Formation of a Three-Dimensional Multicellular Assembly Using

Jan 23, 2009 - Human endothelial progenitor cells and mouse macrophages were magnetically labeled using anionic citrate-coated iron oxide nanoparticle...
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Langmuir 2009, 25, 2348-2354

Formation of a Three-Dimensional Multicellular Assembly Using Magnetic Patterning Guillaume Frasca, Florence Gazeau, and Claire Wilhelm* Laboratoire Matie`re et Syste`mes Complexes (MSC), UMR 7057 CNRS & UniVersite´ Paris-Diderot, Paris Cedex 13, F-75205, France ReceiVed September 19, 2008. ReVised Manuscript ReceiVed December 16, 2008 We demonstrate a facile approach to design three-dimensional cellular assembly of tunable size and controlled geometry with applications for tissue engineering. Three-dimensional cell patterning was performed using external magnetic forces, without the need for substrate chemical or physical modifications. Human endothelial progenitor cells and mouse macrophages were magnetically labeled using anionic citrate-coated iron oxide nanoparticles. Two magnetic tips were designed, and their magnetic field cartographies were calibrated. The focalized magnetic force generated ensured an efficient entrapment of the cells at the tips vicinity. By tuning the magnetic field gradient geometry and intensity, the magnetic cellular load, and the number of cells, we fully described the formation of the three-dimensional multicellular assemblies, and estimated the corresponding packing factor for a large range of experimental conditions.

1. Introduction Controlling spatial organization of a large number of living cells is of utmost importance to make headway in tissue engineering. Indeed, if organ function depends strongly on cell composition, structure is also a key parameter.1 The ongoing challenge is then to achieve reproducible and tuneable patterning of three-dimensional (3D) multicellular assemblies on the way to potential organs. Scaffold-based techniques initiated by Langer and Vacanti2 give access to 3D cell organization and culture by providing a template for cell engraftment. However, most cell seeding approaches lack control of cell density3 and distribution, which are essential for the formation of tissues in vitro. To improve spatial control, cell adhesion guidance over a substrate has been developed following several strategies.4 The cells are passively patterned through a random seeding on structured substrates. Engineered substrates, either physically (by nanostructuration5) or biochemically modified (by ligand linkage6-8 or specific coating tuning surface wettability9,10), can induce a spatial segregation based on cell-substrate interactions differences. The main drawback of this approach is the persistence of differential adhesion properties, which could affect subsequent cell behavior. It cannot guarantee the nonspecificity of cell environment, which is crucial to generate unbiased experimental outcomes. Recently, advanced surface chemistry proposed an electroactive coating allowing reversible cell adhesion.11 None-

theless, these techniques remain expensive and time-consuming, and are limited to two-dimensional patterning. Another approach consists of active patterning through the use of an external force. This constraint can be generated by an electric field, as in the dielectrophoresis technique recently developed by Sebastian et al.,12,13 or by an optical trap.14,15 Alternatively, following the hints of Kimura et al.,16 the pioneering works by Ino et al.17,18 suggested the use of magnetic constraint to induce two-dimensional patterning of magnetically labeled cells on submillimetric scales. Following a similar approach, we applied magnetic forces to create a 3D cell assembly with tuneable size and controlled geometry. Magnetic labeling of living cells is a well-studied nontoxic technique, befitting for a large range of cell types.19 It was widely developed to allow magnetic resonance imaging (MRI) cell detection and monitoring of cell migration, including stem cells.20,21 Beyond detection, cell labeling supplied sufficient magnetization to apply magnetic forces when labeled cells are submitted to an inhomogeneous magnetic field. It initiated a variety of biological applications, from cell sorting in microfluidic devices22 to controlled cell migration through magnetotaxis23 or confined morphogenesis.24 In our study, we applied a temporary magnetic force on a suspension of magnetically labeled cells to form a 3D multicellular aggregate. The magnetic field characteristics (magnitude, geometry, duration of application) will impose the geometry of the so-formed assembly. This technology can be applied with no

