JOURNAL OF BONE A N D MINERAL RESEARCH Volume 6, Number 12, 1991 Mary Ann Liebert, Inc., Publishers
Osteoclast-Specific Monoclonal Antibodies Coupled to Magnetic Beads Provide a Rapid and Efficient Method of Purifying Avian Osteoclasts PATRICIA COLLIN-OSDOBY, MERRY JO OURSLER, DAVID WEBBER, and PHILIP OSDOBY
ABSTRACT Osteoclasts are the major cell type responsible for normal and pathologic bone resorption. Obtaining highly purified populations of these multinucleated cells has been problematic, although such populalions would greatly facilitate investigations of osteoclast regulation and activity. A new immunomagnetic protocol has been devised to surmount these difficulties, employing avian osteoclast-directed monoclonal antibodies (designated 121F,35L, and 75B) surface coupled to uniformly small, magnetic polystyrene beads covalently conjugated with sheep antimouse IgC. Presentation of these antiosteoclast antibody-coated beads to mixed cell preparations derived from marrow-depleted, collagenase- and/or trypsin-treated chick tibiae and wing bones, followed by magnetic separation and washing, results in efficient and selective binding of osteoclasts to the immunomagnetic beads within minutes. The specific nature of this bead-cell interaction i s further demonstrated by the progressive decline in antiosteoclast antibody-coated bead binding to osteoclasts pretreated with the soluble antiosteoclast antibody and also by the absence of binding to osteoclasts by uncoated beads or beads coated with an irrelevant antibody. Under optimal conditions, these isolations typically yield more than a 100-fold enrichment and greater than a 90% purification of osteoclasts from subpopulations of either predominantly nonviable or viable osteoclasts. Although scanning electron microscopy reveals that immunomagnetically purified and cultured osteoclasts internalize large numbers of the antibodycoated beads, such cells appear unimpaired in their ability to attach to tissue culture plastic or devitalized cortical bone slices and to produce resorption pits characteristic for osteoclasts. Additional studies to ascertain the most effective method for removal (desorption) of antibody-coated beads from magnetically isolated osteoclasts demonstrate that moderate physical agitation i s at present the most effective protocol to dislodge antibody-coated beads from the cell surface while maintaining osteoclast viability and function. This immunomagnetic technique therefore provides a gentle method for the isolation of highly purified poplations of osteoclasts from heterogeneous bone cell populations in a rapid, efficient, and selective manner.
INTRODUCTION
B
ONE FORMATION AND DEGRADATION are normally tightly coupled and carefully regulated processes designed to maintain skeletal and physiologic homeostasis.") The mechanisms by which the bone-forming osteoblast and bone-resorbing osteoclast achieve this coordinated activity have constituted a challenging problem for research inves-
tigators. Given the complex, interactive, and heterogeneous nature of the bone environment, isolated cell populations provide an effective approach for dissecting the individual events involved. Despite progress in isolating osteoclasts from a variety of species however, it has proven difficult to obtain sufficiently pure populations of isolated osteoclasts containing reasonable numbers of viable cells for physiological, biochemical, and molecular investiga-
Department of Biology, Washington University, Division of Biological Sciences and Division of Bone and Mineral Metabolism, St. Louis, Missouri.
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COLLIN-OSDOBY ET AL.
Typically, 50 pl (2 x lo7 beads) of Dynabeads M-450 tions. Recently, our laboratory described an avian osteoclast isolation protocol yielding three osteoclast pop- suspension were transferred to a microfuge tube, washed ulations from chick hatchlings maintained on a low-cal- twice with PBS via magnetic sorting, and resuspended in cium diet. 1 6 ) Partial purification of the isolated osteoclasts 200 pl PBS. An aliquot of partially purified monoclonal was accomplished by using a combination of buoyant den- antibody (MAb) was directly added ( 5 pl of 121F MAb at sity and unit gravity separation techniques. The first two 3.9 mg/ml, 0.5 pI of 75B MAb at 24.2 mg/ml, or 5 pI of populations contained approximately 40% osteoclasts, the 35L MAb at 3.1 mg/ml). Magnetic beads were routinely vast majority of which were nonviable, whereas the third incubated with MAb overnight at 4°C with end-over-end population sequentially derived from the bone contained mixing, followed the next day by two to three rinses with fewer osteoclasts but the majority of these cells were PBS containing 1% fetal calf serum (FCS, GIBCO) and chilled on ice just before use in immunosorting of osteoviable. As an alternative to such standard cell fractionation pro- clasts. In initial experiments and periodically thereafter, cedures as density gradient centrifugation and fluores- antiosteoclast antibody coupling to the magnetic beads was cence-activated cell sorting,(7-9)morerapid, yet efficient, assessed by several means. After overnight coupling and physical separations of cells have been achieved using cell rinsing, aliquots of the beads were reacted for 20 minutes surface-directed antibodies and magnetic particles or im- with a 1: 100 dilution of fluorescein-conjugated goat antimunobeads.'l0.l1)In general, favorable yields and cell puri- mouse IgG (Cappel) in PBS and 1% FCS and washed ties, combined with the speed of isolation and mild condi- briefly, and bead-associated fluorescence was viewed on a tions that sustain cell viability, have made immunomag- Leitz Diaplan microscope. In addition, the unbound supernetic sorting a popular choice for cell f r a ~ t i o n a t i o n . ' ~ ~ , " )natant was analyzed for remaining antibody by determinThe prime requirement for selective isolation of cell sub- ing protein levels(17)and by assessing the relative antibody sets from mixed populations is the availability of cell sur- reactivity present against osteoclasts by enzyme-linked imface-directed antibodies specific for the chosen target cell. munosorbent assay (ELISA)I6' in comparison with the Previous reports from this laboratory described the genera- levels and reactivity of the original antibody solution. tion of monoclonal antibodies raised against avian osteoclasts, some of which (including 121F, 75B, and 35L) have Isolation of osteoclasts restricted or unique specificity for this bone cell type.'L4-161 A group of 15 white Leghorn chick hatchlings, which These select antibodies are therefore ideal candidates for use in cell separation immunomagnetic protocols. This re- were maintained on a low-calcium (< 0.1 Vo calcium) diet port describes rapid immunomagnetic harvesting of osteo- (Purina) for a minimum of 28 days, were routinely used clasts from heterogeneous mixed bone cell populations, for each osteoclast preparation. Three sequentially derived yielding purified preparations of isolated osteoclasts that osteoclast populations (1, 2, and 3) were isolated from display no apparent loss of cell integrity, viability, or func- each preparation, representing cells released from bone either before (population 1) or following (population 2) tion. collagenase treatment of bones or from bones treated with both collagenase and trypsin (population 3) as described by METHODS Oursler et a1.'61 Osteoclasts within each population were Preparation of irnrnunornagnetic beads sometimes enriched, when indicated, by Percoll (PharMagnetic polystyrene beads (4.5 pm in diameter), cova- macia) density gradient fractionation over 35% gradients lently coated with affinity-purified sheep antimouse IgG, according to Oursler et aI.I6) were purchased from Dynal, Inc. and stored at 4°C. Primary monoclonal antibodies to be coupled to the beads Irnrnunornagnetic isolation of osteoclasts were partially purified from mouse ascitic fluids, obtained In general, a portion of an osteoclast population ( 2onevia injection of hybridomas raised against isolated avian osteoclasts as described previously. 14) Debris was removed quarter of a preparation) was mixed immediately after isolation with a prepared sample of antibody-coated magnetic from collected ascitic fluids by centrifugation at 23,750 x g for 15 minutes at 4"C, and antibodies were precipitated beads (washed and reserved briefly on ice) in a volume of three times on ice by addition of an equal volume of satu- 5- 10 ml calcium and magnesium (Ca,Mg)-free Tyrode's rated ammonium sulfate (for 30, 5 , and 5 minutes, respec- balanced salt solution, pH 7.4 (TBSS, for population 3 tively) followed by centrifugation at 11,950 x g for 5 min- cells), or in a sorting buffer of Ca,Mg-free TBSS containutes at 4°C each time. The pellets were resuspended in one- ing 0.1% gelatin, 0.8% nonfat dry milk, and 0.001% boto reduce intercellular adhesiveness quarter of the original volume of phosphate-buffered sa- vine serum line (PBS, typically 2 ml) and applied to a CM-Affi-Gel (for population 1 and/or 2 cells). Bead-cell mixtures were Blue (BioRad) dye affinity column (35 ml) preequilibrated incubated in 50 ml polypropylene conical tubes placed at with PBS. The antibody-containing unbound fraction and approximately 45" angles in ice and gently mixed on a platPBS rinses of the column were pooled, concentrated by a form shaker for 2-30 minutes as indicated in the figures single 50% ammonium sulfate precipitation on ice, and re- and tables. Bead-bound cells were collected from populasuspended in a volume of PBS equivalent to two-thirds of tions 1 and/or 2 by applying a magnet for 1-2 minutes to the original ascitic fluid volume. Partially purified anti- the middle to lower outside of the incubation tube still in bodies were stored frozen at -20°C in small aliquots to ice, withdrawing the unbound cell suspension, and washing the bead-bound cells several times with fresh sorting bufminimize repeated antibody thawing and freezing.
HIGH PURITY IMMUNOMAGNETIC ISOLATION OF OSTEOCLASTS fer. Population 3 cells were more difficult to sort, owing to the increased viscosity resultant from repolymerizing collagen. These bead-cell mixtures were transferred to plastic beakers tilted at an angle on ice for magnetic sorting, the volumes were increased 10- to 20-fold, and the sorting times in the magnetic field were increased 5- t o 10-fold to partially overcome these difficulties and allow efficient recovery of magnetic beads and osteoclasts. Magnetically isolated cells were resuspended in a-minimum essential media (MEM) containing 10% fetal calf serum (GIBCO), and 2% antibiotic-antimycotic (GIBCO) and either examined directly or plated on tissue culture dishes (Falcon), which were maintained at 37°C in a 95% air and 5% CO, moist atmosphere.
