet minimize the problems associated with theagglutination process, we covalently attached SBA toderivitized polystyrene tissue culture flasks and usedthese devices to separate cells. Theoretically, the SBAdevices should be able to bind the same SBA+ cells whileleaving the SBA- cells in the nonadherent cell fraction.</p><p>In this report, we compare SBA- cell populationsgenerated by either a standard agglutination procedure, orby flasks with covalently immobilized SBA. To assess theequivalence of these two populations, bone marrowmononuclear cells were isolated from normal donors andsubjected to either soluble soybean agglutination or tocapture on the SBA devices. The two SBA- populationsproved to be similar in size, phenotype, and hematopoie-tic function.</p><p>MATERIALS AND METHODS</p><p>Cell preparationBone marrow was collected into heparin from the</p><p>posterior iliac crest of normal adult volunteers by standardprocedures. The marrow was then diluted 1:16 withDulbecco's phosphate-buffered salineCa2+-Mg2+ free(DPBS-CMF; Life Technologies, Grand Island, NY)1mM ethylenediaminetetraacetic acid (EDTA; SigmaChemical Co., St. Louis, MO), and low-density bonemarrow mononuclear cells (BMMC) were isolated onFicoll-Hypaque density gradients after centrifugation atlOOOg for 20 min at room temperature.</p><p>Preparation offlasks with covalently immobilizedsoybean agglutinin</p><p>The devices containing covalently immobilized SBA(AIS MicroCELLector SBA and AIS CELLector SBA)were prepared by first chemically modifying the surfaceof untreated polystyrene T-25 flasks (Corning, Coming,NY), and then covalently attaching the soybean lectin. Toderivitize the surface polystyrene, the surface Styrolgroups were substituted with bromoacetamide groups bythe amidoalkylation reaction of /V-(hydroxymethyl)-2-bromoacetamide (0.1 M) and trifluoromethanesulfonicacid (1.0 M) (Aldrich Chemical, Milwaukee, WI) intetramethylene sulfone (Phillips Petroleum, Bartlesville,OK) for 2 hr at room temperature. The flasks wererepeatedly washed with large volumes of water, thenrinsed several times with ethanol, and air dried. Becausethe bromoacetamide group is stable at room temperaturefor several months, 25-50 "activated" flasks were pre-pared at a time and stored for coupling until needed.</p><p>Soybean agglutinin was covalently attached to thebromoacetamide activated T-25 flasks by coating eachflask with 5 ml of a solution containing 50 pg/ml SBA(Vector Labs, Burlingame, CA) in DPBS for 2 hr at roomtemperature. After coupling, the solution was removedfrom each device and the flasks were rinsed 10 times withDPBS. The remaining activated sites were blocked withhuman serum albumin. The excess blocking solution waspoured off and the flasks were dried under vacuum.Sterilization of the devices was achieved by electron beamirradiation totaling 2.7 Mrad (IRT, San Diego, CA).Flasks were then stored at 4C.</p><p>Just prior to use, each SBA device was rehydrated withfour 10 ml rinses with DPBS. The last wash was left on thebinding surface until the flask was ready to be used.</p><p>SBA+ cell capture</p><p>BMMC were suspended in a solution of DPBS contain-ing 0.5% Gamimune (Cutter Biological, West Haven,CT) at a concentration of 5 x 106 cells/ml and incubatedfor 30 min at room temperature.</p><p>Immediately following removal of the last rehydrationwash from the SBA devices, 4 ml of cells (2 x 107 cells)was loaded into each SBA flask and the devices wererocked to coat the binding surface. To allow cell capture,the flasks were then incubated for 1 hr at room tempera-ture on a level, vibration-free surface.</p><p>Collection of nonadherent SBA</p><p>cells</p><p>After the 1-hr cell capture, the SBA flasks were gentlyrocked, allowing the buffer to flow across the bindingsurface and the nonadherent cells were collected. Each</p><p>38</p></li><li><p>SBA SEPARATION OF BONE MARROW</p><p>flask was then washed gently two times with 4 ml DPBS.For each wash, the DPBS was pipetted down a nonbind-ing surface in order to avoid disruption of the adherent celllayer. The flask was then capped and the fluid was gentlyrocked over the binding surface as before. Each wash waspooled with the nonadherent cells. The adherent SBA+cells can be recovered after incubation of these cells withRPMI-1640 containing 200 mM Af-acetylgalactosaminefor 5-30 min at 37C. These SBA+ cells can be used forfurther experimentation.</p><p>Soluble soybean agglutinationBMMC were suspended in DPBS at a concentration of</p><p>3.0 x 108 cells/ml. An equal volume of SBA (2 mg/ml)was added to the cells and the suspension was agitated byhand for 2 min at room temperature. The cell suspensionwas then overlaid onto 8 ml of DPBS containing 5% BSA(Sigma). The agglutinated cells were allowed to settle bygravity for 5-10 min at room temperature on a vibration-free surface. The SBA- cells were then collected from thetop of the tube, leaving behind the interface and theagglutinated cells penetrating the BSA layer. The SBA-cells were then washed once with 10 mM D-(+)-galactose(Sigma) in DPBS and resuspended in DPBS.</p><p>Cell phenotypingCell populations (5 x 104-1 x 106 cells/sample) were</p><p>stained with various fluorochrome-labeled antibodies bystandard methods. Samples were analyzed on an OrthoCytofluorograf Ils optical bench with a 2151 computer(Ortho Diagnostic Systems, Westwood MA). A Lexel 75argon laser (Cooper LaserSonics, Santa Clara, CA),emitting in light mode 50 mW of power at 488 nm was anillumination source.