* Corresponding author. E-mail: [email protected]. (1) Isenberg, B. C.; Wong, J. Y. Mater. Today 2006, 9, 54–60. (2) Langer, R.; Vacanti, J. P. Science 1993, 260, 920–926. (3) Bueno, E. M.; Laevsky, G.; Barabino, G. A. J. Biotechnol. 2007, 129, 516–531. (4) Falconnet, D.; Csucs, G.; Grandin, H. M.; Textor, M. Biomaterials 2006, 27, 3044–3063. (5) Curtis, A.; Wilkinson, C. Biomaterials 1997, 18, 1573–1583. (6) Zhang, S.; Yan, L.; Altman, M.; Lassle, M.; Nugent, H.; Frankel, F.; Lauffenburger, D. A.; Whitesides, G. M.; Rich, A. Biomaterials 1999, 20, 1213– 1220. (7) Veiseh, M.; Wickes, B. T.; Castner, D. G.; Zhang, M. Biomaterials 2004, 25, 3315–3324. (8) Dillmore, W. S.; Yousaf, M. N.; Mrksich, M. Langmuir 2004, 20, 7223– 7231. (9) Tan, J. L.; Liu, W.; Nelson, C. M.; Raghavan, S.; Chen, C. S. Tissue Eng. 2004, 10, 865–872. (10) Yamazoe, H.; Uemura, T.; Tanabe, T. Langmuir 2008, 24, 8402–8404. (11) Yeo, W. S.; Yousaf, M. N.; Mrksich, M. J. Am. Chem. Soc. 2003, 125, 14994–14995.

(12) Sebastian, A.; Buckle, A. M.; Markx, G. H. Biotechnol. Bioeng. 2007, 98, 694–700. (13) Venkatesh, A. G.; Gerard, H. M. J. Phys. D: Appl. Phys. 2007, 106. (14) Nahmias, Y.; Schwartz, R. E.; Verfaillie, C. M.; Odde, D. J. Biotechnol. Bioeng. 2005, 92, 129–136. (15) Zhang, H.; Liu, K. K. J. R. Soc. Interface 2008, 5, 671–690. (16) Kimura, T.; Sato, Y.; Kimura, F.; Iwasaka, M.; Ueno, S. Langmuir 2005, 21, 830–832. (17) Ino, K.; Ito, A.; Honda, H. Biotechnol. Bioeng. 2007, 97, 1309–1317. (18) Ino, K.; Okochi, M.; Konishi, N.; Nakatochi, M.; Imai, R.; Shikida, M.; Ito, A.; Honda, H. Lab Chip 2008, 8, 134–142. (19) Wilhelm, C.; Gazeau, F. Biomaterials 2008, 29, 3161–3174. (20) Bulte, J. W.; Kraitchman, D. L. NMR Biomed. 2004, 17, 484–499. (21) Arbab, A. S.; Frank, J. A. Regener. Med. 2008, 3, 199–215. (22) Pamme, N.; Wilhelm, C. Lab Chip 2006, 6, 974–980. (23) Wilhelm, C.; Riviere, C.; Biais, N. Phys. ReV. E: Stat., Nonlinear, Soft Matter Phys. 2007, 75, 041906. (24) Frasca, G.; Raynaud, F.; Bacri, J.-C.; Gazeau, F.; Wilhelm, C. J. Phys.: Condens. Matter 2008, 20, 204149.

10.1021/la8030792 CCC: $40.75  2009 American Chemical Society Published on Web 01/23/2009

Magnetic Patterning to Form 3D Cellular Assemblies

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restriction regarding the physicochemical nature of the substrate, the cell type, or the geometry of the imposed magnetic constraint.

2. Material and Methods 2.1. Cell Culture of Endothelial Progenitor Cells (EPCs) and RAW Macrophages. CD34+ cells were isolated from human cord blood samples (Maco-Pharma, Tourcoing, France), and expanded in endothelial basal medium-2 (EBM2) supplemented with EGM2MV SingleQuots (Clonetics Cambrex, Ermerainville, France). After 10 days of incubation, CD34+ cells are identified as EPCs.25,26 Cells were not passaged more than 10 times. Mouse RAW macrophages were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% of heatinactivated fetal calf serum, 50 U/mM of penicillin, 40 mg/mL of streptomycin and 0.3 mg/mL of (L)-glutamine. Both EPCs and macrophages are cultured as adherent cells in flasks. Cell diameters measured on spherical cells in suspension were 13.9 ( 1.9 µm for EPCs and 10.3 ( 1.2 µm for RAW macrophages. 2.2. Magnetic Cell Labeling and Quantification of Iron Uptake. Cells were magnetically labeled using anionic magnetic nanoparticles (AMNPs) of maghemite (γ-Fe2O3) of diameter 7 nm and polydispersity index 0.35 synthesized by Massart’s process.27 The net negative charge is due to absorption of citrate anions to the nanoparticle surface, leading to a zeta potential of about -35 mV. Cell labeling was performed by incubating adherent, confluent cells with a serum-free culture medium (RPMI) containing AMNPs at a concentration of 2 mM. The labeling medium was supplemented with free citrate anions at a concentration of 5 mM to avoid citrate desorption and subsequent AMNP flocculation. The cultured cells were kept for 2 h in favorable growth conditions (37 °C, 5%CO2 in a humidified atmosphere). The AMNP-charged medium was then flushed, and the cells were rinsed once with culture medium, then left for a 1 h chase in culture medium at 37 °C to allow complete internalization of nanoparticles. The magnetic uptake was measured through single-cell magnetophoresis,28 giving access to the distribution of iron load per cell. Iron load quantification through magnetophoresis is based on the measure of the velocity of a magnetically labeled cell moved by a constant magnetic gradient of known intensity. By balancing the magnetic force, MgradB, and viscous force, 6πηRV, one has access to the cell magnetic moment through eq 1:

