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Jul 29, 2014 - High-Throughput Generation of Multicellular Spheroids ... Institute of Polymer Chemistry, College of Chemistry, Nankai University,. Tia...
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Hydrogel Thin Film with Swelling-Induced Wrinkling Patterns for High-Throughput Generation of Multicellular Spheroids Ziqi Zhao,† Jianjun Gu,† Yening Zhao,† Ying Guan,† X. X. Zhu,‡ and Yongjun Zhang*,† †

State Key Laboratory of Medicinal Chemical Biology and Key Laboratory of Functional Polymer Materials, The Co-Innovation Center of Chemistry and Chemical Engineering of Tianjin, Institute of Polymer Chemistry, College of Chemistry, Nankai University, Tianjin 300071, China ‡ Department of Chemistry, Université de Montréal, C. P. 6128, Succursale Centre-ville, Montreal, Quebec H3C 3J7, Canada ABSTRACT: Three-dimensional (3D) multicellular spheroids (MCSs) mimic the structure and function of real tissue much better than the conventional 2D cell monolayers, however, their application was severely hindered by difficulties in their generation. An ideal method for MCS fabrication should produce spheroids with narrow size distribution and allow for control over their size. The method should also be simple, cheap, and scalable. Here, we use patterned nonadhesive poly(2-hydroxyethyl methacrylate) hydrogel films to guide the self-assembly of cells. The films were fabricated directly in the wells of cell culture plates. They were patterned spontaneously by swelling in water, without the use of any template or specialized facilities. When cell suspension is added, the cells settle down by gravity to the bottom. Because of the presence of the wrinkling pattern composed of uniformed microcaves, the cells accumulate to the center of the microcaves and gradually self-assemble into MCSs. Using this method, monodisperse MCSs were generated. The size of the spheroids can be facilely controlled by the number of cells seeded. The method is compatible with the conventional monolayer cell culture method. Thousands of spheroids can be generated in a single well. We expect this method will pave the way for the application of MCSs in various biomedical areas.

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INTRODUCTION The majority of human cells live in a 3D environment surrounded by other cells and ECM (extracellular matrix) and the cell−cell and cell−ECM interactions play a significant role in modulating their differentiation, proliferation, and migration. When cultured in vitro as 2D cell monolayers, however, these interactions are largely lost. Therefore, tissue-specific properties are often lost.1 In contrast, complex in vivo cell phenotypes can be preserved in 3D multicellular spheroids (MCSs) due to the improved cell−cell and cell−matrix interactions in these models.2,3 Because MCS can model the in vivo environment more accurately, they found many important applications in biomedical researches, including drug screening4−6 and tumor studies.1,7,8 In addition, they hold great promise to be used as building blocks in organ printing for the construction of hybrid artificial organs.9,10 Unfortunately the application of MCSs was severely hindered by difficulties in their generation.1,10−12 Up to now, a lot of techniques have been developed to generate MCSs.1 For many of their applications, it is important that the MCSs generated are uniform in shape and size.10,13,14 Previous studies show that spheroid size of embryonic stem cell is an important parameter in their differentiation.15 Control of spheroid size is also extremely important from the viewpoint of their central necrosis.16 Some methods,11,17−21 for example, the nonadhesive substratum method,11,17 or the method using an intercellular linker,20,21 afford little control over the size and © 2014 American Chemical Society