Quantitative assessment of osteoclast purity and viability Magnetically isolated cells were cultured for 5 h before determination of osteoclast purity and viability. In the first method, nonadherent and adherent cells were collected from the dishes and stained with trypan blue, and the number of total cells, osteoclasts, and red blood cells, as well as their respective viabilities (trypan blue exclusion), were scored for each sample as described in Oursler et al.(b)In selected cases, these results were compared with those obtained from viability assessments based on fluorescein diacetate and ethidium bromide staining according to the method of Gray and Morris.('8) Cell counts were extrapolated to provide yield determinations. Alternatively, purity and viability were determined for cells cultured for 5 h by rinsing off nonadherent cells (of which less than 5% represent viable osteoclasts), briefly fixing the attached cells with 1% paraformaldehyde in Hanks' balanced salt solution (HBSS, GIBCO), and counting the number of total cells and osteoclasts in various randomly chosen fields by light microscopy. Since at least 95% of adherent osteoclasts are viable by other criteria,(6)attachment to plastic by 5 h in culture itself constitutes an accurate measure for osteoclast viability.
Specifcity studies Population 3 cells were suspended in 12 ml a-MEM with 10% FCS and equally divided among six polypropylene tubes containing 0, 0.25, 0.5, or 1 pl 121F MAb (at 3.9 mg/ml) on ice. Cells were preincubated on ice for 30 minutes with moderate shaking, 10 ml Ca,Mg-free TBSS was added to each tube, followed by 10 pl prepared 121F MAb coupled magnetic beads, and the mixture was further incubated for 20 minutes on ice with shaking. Bead-bound cells were magnetically sorted from unbound cells on ice, washed twice with Ca,Mg-free TBSS, resuspended in aMEM with 10% FCS, and cultured for 5 h before analysis of purity, viability, and yield as described earlier.
Desorption of immunomagnetic beads from osteoclasts Removal of beads from immunomagnetically isolated population 1 and/or 2 osteoclasts was attempted by physi-
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cal, chemical, or enzymatic treatments. Following incubation on ice of antibody-coated beads with an osteoclast population, the mixture was magnetically washed several times with sorting buffer, resuspended in the same, divided into equivalent portions, and one portion reserved on ice as an untreated control. Other portions were treated as follows. For low pH, 3.5 ml sorting buffer was added to a 50 p1 bead-cell aliquot, acidified to pH 4.5 with 0.1 N HCI for 2 minutes, and rapidly neutralized by addition of 50 ml Ca,Mg-free TBSS. For trypsin, bead-cell mixtures were incubated with 10 ml Ca,Mg-free TBSS containing 0.1 mg/ ml of EDTA and either 0.1 mg/ml (low) or 1 mg/ml (high) trypsin for 15 minutes at 37°C. For trypsinIDNAase, a bead-cell aliquot (1 ml in sorting buffer) was preincubated in 25 pg/ml of DNAase (Sigma Chemical Co.) for 10 minutes at 37°C and then in 1 mg/ml of trypsin and 25 pg/ml DNAase for a further 10 minutes at 37"C, followed by addition of 1 ml fetal calf serum to inactivate trypsin before magnetic sorting. For physical removal, 9 ml sorting buffer was added to a 1 ml bead-cell aliquot and then subjected to 3 minutes of pulsed vortexing at a high setting (7-8) on a Vortex Genie 2 (Fisher Scientific Co.). For papain, a beadcell aliquot was treated with 1 mg/ml papain in 0.01 M EDTA, 0.06 M 0-mercaptoethanol, and 0.05 M cysteine HCI for 30 minutes at 4°C with end-over-end mixing. lmmediately after each treatment, mixtures were magnetically sorted on ice. Released cells were collected by centrifugation at loo0 x g for 5 minutes, washed, resuspended in a-MEM containing 15% FCS, and cultured for 5 h before purity and viability analyses. Similarly, remaining beadbound cells were washed, resuspended, and cultured so that relative osteoclast purity and viability could be directly compared between released versus bound cells or untreated versus treated samples. In separate experiments, vortex desorption of population 3 osteoclasts from mixed antibody immunomagnetic beads was tried. The mixed antibody beads (150 pl, lo7 beads) were coupled with confluent hybridoma culture supernatants from two sources: 50 pl 121F antiavian osteoclast antibody and 100 pl A12E antialgal antibody,(") the latter serving as an osteoclast-nonreactive an1 ibody. For comparison, lo7beads were also prepared with 150 pl 121F hybridoma supernatant (undiluted by nonspecific antibody) or 150 pl A12E hybridoma supernatant. 'The following day, beads were washed, exposed to population 3 cells, incubated, sorted, portions desorbed by vortexing, and the released and bead-bound cells examined separately by light microscopy as before.
Light microscopy and transmission electron microscopy For light microscopy, freshly isolated cells were fixed in 1% formalin in HBSS (GIBCO) for 30 minutes at 4"C, rinsed in HBSS, and mounted on slides that were viewed on a Leitz Diaplan microscope using phase microscopy. Isolated osteoclasts were also prepared for transmission electron microscopy using the methods described by Osdoby et al.('O1 Plastic sections (1 pm) were cut and stained with toluidine blue and select areas photographed. Ultrathin sections were placed on grids, stained with
COLLIN-OSDOBY ET AL.
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uranyl acetate and lead citrate, and viewed on a Philips 300 electron microscope at 80 kV.
Osteoclast resorption of bone slices Adult bovine cortical bone slices (0.4mm thick by 5 mm* pieces) were prepared, cultured with magnetically isolated osteoclasts, and processed for scanning electron microscopy as described by Webber et al.'211
Scanning electron microscopy Samples for scanning electron microscopy were prepared and viewed as outlined in Webber et al.l2l1Samples were gold sputter coated and viewed on a Philips 501 scanning electron microscope at 15 kV with a tilt angle of 35".
RESULTS
Optimal immunosorting of osteoclasts
polystyrene beads precoated with osteoclast-directed monoclonal antibodies (121F, 35L, or 75B) to purify osteoclasts from mixed cell populations. Magnetic polystyrene beads coated with monoclonal antibody 121F were found to readily bind to isolated osteoclasts present in the population 1-2 and population 3 mixed cell suspensions. These beads bound both quickly and selectively to osteoclasts whether the bead-cell mixture was maintained on ice or not. Beads not conjugated with osteoclast antibodies displayed little, if any, nonspecific affinity ( 90Vo) nonviable. osteoclasts (in 1 ml) at varying bead to cell ratios for 2 minutes on The third population contains 10% osteoclasts; however, ice. The standard ratio was calculated as 12:1 based on incubation 60% of these are viable and, more importantly, 90% of all of 2 x lo' beads with one-sixth of a population 2 osteoclast (OC) the viable osteoclasts released from the bones are recov- prearation (typically 1.7 x 10' OCs). Bead-bound cells were magnetically sorted and washed, and the OCs in aliquots of the sorted ered in this third population. Both the nonviable (popula- samples were counted by light microscopy using a hemacytometer. tions 1 and/or 2) and the predominantly viable (population The remaining sorted cells were plated in tissue culture dishes for 5 3) osteoclasts were employed in studies using magnetic h to determine OC purity (92%) and viability (10-12%). ~
~~
HIGH PURITY IMMUNOMAGNETIC ISOLATION OF OSTEOCLASTS
Yield, viabilit.y, and morphology of isolated osteoclasts Population 2 cells (collagenase-released, primarily nonviable) that were immunomagnetically isolated were reproducibly 80-99% pure for osteoclasts. The higher range purities were routinely derived from populations that had been partially purified by density gradient separations over 35% Percol116)before bead incubation and magnetic sorting. lmmunosorted cells were consistently 10-15% viable, representing the same proportion of live osteoclasts known to be present in this starting materialI6’ and demonstrating no additional diminution in osteoclast viability associated with immunomagnetic sorting. The viable osteoclasts often produced extended processes on plastic tissue culture dishes. Light and transmission electron microscopic examination of these sorted cells showed that numerous beads were attached t o each osteoclast cell surface (Fig. 1). In accordance with viability measures, many of the isolated multinucleated cells from this population exhibited degenerative morphologies, including nuclear pyknoses (Fig. 1). Similarly, population 3 cells (trypsin-released, high viability) were successfully sorted by immunomagnetic means, as illustrated in Fig. 2. Large numbers of highly enriched and viable osteoclasts were obtained from this population using the magnetic sorting procedure. More than 90% (4.86 x lo6) of the osteoclasts present in the original pop-
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ulation 3 cells (5.3 x lo6) were retrieved by the immunobeads. When these magnetically sorted multinucleated cells were examined by transmission electron microscopy (Fig. 2C), many of the cells appeared devoid of such nuclear, organellar, or membrane abnormalities as generally ascribed to degenerative cells. Occasionally, matrix material and small aggregates of mononuclear cells (perhaps also associated with matrix fragments) copurrfied with the immunomagnetically sorted osteoclasts from population 3 . In these instances, close examination of the magnetic beadbound matrix or cell aggregate revealed that they possessed osteoclasts, which were presumably responsible for their coincidental sorting with the free osteoclasts released from the bone matrix. The microscopic observations not only illustrated the tight binding of the antibody-coated magnetic beads to the osteoclast plasma membrane but, further, showed that the beads could be readily phagocytosed by the cells (Figs. 2 and 3). Quantitative assessment of the purity and viability of magnetically sorted osteoclasts derived from population 3, in comparison with those isolated from population 2, was performed. Immunomagnetic separation of osteoclasts from population 2 bone cells typically yielded preparations greater than 90% pure for osteoclasts, in which only 10% of these cells were viable as determined by trypan blue dye exclusion, fluorescein diacetate staining, or cell attachment to culture dishes. In comparison, immunomagnetic sorting
FIG. 1. (A) Immunomagnetic beads (arrows) become associated with large multinucleated osteoclasts (OC) and some cell and matrix debris (asterisks) as shown in the 1 gm section of Epon-embedded population 2 cells. Magnification x 960. (B) A transmission electron micrograph demonstrating immunomagnetic beads associated with the osteoclast plasma membrane (population 2). Note the pyknotic nucleus (arrow). Magnification x 2925.
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COLLIN-OSDOBY ET AL.
