pubs.acs.org/Langmuir © 2009 American Chemical Society
Micropatterning of Hydrogels by Soft Embossing† Stefan Kobel,‡ Monika Limacher,‡ Samy Gobaa,‡ Thierry Laroche,§ and Matthias P. Lutolf*,‡ ‡
Laboratory of Stem Cell Bioengineering and Institute of Bioengineering, Ecole Polytechnique F ed erale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland, and §Swiss Institute of Technology (EPFL), Faculty of Life Sciences Bioimaging and Optics - BIOP, Station 15, CH-1015 Lausanne, Switzerland Received January 18, 2009. Revised Manuscript Received March 5, 2009 Conventional in situ hydrogel micropatterning techniques work successfully for relatively stiff hydrogels, but they often result in locally damaged surfaces upon demolding in the case of soft and fragile polymer networks formed at low precursor concentration. To overcome this limitation, we have developed a versatile method, termed soft embossing, for the topographical micropatterning of fragile chemically cross-linked polymer hydrogels. Soft embossing is based on the imprinting of a microstructured template into a gel surface that is only partially cross-linked. Free functional groups continue to be consumed and upon complete cross-linking irreversibly confine the microstructure on the gel surface. Here we identify and optimize the parameters that control the soft embossing process and show that this method allows the fabrication of desired topographies with good fidelity. Finally, one of the produced gel micropatterns, an array of microwells, was successfully utilized for culturing and analyzing live single hematopoietic stem cells. Confining the stem cells to their microwells allowed for efficient quantification of their growth potential during in vitro culturing.
Introduction Micropatterned hydrogels are gaining increasing attention in biomedicine, for example, as substrates for cell culture, as scaffolds for tissue engineering, and as high-throughput analytical platforms.1 Because hydrogels imbibe large amounts of water and possess near-physiological physicochemical characteristics, they are often advantageous compared to conventional materials in applications that involve cells. Hydrogel micropatterning has been achieved with a wide range of hydrophilic polymers and methodologies. For example, photopolymerizable hydrogels have been directly patterned via photolithography using UV light exposure through a photomask.2-4 This method is simple and robust but requires repeated patterning steps when applied to 3D structures.5 Better control over the gel topography can be achieved with micromolding and related soft-lithography-based techniques, whereby a liquid precursor solution is polymerized in situ against an elastic, microstructured poly(dimethylsiloxane) (PDMS) stamp, and the patterned gel is obtained by demolding.6 A majority of currently used micromolding methods have employed the photopolymerization of various synthetic hydrogel precursors such as poly(ethylene glycol) (PEG)1,4,7 and functionalized polysaccharides such as chitosan8 or hyaluronic acid.9 † Part of the Gels and Fibrillar Networks: Molecular and Polymer Gels and Materials with Self-Assembled Fibrillar Networks special issue. *Corresponding author. E-mail:
[email protected].
(1) Khademhosseini, A.; Langer, R. Biomaterials 2007, 28, 5087. (2) Beebe, D. J.; Moore, J. S.; Bauer, J. M.; Yu, Q.; Liu, R. H.; Devadoss, C.; Jo, B. H. Nature (London) 2000, 404, 588. (3) Revzin, A.; Russell, R. J.; Yadavalli, V. K.; Koh, W. G.; Deister, C.; Hile, D. D.; Mellott, M. B.; Pishko, M. V. Langmuir 2001, 17, 5440. (4) Koh, W. G.; Itle, L. J.; Pishko, M. V. Anal. Chem. 2003, 75, 5783. (5) Papavasiliou, G.; Songprawat, P.; Perez-Luna, V.; Hammes, E.; Morris, M.; Chiu, Y. C.; Brey, E. Tissue Engineering Part C, Methods, 2008 14(2), 129–140. (6) Khademhosseini, A.; Yeh, J.; Jon, S.; Eng, G.; Suh, K. Y.; Burdick, J. A.; Langer, R. Lab Chip 2004, 4, 425. (7) Moeller, H. C.; Mian, M. K.; Shrivastava, S.; Chung, B. G.; Khademhosseini, A. Biomaterials 2008, 29, 752. (8) Fukuda, J.; Khademhosseini, A.; Yeo, Y.; Yang, X.; Yeh, J.; Eng, G.; Blumling, J.; Wang, C. F.; Kohane, D. S.; Langer, R. Biomaterials 2006, 27, 5259. (9) Yeh, J.; Ling, Y.; Karp, J. M.; Gantz, J.; Chandawarkar, A.; Eng, G.; Blumling, J.; Langer, R.; Khademhosseini, A. Biomaterials 2006, 27, 5391.
