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Journal of Magnetism and Magnetic Materials 321 (2009) 1635–1638

Contents lists available at ScienceDirect

Journal of Magnetism and Magnetic Materials

0304-88

doi:10.1

� Corr

E-m

journal homepage: www.elsevier.com/locate/jmmm

Application of magnetic poly(styrene–glycidyl methacrylate) microspheresfor immunomagnetic separation of bone marrow cells

Ting-Hao Chung, Jing-Yi Chang, Wen-Chien Lee �

Department of Chemical Engineering, National Chung Cheng University, Chiayi 621, Taiwan

a r t i c l e i n f o

Available online 21 February 2009

Keywords:

Magnetic microsphere

PS–GMA

Antibody immobilization

Immunomagnetic cell separation

Bone marrow cells

53/$ - see front matter & 2009 Elsevier B.V. A

016/j.jmmm.2009.02.103

esponding author. Tel.: +886 5 2428152; fax:

ail address: chmwcl@ccu.edu.tw (W.-C. Lee).

a b s t r a c t

Surface-functionalized magnetic poly(styrene–glycidyl methacrylate) (PS–GMA) microspheres were

prepared and coupled with Sca-1 antibody for cell selection from murine bone marrow mononuclear

cells (MNCs). Biotinylated Sca-1 antibody could be directly coupled to avidin-bound magnetic

microspheres. Alternatively, oxidized goat anti-mouse antibody was covalently bound onto the amino

group-containing magnetic microspheres in a site-directed manner, and the resultant conjugate was

coupled with non-modified Sca-1 antibody. Using the indirect antibody-bound magnetic microspheres,

the purity of isolated Sca-1+ cells increased with bead-to-cell ratio. Using a bead-to-cell ratio of 10

beads/cell, a purity of 85% Sca-1+ cells corresponding to a 17-fold enrichment was achieved.

& 2009 Elsevier B.V. All rights reserved.

0. Introduction

Bone marrow is rich of stem cell populations includinghematopoietic stem cells (HSCs) and mesenchymal stem cells(MSCs). These two stem cell populations are conventionallycharacterized as suspended and adherent mononuclear cells(MNCs), respectively. Both are able to self-renew and to dif-ferentiate towards specific cell lineages. HSCs are commonlyisolated from the bone marrow based on specific proteins on cellsurface [1]. For example, mouse HSCs can be characterized bySca-1+Lin�c-kit+ cells. Stem cells which are selected based onthese surface markers are long-term HSCs [2,3]. Similarly, surfacemarkers for human HSCs have also been reported [4]. Purificationof HSCs from murine bone marrow is conventionally achieved by aprocedure based on the their expression of cell surface antigen-1(Sca-1) and the lack of expression of cell surface antigensassociated with differentiated hematopoietic cell lineages typi-cally for B cells, myelomonocytic cells, and T cells. The purificationprocedure results in obtaining Sac-1-positive and lineage-nega-tive (Sca-1+Lin�) cells, which are widely used for transplantationand in vitro differentiation study [5–10]. Enriched Sca-1+ cells inthe population of HSCs are useful for transplantation in studies ofHSC-based gene or cell therapies, because of their propensity ofhoming to bone marrow [11]. In the present study, murine Sca-1+

cells were enriched from whole bone marrow by immunomag-netic separation. Most of the isolated Sca-1 positive cells werefound to express also c-kit antigen.

The immunomagnetic cell separation method has becomepopular among not only cell biologists, but also medical profes-

ll rights reserved.

+886 5 2721206.

sionals. In this approach, a magnetic field is used for selectiveisolation of cells labeled with magnetic particles [12–14]. Cells areselectively bound onto magnetic particles via the cell surfaceprotein recognized by its antibody, which has been conjugated onthe magnetic particles. On the coupling of antibody to magneticparticles, oriented immobilization of antibody is very importantfor the specific recognition of antigen on cell surface. Theimmobilization via unique sites in the Fc portion of the antibodymolecule could ensure orientation of the antigen-combining sitesof antibody towards the mobile phase [15,16]. The orientedimmobilization of biologically active proteins like antibodyprovides advantages of good steric accessibilities of active bindingsites and increased stability [17]. The present paper describes theuse of magnetic poly(styrene–glycidyl methacrylate) (PS–GMA)microspheres in the micro-size range, as the carriers for antibodycoupling. Functional groups of the polymer provide the surfacechemistry for oriented immobilization of antibody to recognizeand bind the surface proteins of particular cells. The conventionalmethod to introduce functional groups on the surface of magneticpolymer particles is to form the polymer layer using copolymer-ization of monomers, which include at least one functionalmonomer. In addition to GMA, methacrylic acid [18], methacrylate[19], and the combination of these [20] are often used as thefunctional monomers for the preparation of styrene- anddivinylbenzene-based magnetic microparticles.