homogeneity of the MCSs, despite that they show advantages such as simple to perform and ease to scale up. In contrast, control of spheroid size is easy for the hanging drop method; however, it is cumbersome for massive production.1 To control the spheroid size, many authors used microstructures to guide the multicellular self-assembly.22 As an example, Kotov et al.23,24 fabricated hydrogel scaffolds with inverted colloidal crystal structure in which HepG2 spheroids with a narrow size distribution were successfully generated. A more widely exploited microstructure is microwell, which is fabricated using microfabrication techniques, such as micromolding,25 soft lithography,26,27 and contact-lithographic process.28 Using this method, MCSs with well-controlled size, cell composition, and geometry were generated from various types of cells, including hepatocyte,29,30 human embryonic stem cells (hESCs), 31 HepG2, 25,27,32 embryonic stem (ES) cells,15,26,33−35 human umbilical vein endothelial cells (HUVECs),36 pancreatic islet cells,28,37 human and murine pluripotent stem cells,38 etc. Although this method is extremely powerful,1 it still suffers from some limits. For example, the microstructures were usually fabricated using a template (mask or mold), and the fabrication of the template is complicated Received: May 19, 2014 Revised: July 29, 2014 Published: July 29, 2014 3306

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300000) was purchased from Heowns. HEMA was distilled under reduced pressure. Other reagents were used as received. Hydrogel Film Fabrication. PHEMA hydrogel films with gradient cross-linking density were fabricated according to M. Guvendiren et al.45 When using glass slides as substrate, they were first cleaned by soaking in boiling piranha solution (3:7 v/v H2O2−H2SO4 mixture) (Caution: this solution is extremely corrosive!) for 4 h, rinsed thoroughly with deionized (DI) water, and dried. Vinyl groups were introduced by immersing the slides in a 1.0 wt % ethanolic solution of 3(trimethoxysilyl)propyl methacrylate for 12 h, followed by washing with 95% ethanol and blow-drying.51 To prepare precursor solution for the hydrogel film, 2.0 mL of HEMA and 60 μL of Darocur 1173 were added to a 10 mL beaker and mixed. The mixture was irradiated with a 6W UV lamp for 190s to obtain a viscous, partially polymerized solution. Then additional Darocur 1173 (40 μL) and cross-linker EGDMA were added to form the photocurable precursor solution. The precursor solution was coated onto the glass slides and irradiated with a 500W UV lamp for 4 min to obtain the PHEMA films. To modify 12-well cell culture plates, the precursor solution was added to the wells. Then, the solution was cured in the same way. Swelling-Induced Wrinkling Pattern. DI water was applied onto the surface of the PHEMA films. Fully developed swelling-induced wrinkling patterns were obtained in ∼1 h. Cell Culture Plates Modified with Flat PHEMA Layer. To each well of a 12-well cell culture plate, 1.0 mL of 0.5 wt % PHEMA(average Mv ∼ 300 000) solution in 95% ethanol was added. The solution was allowed to air-dry at 37 °C, leaving a flat layer of hydrophilic, nonadhesive PHEMA on the bottom.11 Multicellular Spheroid Generation. The modified cell culture plates were sterilized under UV light and washed with PBS twice. NIH 3T3 cells grown as a monolayer were detached with trypsin-EDTA to generate single-cell suspension. The cell suspension was diluted to various concentrations with culture media, which contains 90% Dulbecco’s modified Eagle medium (DMEM, Gibco), 10% heatinactivated fetal calf serum, and 100 units/mL of penicillin/ streptomycin. To each well of the cell culture plate, 3.0 mL of cell suspension was added. The cultures were maintained statically in an incubator at 37 °C with a humidified atmosphere of 5% CO2. Real-Time Monitoring of Spheroid Formation. Spheroid formation was monitored using a Zeiss Observer Z1 live imaging unit. During the imaging the cultures were maintained at 37 °C with a humidified atmosphere of 5% CO2. Cell Morphology. The cells (both 3D multicellular spheroids and 2D cell monolayers) were washed thoroughly with PBS. They were fixed in 4% paraformaldehyde at 37 °C for 20 min and washed with PBS. They were then permeabilized in 0.1% Triton X-100 for 5 min and washed again with PBS. The samples were then treated with 1% bovine serum albumin at 37 °C for 30 min. Finally, they were incubated in PBS containing 5 μg/mL isothiocyanate (FITC)phalloidin and 0.2 mg/mL 4′,6-diamidino-2-phenylindole (DAPI) for 20 min. The samples were washed with PBS and imaged using a confocal microscope (Leica TSC SP8). Cell Viability. To assess the viability of the cells, after cultured for a predetermined time, 3 drops of solution containing 50 μg/mL acridine orange (AO) and 50 μg/mL ethidium bromide (EB) were added. After incubated for 1 h, the samples were observed in situ using an inverted fluorescence microscope (Olympus LX70-140). The stained spheroids were also separated from the media by centrifugation, redispersed in PBS, and observed with a confocal microscope. Statistical Analysis. Data was shown as the mean and standard deviation (sd). Statistical analysis was performed with an unpaired student’s t test and p < 0.05 was considered as significant.