FIG. 2. (A) Phase-contrast micrograph of population 3 viable osteoclasts placed in culture immediately following their isolation via immunomagnetic beads coated with 121F monoclonal antibody. The dark images represent bead clusters bound to osteoclasts, whereas the very small refractile elements are individual beads not associated with cells. A small amount of matrix debris is also observed (M). Magnification x 110. (B) A higher magnification of an osteoclast population 3 preparation demonstrating the high concentration of beads associated with the immunomagnetically sorted cells reflected by the dark regions surrounding and enveloping the osteoclasts (arrows). Magnification x 440. (C) A transmission electron micrograph of an osteoclast isolated with 121F monoclonal antibody-coated magnetic beads (b). Note the partial phagocytosis of an immunobead and the close association between beads and the plasma membrane. Magnification x 5863.
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HIGH PURITY IMMUNOMAGNETIC ISOLATION OF OSTEOCLASTS of population 3 cells resulted in a slightly lower osteoclast purity (86Vo) than attained from population 2 cells. However, both the viability (99Vo) and yield (4.9 x lo6 from three-fifths of a preparation) of osteoclasts from this third population were quite high. The slight reduction in osteoclast purity from sorted population 3 cells is likely to have been a consequence of other matrix-associated cells being partitioned along with those osteoclasts associated with matrix fragments and/or cell aggregates during magnetic sorting. Attempts to reduce the solution viscosity caused by repolymerizing collagen during immunosorting by inclusion of 0.05 U chondroitinase ABC were without effect.
Specificity of immunomagnetic isolation To evaluate the restricted nature of binding to osteoclasts by antiosteoclast antibody-coupled magnetic beads, population 3 cells were treated with varying levels of the 121F antibody before their incubation with 121F antibodyconjugated beads. Table 2 presents findings from such an experiment, demonstrating a progressive decline in the number of osteoclasts isolated by the immunomagnetic beads in direct proportion to the concentration of 121F antibody to which osteoclasts were preexposed. Therefore, preincubation with antiosteoclast antibody blocked access of the matching antibody-coated beads to the osteoclast cell surface. At the higher antibody pretreatment levels, no viable osteoclasts were recovered. Clearly, then, the attraction of the osteoclasts for the immunomagnetic beads is mediated by the 121F antiosteoclast antibody in a concentration-dependent fashion. Specificity attributed to the immunomagnetic isolation technique exists both at the cellular and antibody levels. O n the one hand, osteoclasts are readily captured by antiosteoclast antibody (121F, 75B, or 35L)-conjugated immunonmagnetic beads but are unresponsive toward beads devoid of primary antibody or coupled with an irrelevant antibody (A12E) directed against algal flagellar proteins. Moreover, the latter bead types are never internalized by the osteoclasts, demonstrating that ingestion of beads is not a non-
specific phenomenon. Conversely, antiosteoclast antibodycoupled beads are specifically attracted to osteoclasts as opposed to other cells present in the bone cell populations, since the latter are never observed to accumulate any beads on their surfaces.
Functional integrity of immunomagnetically isolated osteoclasts Retention of osteoclast viability and functional properties in cells subjected to immunomagnetic isolation was further documented by experiments in which magnetically sorted osteoclasts were cultured on devitalized bovine cortical bone slices. Figures 3A and B depict magnetically sorted osteoclasts that attached to bone slices and within a 48 h period created well-defined resorption pits. Along with cells displaying characteristic osteoclast morphologies on bone, occasional elongated, spindle-shaped cells, as well as flattened large cells, were associated with the beads. These bead-bound, flattened cells were often observed closely associated with resorption pits. In each panel of Fig. 3, it is striking to note that cultured osteoclasts were capable of internalizing large numbers of the antibodycoated beads without suffering a reduced ability to attach to either bone matrix or the tissue culture plastic substratum. Osteoclast internalization of antibody-coated magnetic beads was not a unique phenomenon related to the 121F monoclonal antibody, since two other monoclonal antibodies that recognize osteoclast-restricted cell surface antigens, 35L and 75B, were similarly internalized via the magnetic beads once the magnetically sorted cells were cultured (Fig. 3D and E) or removed from ice. By 96 h of culture on bone slices, osteoclast morphology appeared altered to a less active phenotype.
Desorption of beads from sorted cells For some applications, isolated osteoclasts no longer bound to antibody-coated magnetic beads are desirable, and so methods for desorbing beads off the osteoclasts
TABLE2. DECREASED IMMUNOBEAD BINDING TO ANTIBODY-PRETREATED OSTEOCLASTS~ ~
IZIF MAb prerncubation (pg)
~~
OC yield Total OCs
Viable OCs
To Viable OCs
x x x x
1.6 x lo6 2.0 x 106 0 0
94 87
1.7 2.3 5.5 5.5
lo6 lo6 105 105
-
-
aPopulation 3 osteoclasts (two-thirds preparation) were equally divided between four tubes (each in 2 ml Ca,Mg-free TBSS) and preincubated with shaking for 30 minutes with ice-cold 121F antibody (0-4 pg). 121F antibody-conjugated magnetic beads (3 x 10") were then added to each sample and the mixtures further incubated 30 minutes while shaking on ice. Bead-bound cells were magnetically sorted, washed, and cultured for 5 h before yield, purity, and viability determinations as described in Materials and Methods. Purity of the viable OCs was unaffected by 1 pg antibody preincubation (90%) in comparison with n o antibody pretreatment (89%).
1360 were sought. Other investigators have reported success with physically dislodging the beads from cells by stirring or vortexing the complexes in large volumes to discourage reassociation.'") To avoid rapid ingestion of the immunobeads by the osteoclasts, steps were performed, when possible, at ice-cold temperatures. Physical disruption (vortexing), proteolytic cleavage of antibodies and/or osteoclast antigens (papain or trypsin), or chemical uncoupling of antibody-antigen complexes (low pH) were individually investigated, and their relative effectiveness at removing beads without adversely affecting osteoclast viability was ascertained. In the studies reported here, vigorous vortexing was the most effective method for desorbing beads (90%)from the magnetically isolated osteoclasts (Table 3). Although a high percentage of immunomagnetic beads were removed, many released osteoclasts frequently retained a small number of beads (three to five), representing one-tenth to one-quarter of the original number of bound beads, on their surfaces (Fig. 4). Some osteoclasts were completely stripped of associated beads; others remained well coated. This brief mechanical treatment did not reduce osteoclast viability or function. However, repetitive or prolonged vortexing led to a marked deterioration of osteoclast structure and was avoided. Physically desorbed cells were capable of rapid attachment to bone (within 30 minutes), as well as production of resorption pits (within 24 h). Scanning electron microscopic examination of osteoclasts on bone slices demonstrated that all osteoclasts attached to bone were capable of producing resorption pits. Alternative desorption methods (Table 3) were less promising. Brief exposure to low pH (54.5) to disrupt antibody-antigen interactions yielded good initial dissociation of beads from the osteoclasts, but as revealed in other experiments, these bonds were suspected of rapidly reforming once the acid pH was neutralized. Papain digestion was generally as effective in releasing osteoclasts (7080%) from the immunomagnetic beads as low pH treatment (data not shown). Desorption was not facilitated by decreasing the bead to cell ratio, immunosorting osteoclasts with mixed antibody beads partly conjugated with an irrelevant antibody (A12E), pretreating osteoclasts with 121F antibody before bead incubation, or employing alternative antiosteoclast antibodies (35L or 75B) for bead coupling.
Reproducibility and other considerations The immunomagnetic separation procedure for obtaining isolated osteoclasts has been performed repeatedly with
COLLIN-OSDOBY ET AL.
a consistently high level of success. It has proven equally effective at isolating osteoclasts from normal diet chicks as from the low-calcium chicks described here. As with any other primary cell isolation procedure, a certain variability arises between trials, which is a consequence of such factors as the composition of the starting cell population and the technical proficiency of the individuals carrying out the procedure. Notwithstanding, the rapid purification of osteoclasts from the population of 1 and/or 2 cells is highly reproducible. To achieve comparable results with population 3 cell preparations, it was necessary to increase the volume of the bead-cell reaction mixture 10- to 20-fold and to lengthen the sorting time in the magnetic field 5- to 10-fold (10 minutes, as opposed to 1-2 minutes) to overcome the solution viscosity experienced from repolymerizing collagen. Under these conditions, excellent osteoclast purities and recovery yields were obtained from each starting population.
DISCUSSION This report describes a new osteoclast purification procedure in which osteoclast-directed monoclonal antibodi e ~ " ~ - are ' ~ ) coupled to magnetic polystyrene beads and used to immunomagnetically separate these multinucleated cells from mixed bone cell populations. The present isolation scheme builds on previously published isolation methods, which involve sequential physical and enzymatic steps to obtain three osteoclast populations, having somewhat different osteoclast purities and viabilities associated with them, from chickens maintained for 4 weeks on low-calcium diets.'b) Further purification of viable osteoclasts is difficult to achieve because of the wide-ranging sizes and buoyant densities associated with the viable osteoclast population. In addition, the comparable densities of red blood cells and some small bone matrix fragments permit these elements to copurify with the osteoclasts. Therefore, an alternative approach was sought to obtain highly enriched osteoclast preparations for use in physiological biochemical, and molecular investigations focused on osteoclast activity and regulation. In recent years, various techniques have been employed to study osteoclast function and regulation in individual cells or populations of isolated osteoclasts. The well-characterized resorption pit assay, developed initially by Boyde and Chambers et al.'25,261 has been successfully et al.'23.*4) used for both qualitative and quantitative investigations of isolated osteoclast activity. Enzyme histochemistry and in situ nucleic acid hybridization analysis have enabled prob-
FIG. 3. (A) A scanning electron micrograph of osteoclasts separated and purified using 121F monoclonal antibodycoated magnetic beads. Immunosorted osteoclasts were cultured for 48 h on bovine cortical bone slices. Note the formation of well-defined resorption pits and the obvious internalization of antibody-coated beads (arrowheads). Other osteoclasts are completely covered with beads (asterisks). A few elongated cells are also observed. Magnification x 960. (B) A higher magnification of a representative immunomagnetically sorted osteoclast cultured on a bovine cortical bone slice. Numerous beads have been internalized, and filamentous processes (arrowheads) emanate from the cell near its attachment site to bone and also by the excavted resorption pit. Bars represent 10 pm. Magnification x 1875. (C-E) Scanning electron micrographs of osteoclasts isolated with magnetic beads coupled to 121F, 75B, or 35L antiosteoclast monoclonal antibodies, respectively. Isolated cells were cultured on tissue culture plastic for 24 h. Each type of antibodycoated bead was internalized. However, neither bead binding nor their internalization appeared to negatively influence cell attachment. Bars 10 pm. Magnification x 1OOO.