1. Materials and methods

1.1. Preparation of functionalized PS–GMA magnetic microspheres

Functionalized PS–GMA magnetic microspheres were preparedby two different schemes (Fig. 1). In the first scheme, magnetic

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PS–GMA microspheres were prepared by a swelling and penetra-tion process [21] and then chemically modified to introduceamino groups on their surface. Briefly, pre-made, non-porousPS–GMA particles of micron size (ca. 2.3mm) were swollen by amixture of N-methyl-2-pyrrolidone (NMP), sodium dodecylsulfate (SDS) and water, and then incubated with a dispersion ofsuperparamagnetic nanoparticles in deionized (DI) water. Themagnetic nanoparticles were allowed to diffuse into polymer micro-spheres during the incubation. Finally, the superparamagneticnanoparticles were entrapped and the PS–GMA microspheresbecame superparamagnetic. To yield amino group-containingmagnetic microspheres, 1 g magnetic PS–GMA microsphereswere suspended in 18 ml of methanol and mixed with 2 gethylenediamine and the resultant mixture was allowed forreaction at 60 1C for 24 h.

The superparamagnetic nanoparticles were synthesized byco-precipitating Fe(II) and Fe(III) ions in a molar ratio of 1:2 in analkaline medium. After a treatment with HCl, the precipitate wascollected by centrifugation and dispersed in deionized water toyield superparamagnetic iron oxide (magnetite) nanoparticleswith an average particle size of about 10 nm. This method wassimilar to the one for the preparation of cobalt ferrite nanopar-ticles, but no step for passivation, peptization, or surface coatingwas taken [22,23].

According to Scheme 2, PS–GMA microspheres were firstchemically modified in order to introduce functional groups ontheir surface and then handled to form magnetic microspheres bythe swelling and penetration process. To yield carboxyl group-containing magnetic microspheres, non-magnetic PS–GMA micro-spheres were chemically modified with ethylenediamine to yieldamino groups on their surface, which were later converted tocarboxyl groups by reacting to succinic anhydride. For the latterreaction, 0.5 g ethylenediamine-modified PS–GMA particles weremixed with 1 g succinic anhydride and 50 ml methanol. Theresultant mixture was purged by N2 to remove oxygen andallowed to react at 50 1C for 48 h. The resulting non-magnetic,carboxyl group-containing microspheres were then swollen by amixture of NMP, SDS, and water and incubated with the aqueousdispersion of superparamagnetic nanoparticles. Finally, magneticmicrospheres containing carboxyl groups on the surface wereobtained.

1.2. Coupling of antibody on magnetic microspheres

Before coupling onto the functionalized, magnetic PS–GMAmicrospheres, the secondary antibody (goat anti-mouse IgG) wasoxidized using NaIO4 to yield aldehyde groups on their carbohy-drate moiety. For the oxidization, the antibody solution was mixedwith 0.2 M NaIO4 (pH 5) by a ratio of 1:1 (v/v) and the resulting

PS-GMA

PS-GMA

NMP

NMP

Fe3O4

Fe3O4

Scheme 1

Scheme 2

NH2

NH2COOH COOH

ethylenedimine

ethylenediminesuccinicanhydride

Fig. 1. Reaction schemes for the preparation of surface-functionalized, magnetic

PS–GMA microspheres.

mixture was allowed for reaction in the dark and ultrasonicconditions at the room temperature (25–30 1C) for 40 min. Afterremoval of oxidation agent by dialysis, the oxidized secondaryantibody was incubated with amino group-containing magneticPS–GMA microspheres at 4 1C for 24 h for covalent binding. Theantibody was thus immobilized by an oriented manner [24,25].These secondary antibody-bound magnetic microspheres werethen coupled with non-modified Sca-1 antibody (primary anti-body) to form an indirect structure of Sca-1 antibody-anti-Sca-1antibody on magnetic microspheres.

The Sac-1 antibody-bound magnetic microspheres could alsobe obtained by the direct coupling of biotinylated Sca-1 antibodyonto avidin-bound magnetic PS–GMA microspheres. In order toyield the avidin-bound magnetic microspheres, 100 mg carboxylgroup-containing magnetic PS–GMA microspheres were mixedwith N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydro-chloride (EDC) and N-hydrosuccinimide (NHS) and the resultingmixture was allowed to react for 15 min. After washing withphosphate buffered saline (PBS) buffer, the magnetic micro-spheres were incubated with 2 mg avidin (in 200ml DI water)and 3.8 ml PBS buffer to react at room temperature for 60 min.Washed products were finally incubated with an aqueous solutionof ethanolamine for 60 min to block unreacted functional groups.