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Moreover, the successful in situ patterning of alginate by complexation10 and the thermomolding of agarose11 or collagen hydrogels12 has been demonstrated. Whereas the above techniques have allowed the patterning of a variety of synthetic hydrogel types, they seem to be limited to substrates that are densely cross-linked and thus relatively stiff. Typically, precursor solid contents above 10% and up to 50% or more have been used,9,13 having elastic moduli on the order of hundreds of kPa. That is to say, loosely cross-linked hydrogels formed at relatively low solid concentration (i.e., below 5%, with elastic moduli from tens of Pa to a few kPa), which arguably better mimic the physicochemical characteristics of biological tissues, have been described to be too fragile and thus tend to be damaged during demolding from the sticky PDMS template9,12 (Supporting Information Figure S1). Indeed, using a PEG-based hydrogel system formed via conjugate addition reaction,15 we encountered the same problems (Lutolf et al., unpublished). What is more is that the extensive swelling of many hydrogels formed at high precursor concentration has been described to lead to feature widening and to the detachment of hydrogels from their substrate.3,7,16,17 Therefore, to overcome these complexities and pattern synthetic hydrogels with stiffness ranges that approach those of soft tissues,18 alternative micropatterning methods are desirable. We present a gentle and facile method, termed soft embossing, to reliably pattern PEG-based hydrogels that are formed at (10) Gillette, B.; Jensen, J.; Tang, B.; Yang, G.; Bazargan-Lari, A.; Zhong, M.; Sia, S. Nat. Mater. 2008, 7, 636. (11) Cheng, S. Y.; Heilman, S.; Wasserman, M.; Archer, S.; Shuler, M. L.; Wu, M. Lab Chip 2007, 7, 763. (12) Tang, M. D.; Golden, A. P.; Tien, J. J. Am. Chem. Soc. 2003, 125, 12988. (13) Albrecht, D. R.; Underhill, G. H.; Wassermann, T. B.; Sah, R. L.; Bhatia, S. N. Nat. Methods 2006, 3, 369. (14) Elbert, D. L.; Pratt, A. B.; Lutolf, M. P.; Halstenberg, S.; Hubbell, J. A. J. Controlled Release 2001, 76, 11. (15) Lutolf, M. P.; Hubbell, J. A. Biomacromolecules 2003, 4, 713. (16) Wang, Y.; Salazar, G.; Pai, J.; Shadpour, H.; Sims, C.; Allbritton, N. Lab Chip 2008, 8, 734. (17) Albrecht, D. R.; Tsang, V.; Sah, R. L.; Bhatia, S. N. Lab Chip 2005, 5, 111. (18) Engler, A. J.; Sen, S.; Sweeney, H. L.; Discher, D. E. Cell 2006, 126, 677.
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relatively low precursor concentration and possess elastic moduli from tens of Pa to a few kPa. Analogous to hot embossing,19 whereby a thermoplastic material is heated above its glasstransition temperature and then embossed, here we hypothesized that hydrogels that are not fully cross-linked could be embossed via PDMS templates to comprise a desired microstructure. Indeed, we found that the progressive cross-linking of a network structure during embossing allows turning local microscopic gel deformation into irreversible surface topography. In this article, we identify and optimize the parameters that control the soft embossing process, and we present one example of a biological application of this novel technique.
