Monodisperse Cell-Encapsulating Peptide Microgel Beads for 3D Cell

Oct 21, 2009 - (a) Schematic illustration of the cell-encapsulating SAP microgel bead formation process by using an external gelation method. The disc...
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Monodisperse Cell-Encapsulating Peptide Microgel Beads for 3D Cell Culture Yukiko Tsuda,†,‡ Yuya Morimoto,† and Shoji Takeuchi*,†,‡ †

Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8505, Japan, and ‡ Life BEANS Center, BEANS Project, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8505, Japan Received July 31, 2009. Revised Manuscript Received September 25, 2009

This paper describes a method to produce monodisperse cell-encapsulating microgel beads composed of a selfassembling peptide gel for three-dimensional (3D) cell culture. We used a 3D microfluidic axisymmetric flow-focusing device with an external gelation method. The finely powdered salts were dispersed into a continuous phase, and the salts induced the gelation when in contact with the peptide solution. Over 93% of the cells survived after the encapsulation, and the cells migrated and grew within the gels. Applications of our cell-encapsulating beads include bead-based cell assays in drug testing and engineering tissue constructs.

Introduction An in vitro three-dimensional (3D) cell culture system that mimics in-vivo-like cellular microenvironments is essential in tissue engineering and drug testing using engineered tissues.1-5 Of the available 3D cell culture methods (e.g., cellular aggregates and spheroids),6 cell encapsulation within hydrogels3,7 facilitates the continuous supply of nutrients and metabolic substances during cell growth and organization into tissues. While bioderived8 and synthetic3,9 materials have been widely used for 3D cell culture, a self-assembling peptide (SAP) hydrogel10,11 is particularly attractive because of the following advantages seen in both type of materials: (i) SAP has a 3D nanofiber structure similar to the natural extracellular matrices (ECM), allowing cells to adhere, grow, and differentiate, and (ii) we can precisely control both the physical and the chemical factors inside the hydrogel. Indeed, we can change the stiffness of the gel for various cell types7 and can functionalize the hydrogel with cytokines to induce cell growth or cell attachment.12 However, cell encapsulation using SAP hydrogels still relies on a traditional cell encapsulation technique that involves the preparation of millimeter-scaled gels.13 These gels cause low reproducibility of the cell culture conditions because the chemical gradient within the millimeter-scaled gel exhibits larger than microscaled gel. Moreover, because a millimeter-scaled SAP *Corresponding author. Tel: þ81-3-5452-6650. Fax: þ81-3-5452-6649. E-mail: [email protected].

(1) Griffith, L. G.; Swartz, M. A. Nat. Rev. Mol. Cell Biol. 2006, 7, 211–224. (2) McGuigan, A. P.; Sefton, M. V. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 11461–11466. (3) Albrecht, D. R.; Underhill, G. H.; Wassermann, T. B.; Sah, R. L.; Bhatia, S. N. Nat. Methods 2006, 3, 369–375. (4) Khademhosseini, A.; Langer, R. Biomaterials 2007, 28, 5087–5092. (5) Yang, J.; Yamato, M.; Sekine, H.; Sekiya, S.; Tsuda, Y.; Ohashi, K.; Shimizu, T.; Okano, T. Adv. Mater. 2009, 21, 1–6. (6) Fukuda, J.; Sakai, Y.; Nakazawa, K. Biomaterials 2006, 27, 1061–1070. (7) Caplan, M. R.; Schwartzfarb, E. M.; Zhang, S.; Kamm, R. D.; Lauffenburger, D. A. J. Biomater. Sci., Polym. Ed. 2002, 13, 225–236. (8) McGuigan, A. P.; Bruzewicz, D. A.; Glavan, A.; Butte, M.; Whitesides, G. M. PLoS One 2008, 3, e2258. (9) Cao, Y.; Vacanti, J.; Paige, K.; Upton, J.; Vacanti, C. Plast. Reconstr. Surg. 1997, 100, 297–302. (10) Zhang, S.; Holmes, T.; Lockshin, C.; Rich, A. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 3334–3338. (11) Kim, M. S.; Park, J.-K. JALA 2006, 11, 352–359. (12) Horii, A.; Wang, X.; Gelain, F.; Zhang, S. PLoS One 2007, 2, e190. (13) Hamada, K.; Hirose, M.; Yamashita, T.; Ohgushi, H. J. Biomed. Mater. Res., Part A 2008, 84, 128–136.