1.3. Immunomagnetic cell separation

Bone marrow cells from mice BALB/C were obtained byflushing femurs with a-MEM medium. This cell suspension wasseparated using Ficoll-Paque to yield mononucleated cells . Forfurther cell selection, MNCs were suspended in PB buffer (1�106

cells/100ml) and incubated with the Sac-1 antibody-boundmagnetic microspheres for 30 min. Sca-1 antibody-bound mag-netic microspheres prepared by either indirect coupling (Sca-1antibody-anti-Sca-1 antibody) or direct coupling (avidin–biotinlinkage) were used here for the immunomagnetic cell separation.The tube containing the mixture of MNCs and antibody-boundmagnetic microspheres was then placed in the MACSiMAGseparator (Miltenyi Biotech) and the cell-bound microsphereswere allowed to adhere to the wall of the tube. Sca-1+ cells wereselectively captured through the affinity of Sca-1 antibody onmagnetic microspheres and cell surface protein Sca-1. Isolatedcells were characterized using flow cytometric analysis withfluorescent-tagged antibodies against Sca-1 and c-kit.

2. Results and discussion

2.1. Surface functionalization and antibody immobilization on

magnetic microspheres

Prior to antibody immobilization, magnetic polymer micro-spheres were chemically modified to introduce a proper functionalgroup, through which the antibody molecule could be tightly boundon their surface. The surface functionalization on magnetic micro-psheres was achieved by two different reaction schemes (Fig. 1).Reactions for the surface functionalization could occur on themagnetic polymer microspheres. Alternatively, the micro-sizePS–GMA particles could be chemically modified to introduce desiredfunctional groups on the surface, and the resulting functionalizedmicrospheres were then filled with magnetic nanoparticles by theswelling and penetrating process. Both approaches resulted infunctionalized magnetic microspheres, which were ready to coupleantibody. The amination with ethylenediamine worked equally wellon both magnetic and non-magnetic PS–GMA microspheres. Theamino group density on the magnetic microspheres according to

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Scheme 1 was almost the same as that on the non-magneticmicrospheres prior to further derivation and filling with magneticnanoparticles according to Scheme 2.

The indirect coupling resulted in the structure of Sca-1antibody-anti-Sca-1 antibody on magnetic microspheres. Toobtain this kind of Sca-1 antibody-bound magnetic carriers, anoxidized goat anti-mouse antibody (secondary antibody) was firstcovalently immobilized onto the amino group-containing mag-netic microspheres and then coupled with non-modified Sca-1antibody (primary antibody). The immobilized density of second-ary antibody on the amino group-containing magnetic micro-spheres increased with the amount of oxidized secondaryantibody applied for immobilization. As shown in Fig. 2, theimmobilized density of goat anti-mouse antibody increasedlinearly with applied amount of antibody and reached the highestvalue of 13.3 mg/g. In this study, the site-directed immobilization

20

15

10

5

00 20 40 60 80 100 120

Imm

obili

zed

dens

ity o

f

seco

ndar

y A

b (µ

g/m

g pa

rticl

e)

Applied amount of secondary Ab (µg)

Fig. 2. Influence of the applied amount of oxidized goat anti-mouse IgG on the

immobilized density of secondary antibody on magnetic microspheres. The dashed

line indicates a 100% binding.

SSC

FS0

0

1023

104

103

102

101

100

104103102101100

c-K

it/PE

0.25%

1.84%94.11%

Sca-1/FITC

3.79%

Fig. 3. Bone marrow cells with large forward scatter are gated for two-color analysis

fluorescent antibodies of Sca-1 and c-kit (b) before and (c) after Sca-1 positive selectio

method was employed for covalent binding of Fc region inantibodies onto the magnetic microspheres. Antibodies immobi-lized in such way are able to expose their Fab region in the liquidphase and can easily bind the antigens. Before immobilization, thecarbohydrate moiety of Fc region was oxidized to form a reactivealdehyde group. According to a previous study, the oxidation onan antibody could reduce the protein activity by 15% on average[24]. The immobilized antibody, however, remained at a highaffinity for binding to its antigen.