Experimental Section Hydrogel Precursor Synthesis. Pentaerythritol tetra(mercaptoethyl) polyoxyethylene (4-arm PEG-thiol), (mol wt 10 063 g/mol, 98.9% substitution as indicated by the manufacturer) and hexaglycerol polyethyleneglycol ether (8-arm PEGOH, mol wt 40 000 g/mol, Mw/Mn= 1.1, 99% substitution as indicated by the manufacturer) were obtained from NOF Corporation (Japan). Divinyl sulfone was purchased from Aldrich (Buchs, Switzerland). 8-arm PEG-vinylsulfones (8-arm PEGVS) were produced and characterized as described elsewhere.15 The final product was dried under vacuum and stored under argon at -20 °C. The product was analyzed by gel permeation chromatography (GPC) using a Waters separation module equipped with a 515 HPLC pump, a series of Styragel columns (HR2, HR3, and HR4 with pore sizes of 102, 103, and 104 A˚, respectively), and a Waters 410 differential refractometer for detection. THF was used as the eluent at a flow rate of 1 mL/min at 40 °C to confirm the identical molecular weight distributions of PEG-OH and PEG-VS. The degree of end group conversion was 88.8% as determined via 1H NMR (CDCl3) on a Bruker (400 MHz) instrument: 3.6 ppm (PEG backbone), 6.1 ppm (d, 1H, dCH2), 6.4 ppm (d, 1H, dCH2), and 6.8 ppm (dd, 1H, -SO2CHd). To obtain PEG precursor solutions, 8-arm PEG-VS and 4-arm PEG-thiol were dissolved at 5% (w/v) in 0.3 M triethanolamine (TEA) (Fluka, Switzerland) and in bidistilled water, respectively. The stock solutions were stored at -20 °C until further use. All hydrogels were cast at 5% (w/v) concentration. In Situ Rheometry to Assess the Extent and Kinetics of Gel Cross-Linking. Gelation kinetics was studied by performing small-strain oscillatory shear experiments on a Bohlin CVO 120 high-resolution rheometer with plate-plate geometry at room temperature.15 The 8-arm PEG-VS and 4-arm PEGthiol were mixed in stoichiometrically balanced amounts, and the mixture was immediately pipetted onto the center of the bottom plate of the rheometer. The upper plate (2 cm in diameter) was lowered to a gap size of 0.1 mm, and the dynamic oscillating measurement was started. The evolution of storage (G0 ) and loss (G00 ) moduli and phase angle δ (deg) at a constant frequency of 0.5 Hz and a constant strain of 0.05 was recorded as a function of time. The gel time (tG), as a kinetic readout for network formation, was defined as the time of crossover of G0 and G00 or at δ = 45°.15
Ellman’s Reagent to Quantify the Content of Free Thiols in Gel Networks. The Ellman reagent20 was dissolved in 0.1 M phosphate buffer at pH 8 according to the manufacturer recommendations. One hundred microliter PEG-hydrogel precursor solutions (5% w/v) were prepared and immediately dispensed into each well of a 24-well Falcon plate (Becton Dickinson, Franklin Lakes, NJ). The Ellman reagent solution (300 μL) was added at t = 15, 30, 45, 60, 75, 90, 105, and 120 min. :: (19) Becker, H.; Gartner, C. Anal. Bioanal. Chem. 2008, 390, 89. (20) ELLMAN, G. L. Arch. Biochem. Biophys. 1958, 74, 443.
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The amount of free thiols was quantified the same way in hydrogel solution where the PEG-VS precursor solution was replaced with water (t = 0 min) and in hydrogels incubated overnight at 37 °C (t = 24 h). As soon as the Ellman reagent solution was added, the plate was placed under constant agitation. After 20 min of incubation, 100 μL of supernatant was collected, and absorbance at 412 nm was assessed with a Safire2 :: microplate reader (Tecan, Mannedorf, Switzerland). A standard curve was made with five dilutions of PEG-thiol (0 to 0.250 μM thiol groups). Ellman reagent solution (300 μL) was added, and absorbance at 412 nm was assessed after 20 min of incubation. The best-fit regression (R2 = 0.99) was determined using the Origin 8.0 software. The corresponding regression parameters were used to determine the free thiol concentration in the assayed samples.