M)

6πηRV gradB

(1)

where η is the viscosity of the carrier fluid, R is the cell radius, V is its velocity, and M is its magnetic moment. It then leads to the mass of iron internalized by one individual cell.28 In parallel, intracellular localization of magnetic particles was assessed by electron microscopy (Zeiss EM902 microscope, 70 nm sections of Epon embedded cells), performed at INRA (Laboratoire de Ge´nomique et Physiologie de la Lactation, Jouy en Josas, France). 2.3. Calibration of Magnetic Field Gradients. Two magnetic devices have been used to create magnetic field gradients: a 750 µm diameter cylinder, and a truncated cone of same basal diameter, in soft iron, both being magnetized at saturation by a permanent magnet generating a magnetic field of 0.32 T at the surface. Both devices display cylindrical symmetry. Magnetic field gradients generated by the devices were calibrated experimentally, by tracking the movement of fluorescent magnetic beads of 4.6 µm diameter of well-defined magnetic moment (M ) 2.0 ( 0.4 × 10-13 A · m2) in a solvent of known viscosity (98% (v/v) glycerol/water, η ) 0.61 (25) Bompais, H.; Chagraoui, J.; Canron, X.; Crisan, M.; Liu, X. H.; Anjo, A.; Tolla-Le Port, C.; Leboeuf, M.; Charbord, P.; Bikfalvi, A.; Uzan, G. Blood 2004, 103, 2577–2584. (26) Yoder, M. C.; Mead, L. E.; Prater, D.; Krier, T. R.; Mroueh, K. N.; Li, F.; Krasich, R.; Temm, C. J.; Prchal, J. T.; Ingram, D. A. Blood 2007, 109, 1801–1809. (27) Massart, R. IEEE Trans. Magn. 1981, 17, 1247–1248. (28) Wilhelm, C.; Gazeau, F.; Bacri, J. C. Eur. Biophys. J. 2002, 31, 118–125.

Pa · s at 25 °C). The inertial force being negligible, magnetic force on the bead is directly balanced by viscous force as expressed in eq 1. By tracking bead movement with a camera (Micromax, Princeton Instruments) and averaging the velocities of 40 magnetic beads crossing each 0.2 mm × 0.2 mm square, a magnetic field gradient map was calculated for both tips. 2.4. Formation of 3D Multicellular Assembly by Magnetic Constraint. For cell patterning, a laboratory-made square chamber with a side of 50 mm was set above the magnetic device. The chamber was designed to allow both front and top observations. Its bottom was sealed by 100 or 200 µm thick glass slides. To tune surface thickness toward lower values, mylar sheets (polyester film, DuPont Teijin Films) of various thicknesses from 8 to 75 µm were used as well. Cells were magnetically labeled as described earlier. After the 1 h chase, cells were detached using cold EDTA, centrifuged at 1200 rpm for 5 min, and resuspended in a small volume of culture medium to reach a cell suspension concentration ranging from 106 to 5 × 108 cells/mL. The patterning chamber was filled with culture medium. A small volume, typically 1 µL, of cell suspension was gently deposed at the vicinity of the magnetic tip onto the bottom substrate of the chamber. The magnetically labeled cells were driven to the tip and started piling to form a macroscopic aggregate on the substrate. The multicellular assembly obtained was kept for 10 min under magnetic constraint before the removal of the magnetic tip. The aggregate profile was then observed with a camera Canon ESO 20D equipped with a high-magnification lens. Its projection was visualized through a microscope Leica DM IRB 1.6×, and the images were captured by a Qimaging QiCam Fast 1394 digital camera. 2.5. Cell Proliferation and Viability. After 10 min under magnetic constraint, packed cells forming the aggregate were resuspended in culture medium and reseeded into six-well plates. The number of cells was measured every day from day 0 to day 4. The growth rate thus obtained was compared to unlabeled and nonconstrained cells. In some cases, the so-formed aggregate was observed after 24 h in the incubator in growth conditions, to observe how cells recover from their intense magnetic exposure and 3D packing. A viability test was conducted in parallel at day 0 by live-dead staining with calcein-AM. After a 10 min magnetic exposure, cells were resuspended in a well of a six-well plate containing 6 mL of phosphate-buffered saline (PBS) and 1.5 µL of 1 mM calcein-AM. After a 30-min incubation at 37 °C, the plate was set on ice. Cells were observed with fluorescence microscopy to quantify the living cells in green (calcein staining). The percentage of cells with exclusively green fluorescence (interpreted as viable cells) was calculated.