COLLIN-OSDOBY ET AL.
1362 TABLE3 . OSTEOCLAST VIABILITYA N D PURITYAS OF DESORPTION METHOD^
A
FUNCTION
Number of viable OC/field Desorption method Untreated Vortexed Low pH Low trypsin High trypsin DNAase/trypsin
Released
Bead-bound ocs
-
45 4 20 23 28 19
ocs 35 17 1
0 6
% OC purity
Released ocs
Bead-bound ocs
-
92 80 67 88 88 79
96 71 33 -
60
a I21 F antibody-coated immunomagnetic beads were incubated with trypsin-released cells on ice, magnetically sorted, and equivalent portions of the bead-bound cells subjected to desorption trials as detailed in Materials and Methods. Cells retained on the beads following desorption were magnetically separated from released cells, and both portions were cultured for 5 h before viability and purity determinations. Results from a representative desorption series are presented. Vortexing was the most effective desorption protocol (90% OCs released), followed by low-pH treatment (85% OCs released).
ing at the single-cell level. Advanced technologies, such as patch clamp a n a l y s i ~ ' ~ ' - ' ~and ' improved image analys ~ s , ( ~ Ohave . ~ ' ) provided valuable procedural means to examine the function of individual osteoclasts. Despite the obvious strengths of such methods, there are notable limitations restricting their use or the conclusions drawn from studies employing these strategies. On the one hand, inaccuracies can arise and be magnified when results are extrapolated from individual cells to a population as a whole. On the other hand, isolated populations generated by protocols available to date are typically impure for osteoclasts and thus highly susceptible to influences exerted by contaminating cell types. Among the advantages of the immunomagnetic isolation procedure are the relatively short times required, the gentle manipulations of the cell preparations possible, the high cell purities achieved, and the efficient recovery yields of osteoclasts obtained. In particular, when the yield and purity of immunomagnetically isolated osteoclasts are compared with those associated with osteoclasts purified by size and buoyant density dependent methods,'6) it is clear that the immunomagnetic protocol reflects a dramatic improvement in the purity of the population 1-2 osteoclasts, as well as an improvement in both the purity and yield of the viable population 3 osteoclasts. The enhanced yields from immunomagnetic isolations over alternative protocols are likely due to the avoidance of steps based on cell size and buoyant density, since both these vary considerably among osteoclasts residing in population 3 cells. Instead, immunomagnetic cell isolation relies upon a possibly more uniform osteoclast parameter, namely, expression levels of surface antigens reactive with osteoclast-specific monoclonal antibodies. The ultimate cell purity, ease of separation, and final recovery yield achievable by immunomagnetic protocols are a function of the specificity and avidity of the antibodies employed, the proportion of target cells in the original population, and, to a lesser extent, the manipulation regimen followed. Since partitioning of
osteoclasts apart from other bone cells is performed easily and quickly with immunomagnetic beads, omitting lengthy and multistep handling of the cells, the viability and biological integrity of the osteoclasts are maintained. One potential disadvantage of the immunomagnetic procedure relates to observations that plasma membrane antigens may be activated through binding of antibodies in a manner analogous to receptor-ligand responses."') Therefore, a degree of caution is necessary when proposing experiments to examine physiologic responses of osteoclasts using cells isolated in this manner. However, immunomagnetic isolation of other cell types has not resulted in their it is clear from the scanning a ~ t i v a t i o n . ' ~Furthermore, ~) electron microscopic observations that immunomagnetically purified osteoclasts can attach to bone and generate characteristic resorption pits, even when no attempts are made to remove the beads. In fact, each attached osteoclast was found to excavate a resorption pit. In other experiments, immunomagnetically isolated avian osteoclasts were shown to respond to estrogen by decreased bone resorption and increased nuclear protooncogene mRNA levels.(34)A further consideration for some applications is the observation that once the cells are released from lowtemperature conditions they are capable of internalizing the beads. Therefore, if later removal of the beads is desirable, it is best to carry out all the prior incubation and magnetic isolation steps on ice. Cold temperature itself causes no apparent decrease in the number or strength of beads interacting with osteoclasts or in the ultimate purity or yield achieved. Although this may be a feature associated with the affinity of a particular antibody for the target antigen, similar results have been obtained using the 121F, 75B. or 35L antiosteoclast monoclonal antibodies, suggesting that these all exhibit roughly comparable beadcell interactions. The concern for antibody and/or bead effects on osteoclasts can partially be overcome by removal of the beads from the cells before their use in physiological studies. Un-
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FIG. 4. (A) A low-power scanning electron micrograph of osteoclasts cultured on cortical bone slices after immunomagnetic separation with 121F monoclonal antibody beads and subsequent bead desorption through vortexing. Stripped osteoclasts display fewer beads associated with their surfaces. Many resorption pits formed by the desorbed osteoclasts are seen (asterisks). Magnification x 576. (B) A higher power scanning electron micrograph of a preparation similar to A. Note the decreased number of beads (arrows) associated with the osteoclast, as well as a few internalized beads (arrowheads). Part of a resorption pit can also be observed. Magnification x 1152.
COLLIN-OSDOBY ET AL.
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like some other cell types, such as lymphocytes, osteoclasts Anderson for his expert technical assistance in cell prepain culture d o not shed antibody-coated beads from their ration, and Ms. Terri Moulton for her patient typing of surfaces. Quite the contrary, these multinucleated cells ac- this manuscript. This work was supported by NIH Grants tively internalize beads bound to their membranes, pre- AR32927 and AR32087 and a Research Career Developventing their removal. As a further complication, more ment Award K04-AM1474 to Dr. Philip Osdoby. than five beads per osteoclast were required to attain effective magnetic sorting of these large cells, although bead to REFERENCES cell ratios of 1: 1 or 2: 1 are recommended for desorption trials (Dynal, Inc.). The most effective desorption proceI . Rodan G , Martin T 1981 Role of osteoblasts in hormonal dure, with simultaneously the least adverse effect on osteocontrol of bone resorption-a hypothesis. Calcif Tissue Int clast yield and viability, was mechanical vortexing, which 33:349-35 I . stripped the majority of beads from the osteoclast surface. 2. DeVernejoul M, Horowitz M. Demignon J , Neff L. Baron R Complete removal of every bead from all target cell sur1988 Bone resorption by isolated chick osteoclasts in culture faces has not yet been reported for any system. I f cells is stimulated by murine spleen cell supernatant fluids (osteototally devoid of beads are required, the opposite strategy clast-activating factor) and inhibited by calcitonin and prosbased on negative immunomagnetic selection to deplete taglandin E,. J Bone Miner Res 3:69-80. contaminating cells from a preparation, leaving the desired 3 . Zambonin-Zallone A, Teti A , Primavera M I982 Isolated cell population, is the preferred route. osteoclasts in primary culture: First observations o n structure The high purity and yield of osteoclasts obtained by this and survival in cultured media. Anat Embryo1 (Berl) 165: 405-41 3 . procedure has the potential to facilitate sensitive physio4. Osdoby P, Martini M, Caplan A 1982 Isolated osteoclasts logical studies, such as those to determine whether a horand their presumed progenitor, the monocyte, in culture. J mone, cytokine, or other modulator of bone remodeling Exp Zoo1 224:33 1-334. acts directly on the osteoclast or, alternatively, upon other 5 . Hefly T , Stern P 1982 Isolated osteoclasts from fetal rat long cells in the bone environment, which in turn influence bones. Calcif Tissue Int 34:480-487. osteoclast function. Another powerful application of this 6. Oursler M J , Collin-Osdoby P , Anderson F, Li L, Webber D, procedure is to provide cellular material for biochemical Osdoby P 1991 Isolation of avian osteoclasts: Improved techand molecular characterization of the osteoclast phenoniques to preferentially purify viable cells. J Bone Miner Res type. Using such highly purified cells, it may be possible 6:375-385. to perform detailed protein fingerprinting on structural, 7. Hutchins D, Steel CM 1979 Separation of human lymphocytes on the basis of volume and density. In: Peeters H (ed.) metabolic, and secretory proteins involved in osteoclast acSeparation of Cell9 and Subcellular Element\. Pergamon tivity and cell-cell or cell-matrix interactions. Beyond the Press, Oxford, pp. 28-43. physiological and biochemical approaches, i t is possible to 8. Harris R, Ukaejiofo E O 1970 Tissue typing using a routine confidently pursue molecular studies on pure populations one-step lymphocyte separation technique. Br J Haematol of osteoclasts isolated in this manner. The immunomag18~229-235. netic isolation scheme has already proven vital for identify9. Sharpe P T 1988 Methods of Cell Separation. Elsevier, New ing estrogen-responsive receptors on avian o s t e o c l a s t ~ ~ ~ ~ York. ~~ and for successfully obtaining sufficient intact osteoclast 10. Gaudernack G , Leivestad T, Ugelstad J , Thorsby E 1986 mRNA for construction of an osteoclast cDNA library Positive 5election with monoclonal antibodies directly conju(unpublished observations). In addition, consistent with gated t o monosiled magnetic microspheres. J lmmunol Methods 90:179- 189. our demonstration that the 121F monoclonal antibody recI . Padmanabhan R, Corsico C D , Howard T H , Holter W , ognizes a complementary antigen on human osteoclast-like Fordis C M , Willingham M, Howard BH 1988 Purification of cells, this immunomagnetic purification procedure has transiently transfected cells by magnetic affinity cell sorting. enabled purification of functional human osteoclasts as Anal Biochem 170:341-348. well (unpublished observations). In a different vein, unlike 2. Vaccaro DE 1990 Applications of magnetic separation: Cell the positive selections for osteoclasts, this technique could sorting. Am Biotech Lab 8:30-35. be utilized to selectively deplete a bone cell preparation of 3. Lea T, Smeland E, Funderud S, Vartdal F, Davies C , Beiske osteoclasts for study of the remaining cells or for subseD, Ugelstad J 1986 Characterization of human mononuclear quent addition of experimental cells or substances. Because cells after positive
Osteoclast-Specific Monoclonal Antibodies Coupled to Magnetic Beads Provide a Rapid and Efficient Method of Purifying Avian Osteoclasts PATRICIA COLLIN-OSDOBY, MERRY JO OURSLER, DAVID WEBBER, and PHILIP OSDOBY
ABSTRACT Osteoclasts are the major cell type responsible for normal and pathologic bone resorption. Obtaining highly purified populations of these multinucleated cells has been problematic, although such populalions would greatly facilitate investigations of osteoclast regulation and activity. A new immunomagnetic protocol has been devised to surmount these difficulties, employing avian osteoclast-directed monoclonal antibodies (designated 121F,35L, and 75B) surface coupled to uniformly small, magnetic polystyrene beads covalently conjugated with sheep antimouse IgC. Presentation of these antiosteoclast antibody-coated beads to mixed cell preparations derived from marrow-depleted, collagenase- and/or trypsin-treated chick tibiae and wing bones, followed by magnetic separation and washing, results in efficient and selective binding of osteoclasts to the immunomagnetic beads within minutes. The specific nature of this bead-cell interaction i s further demonstrated by the progressive decline in antiosteoclast antibody-coated bead binding to osteoclasts pretreated with the soluble antiosteoclast antibody and also by the absence of binding to osteoclasts by uncoated beads or beads coated with an irrelevant antibody. Under optimal conditions, these isolations typically yield more than a 100-fold enrichment and greater than a 90% purification of osteoclasts from subpopulations of either predominantly nonviable or viable osteoclasts. Although scanning electron microscopy reveals that immunomagnetically purified and cultured osteoclasts internalize large numbers of the antibodycoated beads, such cells appear unimpaired in their ability to attach to tissue culture plastic or devitalized cortical bone slices and to produce resorption pits characteristic for osteoclasts. Additional studies to ascertain the most effective method for removal (desorption) of antibody-coated beads from magnetically isolated osteoclasts demonstrate that moderate physical agitation i s at present the most effective protocol to dislodge antibody-coated beads from the cell surface while maintaining osteoclast viability and function. This immunomagnetic technique therefore provides a gentle method for the isolation of highly purified poplations of osteoclasts from heterogeneous bone cell populations in a rapid, efficient, and selective manner.