and often requires specialized facilities (inexpensive masks for microwell construction emerged recently).25,26,33 Fitting the microstructures with commercially available cell culture plates also represents a problem. Here, we propose a new method for the fabrication of microstructures for MCS generation. Most of the microstructures used for MCS generation are patterned hydrogel films of cell-nonadhesive inert materials such as agarose36,38 or polyethylene glycol.26,28 Here, instead of using template and microfabrication technique, patterned hydrogel films were obtained by the swelling-induced wrinkling of the film. Pattern formation has long been observed when a substrate-attached hydrogel film swells in water.39 Because the film is mechanically confined by the substrate, it can only expand along the direction normal to the substrate. The anisotropic swelling generates in-plane compressive stress within the gel. When the stress is sufficiently large, the free surface of the gel will locally buckle and fold against itself to form various wrinkling patterns.40(Scheme 1A) Swelling-induced wrinkling patterns Scheme 1. (A) Swelling-Induced Wrinkling of a Hydrogel Film: (1) Anisotropic Swelling of a Substrate-Attached Hydrogel Film Results in Equibiaxial Compressive Stress within the Film; (2) The Film Surface Buckles When the Stress Is above a Critical Threshold.39 (B) Generation of Multicellular Spheroids Guided by the Wrinkling Patterns

have been observed from various hydrogels,39−47 including polyacrylamide39,41 and poly(N-isopropylacrylamide).42−44 Particularly, M. Guvendiren et al.45,46 synthesized poly(2hydroxyethyl methacrylate) (PHEMA) hydrogels with gradient cross-linking density. Swelling of the films in water produces a wide range of surface patterns, including random, lamellar, peanut, and hexagonal structures. Here, we demonstrate that the in situ-formed wrinkling patterns can be used to guide the aggregation of cells. MCSs with narrow size distribution were generated. It is noteworthy the new method developed here does not require any template or specialized facilities for the patterning of the hydrogel films. In addition the microstructures were developed in situ in the wells of commercial cell culture plates. It is noteworthy that wrinkling patterns have previously been used to control cell spreading,48 align myoblasts,49 and control the differentiation of stem cells.50

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RESULT AND DISCUSSION Fabrication Patterned Hydrogel Films. In this work, we used swelling-induced wrinkling patterns to guide the generation of MCSs (Scheme 1). Unlike other patterning methods, swelling-induced wrinkling patterns form spontaneously on the surface of hydrogel films. As shown in Scheme

EXPERIMENTAL SECTION

Materials. 2-Hydroxyethyl methacrylate (HEMA) was purchased from Alfa Aesar. Ethylene glycol dimethacrylate (EGDMA), Darocur 1173 (2-hydroxy-2-methyl-1-phenyl-1-propanone), and 3(trimethoxysilyl)propyl methacrylate were purchased from Aldrich. Linear poly(2-hydroxyethyl methacrylate) (PHEMA, average Mv ∼ 3307