3. Results and Discussion 3.1. Cell Magnetic Labeling. After the magnetic labeling procedure, AMNPs were concentrated within intracellular vesicles as shown in Figure 1a,b, for both EPCs and macrophages. The organelles containing nanoparticles were identified as late endosomes or lysosomes in previous works.19,29 Indeed, AMNPs first spontaneously adsorb on the plasma membrane through electrostatic interactions. Unlike other surface modifications currently used for maghemite nanoparticles, such as dextrancoated superparamagnetic iron oxide (SPIO) showing no cell affinity, the negatively charged citrate coating interacts with the plasma membrane. No transfection agent is thus required to promote cell internalization of the nanoparticles, which is carried through the endocytosis pathway, a natural, nonspecific mechanism, thanks to their nanometric scale. This magnetic labeling technique has been demonstrated to be applicable to a broad range of cell types. Intracellular confinement of nanoparticles inside lysosomes protects the cells from eventual effect of free iron species, resulting in a nontoxic labeling.19 In particular, (29) Loube´ry, S.; Wilhelm, C.; Hurbain, I.; Neveu, S.; Louvard, D.; Coudrier, E. Traffic 2008, 9, 492–509.

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Figure 1. Magnetic labeling of two cell types: RAW macrophages and EPCs. (a,b) Electron micrographs of macrophages (a) and EPCs (b) after labeling for 2 h with maghemite nanoparticles at an extracellular iron concentration of [Fe] ) 2 mM, followed by a 1 h chase. For both cell types, the AMNPs appear as black points, confined within intracellular organelles, known as endosomes. Bar ) 1 µm. (c,d) Single-cell magnetophoresis gives the distribution among the cell population of cell magnetic velocity (expressed in µm · s-1) in a uniform magnetic field gradient of 17 mT/mm. The distribution of iron load per cell (expressed in pg of iron) (d) is then directly deduced by balancing the viscous drag force with the magnetic force. Iron load is distributed around a mean value of 5 pg and 12.1 pg for macrophages and EPCs, respectively.

AMNP-labeling of EPCs has been studied specifically,30 showing no adverse effect on cell proliferation, specific endothelial markers expression, or capacity to differentiate and self-organize into capillary-like networks. Internalized magnetic nanoparticles confer mobility to cells submitted to a magnetic field gradient. In the setup of single cell magnetophoresis, the magnetic field gradient is uniform (17 mT/mm), and the magnetic field (175 mT) is sufficient for AMNPs to attain 75% of their saturation magnetization value (2.23 × 105 A/m). Cell velocity ranges from 20 to 110 µm/s for labeled macrophages and from 40 to 180 µm/s for EPCs after a 2 h incubation with AMNPs ([Fe] ) 2 mM) as shown in Figure 1c. The dispersion in cell velocity reflects the variability of internalization capability among the cell population. As expressed by eq 1, cell velocity is proportional to the cell magnetization (proportional to cell iron load) and to the inverse of the cell diameter. Iron load (Figure 1d) can thus be deduced unambiguously for each cell after measurements of cell velocity and diameter. EPCs and macrophages show a mean iron load of 12.1 ( 3.6 pg and 5.0 ( 1.6 pg, respectively, after labeling for 2 h ([Fe] ) 2 mM), further called normal labeling conditions. To vary iron uptake, EPCs were also labeled for 30 min ([Fe] ) 0.5 mM), resulting in a lower iron load of 4.2 ( 1.7 pg. These results are in good agreement with saturating nanoparticle uptake previously observed19,30 as a function (30) Wilhelm, C.; Bal, L.; Smirnov, P.; Galy-Fauroux, I.; Clement, O.; Gazeau, F.; Emmerich, J. Biomaterials 2007, 28, 3797–3806.

of the incubation time. Differences of nanoparticle uptake between macrophages and EPCs are linked to their relative sizes (the larger the cell, the higher the AMNP uptake), the overall particles uptake being essentially governed by the external membrane surface (varying as the square of the cell diameter19). Importantly, as this magnetic labeling procedure allows reproducible and quantitatively predictable iron uptake for any cell type, the magnetic force experienced by cell (for a couple of magnetic fields and magnetic field gradients) can be considered as a tuneable parameter. 3.2. Magnetic Forces Used for Cell Patterning. The mapping of the magnetic field gradient, deduced from the averaging of magnetic beads velocities, is presented in Figure 2 for both magnetic device geometries. Magnetic field gradient was inhomogeneous, increasing when approaching the tip. The gradient generated by the cylindrical tip on its surface (i.e., its maximal value) was slightly lower than that for the truncated tip, but it decreases slower the further away from the tip it becomes. Therefore the truncated tip should be more suitable to apply large forces on a very local scale (distance inferior to 400 µm), whereas the cylindrical tip may entrap more distant cells. From calibration, one can estimate the magnetic force dragging a labeled cell to the magnetic device. As an example, the gradient was about 1000 mT/mm at 500 µm from the cylindrical tip, leading to a magnetic force of M · gradB ) 900 pN for an EPC labeled with 12.1 pg of iron. This force is by several orders of magnitude higher than other forces experienced by a cell in suspension, including Brownian motion (around 10-16 N) or buoyancy.