INTRODUCTION
B
ONE FORMATION AND DEGRADATION are normally tightly coupled and carefully regulated processes designed to maintain skeletal and physiologic homeostasis.") The mechanisms by which the bone-forming osteoblast and bone-resorbing osteoclast achieve this coordinated activity have constituted a challenging problem for research inves-
tigators. Given the complex, interactive, and heterogeneous nature of the bone environment, isolated cell populations provide an effective approach for dissecting the individual events involved. Despite progress in isolating osteoclasts from a variety of species however, it has proven difficult to obtain sufficiently pure populations of isolated osteoclasts containing reasonable numbers of viable cells for physiological, biochemical, and molecular investiga-
Department of Biology, Washington University, Division of Biological Sciences and Division of Bone and Mineral Metabolism, St. Louis, Missouri.
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COLLIN-OSDOBY ET AL.
Typically, 50 pl (2 x lo7 beads) of Dynabeads M-450 tions. Recently, our laboratory described an avian osteoclast isolation protocol yielding three osteoclast pop- suspension were transferred to a microfuge tube, washed ulations from chick hatchlings maintained on a low-cal- twice with PBS via magnetic sorting, and resuspended in cium diet. 1 6 ) Partial purification of the isolated osteoclasts 200 pl PBS. An aliquot of partially purified monoclonal was accomplished by using a combination of buoyant den- antibody (MAb) was directly added ( 5 pl of 121F MAb at sity and unit gravity separation techniques. The first two 3.9 mg/ml, 0.5 pI of 75B MAb at 24.2 mg/ml, or 5 pI of populations contained approximately 40% osteoclasts, the 35L MAb at 3.1 mg/ml). Magnetic beads were routinely vast majority of which were nonviable, whereas the third incubated with MAb overnight at 4°C with end-over-end population sequentially derived from the bone contained mixing, followed the next day by two to three rinses with fewer osteoclasts but the majority of these cells were PBS containing 1% fetal calf serum (FCS, GIBCO) and chilled on ice just before use in immunosorting of osteoviable. As an alternative to such standard cell fractionation pro- clasts. In initial experiments and periodically thereafter, cedures as density gradient centrifugation and fluores- antiosteoclast antibody coupling to the magnetic beads was cence-activated cell sorting,(7-9)morerapid, yet efficient, assessed by several means. After overnight coupling and physical separations of cells have been achieved using cell rinsing, aliquots of the beads were reacted for 20 minutes surface-directed antibodies and magnetic particles or im- with a 1: 100 dilution of fluorescein-conjugated goat antimunobeads.'l0.l1)In general, favorable yields and cell puri- mouse IgG (Cappel) in PBS and 1% FCS and washed ties, combined with the speed of isolation and mild condi- briefly, and bead-associated fluorescence was viewed on a tions that sustain cell viability, have made immunomag- Leitz Diaplan microscope. In addition, the unbound supernetic sorting a popular choice for cell f r a ~ t i o n a t i o n . ' ~ ~ , " )natant was analyzed for remaining antibody by determinThe prime requirement for selective isolation of cell sub- ing protein levels(17)and by assessing the relative antibody sets from mixed populations is the availability of cell sur- reactivity present against osteoclasts by enzyme-linked imface-directed antibodies specific for the chosen target cell. munosorbent assay (ELISA)I6' in comparison with the Previous reports from this laboratory described the genera- levels and reactivity of the original antibody solution. tion of monoclonal antibodies raised against avian osteoclasts, some of which (including 121F, 75B, and 35L) have Isolation of osteoclasts restricted or unique specificity for this bone cell type.'L4-161 A group of 15 white Leghorn chick hatchlings, which These select antibodies are therefore ideal candidates for use in cell separation immunomagnetic protocols. This re- were maintained on a low-calcium (< 0.1 Vo calcium) diet port describes rapid immunomagnetic harvesting of osteo- (Purina) for a minimum of 28 days, were routinely used clasts from heterogeneous mixed bone cell populations, for each osteoclast preparation. Three sequentially derived yielding purified preparations of isolated osteoclasts that osteoclast populations (1, 2, and 3) were isolated from display no apparent loss of cell integrity, viability, or func- each preparation, representing cells released from bone either before (population 1) or following (population 2) tion. collagenase treatment of bones or from bones treated with both collagenase and trypsin (population 3) as described by METHODS Oursler et a1.'61 Osteoclasts within each population were Preparation of irnrnunornagnetic beads sometimes enriched, when indicated, by Percoll (PharMagnetic polystyrene beads (4.5 pm in diameter), cova- macia) density gradient fractionation over 35% gradients lently coated with affinity-purified sheep antimouse IgG, according to Oursler et aI.I6) were purchased from Dynal, Inc. and stored at 4°C. Primary monoclonal antibodies to be coupled to the beads Irnrnunornagnetic isolation of osteoclasts were partially purified from mouse ascitic fluids, obtained In general, a portion of an osteoclast population ( 2onevia injection of hybridomas raised against isolated avian osteoclasts as described previously. 14) Debris was removed quarter of a preparation) was mixed immediately after isolation with a prepared sample of antibody-coated magnetic from collected ascitic fluids by centrifugation at 23,750 x g for 15 minutes at 4"C, and antibodies were precipitated beads (washed and reserved briefly on ice) in a volume of three times on ice by addition of an equal volume of satu- 5- 10 ml calcium and magnesium (Ca,Mg)-free Tyrode's rated ammonium sulfate (for 30, 5 , and 5 minutes, respec- balanced salt solution, pH 7.4 (TBSS, for population 3 tively) followed by centrifugation at 11,950 x g for 5 min- cells), or in a sorting buffer of Ca,Mg-free TBSS containutes at 4°C each time. The pellets were resuspended in one- ing 0.1% gelatin, 0.8% nonfat dry milk, and 0.001% boto reduce intercellular adhesiveness quarter of the original volume of phosphate-buffered sa- vine serum line (PBS, typically 2 ml) and applied to a CM-Affi-Gel (for population 1 and/or 2 cells). Bead-cell mixtures were Blue (BioRad) dye affinity column (35 ml) preequilibrated incubated in 50 ml polypropylene conical tubes placed at with PBS. The antibody-containing unbound fraction and approximately 45" angles in ice and gently mixed on a platPBS rinses of the column were pooled, concentrated by a form shaker for 2-30 minutes as indicated in the figures single 50% ammonium sulfate precipitation on ice, and re- and tables. Bead-bound cells were collected from populasuspended in a volume of PBS equivalent to two-thirds of tions 1 and/or 2 by applying a magnet for 1-2 minutes to the original ascitic fluid volume. Partially purified anti- the middle to lower outside of the incubation tube still in bodies were stored frozen at -20°C in small aliquots to ice, withdrawing the unbound cell suspension, and washing the bead-bound cells several times with fresh sorting bufminimize repeated antibody thawing and freezing.
HIGH PURITY IMMUNOMAGNETIC ISOLATION OF OSTEOCLASTS fer. Population 3 cells were more difficult to sort, owing to the increased viscosity resultant from repolymerizing collagen. These bead-cell mixtures were transferred to plastic beakers tilted at an angle on ice for magnetic sorting, the volumes were increased 10- to 20-fold, and the sorting times in the magnetic field were increased 5- t o 10-fold to partially overcome these difficulties and allow efficient recovery of magnetic beads and osteoclasts. Magnetically isolated cells were resuspended in a-minimum essential media (MEM) containing 10% fetal calf serum (GIBCO), and 2% antibiotic-antimycotic (GIBCO) and either examined directly or plated on tissue culture dishes (Falcon), which were maintained at 37°C in a 95% air and 5% CO, moist atmosphere.