Microfabrication of PDMS Molds for Soft Embossing. PDMS molds for soft embossing were fabricated by soft lithography as previously described.21 In brief, a photolithography mask was designed as a negative pattern using CleWin (PhoeniX, Netherlands) and written with a laser writer onto a chrome mask. This mask was then developed and etched in a chrome etch bath. SU8 GM1070 (Gersteltec, Switzerland) was spin-coated onto a 4 in. silicon wafer to a thickness of 50 μm on a spin coater and soft-baked for 15 min at 130 °C (4 °C/min) on a programmable hot plate. The wafer was then exposed to the previously fabricated mask on a mask aligner for 3 13 s. After the postexposure bake (40 min at 105 °C), the wafer was developed in propylene glycol monomethyl ether acetate for 2 2 min, cleaned with isopropyl alcohol, and air dried. The thickness of the SU8 was confirmed with a surface profiler (Alpha-Step 500, Tencor). The structured wafer was then used to mold PDMS (Sylgard 184 silicone elastomer, Dow Corning Corporation, Oftringen, Switzerland). The components were mixed in a weight ratio of 10:1 base/curing agent, poured onto the wafer to a thickness of about 4 mm, and baked for 2 h at 60-70 °C. The PDMS replicas were carefully peeled off and cut to the desired size. All microfabrication work was carried out in the clean room facility of EPFL. PDMS was replicated under a sterile fume hood. Surface Modification of Glass Slides. To covalently graft hydrogel films to glass substrates, microscopy glass slides were silanized to expose free thiol groups that could covalently react with VS groups of the forming PEG hydrogel. The silanization was performed as previously described using 3-mercaptopropyltrimethoxylsilane (MPS) (Falcone, Switzerland).22 Briefly, glass slides were cleaned with detergent, bidistilled water. and ethanol and dried in air. MPS (1.5 mL) and 5 drops (∼0.2 mL) of acetic acid were mixed in 150 mL of toluene for 30 min. The glass slides were immersed for 30 min in this solution, rinsed with toluene, air dried, and baked for 1 h at 110 °C. Prior to use, the slides were treated for 10 min with a 10 mM dithiothreitol solution to reduce disulfide bonds, washed with bidistilled water, and dried with an air gun. Hydrogel Patterning by Soft Embossing. After mixing, the precursors of 5%, stoichiometrically balanced hydrogels were cast in a clamped sandwich structure consisting of a MPS slide, a glass slide treated with Sigmacote (Sigma, Switzerland), and a shaped 100-μm-thick plastic spacer. After 30 min of crosslinking (or 20, 40, 90, 120, or 180 min, respectively, for the data shown in Figure 2), the glass slide was removed, and the previously cut PDMS molds were quickly pressed into the hydrogel film to ensure firm contact with the hydrogel. The PDMS molds were left on the hydrogel for 90 min and then carefully removed.
Confocal Microscopy to Assess the Quality of Embossed Microstructures. To qualitatively assess the outcome of the (21) Xia, Y. N.; Whitesides, G. M. Annu. Rev. Mater. Sci. 1998, 28, 153. (22) Huang, L.; Nair, P. K.; Nair, M. T. S.; Zingaro, R. A.; Meyers, E. A. Thin Solid Films 1995, 268, 49.
