separation of lectin-binding cells using polystyrene culture devices with covalently immobilized...
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JOURNAL OF HEMATOTHERAPY 3:37-46 (1994)Mary Ann Liebert, Inc., Publishers
Separation of Lectin-Binding Cells Using Polystyrene CultureDevices with Covalently Immobilized Soybean Agglutinin
LISA R. SCHAIN, DAVID OKRONGLY, THOMAS B. OKARMA, andJANE S. LEBKOWSKI
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.
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).
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
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).
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).
Applied Immune Sciences, Inc., Santa Clara, CA 95054.
SCHAIN ET AL.
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.
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.
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.
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.
MATERIALS AND METHODS
Cell preparationBone marrow was collected into heparin from the
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.
Preparation offlasks with covalently immobilizedsoybean agglutinin
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.
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.
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.
SBA+ cell capture
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.
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.
Collection of nonadherent SBA
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
SBA SEPARATION OF BONE MARROW
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.
Soluble soybean agglutinationBMMC were suspended in DPBS at a concentration of
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.
Cell phenotypingCell populations (5 x 104-1 x 106 cells/sample) were
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.
The phenotyping panel consisted of the followingphycoerythrin and fluorescein isothiocyanate-conjugatedantibodies: mouse immunoglobulin control, anti-CD3clone