Quantitative assessment of osteoclast purity and viability Magnetically isolated cells were cultured for 5 h before determination of osteoclast purity and viability. In the first method, nonadherent and adherent cells were collected from the dishes and stained with trypan blue, and the number of total cells, osteoclasts, and red blood cells, as well as their respective viabilities (trypan blue exclusion), were scored for each sample as described in Oursler et al.(b)In selected cases, these results were compared with those obtained from viability assessments based on fluorescein diacetate and ethidium bromide staining according to the method of Gray and Morris.('8) Cell counts were extrapolated to provide yield determinations. Alternatively, purity and viability were determined for cells cultured for 5 h by rinsing off nonadherent cells (of which less than 5% represent viable osteoclasts), briefly fixing the attached cells with 1% paraformaldehyde in Hanks' balanced salt solution (HBSS, GIBCO), and counting the number of total cells and osteoclasts in various randomly chosen fields by light microscopy. Since at least 95% of adherent osteoclasts are viable by other criteria,(6)attachment to plastic by 5 h in culture itself constitutes an accurate measure for osteoclast viability.
Specifcity studies Population 3 cells were suspended in 12 ml a-MEM with 10% FCS and equally divided among six polypropylene tubes containing 0, 0.25, 0.5, or 1 pl 121F MAb (at 3.9 mg/ml) on ice. Cells were preincubated on ice for 30 minutes with moderate shaking, 10 ml Ca,Mg-free TBSS was added to each tube, followed by 10 pl prepared 121F MAb coupled magnetic beads, and the mixture was further incubated for 20 minutes on ice with shaking. Bead-bound cells were magnetically sorted from unbound cells on ice, washed twice with Ca,Mg-free TBSS, resuspended in aMEM with 10% FCS, and cultured for 5 h before analysis of purity, viability, and yield as described earlier.
Desorption of immunomagnetic beads from osteoclasts Removal of beads from immunomagnetically isolated population 1 and/or 2 osteoclasts was attempted by physi-
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cal, chemical, or enzymatic treatments. Following incubation on ice of antibody-coated beads with an osteoclast population, the mixture was magnetically washed several times with sorting buffer, resuspended in the same, divided into equivalent portions, and one portion reserved on ice as an untreated control. Other portions were treated as follows. For low pH, 3.5 ml sorting buffer was added to a 50 p1 bead-cell aliquot, acidified to pH 4.5 with 0.1 N HCI for 2 minutes, and rapidly neutralized by addition of 50 ml Ca,Mg-free TBSS. For trypsin, bead-cell mixtures were incubated with 10 ml Ca,Mg-free TBSS containing 0.1 mg/ ml of EDTA and either 0.1 mg/ml (low) or 1 mg/ml (high) trypsin for 15 minutes at 37°C. For trypsinIDNAase, a bead-cell aliquot (1 ml in sorting buffer) was preincubated in 25 pg/ml of DNAase (Sigma Chemical Co.) for 10 minutes at 37°C and then in 1 mg/ml of trypsin and 25 pg/ml DNAase for a further 10 minutes at 37"C, followed by addition of 1 ml fetal calf serum to inactivate trypsin before magnetic sorting. For physical removal, 9 ml sorting buffer was added to a 1 ml bead-cell aliquot and then subjected to 3 minutes of pulsed vortexing at a high setting (7-8) on a Vortex Genie 2 (Fisher Scientific Co.). For papain, a beadcell aliquot was treated with 1 mg/ml papain in 0.01 M EDTA, 0.06 M 0-mercaptoethanol, and 0.05 M cysteine HCI for 30 minutes at 4°C with end-over-end mixing. lmmediately after each treatment, mixtures were magnetically sorted on ice. Released cells were collected by centrifugation at loo0 x g for 5 minutes, washed, resuspended in a-MEM containing 15% FCS, and cultured for 5 h before purity and viability analyses. Similarly, remaining beadbound cells were washed, resuspended, and cultured so that relative osteoclast purity and viability could be directly compared between released versus bound cells or untreated versus treated samples. In separate experiments, vortex desorption of population 3 osteoclasts from mixed antibody immunomagnetic beads was tried. The mixed antibody beads (150 pl, lo7 beads) were coupled with confluent hybridoma culture supernatants from two sources: 50 pl 121F antiavian osteoclast antibody and 100 pl A12E antialgal antibody,(") the latter serving as an osteoclast-nonreactive an1 ibody. For comparison, lo7beads were also prepared with 150 pl 121F hybridoma supernatant (undiluted by nonspecific antibody) or 150 pl A12E hybridoma supernatant. 'The following day, beads were washed, exposed to population 3 cells, incubated, sorted, portions desorbed by vortexing, and the released and bead-bound cells examined separately by light microscopy as before.
Light microscopy and transmission electron microscopy For light microscopy, freshly isolated cells were fixed in 1% formalin in HBSS (GIBCO) for 30 minutes at 4"C, rinsed in HBSS, and mounted on slides that were viewed on a Leitz Diaplan microscope using phase microscopy. Isolated osteoclasts were also prepared for transmission electron microscopy using the methods described by Osdoby et al.('O1 Plastic sections (1 pm) were cut and stained with toluidine blue and select areas photographed. Ultrathin sections were placed on grids, stained with
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uranyl acetate and lead citrate, and viewed on a Philips 300 electron microscope at 80 kV.
Osteoclast resorption of bone slices Adult bovine cortical bone slices (0.4mm thick by 5 mm* pieces) were prepared, cultured with magnetically isolated osteoclasts, and processed for scanning electron microscopy as described by Webber et al.'211
Scanning electron microscopy Samples for scanning electron microscopy were prepared and viewed as outlined in Webber et al.l2l1Samples were gold sputter coated and viewed on a Philips 501 scanning electron microscope at 15 kV with a tilt angle of 35".
RESULTS
Optimal immunosorting of osteoclasts
polystyrene beads precoated with osteoclast-directed monoclonal antibodies (121F, 35L, or 75B) to purify osteoclasts from mixed cell populations. Magnetic polystyrene beads coated with monoclonal antibody 121F were found to readily bind to isolated osteoclasts present in the population 1-2 and population 3 mixed cell suspensions. These beads bound both quickly and selectively to osteoclasts whether the bead-cell mixture was maintained on ice or not. Beads not conjugated with osteoclast antibodies displayed little, if any, nonspecific affinity ( 90Vo) nonviable. osteoclasts (in 1 ml) at varying bead to cell ratios for 2 minutes on The third population contains 10% osteoclasts; however, ice. The standard ratio was calculated as 12:1 based on incubation 60% of these are viable and, more importantly, 90% of all of 2 x lo' beads with one-sixth of a population 2 osteoclast (OC) the viable osteoclasts released from the bones are recov- prearation (typically 1.7 x 10' OCs). Bead-bound cells were magnetically sorted and washed, and the OCs in aliquots of the sorted ered in this third population. Both the nonviable (popula- samples were counted by light microscopy using a hemacytometer. tions 1 and/or 2) and the predominantly viable (population The remaining sorted cells were plated in tissue culture dishes for 5 3) osteoclasts were employed in studies using magnetic h to determine OC purity (92%) and viability (10-12%). ~
~~
HIGH PURITY IMMUNOMAGNETIC ISOLATION OF OSTEOCLASTS
Yield, viabilit.y, and morphology of isolated osteoclasts Population 2 cells (collagenase-released, primarily nonviable) that were immunomagnetically isolated were reproducibly 80-99% pure for osteoclasts. The higher range purities were routinely derived from populations that had been partially purified by density gradient separations over 35% Percol116)before bead incubation and magnetic sorting. lmmunosorted cells were consistently 10-15% viable, representing the same proportion of live osteoclasts known to be present in this starting materialI6’ and demonstrating no additional diminution in osteoclast viability associated with immunomagnetic sorting. The viable osteoclasts often produced extended processes on plastic tissue culture dishes. Light and transmission electron microscopic examination of these sorted cells showed that numerous beads were attached t o each osteoclast cell surface (Fig. 1). In accordance with viability measures, many of the isolated multinucleated cells from this population exhibited degenerative morphologies, including nuclear pyknoses (Fig. 1). Similarly, population 3 cells (trypsin-released, high viability) were successfully sorted by immunomagnetic means, as illustrated in Fig. 2. Large numbers of highly enriched and viable osteoclasts were obtained from this population using the magnetic sorting procedure. More than 90% (4.86 x lo6) of the osteoclasts present in the original pop-
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ulation 3 cells (5.3 x lo6) were retrieved by the immunobeads. When these magnetically sorted multinucleated cells were examined by transmission electron microscopy (Fig. 2C), many of the cells appeared devoid of such nuclear, organellar, or membrane abnormalities as generally ascribed to degenerative cells. Occasionally, matrix material and small aggregates of mononuclear cells (perhaps also associated with matrix fragments) copurrfied with the immunomagnetically sorted osteoclasts from population 3 . In these instances, close examination of the magnetic beadbound matrix or cell aggregate revealed that they possessed osteoclasts, which were presumably responsible for their coincidental sorting with the free osteoclasts released from the bone matrix. The microscopic observations not only illustrated the tight binding of the antibody-coated magnetic beads to the osteoclast plasma membrane but, further, showed that the beads could be readily phagocytosed by the cells (Figs. 2 and 3). Quantitative assessment of the purity and viability of magnetically sorted osteoclasts derived from population 3, in comparison with those isolated from population 2, was performed. Immunomagnetic separation of osteoclasts from population 2 bone cells typically yielded preparations greater than 90% pure for osteoclasts, in which only 10% of these cells were viable as determined by trypan blue dye exclusion, fluorescein diacetate staining, or cell attachment to culture dishes. In comparison, immunomagnetic sorting
FIG. 1. (A) Immunomagnetic beads (arrows) become associated with large multinucleated osteoclasts (OC) and some cell and matrix debris (asterisks) as shown in the 1 gm section of Epon-embedded population 2 cells. Magnification x 960. (B) A transmission electron micrograph demonstrating immunomagnetic beads associated with the osteoclast plasma membrane (population 2). Note the pyknotic nucleus (arrow). Magnification x 2925.
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FIG. 2. (A) Phase-contrast micrograph of population 3 viable osteoclasts placed in culture immediately following their isolation via immunomagnetic beads coated with 121F monoclonal antibody. The dark images represent bead clusters bound to osteoclasts, whereas the very small refractile elements are individual beads not associated with cells. A small amount of matrix debris is also observed (M). Magnification x 110. (B) A higher magnification of an osteoclast population 3 preparation demonstrating the high concentration of beads associated with the immunomagnetically sorted cells reflected by the dark regions surrounding and enveloping the osteoclasts (arrows). Magnification x 440. (C) A transmission electron micrograph of an osteoclast isolated with 121F monoclonal antibody-coated magnetic beads (b). Note the partial phagocytosis of an immunobead and the close association between beads and the plasma membrane. Magnification x 5863.