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Figure 1. Illustration of the soft embossing concept (A). A PEG hydrogel film is cast from multiarm PEG precursors (for clarity, only four arms are shown) (1). After gelation, but before completion of cross-linking, a microfabricated PDMS mold is embossed into the surface (2). Further cross-linking irreversibly confines the embossed micropattern into the hydrogel surface (3) and the PDMS stamp can be removed (4). Examples of micropatterning by soft embossing: 100-μm-diameter microwell arrays (B) and the EPFL logo embossed using a 50-μm-deep template (C). micropatterning process and to determine the dimensions of the microstructures, the micropatterned hydrogels were fluorescently labeled with fluorescein isothiocyanate (FITC)-conjugated BSA (Sigma, Buchs, Switzerland) and imaged by confocal laser scanning microscopy. FITC-BSA was prereacted for 30 min at room temperature with a 10-fold molar excess of a heterofuntional NHS-PEG-VS PEG linker (Nektar, Huntsville, AL). The PEGylated protein was mixed with the precursor solutions to graft to the termini of the thiolated PEG macromer. Images were acquired using a Leica SP5 motorized upright confocal laser scanning microscope (Leica, Germany). Typically, z stacks were acquired with a constant slice thickness of 2 μm, reconstructing a cross-sectional profile of approximately 100-150 μm. Cross-sectional analysis and surface plots of the 3D stacks were made using Imaris 6.0 software (Bitplane, Switzerland).
Isolation and Purification of Hematopoietic Stem Cells by Flow Cytometry. Hematopoietic stem cells were isolated from the bone marrow of 8- to 12-week-old C57BL/6-Ly5.1 mice, stained and purified as described in detail in ref 23. The lineagedepleted bone marrow fraction was separated by flow cytometry on FACSDiVa (BD Bioscience, Switzerland) at the EPFL FACS core facility. Single viable (i.e., propidium iodide negative) Lin-ckit+Sca1+CD150+ cells (LSK-CD150+) were triple sorted.
Time-Lapse Microscopy and Image Analysis to Assess Single Cell Proliferation Kinetics. LKS-CD150+ cells were directly sorted into basal medium containing Stemline II hematopoietic expansion medium (Sigma, Buchs, Switzerland) (23) Lutolf, M. P.; Doyonnas, R.; Havenstrite, K.; Koleckar, K.; Blau, H. M. Integr. Biol. 2009, 1, 59.
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supplemented with 100 ng/mL SCF, 2 ng/mL Flt-3 ligand, and 100 ng/mL TPO (all from R&D Systems, Minneapolis, MN), seeded onto the microwell array at a density of 1.5 104 cells/mL and allowed to settle for 1 h. The array was then placed on an inverted microscope (Zeiss Observer) equipped with a motorized (xyz) stage and an environmental chamber set to 37 °C and 5% (v/v) CO2. Automated image acquisition of the entire array was performed at time intervals of 4 h for up to 4 days using MetaMorph (Visitron Systems, Germany). Images were analyzed with the open-source program ImageJ (http://rsbweb.nih. gov) to quantify cell numbers in the microwells at each time point. Only microwells with a single cell at the beginning were included in the analysis. The time to the first cell division was defined as the first appearance of 2 cells in a microwell, the second division was defined as 3 or 4 cells per microwell, the third division was defined as 5-8 cells per microwell, and the fourth division was defined as 9-16 cells per microwell. Cells were scored dead when they ceased to move on the microwell surface, shrunk markedly in size, and lost their shiny appearance. This viability criterium was verified using propidium iodide staining and fluorescent microscopy.
Results and Discussion We hypothesized that chemically cross-linked PEG hydrogels could be micropatterned by taking advantage of their incomplete but gradually increasing cross-linking density. This hypothesis was tested by developing a novel micropatterning process (Figure 1). First, a 100 μm thin film of a 5% (w/v) PEG hydrogel was formed via conjugate addition reaction15 on the surface of a reactive glass substrate (Figure 1A, 1). In the second step, a PDMS mold was embossed into the incompletely cross-linked hydrogel Langmuir 2009, 25(15), 8774–8779
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Figure 2. Evolution of the elastic shear modulus during PEG hydrogel formation (A). The inset shows both G0 and G00 as a function of time, showing the gel point at ca. 4 min as the crossover of these values. Changes in concentration of free thiol groups during gelation detected by Ellman’s reagent (B). Dependence of the embossed microwell array depth on the elapsed time between mixing the precursors (i.e., no gel at t = 0) and the start of the soft embossing. (C, n = 5). Insets show confocal images of fluorescently stained hydrogels that were embossed with a 50 μm PDMS stamp after gelation times of 25 and 50 min. Scale bars correspond to 20 μm.