</p><p>The phenotyping panel consisted of the followingphycoerythrin and fluorescein isothiocyanate-conjugatedantibodies: mouse immunoglobulin control, anti-CD3clone #SK7, anti-CDIO clone #W8E7, anti-CD14 clone#M/-P9, anti-CD 15 clone #MMA, anti-CD 16 clone#NKP15, anti-CD19 clone #467, anti-CD20 clone#L27, anti-CD33 clone #P676, anti-HLA-DR clone#L243, anti-CD34 clone #MY10 (Becton Dickinson,San Jose, CA), sheep F(ab)2-anti-mouse IgG (New En-gland Nuclear, Boston, MA).</p><p>Colony assaysCells (5 x 105) were suspended in 6 ml of Iscoves</p><p>modified Dulbecco's medium (IMDM) (Life Technolo-gies) containing 20% fetal bovine serum, penicillin/streptomycin (100 U/100 g/ml; Life Technologies), 5ng/ml interleukin-1, 10 ng/ml interleukin-3, 10 ng/ml</p><p>interleukin-6 (Amgen Biologies, Thousand Oaks, CA),plus 0.3% Bacto Agar (Difco Labs, Detroit, MI). Thissuspension was then plated into 2 wells of a 6-well plate (3ml/well) and incubated at 37C in humidified air contain-ing 5% or 7% C02. Fourteen days later the plates werecounted for CFU-GM by visual inspection with an in-verted microscope.</p><p>More recently, CFUs were assayed using media con-taining methylcellulose plus 5% PHA-LCM (Terry FoxLaboratory, Vancouver). These cultures were incubatedas above and CFU-GM, BFU-E, and CFU-GEMM werecounted 14 days later.</p><p>Long-term bone marrow culture assaysBone marrow stromal layers were established by seed-</p><p>ing 2-4x 106 BMMC into T-25 tissue culture flasks(Corning) containing McCoy's complete growth media(13). Cultures were incubated at 37C in a humidifiedchamber containing 5% or 7% C02 for 2-4 weeks.Freshly prepared medium was used for complete mediachanges as needed.</p><p>Prior to seeding, well-established stromal cultures wereirradiated with 25 Gy and their media was changed.Within 48 hr after irradiation, 2-10 x 105 cells wereseeded into each irradiated stromal culture flask andincubated at 37C in 5% or 7% C02. Equal numbers ofeach cell population were used to seed the stromal layers.Each cell population was seeded in triplicate. Once aweek for the following 6 weeks, nonadherent cells werecollected; cells from similar populations were then pooledand counted. Cells (4 x 105) from each population wereplated for progenitor colony assays. The remaining cellsfrom each population were distributed to the original threeflasks and returned to the incubator until the followingweek.</p><p>RESULTS</p><p>Bone marrow mononuclear cell samples from 5 healthydonors were split and simultaneously processed usingeither SBA polystyrene devices or soluble SBA aggluti-nation to directly compare these cell separation proce-dures. The number of cells recovered after either SBAtreatment was determined (Table 1). After soluble SBAagglutination, 25% (range 10-33%) of BMMC wererecovered. Likewise, after use of the SBA devices, anaverage of 27% (range 12-40%) of the input cells werecollected as the nonadherent, SBA- cells. These directstudies suggest that these two procedures produced simi-lar SBA- cell recoveries (p = not significant).</p><p>Further support for this conclusion comes from 90additional experiments where either agglutination or SBAflask capture was independently performed. In these tests,</p><p>39</p></li><li><p>SCHAIN ET AL.</p><p>Table 1. Number of Cells Recovered afterSBA Treatment"</p><p>Cell Recovery</p><p>Experiment Agglutination SBA device SABCDE</p><p>Mean</p><p>2410333129</p><p>25</p><p>4012153139</p><p>27</p><p>"The percent of cells recovered for each SBA- cell fraction isshown for both agglutination and SBA device separation. Thesefigures were calculated for five separate experiments and theiraverages are presented at the bottom.</p><p>32.2% of the BMMC (n = 22 experiments, SD = 13.2)were recovered after SBA agglutination. Similarly 26.2%(n = 90 experiments, SD = 15.3) of the BMMC werepresent in the SBA- fraction after flask capture (data notshown).</p><p>The binding of cells to the SBA flask was specific forthe immobilized SBA lectin. Devices coated with anirrelevant protein, human serum albumin, failed to bindcells. Moreover, those cells bound to the SBA flask couldbe completely removed using 200 mM N-acetylgalac-tosamine in RPMI-1640 medium for 10-30 min at 37C.</p><p>The cell size and granularity of each cell populationwere studied by light scatter flow cytometry (Fig. 1). Theinput BMMC had a large number of small, agranularcells, consisting mostly of mature lymphocytes (Fig. 1).These small cells made up 44% (range 35-55%) of allBMMC, but comprised only 27% (range 14-36%) and24% (range 14-31%) of the SBA- cells produced aftersoluble agglutination or device separation, respectively.Moreover, both treatments produced SBA- populationsenriched in large granular cells, which rose from 30%(range 20-40%) of the total BMMC to 46% (range40-52%) of SBA- agglutinated cells and 52% (range43-67%) of SBA device nonadherent cells. Neither treat-ment produced a significant change in the percent ofmedium to large agranular cells. These results indicatethat the morphological characteristics of these two SBApopulations are very similar.</p><p>To determine more conclusively that both procedureslead to the binding of SBA+ cells, the BMMC and finaltwo SBA" fractions were phenotyped using an SBA-FITC conjugate. This analysis was performed to quantifythe specific depletion of SBA+ cells. Figure 2 shows that30% of the input population stain brightly with SBA+ asmeasured by SBA-FITC flow cytometry. After agglutina-tion or flask binding, the frequency of SBA+ cells in theSBA- fractions was red...</p></li></ul>