Magnetic Patterning to Form 3D Cellular Assemblies

Figure 2. Magnetic field gradients generated by a cylindrical tip (top) and a truncated tip (bottom) placed on a permanent magnet. Each tip modifies the magnetic field cartography at a local scale. Magnetic field gradients were mapped on a grid of 200 µm × 200 µm by averaging the velocities of 40 single magnetic beads of known magnetization crossing each square. The gradient created by the cylindrical tip decreases more slowly than for the truncated tip. At the tip proximity, both devices create a magnetic field gradient close to 2000 mT/mm.

3.3. Quantitative Control of Cell Pattern Geometry. 3D cell constructs were spontaneously formed by simply dropping the magnetically labeled cells in culture medium in the vicinity of the magnetic tip placed under the substrate. Cell deposit (constant volume of 1 µL) was performed at a distance of approximately 1 mm of the magnetic tip. As shown in Figure 3, cells progressively stacked to form a 3D aggregate. Cell deposit was complete within 30 s. Cell seeding density was varied from 104 to 4 × 105, whereas the deposit suspension volume was maintained constant. Figure 4 shows lateral and underneath views of 3D constructs obtained for various numbers of EPCs after a 10 min magnetic exposure to the magnetized cylindrical (top row) or truncated (bottom row) tip (thickness of the substrate: 100 µm). Note that we controlled the number of cells in the assembly: contrary to other patterning methods, there is minimal loss of cells during assembly formation (Figure 4, underneath views) since all the cells are attracted to the magnet as a result of both focalized injection of a highly dense cell suspension and the intensity of the magnetic force. This point may be of importance when manipulating stem cells, which are costly and time-consuming to grow. Geometrical dimensions of the aggregate, including radius R, height at the apex h, and volume V, were assessed from both profile and projection images. The aggregate profile was assimilated to a portion of an ellipse characterized by its semimajor

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axis a and semiminor axis b. Assuming axisymmetry, the volume is V ) (π/3)[(ah)/b]2(3b - h). The radius could be assessed from both views: the profile gives R ) a(1 -[(b - h)/b]2)1/2, while the projection allows a direct measurement of the aggregate radius. It was verified that the two measurements were in good agreement (relative error less than 5%). 3.4. Characterization of Multicellular Organization. To investigate the parameters governing the formation of 3D cell assembly, a range of experimental conditions were assessed by modifying the cell type, the cell iron load, the distance of substrate from the tip, and the tip itself. We first probe the impact of the tip geometry and associated map of magnetic field gradient (Figure 2), on the 3D assembly. The radius R of magnetically patterned aggregates was measured as a function of cell number N for EPCs at normal iron load using two different magnetic set-ups: cylindrical tip (thickness e ) 100 µm) and truncated cone (e ) 8 µm) (Figure 5a). For both magnetic devices, the assembly radius remains nearly constant for low cell number. These lower bounds depend on the dimensions of the magnetic tip (375 µm for the cylindrical tip and 140 µm for the truncated cone), the cells being confined on the area of higher magnetic gradient, through vertical piling up. For increasing cell number above a critical value Nc close to 104, the radius starts increasing according to a logarithmic law, following the same trend for both magnetic devices. This spreading over the surface plane then combines with the prior piling up, to ensure both vertical and lateral extensions of the assembly. We observe that the assembly radius remains significantly lower for the truncated tip for the same number of cells deposited on the substrate, corresponding to a more focalized attracting magnetic force, consistent with the overall dimensions of the multicellular assembly: radius R and height h. A magnetically labeled cell approaching the substrate would then experience a local field that would significantly differ from a magnetic device to the other, leading to discrepancies in cell spatial containment. In Figure 5b, the volume of cell assembly V is displayed as a function of deposited cell number N for EPCs with two different iron loads (normal (12.1 pg) and low (4.2 pg)) and for macrophages with 5.0 pg iron load, all other things being equal: the magnetic device chosen was the cylindrical tip, and the substrate was a glass slide of 100 µm thickness. First, notice that the aggregate volume V depends linearly on cell number over 2 orders of magnitude, regardless of cell types or cell labeling. Therefore, if we deduced from Figure 5a that the overall geometrical dimensions of the aggregate depend on N, the internal organization of cell assembly seems independent of the total number of cells composing it and is fully characterized by the slope value of the graph V(N) (Table 1). However, V/N ratios depend on cell type, magnetic load, and magnetic device geometry. In order to make comprehensive comparison between EPCs and macrophages assemblies, one has to take into account the cell size by introducing a new parameter called the packing factor F. Knowing both the number of cells clustered in the assembly and its final volume, one defines the cell packing factor F as the ratio of total volume of cells (assuming spherical cells) over assembly volume: F ) [(π/6)dcell3N]/V. EPCs and macrophages for same magnetic labeling conditions show a similar packing factor, close to 0.52 (Table 1), associated to a 3D simple cubic lattice, set as a lower bound for self-organized cell aggregates by Martin et al.31 It is worthy to note that no experimental conditions tested during our study leads to the higher (31) Martin, I.; Dozin, B.; Quarto, R.; Cancedda, R.; Beltrame, F. Cytometry 1997, 28, 141–146.