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1A, because the hydrogel films are fixed on a rigid substrate, when swelling in water, the films can only expand along the direction normal to the substrate. The unidirectional swelling generates a biaxial compressive stress within the gel. When the stress is sufficiently large, the free gel surface locally buckles and folds against itself to form various wrinkling patterns.41 PHEMA hydrogel films were chosen because PHEMA was previously used as nonadhesive substratum to generate MCSs.11,17 We adopted a method originally developed by Guvendiren et al.45 to fabricate the patterned hydrogel film. First, the precursor solution composed of partially polymerized HEMA, photoinitiator (Darocur 1173), and cross-linker (EGDMA) was coated onto vinyl-modified glass slides and exposed to UV light to form a cross-linked film. As the photopolymerization were carried out when the solution is open to air, the resulting film have a lower cross-linking density at the top surface, which gradually increases with film depth, and levels off after reaching the critical depth for oxygen diffusion.45 We synthesized a series of PHEMA films with different thicknesses and cross-linker concentrations. When swelling in water, various wrinkling patterns, including peanutlike and hexagonal patterns, form spontaneously on the film surfaces. (Figure 1) Peanut-shaped patterns were formed on

Figure 2. Wrinkling patterns developed on PHEMA films with various thicknesses and EDGMA contents. The films were fabricated on the bottom of the wells of 12-well cell culture plates.

glass was used as substrate, possibly because of the different constraint of the hydrogel film from the substrates. As shown in Figure 2, hexagonal patterns with almost the same size were obtained on the films with a cross-linker concentration of 7.0 wt %. These films were chosen for the following studies. Multicellular Spheroid Generation Guided by Wrinkling Pattern. To study if the wrinkling pattern can guide the generation of MCSs, suspensions of NIH3T3 murine embryonic fibroblasts were added to the wells whose bottom were modified with the patterned PHEMA hydrogel films. The behaviors of the cells were observed in situ. At the beginning, the cells distributed homogeneously in the suspension. Shortly after its addition to the well, the cells settle toward the bottom under the influence of gravity. Because of the presence of wrinkling pattern, they gradually aggregate in the center of the microcaves. This process is relatively fast. As shown in Figure 3A, most of the cells aggregate in the center of the microcaves in ∼10 min. It is noteworthy that the cells still exist as single cells at this stage; however, continued in situ observation reveals that

Figure 1. Wrinkling patterns developed on PHEMA films with various thicknesses and EDGMA contents. The films were fabricated on glass slides.

films with smaller thickness and lower cross-linker concentration. With increasing thickness and/or cross-linker concentration, the patterns become hexagonal. These observations are generally in agreement with that of Guvendiren et al.,45 except that Guvendiren et al. reported that films with cross-linker concentration ≥4.0 wt % do not form wrinkling patterns, while we observed wrinkling patterns from films with a cross-linker concentration as high as 7.0 wt %. The divergence may originate from the different experiment conditions. Despite of the small divergence, our results confirm that the method developed by Guvendiren et al. is rather robust. Next, we fabricated the PHEMA films directly in the wells of 12-well cell culture plates. To enhance the interaction between the substrate and the hydrogel film, the plate surface was first treated with concentrated H2SO4 to make it hydrophilic. Again, swelling of the PHEMA films results in wrinkling patterns on the film surfaces (Figure 2). Similarly, depending on the thickness and/or cross-linker concentration of the film, peanutshaped or hexagonal patterns will be obtained. However, in order to obtain hexagonal pattern which is desirable for MCS generation, the film should have a slightly higher cross-linker concentration compared with the case in which vinyl-modified

Figure 3. In situ observation of the aggregation of the cells on the patterned surface of PHEMA hydrogel film. The cells were maintained at 37 °C with a humidified atmosphere of 5% CO2. (A) Over the initial 10 min period. (B) Over the initial 6 h period. 3308

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Figure 4. (A) Optial images of the multicellular spheroids generated on patterned or flat PHEMA surfaces after 24 h or 48 h culture. (B) Size distribution of the spheroids generated on patterned or flat surfaces by 48 h culture.