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hydrogel is fragile, it is difficult to manipulate.14 Thus, a method capable of producing microscaled and uniform-sized SAP hydrogels is needed to use this material. Microgel beads show a high potential to create a reproducible cellular microenvironment that provides relatively uniform chemical distribution within the hydrogels. In addition, minimizing the size of the gels allows easy manipulation with micropipets or microsyringes. To produce monodisperse microhydrogel beads, microfluidic devices, such as the T-junction device15,16 and flow-focusing,17-19 have been used. With the T-junction device, we have previously reported that an internal gelation method is useful for the formation of monodisperse alginate hydrogel beads that form a gel by ionic gelation.16 This method involves the addition of a cross-linking agent (e.g., ions) in the discontinuous aqueous phase. However, in our preliminary test, we found that this method cannot be applied for the SAP hydrogel even though the SAP solution is an ionic gelation material (see Supporting Information, 1). We believe that this is because SAP is a sol state at pH 3 and immediately forms nanofibers when exposed to a cross-linking agent or at higher pH. In this paper, we propose an alternative gelation method to produce monodisperse SAP microgel beads in a microfluidic device. We used an external gelation method where we initiate gelation after generating droplets of pregel solution by incorporating cross-linking agents (i.e., salts) in the continuous (oil) phase. Figure 1a-d shows our approach used to form the SAP hydrogel beads. We tested finely powdered salts dispersed into the continuous (oil) phase. When the powdered salts make contact with the aqueous phase, they immediately dissolve and form ions that work as cross-linking agents for the gelation of SAPs. Here, we investigated this method using an axisymmetric flow-focusing device (Figure 1c). We also assessed the characteristics of the SAP hydrogel microbeads as a cell encapsulating platform. Finally, we (14) Gelain, F.; Horii, A.; Zhang, S. Macromol. Biosci. 2007, 7, 544–551. (15) Okushima, S.; Nisisako, T.; Torii, T.; Higuchi, T. Langmuir 2004, 20, 9905– 9908. (16) Tan, W.-H.; Takeuchi, S. Adv. Mater. 2007, 19, 2696–2701. (17) Takeuchi, S.; Garstecki, P.; Weibel, D. B.; Whitesides, G. M. Adv. Mater. 2005, 17, 1067–1072. (18) Morimoto, Y.; Tan, W. H.; Tsuda, Y.; Takeuchi, S. Lab Chip 2009, 9, 2217– 2223. (19) Zhang, H.; Tumarkin, E.; Sullan, R. M. A.; Walker, G. C.; Kumacheva, E. Macromol. Rapid Commun. 2007, 28, 527–538.

Published on Web 10/21/2009

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Figure 1. Preparation of cell-encapsulating SAP microgel beads using the AFFD. (a) Schematic illustration of the cell-encapsulating SAP microgel bead formation process by using an external gelation method. The discontinuous (aqueous) phase of the mixed solution of SAPs and cells is focused and broken into w/o droplets at the orifice and then collected in the microtube. Powdered salts as a cross-linking agent dispersed into the continuous (oil) phase induce gelation of the SAP solution droplets. (b) Experimental setup to produce cell-encapsulating SAP microgel beads using the AFFD. (c) A macroscopic view of the AFFD used in this study.21 Scale: 10 mm. (d) Mechanism of external gelation of the SAP droplets using the powdered salts.

performed 3D cell culture and cell viability tests, and assessed the behavior of the generated SAP microbeads.