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Figure 3. Snapshots of a cell suspension (2 × 105 cells in 1 µL) released from a micropipette (time interval ) 2s). (Top) Magnetically labeled cells are massively attracted by the magnetized tip and pile up to form a 3D multicellular assembly of controlled dimensions. (Bottom) By contrast, the same cell suspension spreads over the substrate if no magnetic force is applied. Bar ) 500 µm.

Figure 4. Geometry of magnetically formed 3D assembly as a function of cell number N. Cell assembly is monitored by lateral snapshots with a high-magnification camera and by underneath views with a transmission inverted microscope (4× objective). From left to right, decreasing numbers of cells were deposited (1.4 × 105, 6.8 × 104, 2.7 × 104, 1.5 × 104) under the effect of the cylindrical (top) or truncated (bottom) tips. The underneath views are displayed only for the cylindrical tip, showing almost no cells outside of the patterned assemblies. Image analysis was used to measure aggregate radius R, height h, and volume V through the fitting ellipse of semiaxes a and b, as detailed for the aggregate formed with the cylindrical tip (2.7 × 104 cells/top right). Bar ) 500 µm.

bound corresponding to a face-centered cubic lattice packing of F ) 0.74, the maximum packing efficiency for hard spheres of equal radius. For a lower iron load, EPC assemblies exhibit a looser organization, leading to F ) 0.33. The multicellular organization of the assembly due to the temporary application of a magnetic force appears to vary with cell magnetization. Indeed, instead of iron load, if one compares the volume magnetization of one labeled cell for EPCs and macrophages, one obtains for EPC Mv ) 82 A/m and for macrophages Mv ) 83 A/m. EPC and macrophage suspension at normal iron load have then the same bulk magnetic properties, leading to equal packing factors. In contrast, for EPCs with lower magnetic load, the volume magnetization is Mv ) 28 A/m, leading to a reduced packing factor. For the same cell type, the cell compaction, induced by the temporary application of intense magnetic gradients, seems to depend solely on the magnetic force applied on the cell suspension. It then suggest cell-cell interactions would not intervene in the first 10 min of the formation of 3D aggregate. The less intense the magnetic force, the looser the assembly is. The same experiment was conducted for a truncated tip over a 100 µm thick glass substrate. We noticed the same linear dependence of V on the cell number N (data not shown), whatever the magnetic device is. The subsequent packing factor for normal labeling conditions on EPCs was F ) 0.42 (Table 1), slightly lower than the same experiment conducted with the cylindrical tip. It can be related to the fact that the magnetic gradient, although being stronger at the vicinity of the tip, decreases rapidly as it moves away from the substrate.

We finally analyzed in Figure 5c the influence of the substrate thickness by measuring the aggregate volume V as a function of cell number N for different substrate thicknesses e, with all other things being equal: EPCs at a normal iron load (12.1 pg) and cylindrical tip. The assembly volume V depends linearly on cell number N as demonstrated previously. The packing factor depends on the substrate thickness: F(e ) 100 µm) ) 0.49 ( 0.01 and F(e ) 200 µm) ) 0.25 ( 0.03. As seen in the inset in Figure 5c, the packing factor of EPC assemblies remains constant for e < 100 µm but decreases significantly for e ) 200 µm, when the magnetic gradient ensures a looser 3D confinement of the cell suspension. The thickness of the culture surface can be used as a tuneable parameter, as previously described in ref 18. To conclude on this part, the efficiency of cell aggregation after 10 min of magnetic exposure was characterized by a direct measurement of multicellular assembly packing factor F. It is controlled by the volume magnetic force Fv ) MvgradB via its two terms: the volume cell magnetization, fixed by the labeling conditions, and the magnetic gradient tuned by the geometry of the magnetic device and the substrate thickness. 3.5. Viability and Proliferation of Magnetically Labeled EPC after Patterning. The biological effect of magnetic labeling coupled to magnetic exposure and subsequent cell aggregation was studied for EPCs. First, the viability of EPCs after magnetic exposure was assessed via calcein-AM staining.32 After a magnetic exposure of 10 min, the survival rate reached 97.8% (Figure 6a,b), similar to that of untreated cells.