matrix which facilitate their aggregation.1 This process requires a sufficient long time.14 One advantage of the use of the wrinkling patterned hydrogel surface is that this surface is capable to produce uniform MCSs. Because PHEMA is nonadhesive, NIH 3T3 cells seeded on flat PHEMA surface also aggregate into MCSs.11,17 Figure 4A compares the MCSs produced on flat PHEMA surface and the PHEMA surface with wrinkling patterns. MCSs generate on both surfaces and a longer culture produces tighter MCSs. On a flat surface, the cells aggregate randomly. In addition, the spheroids may move on the flat surface and fuse with neighboring spheroids to afford larger ones. Therefore, the MCSs generated on flat surface exhibit a very wide size distribution, ranging from 70 to 310 μm for those obtained after 48 h culture (Figure 4B). In contrast, the size of MCSs

connections were built up gradually among the cells during the following culture (Figure 3B). The average projected area of the aggregates becomes smaller with time. In addition, the boundary between cells becomes blurred. These observations suggest that the cell aggregates become more compact with time. The formation of tight cell aggreagtes is attributed to the relatively weak cell−substrate interaction. As many previous studies pointed out, relatively weak cell−substrate interaction facilitates the formation of multicellular aggregates.18,23,27,28 As mentioned above, PHEMA hydrogel is nonadhesive for cells.11,17 Indeed the cells remain to be round in shape during the aggreagtion process, suggesting they did not attach to the gel surface because of the weak interation between PHEMA gel and the cells. According to previous studies, when the cells contact with other, they secret autocrine factors to extracellular 3309

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Characterization of Multicellular Spheroids. To study the cell morphology, the cell nuclei were stained blue with DAPI, while F-actin in the cellular cytoskeleton was stained green with FITC-phalloidin. The spheroids were then visualized using confocal microscope. For comparison, the cells cultured as 2D monolayer was also immunohistochemically stained and examined. As shown in Figure 6, the cells in

generated on the patterned surface under the same conditions varies only from 80 to 120 μm, exhibiting a much narrower distribution. The reason lies that the number of cells accumulated in each microcave is almost the same. In addition, under gravity the spheroids will remain at the bottom of the caves and therefore will not fuse with other spheroids. One can control the size of the MCSs facilely by changing the number of cells seeded in the well.13 Figure 5A compares

Figure 6. Confocal images of immunohistochemically stained NIH3T3 2D monolayers and 3D multicellular spheroids. Cell nuclei and F-actin were stained blue and green, respectively.

2D monolayer show a spread-out morphology with an elongated cytoplasm. The cytoskeleton distribution is spread throughout the cytoplasmas stress fibers, which is required for the cells to adhere to the substrate. In contrast, cells in the 3D spheroids display a rounded, clustered morphology. The actin filaments were located around the edges of cells with a cortical distribution, which is typical for 3D cell morphology.4,14,52 To assess the viability of the cells in the spheroids, the spheroids were double stained with acridine orange (AO) and ethidium bromide (EB). In this way, live cells were stained green while dead ones red. The stained samples were first observed using a fluorescence microscope. As shown in Figure 7A, almost all cells are green, indicating high viability of the

Figure 5. (A) Optical images of the multicellular spheroids generated on patterned PHEMA surfaces. The cell density in the seeded cell suspensions and culture time were marked. (B) Size of the spheroids as a function of the cell density in the seeded cell suspensions. Culture time: 48 h. * indicates statistical significance at the 0.05 level. (C) Size distribution of the multicellular spheroids.

Figure 7. Representative fluorescence microscope image (A) and confocal microscopeimage (B and C) of the multicellular spheroids. Live cells were stained green with acridine orange, while dead cells were stained red with ethidium bromide.

the morphology of MCSs generated on the patterned surface at different seeding densities. In all cases, the MCSs generated after 48 h culture is smaller and tighter than the ones generated after 24 h culture. For the MCSs obtained after 24 h culture, the cell boundary is still detectable, but the boundary becomes barely detectable after 48 h culture. More importantly the size of the MCSs increases with increasing seeding density (Figure 5B). It is understandable that the average number of cells accumulated in each microcaves will increase with increasing seeding density, and more cells will fuse into a larger spheroids. Although the size of the MCSs becomes larger with increasing seeding density, the size distribution remains narrow, as shown in Figure 5C.