Materials and Methods Materials. SAPs (PuraMatrix RADA 16) dissolved in water (1 w/v %, pH 3) were kindly provided by 3D Matrix Ltd. (Tokyo, Japan). The synthesis and characterization of RADA 16 was described in the previous report.20 RADA 16 has the amino acid sequence [COCH3]-RADARADARADARADA-[CONH2] and is soluble in salt-free aqueous solution. When RADA 16 solution is exposed to a solution containing salt, such as physiological media, including saline or a higher pH condition, the peptide self-assembles spontaneously to form a hydrogel. Yokoi et al. described that the water content of the hydrogels formed from 1 to 5 mg/mL of SAP solution is 99.5- 99.9%.20 In this study, the SAP solution was degassed by ultrasonication for 30 min prior to use. Cell Culture and Preparation of Cell Suspension. Bovine carotid artery endothelial cells (ECs) were obtained from the Japan Health Science Foundation (HH, JCRB0099, Osaka, Japan). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS) (JBS-5441, Japan Bioserum), 100 U/mL penicillin, and 100 μg/mL streptomycin, at 37 °C under a humidified atmosphere of 5% CO2. The cells were used between the 17th and 25th passages in this study. Subconfluent cultured ECs were harvested by incubation for 5 min at 37 °C with 0.25% trypsin and 0.02% EDTA in phosphate-buffered saline (PBS) and (20) Yokoi, H.; Kinoshita, T.; Zhang, S. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 8414–8419.

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collected by centrifugation. For the cell-encapsulation study, the cell pellet was resuspended in 20% (w/v) sucrose and was washed twice to remove any residual salts that induce self-assembling and gelation of the peptide. Cell morphology was monitored under a phase contrast microscope (IX71, Olympus, Tokyo, Japan). Axisymmetric Flow-Focusing Device Fabrication. The three-dimensional (3D) microfluidic devices, axisymmetric flowfocusing devices (AFFDs), were fabricated as follows. We used a commercial stereolithography prototype (Perfactory3, EnvisionTec, Gladbeck, Germany) to rapidly fabricate our AFFD by sequentially exposing and polymerizing the design, plane-byplane, using a digital micromirror device (DMD) projector. The fabrication process and the structure of the device is the same as that described in our previous report.21 Briefly, we designed the device with a 3D modeling software (Rhinoceros, AppliCraft, Tokyo, Japan), and uploaded the data to the stereolithography prototype to fabricate the 3D structures with photoreactive acrylate resin (R11, EnvisionTec). The fabricated device consists of two concentric hollow cylinders (Figure 1a). Each cylinder has a connection port that separately guides two types of fluid, such as oil and aqueous solution, into the device. Cell Encapsulation within SAP Hydrogel Beads Using the AFFD. We used an axisymmetric flow-focusing device (AFFD) to obtain the cell-encapsulating peptide microgel beads. The fabrication process of the AFFD is described in our previous report.21 Figure 1a illustrates the design of the AFFD and the methods used to prepare the cell-encapsulating microgel beads. (21) Morimoto, Y.; Tan, W. H.; Takeuchi, S. Biomed. Microdevices 2009, 11, 369–377.