(Figures 1A, 2 and 3). Upon further cross-linking, the mold was gently removed from the gel (Figure 1A, 4). Indeed, this process led to the irreversible formation of micropatterns on the hydrogel surface, as assessed by confocal laser scanning microscopy of fluorescently labeled gels (Figure 1B,C). We managed to pattern shapes of various geometries with high pattern fidelity, for example, arrays of microwells (Figure 1B), micrometer-sized squares or grooves (Supporting Information Figure S2), or the logo of our institution, EPFL (Figure 1C). These micropatterns retained their shape even after several weeks of incubation in water. A measurement of the dimensions of the micropatterns revealed a slight undersizing of 6% due to gel swelling. This dimensional change corresponds to the expected swelling ratio of the used gel formulation.24 By comparing (24) Cordey, M.; Limacher, M.; Kobel, S.; Taylor, V.; Lutolf, M. P. Stem Cells 2008, 26(10), 2586–2594.
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embossed grooves of various widths, we determined a reproducible aspect ratio of 2:1, which corresponds to previously reported data on micromolded photopolymerized PEG hydrogels.7,25 Notably, we did not encounter any demolding problems in these relatively loosely cross-linked gels, as reported by others,9,12 and the detachment of gels from the substrate7 was never observed. Therefore, soft embossing is a versatile method of micropatterning fragile synthetic hydrogels. Determining the Parameter Space of Soft Embossing. To find the time window that allows soft embossing of gel microstructures, we first assessed the gelation kinetics in situ by small oscillatory shear rheometry at a constant frequency of 1 Hz.15 The gel point, defined here as the crossover of the elastic and viscous part of the complex modulus (G0 and G00 , respectively), was observed ca. 4 min after mixing the PEG precursors (Figure 2A, inset), and the modulus progressed logarithmically to reach a maximum of 11.3 kPa after 116 min (Figure 2A). The observed cross-linking kinetics are typical of covalently cross-linked PEG-based hydrogels and confirm several prior studies.15,26 We next adapted the Ellman assay to measure the timedependent availability of free sulfhydryl groups retained within the forming networks. Because the hydrogels were cast with balanced stoichiometric ratios (1:1 vinylsulfones/thiols), this assay was performed to assess the relative level of free functional groups across the studied time course (Figure 2B). Decreasing amounts of free thiols were detectable for up to ca. 90 min after mixing, confirming the above in situ rheometry data, showing that cross-linking was still ongoing far beyond the gel point. The clear negative correlation between the evolution of G0 and the availability of free thiols (R2 = 0.98, p < 0.01) demonstrates that, unsurprisingly, the progressing polymerization led to the observed increase in stiffness. These data also show that the delayed cross-linking of a relatively small fraction of PEG chains is sufficient to confine an embossed micropattern irreversibly on the hydrogel surface. On the basis of the measured cross-linking kinetics and the detected amounts of free thiol groups, we expected to be able to generate micropattens over a relatively wide range (i.e., between ca. 10 and 90 min after mixing the precursors). We therefore chose the microwell array pattern of Figure 1B (well diameter 100 μm, well depth 50 μm, and spacing 50 μm) to establish the feasibility, fidelity, and reproducibility of the soft embossing process. To correlate the extent of cross-linking of the hydrogel with the outcome of the micropatterning, we assessed the depth profiles of embossed individual microwells of an array as a function of time. The micropatterned profile was measured by confocal microscopy. Consistent with the above estimations, we found that the depth of the embossed microwell profile decreased as a function of the gelation time (Figure 2C). We obtained a maximum well depth of 43 ((4) μm after 25 min of gelation time, which is close to the effective depth of the mold (48 μm). Using the same PDMS mold, the microwell depth gradually decreased to 7.4 ((1.4) μm after a gelation time of 90 min. Therefore, by controlling the time after initiating the crosslinking we can readily tune the depth of the micropatterns, independent of the PDMS template topography. We believe that the matrix stiffness and number of free sulfhydryl groups play a key role in controlling the micropattern (25) Karp, J. M.; Yeh, J.; Eng, G.; Fukuda, J.; Blumling, J.; Suh, K. Y.; Cheng, J.; Mahdavi, A.; Borenstein, J.; Langer, R.; Khademhosseini, A. Lab Chip 2007, 7, 786. (26) Ehrbar, M.; Rizzi, S. C.; Hlushchuk, R.; Djonov, V.; Zisch, A. H.; Hubbell, J. A.; Weber, F. E.; Lutolf, M. P. Biomaterials 2007, 28, 3856.