cells. The samples were also examined with a confocal microscope. Again almost all cells are stained green, confirming their viability. Figure 7B shows the image of a spheroid obtained after 24 h culture. The individual cells are easy to be identified. After 48 h culture, however, individual cells are no longer recognizable (Figure 7C). Similar gradual formation of tight spheroids was reported previously.23 The viability of the cells in the MCSs is also demonstrated by their ability to attach and respread on the 2D surface of normal cell culture plates.23,53 For this purpose, the MCSs were 3310

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Peking University, China, for his 80th birthday in November 2014.

pipetted out. After centrifugation, the culture media was changed with fresh ones. Then, the MCSs were transferred to a normal cell culture plate. As shown in Figure 8, the structure of

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(1) Lin, R. Z.; Chang, H. Y. Recent advances in three-dimensional multicellular spheroid culture for biomedical research. Biotechnol. J. 2008, 3, 1172−1184. (2) Glicklis, R.; Shapiro, L.; Agbaria, R.; Merchuk, J. C.; Cohen, S. Hepatocyte behavior within three-dimensional porous alginate scaffolds. Biotechnol. Bioeng. 2000, 67, 344−353. (3) Kataoka, K.; Nagao, Y.; Nukui, T.; Akiyama, I.; Tsuru, K.; Hayakawa, S.; Osaka, A.; Huh, N. An organic−inorganic hybrid scaffold for the culture of HepG2 cells in a bioreactor. Biomaterials 2005, 26, 2509−2516. (4) Xia, L.; Sakban, R. B.; Qu, Y.; Hong, X.; Zhang, W.; Nugraha, B.; Tong, W. H.; Ananthanarayanan, A.; Zheng, B.; Chau, I. Y.; Jia, R.; McMillian, M.; Silva, J.; Dallas, S.; Yu, H. Tethered spheroids as an in vitro hepatocyte model for drug safety screening. Biomaterials 2012, 33, 2165−2176. (5) Ho, V. H. B.; Slater, N. K. H.; Chen, R. pH-responsive endosomolytic pseudo-peptides for drug delivery to multicellular spheroids tumour models. Biomaterials 2011, 32, 2953−2958. (6) Friedrich, J.; Seidel, C.; Ebner, R.; Kunz-Schughart, L. A. Spheroid-based drug screen: Considerations and practical approach. Nat. Protoc. 2009, 4, 309−324. (7) Ho, V.; Muller, K. H.; Barcza, A.; Chen, R. J.; Slater, N. Generation and manipulation of magnetic multicellular spheroids. Biomaterials 2010, 31, 3095−3102. (8) Kunz-Schughart, L. A.; Kreutz, M.; Knuechel, R. Multicellular spheroids: A three-dimensional in vitro culture system to study tumour biology. Int. J. Exp. Pathol. 1998, 79, 1−23. (9) Mironov, V.; Kasyanov, V.; Drake, C.; Markwald, R. R. Organ printing: Promises and challenges. Regen. Med. 2008, 3, 93−103. (10) Mironov, V.; Visconti, R. P.; Kasyanov, V.; Forgacs, G.; Drake, C. J.; Markwald, R. R. Organ printing: Tissue spheroids as building blocks. Biomaterials 2009, 30, 2164−2174. (11) Ivascu, A.; Kubbies, M. Rapid generation of single-tumor spheroids for high-throughput cell function and toxicity analysis. J. Biomol. Screen. 2006, 11, 922−932. (12) Girard, Y. K.; Wang, C.; Ravi, S.; Howell, M. C.; Mallela, J.; Alibrahim, M.; Green, R.; Hellermann, G.; Mohapatra, S. S.; Mohapatra, S. A 3D fibrous scaffold inducing tumoroids: A platform for anticancer drug development. PLoS One 2013, 8, e75345. (13) Huang, Y.; Chan, C.; Lin, W.; Chiu, H.; Tsai, R.; Tsai, T.; Chan, J.; Lin, S. Scalable production of controllable dermal papilla spheroids on PVA surfaces and the effects of spheroid size on hair follicle regeneration. Biomaterials 2013, 34, 442−451. (14) Kim, J. A.; Choi, J.; Kim, M.; Rhee, W. J.; Son, B.; Jung, H.; Park, T. H. High-throughput generation of spheroids using magnetic nanoparticles for three-dimensional cell culture. Biomaterials 2013, 34, 8555−8563. (15) Hwang, Y.; Chung, B. G.; Ortmann, D.; Hattori, N.; Moeller, H.; Khademhosseini, A. Microwell-mediated control of embryoid body size regulates embryonic stem cell fate via differential expression of WNT5a and WNT11. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 16978− 16983. (16) Sakai, Y.; Nakazawa, K. Novel microchip technique for the transfer of spheroids as floating cultures to micropatterned-adherent cultures. Biochips Tissue Chips 2011, S4, 001. (17) Landry, J.; Bernier, D.; Ouellet, C.; Goyette, R.; Marceau, N. Spheroidal aggregate culture of rat liver cells: Histotypic reorganization, biomatrix deposition, and maintenance of functional activities. J. Cell Biol. 1985, 101, 914−923. (18) Wang, D.; Cheng, D.; Guan, Y.; Zhang, Y. Thermoreversible hydrogel for in situ generation and release of HepG2 spheroids. Biomacromolecules 2011, 12, 578−584. (19) Wu, Y.; Zhao, Z.; Guan, Y.; Zhang, Y. Galactosylated reversible hydrogels as scaffold for HepG2 spheroid generation. Acta Biomater. 2014, 10, 1965−1974.