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For the inner fluid, a mixture of the cell suspension in 20% (w/v) sucrose solution and 0.5% (w/v) SAP solution at a ratio of 1:1 (v/v) was introduced into the inner channel. We dispsersed finely powdered salts (DMEM powder, Invitrogen) as a cross-linking agent at a concentration of 6.8 mg/mL in mineral oil containing Span 80 (2% w/v). This oil mixture was introduced into the outer channel to form water-in-oil (w/o) droplets containing the SAPs and cells. The droplets were then collected in a microtube with mineral oil containing Span80. To confirm the gelation of the peptide, fluorescently labeled nanobeads (FluoSpheres, yellowgreen fluorescent-conjugated carboxylate-modified microspheres, 2% solids, 0.20 μm, Molecular Probes) were sometimes incorporated with the peptide solution when forming the droplets. On the basis of previously reported methods,16,18 the microgel beads in a collection tube were extracted from the oil into cell culture media. Briefly, hexadecane containing Span 80 (2% w/w) was added to the collection tube, the microbeads were centrifuged, and the oil layer was discarded. The microbeads were washed three times by hexadecane. Washing buffer solution (DMEM containing 0.1% (w/w) Tween 20) was gently added to the collection tube. The collection tube containing microbeads, hexadecane, and washing buffer was then centrifuged to extract the microbeads from the hexadecane into the washing buffer phase. Because hexadecane (organic phase) and DMEM solution (aqueous phase) are immiscible, the cells encapsulated within gel beads do not come into contact with the oil phase. The microgel beads were finally suspended in DMEM, supplemented with 10% FBS for cell culture, at 37 °C with 5% CO2. As organic hexadecane and water are immiscible, hexadecane does not go into aqueous phase in the SAP gel beads. The procedure of “centrifugation” allows transfer of microgel beads from hexadecane into DMEM in centrifuge tubes. For the time-course study of cell behavior within the microgel beads, microphotographs were acquired at predetermined time points (0, 4, and 8 h). Conventional Cell Encapsulation System Using the Cell Culture Insert to Form the Bulk Gel. To compare our encapsulation system using the AFFD with the conventional encapsulation method, the cells were encapsulated in the peptide gels according to the manufacturers’ instructions.13 Briefly, the cell suspension in 20% (w/v) sucrose was combined with 0.5% (w/v) SAP solution at a ratio of 1:1 (v/v), and 200 μL of the mixture was loaded on top of a cell culture insert placed in a 24-well plate (Transwell-clear, Corning, New York, U.S.A.). The cell culture media (700 μL) was gently added to the top of the mixture to induce gelation. The medium was changed twice within 15 min, and the cells encapsulated within the bulk gel were cultured at 37 °C with 5% CO2. Cell Viability Test. Cell viability was assessed after cell encapsulation within the SAP gels using the Live/Dead Viability/Cytotoxicity Kit (Molecular Probes) based on a simultaneous determination of living and dead cells with two probes, calceinbis[(acetyloxy) methyl] ester (Calcein-AM) for intracellular esterase activity and ethidium homodimer-1 (EthD-1) for plasma membrane integrity.13 In brief, the cell-encapsulating hydrogels were incubated in a working solution (2 mM Calcein-AM and 5 mM EthD-1) for 10 min at 37 °C. The stained cells were observed under a fluorescent microscope, and the images were superimposed using Adobe Photoshop (version CS2, Adobe Systems, Inc.). Immunohistochemistry. Cells cultured within the SAP microgel beads were fixed in 4% paraformaldehyde in PBS for 20 min. After permeabilization with 0.5% Triton X-100 in PBS for 2 min, the cells were blocked with 0.1% bovine serum albumin Langmuir 2010, 26(4), 2645–2649

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Figure 2. Microscopic views of the generated SAP hydrogel beads collected in the oil. (a) Bright-field image of the SAP hydrogel beads in the oil. Small crystals indicate powdered salts dispersed into the continuous (oil) phase. (b) Fluorescent microscopy of the SAP gels beads in (a). The gel beads were visualized by incorporating 200 nm diameter fluorescent beads and revealed that the SAPs uniformly self-assembled in the w/o droplets. Scale bars: 100 μm in (a) and (b).

Figure 3. Distribution of droplet diameter at different flow rate ratios. The final concentration of SAP hydrogels is constant at 0.25% (w/v) in this experiment. (a) Qc/Qd = 150/5 (μL/min). (b) Qc/Qd = 150/10 (μL/min). These results indicate that this external gelation method using the AFFD is able to produce monodisperse droplets. Scale bars: 100 μm in (a) and (b).

fraction V (Sigma) in PBS for 1 h. For F-actin staining, cells were stained with a 1:100 dilution of Alexa488-conjugated phalloidin (200 U/mL) (Molecular Probes) at 25 °C for 2 h. For cell nucleus staining, the cells were double-stained with a 1:500 dilution of DNA-binding dye, Hoechst 33342 (1 mg/mL), at 25 °C for 5 min. These stained cells were observed under a confocal laser scanning microscope (CSU 22, Yokogawa Elect. Co., Japan).