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Figure 3. Still images from time-lapse movies of hematopoietic stem cells seeded and cultured on a soft-embossed hydrogel microwell array (colors refer to variable numbers of cell divisions as indicated in B). Distribution of cells per microwell at various time points during the experiment (B). Quantification of the absolute time to the first, second, third, and fourth divisions (C). The scale bar corresponds to 100 μm.
depth. On one hand, matrix stiffness may govern the penetration depth into the surface of a given pattern; increasing stiffness leading to reduced penetration by elastic and/or plastic gel deformation. On the other hand, to generate irreversible micropatterns on the surface, the created topography from template penetration needs to be permanently locked in. This is achieved by covalent cross-linking of the remaining free functional groups. It should be mentioned that G0 reached a plateau while at the same time a relatively large number of free thiols was still detectable. It is not clear whether the evolution of the viscoelastic network parameters (G0 and G00 ) is not sensitive enough to detect the remaining free functional groups (i.e., free thiols continue to get consumed by vinylsulfones) or whether the free thiols would suggest a stoichiometric imbalance of functional groups (i.e., free thiols that will not get consumed by vinylsulfones). If the latter were the case, then it is likely that the residual thiols would react intramolecularly to form disulfide bonds and thus loops that would not significantly contribute to the density of elastically active chains. In that case, the formation of these loops did not have an influence on the soft embossing process. However, the detailed mechanism of micropatterning via soft embossing remains to be further elucidated. Soft-Embossed Hydrogel Microwell Arrays Are Suitable to Culture and Study Single Hematopoietic Stem Cells. Hydrogel microwells have been shown to be an efficient method for single stem cell culturing.7,24 Considering this and the fact that matrix elasticity can influence a stem cell’s fate,18,27 we (27) Gerecht, S.; Burdick, J. A.; Ferreira, L. S.; Townsend, S. A.; Langer, R.; Vunjak-Novakovic, G. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 11298.
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reasoned that soft-embossed hydrogel microwell arrays could be used to culture and study rare hematopoietic stem cells (HSC) at a single-cell level, similar to previously published work by some of us that successfully cultured HSCs on PEGbased gels microfabricated using another method.23 HSCs are somatic stem cells responsible for the life-long production of the entire blood system, which requires them to divide either to produce more of themselves, an ability that is termed selfrenewal,28 or to produce differentiated progeny that later on give rise to all of the specialized blood cell types. HSCs obtained from bone marrow transplants are the first stem cell types to be successfully employed in stem cell-based therapies, mainly to cure leukemia and lymphoma.29 However, the limited supply of donor tissue and the lack of in vitro stem cell expansion methods restrict the availability and complicationfree use of this therapy. We reasoned that soft-embossed hydrogel microwell arrays could serve as an in vitro cultivation system that mimics the native microenvironment in which HSCs are embedded and that maintains the self-renewal capacity of the stem cells. Similar to what had been demonstrated previously on a micromolded gel array that extensively swelled,23 soft-embossed hydrogel microwells should allow the study of the proliferation kinetics of single stem cells in this constrained biomimetic microenvironment, thereby avoiding that slowly dividing cells could be outgrown by other quickly proliferating cells. (28) Morrison, S. J.; Uchida, N.; Weissman, I. L. Annu. Rev. Cell Dev. Biol. 1995, 11, 35. (29) Bryder, D.; Rossi, D. J.; Weissman, I. L. Am. J. Pathol. 2006, 169, 338.