Magnetic Patterning to Form 3D Cellular Assemblies

Langmuir, Vol. 25, No. 4, 2009 2353 Table 1. Packing Factors of 3D Cell Assemblies with Varying Parameters: Cell Type (EPC or RAW Macrophage), Cell Iron Load, or Magnetic Tip Geometry (Cylindrical or Truncated Tip) magnetic device

Figure 5. (a) Radius R of a 3D assembly depends on the number N of seeded cells for the cylindrical tip (substrate thickness e ) 100 µm; filled squares) and for the truncated tip (e ) 8 µm; open circles). The radius remains constant, close to the tip dimensions up to a critical number of seeded cells Nc (Nc ) 104 and 1.2 × 104 for the cylindrical and truncated tips, respectively). Above Nc, the radius increases with N following a logarithmic law. In the first regime (N < Nc), cells pile up in the vertical dimension remaining enclosed in the surface of the tip until the aggregate reaches a critical height. In the second regime (N > Nc), cell addition results in a combination of both stacking and spreading beyond the tip surface. (b): The total volume V of the aggregate (expressed in µL) increases linearly with the number N of seeded cells, as illustrated for three different conditions: macrophages labeled with 5 pg of iron per cell (red squares), EPCs labeled with 12.1 pg of iron (black squares), and EPCs labeled with 4.2 pg of iron (gray squares). In all conditions, the surface thickness is 100 µm. The linear dependence indicates that the arrangement in the aggregate does not depend on its size, the slope being directly related to the packing factor F in the aggregate. The packing factor is found to be close to 0.5 for both macrophages and EPCs, which show comparable magnetization per unit volume (about 80 A/m), and falls to 0.33 for EPCs with lower iron load (volume magnetization of 28 A/m). (c) The volume of cellular assembly depends on the substrate thickness as represented here for EPC seeded on a 100 µm thick substrate (black squares) and 200 µm substrate thickness (open squares) on the cylindrical tip. The packing factor falls from 0.54 for lower thickness to 0.25 for the 200 µm thickness. For thickness below 100 µm, the packing factor is constant (inset).

Second, the growth rate of exposed EPCs was 1.14 ( 0.09 days, to be compared to 1.17 ( 0.05 days for control cells (unlabeled cells, without magnetic exposure) (Figure 6c). The

cell type

slope (10-6µL) packing factor

cylindrical tip RAW macrophage EPC EPC low iron load

1.06 ( 0.28 2.88 ( 0.05 4.31 ( 0.42

0.54 ( 0.14 0.49 ( 0.01 0.33 ( 0.03

truncated cone EPC

3.35 ( 0.12

0.42 ( 0.02

proliferation was thus unaffected by the magnetic labeling and the subsequent magnetic force application over 10 min. Finally, the aggregate of EPCs was observed after 24 h in adequate growth conditions (Figure 6d,e): the cells adhered to the substrate, regaining elongated form. The cells forming the multicellular assembly are therefore viable. Additionally, EPCs were still capable of forming capillary-like networks (data not shown) after brief exposure to magnetic forces and dense packing in the 3D bioconstruct. We thus observe no significant short-term cytotoxic effects of magnetic labeling and patterning on EPCs. 3.6. Breakthroughs and Potential Applications. The use of a focalized magnetic constraint to precisely organize cells displays various advantages compared to other patterning methods. Passive patterning is based on the nanostructuration of surfaces, and thus imposes some restrictions on the physicochemical nature of the culture substrate. Active dielectrophoresis uses, at least temporarily, low-conductivity solutions12 before the so-formed cell aggregate can be cultured in normal growth conditions. The patterning method here presented solely depends on the external magnetic attraction force and is then compatible with any culture surface or medium, particularly with standard cell growth conditions. Additionally, the magnetic labeling is nonspecific and adaptable to any cell type. It has been applied for instance to nonadherent monocytes (data not shown here). This active patterning technique, relying not on intrinsic adhesive cell properties but on the temporary application of an external force, is of great interest to control extensively multicellular organization. The patterning chamber is easy to assemble, requiring no specific preliminary preparation for cell surface or medium (as for example, use of a clean room for microengineered substrate). The culture surface being transparent, it allows direct observation through transmission microscopy, and a second viewpoint for lateral observation gives access to 3D characterization. As seen in Figure 5a, the overall dimensions of the multicellular aggregate depend on the geometry of the magnetic device generating gradients. Therefore, this technique is amenable to scale up or down by changing the size of the magnetic tip; it can then be used for single cell analysis as seen in ref 18 by forming aggregates of about 10 cells. However, the main interest of our technique is its ability to form “mesoscale” cell assemblies, between natural spheroids of a few thousand cells and tissues. We are able to control the formation of 104-4 × 105 cells cluster of precise size and shape and controllable packing factor. The cylindrical tip, of higher dimensions, appears to be more suitable for potential applications, allowing the formation of larger cell assemblies. Any magnet generating high enough magnetic gradient would be appropriate for this study. We achieved the precise patterning formation of arrays of independent multicellular assemblies (Figure 7), allowing us to investigate the potential interactions between those biological entities. All the experimental parameters (magnetic labeling, intensity, and duration of magnetic constraint) can be well chosen to ensure the better long-term