To obtain the Sca-1 antibody-bound magnetic carriers by thedirect coupling manner, biotinylated Sca-1 antibody was coupledonto the magnetic microspheres, which had been previouslybound with avidin on the surface. Immobilization of avidin on thecarboxyl group-containing magnetic PS–GMA microspheres couldbe effectively accomplished by the conventional protocol usingEDC and NHS as the activating reagents. Based on the proteinassay (BCA protein assay kit, Pirece), the avidin density bound onthe magnetic microspheres was determined to be about 50 nmol/g. The coupling of antibody on the magnetic microspheres wasthus via an avidin–biotin linkage, which was able to withstand theoperative forces during magnetic extraction procedures.

Briefly, magnetic PS–GMA microspheres containing eitheramino or carboxyl groups on the surface were prepared andcoupled with antibodies that could specifically recognize thesurface proteins of stem cells in bone marrow. Surface functiona-lization could be performed either before or after the non-magnetic PS–GMA microspheres were made magnetic by aswelling and penetration process.

2.2. Immunomagnetic cell isolation

For the immunomagnetic selection of Sca-1+ cells, antibody-bound magnetic microspheres prepared by both indirect and

C1023

R1

104

103

102

101

100

104103102101100

c-K

it/PE

4.06% 87.73%

8.00% 0.21%

Sca-1/FITC

(a), two-color flow cytometric analysis of murine bone marrow cells stained with

n.

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100

80

60

40

20

01 5 10

beads per cell

Sca

-1 p

ositi

ve c

ells

(%)

Fig. 4. Influence of the bead-to-cell ratio on the purification of Sca-1 positive cells from bone marrow using indirect anti-Sca-1 antibody-bound magnetic microspheres.

Pictures in lower panel show isolated Sca-1 positive cells bound with the direct antibody-bound magnetic microspheres (arrows).

T.-H. Chung et al. / Journal of Magnetism and Magnetic Materials 321 (2009) 1635–16381638

direct coupling were effective. When the indirect antibody-boundmagnetic microspheres were used, the selection of Sca-1 positivecell was through the affinity of primary antibody on the magneticmicrospheres and cell surface protein. Results from flow cyto-metric assay of cell populations before and after immunomagneticisolation suggested that not only the faction of Sca-1 positive cellswas increased, but also the cells with both Sac-1 and c-kit surfacemarkers were enriched by the immunomagnetic isolation throughthe use of anti-Sca-1 antibody (Fig. 3). The purity of Sca-1+ cellswas influenced by the bead-to-cell ratio. We found that the purityincreased when using the indirect antibody-bound magneticmicrospheres for the Sca-1+ selection, with a bead-to-cell ratioup to 10 beads/cell (Fig. 4). Using the direct (avidin–biotin linked)antibody-bound magnetic microspheres, the purity, however, wasaltered slightly by the bead-to-cell ratio. When the bead-to-cellwas 10 beads/cell, an average purity of 85% Sca-1+ cells (83.7% ofthem were Sca-1+c-kit+ cells) and a 17.2 times enrichment wereachieved using the indirect antibody-bound magnetic micro-spheres. The purity was 63.9% (58.8% of them were Sca-1+c-kit+

cells) and the enrichment was 11.4 fold when the direct antibody-bound magnetic microspheres were used with a bead-to-cell ratioof 10 beads/cells.

The number of microspheres bound on cells is dependent on thenumber of Sca-1+ epitopes on the cell surface and particle size ofmicrospheres. Pictures in Fig. 4 show the isolated Sca-1+ cells,which are still coupled with the direct (avidin–biotin linked)antibody-bound magnetic microspheres. The average number ofmagnetic microspheres on each cell was determined to be about 3.Positively selected cells were finally released from the magneticmicrospheres for in vitro cultivation. To obtain Sca-1+Lin� popula-tion of HSCs, the isolated population of Sca-1+ cells was furtherpurified by using anti-Lin surface markers bound on magneticmicrospheres to deplete cells with lineage surface markers (Lin+).In addition to the suspension population of Sca-1 positive cells inMNCs of bone marrow, there is an adherent population of MNCs,which can be well cultured and expanded in MSCs medium.

In summary, for the immunomagnetic separation of Sca-1positive cells from the suspension of MNCs from mice femursbased on the recognition of cell surface marker by Sca-1 antibody,

the selection efficiency was influenced by the method of antibodyimmobilization and the number ratio of microspheres to cells. Thedirect coupling of biotinylated Sca-1 antibody to magnetic avidinmicrospheres was more easy to perform, but yielded a relativelylow purity of target cells. Using the indirect antibody-boundmagnetic microspheres, the purity of isolated Sca-1+ cells washigher based on the same bead-to-cell ratio.

Acknowledgments

This study was supported by the contracts of NSC 94-2522-S-194-001 and NSC 95-2221-E194-069 from the National ScienceCouncil (Taiwan).

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