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HIGH PURITY IMMUNOMAGNETIC ISOLATION OF OSTEOCLASTS of population 3 cells resulted in a slightly lower osteoclast purity (86Vo) than attained from population 2 cells. However, both the viability (99Vo) and yield (4.9 x lo6 from three-fifths of a preparation) of osteoclasts from this third population were quite high. The slight reduction in osteoclast purity from sorted population 3 cells is likely to have been a consequence of other matrix-associated cells being partitioned along with those osteoclasts associated with matrix fragments and/or cell aggregates during magnetic sorting. Attempts to reduce the solution viscosity caused by repolymerizing collagen during immunosorting by inclusion of 0.05 U chondroitinase ABC were without effect.
Specificity of immunomagnetic isolation To evaluate the restricted nature of binding to osteoclasts by antiosteoclast antibody-coupled magnetic beads, population 3 cells were treated with varying levels of the 121F antibody before their incubation with 121F antibodyconjugated beads. Table 2 presents findings from such an experiment, demonstrating a progressive decline in the number of osteoclasts isolated by the immunomagnetic beads in direct proportion to the concentration of 121F antibody to which osteoclasts were preexposed. Therefore, preincubation with antiosteoclast antibody blocked access of the matching antibody-coated beads to the osteoclast cell surface. At the higher antibody pretreatment levels, no viable osteoclasts were recovered. Clearly, then, the attraction of the osteoclasts for the immunomagnetic beads is mediated by the 121F antiosteoclast antibody in a concentration-dependent fashion. Specificity attributed to the immunomagnetic isolation technique exists both at the cellular and antibody levels. O n the one hand, osteoclasts are readily captured by antiosteoclast antibody (121F, 75B, or 35L)-conjugated immunonmagnetic beads but are unresponsive toward beads devoid of primary antibody or coupled with an irrelevant antibody (A12E) directed against algal flagellar proteins. Moreover, the latter bead types are never internalized by the osteoclasts, demonstrating that ingestion of beads is not a non-
specific phenomenon. Conversely, antiosteoclast antibodycoupled beads are specifically attracted to osteoclasts as opposed to other cells present in the bone cell populations, since the latter are never observed to accumulate any beads on their surfaces.
Functional integrity of immunomagnetically isolated osteoclasts Retention of osteoclast viability and functional properties in cells subjected to immunomagnetic isolation was further documented by experiments in which magnetically sorted osteoclasts were cultured on devitalized bovine cortical bone slices. Figures 3A and B depict magnetically sorted osteoclasts that attached to bone slices and within a 48 h period created well-defined resorption pits. Along with cells displaying characteristic osteoclast morphologies on bone, occasional elongated, spindle-shaped cells, as well as flattened large cells, were associated with the beads. These bead-bound, flattened cells were often observed closely associated with resorption pits. In each panel of Fig. 3, it is striking to note that cultured osteoclasts were capable of internalizing large numbers of the antibodycoated beads without suffering a reduced ability to attach to either bone matrix or the tissue culture plastic substratum. Osteoclast internalization of antibody-coated magnetic beads was not a unique phenomenon related to the 121F monoclonal antibody, since two other monoclonal antibodies that recognize osteoclast-restricted cell surface antigens, 35L and 75B, were similarly internalized via the magnetic beads once the magnetically sorted cells were cultured (Fig. 3D and E) or removed from ice. By 96 h of culture on bone slices, osteoclast morphology appeared altered to a less active phenotype.
Desorption of beads from sorted cells For some applications, isolated osteoclasts no longer bound to antibody-coated magnetic beads are desirable, and so methods for desorbing beads off the osteoclasts
TABLE2. DECREASED IMMUNOBEAD BINDING TO ANTIBODY-PRETREATED OSTEOCLASTS~ ~
IZIF MAb prerncubation (pg)
~~
OC yield Total OCs
Viable OCs
To Viable OCs
x x x x
1.6 x lo6 2.0 x 106 0 0
94 87
1.7 2.3 5.5 5.5
lo6 lo6 105 105
-
-
aPopulation 3 osteoclasts (two-thirds preparation) were equally divided between four tubes (each in 2 ml Ca,Mg-free TBSS) and preincubated with shaking for 30 minutes with ice-cold 121F antibody (0-4 pg). 121F antibody-conjugated magnetic beads (3 x 10") were then added to each sample and the mixtures further incubated 30 minutes while shaking on ice. Bead-bound cells were magnetically sorted, washed, and cultured for 5 h before yield, purity, and viability determinations as described in Materials and Methods. Purity of the viable OCs was unaffected by 1 pg antibody preincubation (90%) in comparison with n o antibody pretreatment (89%).
1360 were sought. Other investigators have reported success with physically dislodging the beads from cells by stirring or vortexing the complexes in large volumes to discourage reassociation.'") To avoid rapid ingestion of the immunobeads by the osteoclasts, steps were performed, when possible, at ice-cold temperatures. Physical disruption (vortexing), proteolytic cleavage of antibodies and/or osteoclast antigens (papain or trypsin), or chemical uncoupling of antibody-antigen complexes (low pH) were individually investigated, and their relative effectiveness at removing beads without adversely affecting osteoclast viability was ascertained. In the studies reported here, vigorous vortexing was the most effective method for desorbing beads (90%)from the magnetically isolated osteoclasts (Table 3). Although a high percentage of immunomagnetic beads were removed, many released osteoclasts frequently retained a small number of beads (three to five), representing one-tenth to one-quarter of the original number of bound beads, on their surfaces (Fig. 4). Some osteoclasts were completely stripped of associated beads; others remained well coated. This brief mechanical treatment did not reduce osteoclast viability or function. However, repetitive or prolonged vortexing led to a marked deterioration of osteoclast structure and was avoided. Physically desorbed cells were capable of rapid attachment to bone (within 30 minutes), as well as production of resorption pits (within 24 h). Scanning electron microscopic examination of osteoclasts on bone slices demonstrated that all osteoclasts attached to bone were capable of producing resorption pits. Alternative desorption methods (Table 3) were less promising. Brief exposure to low pH (54.5) to disrupt antibody-antigen interactions yielded good initial dissociation of beads from the osteoclasts, but as revealed in other experiments, these bonds were suspected of rapidly reforming once the acid pH was neutralized. Papain digestion was generally as effective in releasing osteoclasts (7080%) from the immunomagnetic beads as low pH treatment (data not shown). Desorption was not facilitated by decreasing the bead to cell ratio, immunosorting osteoclasts with mixed antibody beads partly conjugated with an irrelevant antibody (A12E), pretreating osteoclasts with 121F antibody before bead incubation, or employing alternative antiosteoclast antibodies (35L or 75B) for bead coupling.
Reproducibility and other considerations The immunomagnetic separation procedure for obtaining isolated osteoclasts has been performed repeatedly with
COLLIN-OSDOBY ET AL.
a consistently high level of success. It has proven equally effective at isolating osteoclasts from normal diet chicks as from the low-calcium chicks described here. As with any other primary cell isolation procedure, a certain variability arises between trials, which is a consequence of such factors as the composition of the starting cell population and the technical proficiency of the individuals carrying out the procedure. Notwithstanding, the rapid purification of osteoclasts from the population of 1 and/or 2 cells is highly reproducible. To achieve comparable results with population 3 cell preparations, it was necessary to increase the volume of the bead-cell reaction mixture 10- to 20-fold and to lengthen the sorting time in the magnetic field 5- to 10-fold (10 minutes, as opposed to 1-2 minutes) to overcome the solution viscosity experienced from repolymerizing collagen. Under these conditions, excellent osteoclast purities and recovery yields were obtained from each starting population.
DISCUSSION This report describes a new osteoclast purification procedure in which osteoclast-directed monoclonal antibodi e ~ " ~ - are ' ~ ) coupled to magnetic polystyrene beads and used to immunomagnetically separate these multinucleated cells from mixed bone cell populations. The present isolation scheme builds on previously published isolation methods, which involve sequential physical and enzymatic steps to obtain three osteoclast populations, having somewhat different osteoclast purities and viabilities associated with them, from chickens maintained for 4 weeks on low-calcium diets.'b) Further purification of viable osteoclasts is difficult to achieve because of the wide-ranging sizes and buoyant densities associated with the viable osteoclast population. In addition, the comparable densities of red blood cells and some small bone matrix fragments permit these elements to copurify with the osteoclasts. Therefore, an alternative approach was sought to obtain highly enriched osteoclast preparations for use in physiological biochemical, and molecular investigations focused on osteoclast activity and regulation. In recent years, various techniques have been employed to study osteoclast function and regulation in individual cells or populations of isolated osteoclasts. The well-characterized resorption pit assay, developed initially by Boyde and Chambers et al.'25,261 has been successfully et al.'23.*4) used for both qualitative and quantitative investigations of isolated osteoclast activity. Enzyme histochemistry and in situ nucleic acid hybridization analysis have enabled prob-
FIG. 3. (A) A scanning electron micrograph of osteoclasts separated and purified using 121F monoclonal antibodycoated magnetic beads. Immunosorted osteoclasts were cultured for 48 h on bovine cortical bone slices. Note the formation of well-defined resorption pits and the obvious internalization of antibody-coated beads (arrowheads). Other osteoclasts are completely covered with beads (asterisks). A few elongated cells are also observed. Magnification x 960. (B) A higher magnification of a representative immunomagnetically sorted osteoclast cultured on a bovine cortical bone slice. Numerous beads have been internalized, and filamentous processes (arrowheads) emanate from the cell near its attachment site to bone and also by the excavted resorption pit. Bars represent 10 pm. Magnification x 1875. (C-E) Scanning electron micrographs of osteoclasts isolated with magnetic beads coupled to 121F, 75B, or 35L antiosteoclast monoclonal antibodies, respectively. Isolated cells were cultured on tissue culture plastic for 24 h. Each type of antibodycoated bead was internalized. However, neither bead binding nor their internalization appeared to negatively influence cell attachment. Bars 10 pm. Magnification x 1OOO.