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Figure 7. Hexagonal array of cylindrical tips generate a similar pattern of 3D assemblies. A different number N of cells were seeded on each tip, from 2.5 × 103 to 1.6 × 105. Bar ) 1 mm.

Figure 6. Effect of the magnetic treatment on EPCs behavior. The viability of the cell population after magnetic labeling and exposure was assessed by calcein AM staining. Viable cells appear fluorescent, while dead cells show no fluorescence. Fluorescence was quantified for 750 cells, and more than 97% cells were stained. A typical evaluation is there illustrated: all 11 cells observed by transmission in (a) are fluorescent (b), and are thus all viable. Bar ) 50 µm. After magnetic patterning, cells were dispersed in culture medium and counted every day to determine cell growth (c, blue circles). Proliferation rate was also measured for cells in normal growth conditions (red squares). Both cell numbers increased as N ) N02D/D0, where D0 is the doubling time in days, equal to 1.14 ( 0.09 days after magnetic aggregation and to 1.17 ( 0.05 days in normal conditions: no alteration in cell proliferation was thus observed. (d,e) Long-term observation of the aggregate shows that, 24 h after magnetic deposition, cells have adhered onto the glass substrate, regaining elongated form for low number of cells in the aggregate (e), while some cells escaped from a larger aggregate (d), adhering as well. Bar ) 1 mm.

viability through sufficient nutrient and oxygen diffusion and extracellular matrix synthesis. Finally, this magnetic patterning technique should be of great interest to investigate the influence of a controlled multicellular organization on cell behavior, for instance, the differentiation capability of mesenchymal stem cells (MSCs). Chondrogenesis is known to only occur when MSCs are displayed as a pellet; with our technique, we should be able to modify the cell packing and volume of a so-formed pellet and observe the eventual modifications in the differentiation pathway. By modifying the packing factor of 3D assemblies and their sizes and shapes, we may probe the impact of cell-cell close connections on tissue

formation, for homotypic assembly (e.g., chondrocytes33) as well as heterotypic assembly (e.g., hepatocytes and fibroblasts34). Moreover, we should be able to quantify these intercellular interactions by monitoring the subsequent dynamic behavior of the assembly after the release of the magnetic constraint.

4. Conclusion We successfully build a 3D multicellular assembly of submillimetric dimensions, with well-defined geometry imposed by the application of a temporary constraint that does not thwart subsequent cell behavior. Overstepping limitations of current passive cell patterning techniques and setting up an easy active apparatus, which can generate intense forces on a localized area, we are moving toward the in vitro construction of parts of functional tissues for in vivo transplants. Acknowledgment. This work was supported by ANR Physique et Chimie du Vivant and Direction Ge´ne´rale de l’Armement (DGA). The authors thank Christine Me´nager for providing them with magnetic nanoparticles, Isabelle Galy-Fauroux for the EPC cell line, Christine Longin and Sophie Chat for TEM images, and Alexandre Lantheaume for mechanical support. LA8030792 (32) Xiu Ming, W.; Terasaki, P. I.; Rankin, G. W., Jr.; Chia, D.; Hui Ping, Z.; Hardy, S. Hum. Immunol. 1993, 37, 264–270. (33) Albrecht, D. R.; Underhill, G. H.; Wassermann, T. B.; Sah, R. L.; Bhatia, S. N. Nat. Methods 2006, 3, 369–375. (34) Lu, H.-F.; Chua, K.-N.; Zhang, P.-C.; Lim, W.-S.; Ramakrishna, S.; Leong, K. W.; Mao, H.-Q. Acta Biomater. 2005, 1, 399–410.