Figure 8. Attachment and disassembly of a NIH3T3 spheroid on the surface of 2D tissue culture plate. The cell spheroid was cultured on 2D substrate for 0 h, 6 h, and 24 h, respectively. Scale bar: 50 μm.

the spheroids remained intact. After being cultured for 6 h, however, the cells at the periphery gradually spread on the substrate. The spheroids were fully disassembled after a 24 h culture. Note the cells in the spheroids exhibit a round morphology; however, they present an irregular shape with protrusions when attached and spread on the substrate. These results indicate that the cells in the spheroids, even those reside in the inner part of the spheroids, are alive. In addition, they retain the capability to migrate.23

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CONCLUSIONS In conclusion, we showed that the PHEMA hydrogel film with swelling-induced wrinkling surface patterns can be used for MCS generation. The resulting spheroids have a uniform size, and the spheroid size can be facilely controlled by the number of cells seeded in the well. The cells in the spheroids remain a high viability. Similar to other microwell approaches, this scaffold-free method avoids unwanted interference from the synthetic or natural components in the scaffold. It allows for simple harvest of the generated spheroids. Unlike the previously reported approaches, here, the patterns were generated spontaneously without the use of any template or specialized facilities. The patterned hydrogel films were fabricated in situ in the wells of commercial cell culture plates. More importantly, the new method allows for massive production of MCSs. For the 12 well plate used in the work, the diameter of the wells is 22.1 mm, while the diameter of the microcaves is ∼300 μm. The number of the microcaves in each well is estimated to be ∼5000. Therefore, one operation will produce ∼5000 MCSs in each well, or ∼60 000 MCSs in each plate. It is expected that these MCSs will be used in cell biology and drug screening and help reduce the use of animals in research.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Tel: 86-22-23501657. Email: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank financial support for this work from the National Natural Science Foundation of China (Grants Nos. 21174070, 21274068, 21228401, and 21374048), Tianjin Public Health Bureau (13KG110), Program for New Century Excellent Talents in University (NCET-11-0264), and PCSIRT program (IRT1257). This work is dedicated to Professor Weixiao Cao, 3311

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dx.doi.org/10.1021/bm500722g | Biomacromolecules 2014, 15, 3306−3312