COLLIN-OSDOBY ET AL.
1362 TABLE3 . OSTEOCLAST VIABILITYA N D PURITYAS OF DESORPTION METHOD^
A
FUNCTION
Number of viable OC/field Desorption method Untreated Vortexed Low pH Low trypsin High trypsin DNAase/trypsin
Released
Bead-bound ocs
-
45 4 20 23 28 19
ocs 35 17 1
0 6
% OC purity
Released ocs
Bead-bound ocs
-
92 80 67 88 88 79
96 71 33 -
60
a I21 F antibody-coated immunomagnetic beads were incubated with trypsin-released cells on ice, magnetically sorted, and equivalent portions of the bead-bound cells subjected to desorption trials as detailed in Materials and Methods. Cells retained on the beads following desorption were magnetically separated from released cells, and both portions were cultured for 5 h before viability and purity determinations. Results from a representative desorption series are presented. Vortexing was the most effective desorption protocol (90% OCs released), followed by low-pH treatment (85% OCs released).
ing at the single-cell level. Advanced technologies, such as patch clamp a n a l y s i ~ ' ~ ' - ' ~and ' improved image analys ~ s , ( ~ Ohave . ~ ' ) provided valuable procedural means to examine the function of individual osteoclasts. Despite the obvious strengths of such methods, there are notable limitations restricting their use or the conclusions drawn from studies employing these strategies. On the one hand, inaccuracies can arise and be magnified when results are extrapolated from individual cells to a population as a whole. On the other hand, isolated populations generated by protocols available to date are typically impure for osteoclasts and thus highly susceptible to influences exerted by contaminating cell types. Among the advantages of the immunomagnetic isolation procedure are the relatively short times required, the gentle manipulations of the cell preparations possible, the high cell purities achieved, and the efficient recovery yields of osteoclasts obtained. In particular, when the yield and purity of immunomagnetically isolated osteoclasts are compared with those associated with osteoclasts purified by size and buoyant density dependent methods,'6) it is clear that the immunomagnetic protocol reflects a dramatic improvement in the purity of the population 1-2 osteoclasts, as well as an improvement in both the purity and yield of the viable population 3 osteoclasts. The enhanced yields from immunomagnetic isolations over alternative protocols are likely due to the avoidance of steps based on cell size and buoyant density, since both these vary considerably among osteoclasts residing in population 3 cells. Instead, immunomagnetic cell isolation relies upon a possibly more uniform osteoclast parameter, namely, expression levels of surface antigens reactive with osteoclast-specific monoclonal antibodies. The ultimate cell purity, ease of separation, and final recovery yield achievable by immunomagnetic protocols are a function of the specificity and avidity of the antibodies employed, the proportion of target cells in the original population, and, to a lesser extent, the manipulation regimen followed. Since partitioning of
osteoclasts apart from other bone cells is performed easily and quickly with immunomagnetic beads, omitting lengthy and multistep handling of the cells, the viability and biological integrity of the osteoclasts are maintained. One potential disadvantage of the immunomagnetic procedure relates to observations that plasma membrane antigens may be activated through binding of antibodies in a manner analogous to receptor-ligand responses."') Therefore, a degree of caution is necessary when proposing experiments to examine physiologic responses of osteoclasts using cells isolated in this manner. However, immunomagnetic isolation of other cell types has not resulted in their it is clear from the scanning a ~ t i v a t i o n . ' ~Furthermore, ~) electron microscopic observations that immunomagnetically purified osteoclasts can attach to bone and generate characteristic resorption pits, even when no attempts are made to remove the beads. In fact, each attached osteoclast was found to excavate a resorption pit. In other experiments, immunomagnetically isolated avian osteoclasts were shown to respond to estrogen by decreased bone resorption and increased nuclear protooncogene mRNA levels.(34)A further consideration for some applications is the observation that once the cells are released from lowtemperature conditions they are capable of internalizing the beads. Therefore, if later removal of the beads is desirable, it is best to carry out all the prior incubation and magnetic isolation steps on ice. Cold temperature itself causes no apparent decrease in the number or strength of beads interacting with osteoclasts or in the ultimate purity or yield achieved. Although this may be a feature associated with the affinity of a particular antibody for the target antigen, similar results have been obtained using the 121F, 75B. or 35L antiosteoclast monoclonal antibodies, suggesting that these all exhibit roughly comparable beadcell interactions. The concern for antibody and/or bead effects on osteoclasts can partially be overcome by removal of the beads from the cells before their use in physiological studies. Un-
HIGH PURITY IMMUNOMAGNETIC ISOLATION OF OSTEOCLASTS
1363
FIG. 4. (A) A low-power scanning electron micrograph of osteoclasts cultured on cortical bone slices after immunomagnetic separation with 121F monoclonal antibody beads and subsequent bead desorption through vortexing. Stripped osteoclasts display fewer beads associated with their surfaces. Many resorption pits formed by the desorbed osteoclasts are seen (asterisks). Magnification x 576. (B) A higher power scanning electron micrograph of a preparation similar to A. Note the decreased number of beads (arrows) associated with the osteoclast, as well as a few internalized beads (arrowheads). Part of a resorption pit can also be observed. Magnification x 1152.
COLLIN-OSDOBY ET AL.
1364
like some other cell types, such as lymphocytes, osteoclasts Anderson for his expert technical assistance in cell prepain culture d o not shed antibody-coated beads from their ration, and Ms. Terri Moulton for her patient typing of surfaces. Quite the contrary, these multinucleated cells ac- this manuscript. This work was supported by NIH Grants tively internalize beads bound to their membranes, pre- AR32927 and AR32087 and a Research Career Developventing their removal. As a further complication, more ment Award K04-AM1474 to Dr. Philip Osdoby. than five beads per osteoclast were required to attain effective magnetic sorting of these large cells, although bead to REFERENCES cell ratios of 1: 1 or 2: 1 are recommended for desorption trials (Dynal, Inc.). The most effective desorption proceI . Rodan G , Martin T 1981 Role of osteoblasts in hormonal dure, with simultaneously the least adverse effect on osteocontrol of bone resorption-a hypothesis. Calcif Tissue Int clast yield and viability, was mechanical vortexing, which 33:349-35 I . stripped the majority of beads from the osteoclast surface. 2. DeVernejoul M, Horowitz M. Demignon J , Neff L. Baron R Complete removal of every bead from all target cell sur1988 Bone resorption by isolated chick osteoclasts in culture faces has not yet been reported for any system. I f cells is stimulated by murine spleen cell supernatant fluids (osteototally devoid of beads are required, the opposite strategy clast-activating factor) and inhibited by calcitonin and prosbased on negative immunomagnetic selection to deplete taglandin E,. J Bone Miner Res 3:69-80. contaminating cells from a preparation, leaving the desired 3 . Zambonin-Zallone A, Teti A , Primavera M I982 Isolated cell population, is the preferred route. osteoclasts in primary culture: First observations o n structure The high purity and yield of osteoclasts obtained by this and survival in cultured media. Anat Embryo1 (Berl) 165: 405-41 3 . procedure has the potential to facilitate sensitive physio4. Osdoby P, Martini M, Caplan A 1982 Isolated osteoclasts logical studies, such as those to determine whether a horand their presumed progenitor, the monocyte, in culture. J mone, cytokine, or other modulator of bone remodeling Exp Zoo1 224:33 1-334. acts directly on the osteoclast or, alternatively, upon other 5 . Hefly T , Stern P 1982 Isolated osteoclasts from fetal rat long cells in the bone environment, which in turn influence bones. Calcif Tissue Int 34:480-487. osteoclast function. Another powerful application of this 6. Oursler M J , Collin-Osdoby P , Anderson F, Li L, Webber D, procedure is to provide cellular material for biochemical Osdoby P 1991 Isolation of avian osteoclasts: Improved techand molecular characterization of the osteoclast phenoniques to preferentially purify viable cells. J Bone Miner Res type. Using such highly purified cells, it may be possible 6:375-385. to perform detailed protein fingerprinting on structural, 7. Hutchins D, Steel CM 1979 Separation of human lymphocytes on the basis of volume and density. In: Peeters H (ed.) metabolic, and secretory proteins involved in osteoclast acSeparation of Cell9 and Subcellular Element\. Pergamon tivity and cell-cell or cell-matrix interactions. Beyond the Press, Oxford, pp. 28-43. physiological and biochemical approaches, i t is possible to 8. Harris R, Ukaejiofo E O 1970 Tissue typing using a routine confidently pursue molecular studies on pure populations one-step lymphocyte separation technique. Br J Haematol of osteoclasts isolated in this manner. The immunomag18~229-235. netic isolation scheme has already proven vital for identify9. Sharpe P T 1988 Methods of Cell Separation. Elsevier, New ing estrogen-responsive receptors on avian o s t e o c l a s t ~ ~ ~ ~ York. ~~ and for successfully obtaining sufficient intact osteoclast 10. Gaudernack G , Leivestad T, Ugelstad J , Thorsby E 1986 mRNA for construction of an osteoclast cDNA library Positive 5election with monoclonal antibodies directly conju(unpublished observations). In addition, consistent with gated t o monosiled magnetic microspheres. J lmmunol Methods 90:179- 189. our demonstration that the 121F monoclonal antibody recI . Padmanabhan R, Corsico C D , Howard T H , Holter W , ognizes a complementary antigen on human osteoclast-like Fordis C M , Willingham M, Howard BH 1988 Purification of cells, this immunomagnetic purification procedure has transiently transfected cells by magnetic affinity cell sorting. enabled purification of functional human osteoclasts as Anal Biochem 170:341-348. well (unpublished observations). In a different vein, unlike 2. Vaccaro DE 1990 Applications of magnetic separation: Cell the positive selections for osteoclasts, this technique could sorting. Am Biotech Lab 8:30-35. be utilized to selectively deplete a bone cell preparation of 3. Lea T, Smeland E, Funderud S, Vartdal F, Davies C , Beiske osteoclasts for study of the remaining cells or for subseD, Ugelstad J 1986 Characterization of human mononuclear quent addition of experimental cells or substances. Because cells after positive