Results and Discussion We demonstrated the formation of monodisperse water-in-oil (w/o) droplets comprising SAP using the AFFD. Our method successfully generated uniform-sized droplets containing SAPs (Figure 2a,b), indicating that the powdered salts in the oil mixture successfully works as a cross-linking agent for the gelation of SAPs. To examine the degree of gelation of the peptide fibers, we incorporated fluorescent nanoparticles into the inner fluids. We observed that the Brownian motion of the nanoparticles in the droplets stopped within 1 min after collecting the droplets in the microtube. This result indicates that the gelation within the droplets is gradual and that the powdered salts in the mixture oil successfully work as a cross-linking agent for the gelation of SAPs. We achieved a high degree of control over the diameter of droplets by varying the flow rate ratio. The droplets formed at Qc/Qd = 150/5 (μL/min) had an average diameter of 113.9 ( 4.65 μm (Figure 3a). Increasing the flow rate of the discontinuous phase to Qd = 10 μL/min produced larger droplets 145.5 ( 2.95 μm (Figure 3b). The coefficient of variation (% CV), defined as the standard deviation to the mean, of the droplets was 4.0% and 2.7%, respectively, indicating that they were monodisperse. Although we altered only the flow rate ratio in the bead generation process, the diameter, monodispersity, DOI: 10.1021/la902827y

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Figure 4. Phase contrast microscopies of the cell-encapsulating SAP microgel beads after being transferred from the oil to the cell culture media. Cells are stained with red CellTracker Orange in (b). Scale bars: 100 μm in (a) and 50 μm in (b).

and properties of gels (i.e., porosity, fiber size, mechanical properties, gelation time) can also be tuned by the concentration of SAP solution and the type of the organic phase. To produce monodisperse microgel beads, the cross-linking agents must be uniformly dispersed within the oil phase, which we accomplished by using fine DMEM powders. As SAP solution forms gel in the presence of salts, other materials, such as finely crushed CaCl2 and NaCl, could be used as substitutes for DMEM powder. In addition, the monodispersity and the diameter of the microgel beads did not change when DMEM powders were incorporated in the oil phase at concentrations greater than 6.8 mg/mL. The cell-encapsulating gel beads maintained their spherical structure, even after extracting from the oil to the cell culture media (Figure 4a,b). In our current system, the number of cells encapsulated within each microgel bead can be roughly controlled by varying the cell number density in the SAP solution infused into the AFFD. Although it is still difficult to control the position of cells in the microgel beads precisely, we believe that the conditions within the beads (i.e., chemical gradient and gap between neighboring cells) are more uniform compared with bulk gels because the variation of these factors decreases as the diameter of the beads decreases. Moreover, the distance between neighboring cells in microgel beads could be minimized compared with the distances within bulk gel. Recently, advances in MEMS technologies have enabled the incorporation of individual cells within individual microdroplets.22 Application of this method could enable us to control the position of cells within the microgel beads. To show that the cells encapsulated within the SAP hydrogels remained alive, we evaluated cell viability using the Live/ Dead assay; this assay stains dead cells red and living cells green. Figure 5 shows microphotographs of the cell-encapsulating millimeter-scaled bulk gels prepared by a conventional method and the microgel beads prepared by the AFFD method. The cell viability was 95.6 ( 0.1% in the bulk gels and 93.9 ( 0.9% in the microgel beads. On the basis of the manufacturer’s protocol, the viability of the fibroblast cells is more than 80% when encapsulated in the bulk condition. We showed that our method was mild enough to encapsulate mammalian cells (endothelial cells). Note that, to maintain the viability of the cells using this encapsulation system, the entire process from the reagent introduction to the extraction of beads into the cell culture medium should be completed within 15 min because the SAP solution is acidic (pH 3). Longer processing time affects cell viability in this system. For the mass production of cell-encapsulating microgel beads in the future, we need to modify the device that mixes both the SAP solution and the cells immediately prior to generating the w/o droplets at an orifice (250 μm in diameter). (22) Edd, J. F.; Di Carlo, D.; Humphry, K. J.; Koster, S.; Irimia, D.; Weitz, D. A.; Toner, M. Lab Chip 2008, 8, 1262–1264.