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To demonstrate that the proliferation kinetics of HSCs can be studied at the single-cell level using soft-embossed hydrogel microwell arrays, several thousand HSCs were freshly isolated from the bone marrow of a mouse, purified by fluorescence-activated cell sorting (FACS), seeded onto a microwell array by gravitational sedimentation, and then cultured on the array in the presence of a cocktail of growth factors/cytokines in serum-free medium. Several hundred of these rare individual HSCs were efficiently trapped in microwells patterned on the bottom of one well of a 96-well plate. Because of the nonadhesive properties of PEG, we never observed that single cells migrated out of the microwell, facilitating efficient long-term time-lapse videomicroscopy (Figure 3A and Supporting Information movie 1). This behavior was in marked contrast to HSCs grown on conventional plastic culture dishes that migrated extensively and that were frequently lost from an area of interest (Supporting Information movie 2). As a first readout of this time-lapse experiment, we assessed the number of cells per microwell generated from a single stem cell as a function of time in culture (Figure 3B). These data indicated that about 50% of the starting population underwent at least one cell division within the first 24 h and that proliferation over the following days occurred in a highly heterogeneous manner. As a result, a broad distribution of cells per microwells was obtained at t = 72 h, consistent with other data.23 Notably, we identified single stem cells that never divided over the course of the experiment and cells that divided more than six times within 3 days. About 25% of all single cells seeded on the array died during this experiment. These data therefore reveal that soft-embossed microwell arrays are valuable substrates to assess the growth of individual HSCs in highthroughput experiments. To further dissect the proliferation pattern of a population of single HSCs, we calculated the time to the first, second, third, and fourth division (Figure 3C). The average time to the first division was ca. 20 h (and 34.5 h to the second), but statistical analysis revealed that the rates to the first and second divisions were not normally distributed, as could be expected from homogeneously proliferating cells. One possible explanation for these findings is that different subpopulations of HSCs show a distinct proliferation pattern, as was shown by
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others in vivo.30 It remains to be shown whether the generated proliferating progeny of single microwell-cultured stem cells retained their stem cell potential and could therefore be used to reconstitute the blood over prolonged periods of time.
Conclusions We have developed a novel hydrogel microfabrication technique termed soft embossing. This technique allows to micropattern relatively soft and low-swelling chemically cross-linked PEG hydrogel surfaces. The method is based on the principle that microscopically deformed surfaces of incompletely cross-linked hydrogels can be irreversibly embossed upon complete crosslinking. We have proven that free reactive groups are crucial for successful patterning. In addition, we have shown that the stiffness of the hydrogel increases and the subsequent depth of the embossed patterned decreases with progressive degree of polymerization. We believe that soft embossing can be successfully adapted to a variety of gel cross-linking chemistries that occur in a time frame of minutes to hours. We have further demonstrated the versatility of the method by embossing structures having various topographies. One of the gel micropatterns, an array of microwells, was successfully utilized for culturing and analyzing live single hematopoietic stem cells. Confining the stem cells to their microwells allows for efficient quantification of their growth potential and could be applied, for example, to identify rare subpopulations within a heterogeneous mixture of stem and progenitor cells. Acknowledgment. This work was supported by SNSF grant FN 205321-112323/1 and by a EURYI award to M.P.L. Supporting Information Available: Two figures and two time-lapse movies of single HSC culturing on soft-embossed hydrogel microwell arrays and conventional plastic dishes, respectively. This material is available free of charge via the Internet at http://pubs.acs.org. (30) Wilson, A.; Laurenti, E.; Oser, G.; van der Wath, R. C.; Blanco-Bose, W.; Jaworski, M.; Offner, S.; Dunant, C. F.; Eshkind, L.; Bockamp, E.; Lio, P.; MacDonald, H. R.; Trumpp, A. Cell 2008, 135, 1118.
DOI: 10.1021/la9002115
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