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Figure 5. Cell viability in the cell-encapsulating SAP gels. (a, b) Cells were encapsulated in the SAP hydrogels by a conventional method using a cell culture insert. (c, d) Cells were encapsulated in the SAP microgel beads by the AFFD. Observation of the cells under bright-field (a, c) and fluorescent microscopies (b, d). Dead cells are stained red, and living cells are stained green. The cell viability was 95.6 ( 0.1% in the bulk gels and 93.9 ( 0.9% in the microgel beads. Data are calculated as the mean of three samples with standard error of the mean. Scale bars: 50 μm.

We have also demonstrated the possibility of performing 3D cell culture within the SAP hydrogel beads. We incubated the cellencapsulating microgel beads in cell culture media, supplemented with 10% FBS, at 37 °C in a humidified atmosphere with 5% CO2. We observed that the cells migrate within the SAP microgel beads (Figure 6a), as usually shown in bulk gels.23 After a 3-day cultivation, we also observed that endothelial cells within the SAP microgel beads migrated and formed cell-to-cell contacts within the gels (Figure 6b). These results indicate that our SAP microgel beads successfully allow diffusion of the nutrients and facilitate migration, growth, and differentiation of cells. Because the SAP gels used in this study acted as cell adhesive scaffolds, different cell types can be cultured on the outer surface of the gel beads to achieve a hierarchic 3D coculture system to increase cellular functions for fabrication of engineered tissues or heterotypic cellular interactions within 3D coculture systems (see Supporting Information, 2). We found that microgel beads offered easy manipulation without breakage by normal micropipetting operations. We believe that monodispersity of cell-encapsulating SAP microgel beads will be useful for a beadbased assay, such as on the microfluidic dynamic arrays24 for high-throughput studies of pathological and physiological phenomena in 3D cultured cells.

Conclusions We have developed an external gelation method to produce monodisperse cell-encapsulating SAP microgel beads by incor(23) Narmoneva, D. A.; Vukmirovic, R.; Davis, M. E.; Kamm, R. D.; Lee, R. T. Circulation 2004, 110, 962–968. (24) Tan, W. H.; Takeuchi, S. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 1146– 1151.

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Figure 6. Microscopic views of the 3D cultured endothelial cells in SAP microgel beads. (a) Time-lapse images of the 3D cultured endothelial cells after encapsulation indicate that the cells are able to migrate through the gel. (b) Fluorescent confocal microscopy of the encapsulated endothelial cells after culture for 3 days. The cells were stained with Alexa488-conjugated phalloidin to label F-actin (green) and Hoechst 33342 to label the nuclei (blue). The cells migrated and formed cell-to-cell contact within the gel beads. Scale bars: 50 μm in (a) and 10 μm in (b).

porating finely powdered salts in continuous (oil) phase using the AFFD. This method offers easy control over the size of beads at a micrometer scale by tuning the flow rate ratio, creating a reproducible and uniform cellular microenvironment. Also, this method would be applicable to other materials that gelate by ionic gelation. Because the synthetic peptides used here can be functionalized with short biologically active motifs that promote cell adhesion, cell spreading, and cell differentiation, it is possible to design scaffolds for a specific tissue and properties that are useful for tissue engineering and regenerative medicine.25 Furthermore, our size-controlled cell/biomolecule-encapsulating microgel beads have the potential for high-throughput screening, such as cell-based diagnostic applications, drug testing, and toxicology, using a dynamic microarray platform.24 We believe that cell-encapsulating microbeads will be useful for the fabrication (25) Genove, E.; Shen, C.; Zhang, S.; Semino, C. E. Biomaterials 2005, 26, 3341– 3351.

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of 3D tissue architectures to mimic tissues by combining various beads. Acknowledgment. This work was partly supported by CREST from the Japan Science and Technology Agency, and by New Energy and Industrial Technology Development Organization (NEDO) of Japan. We thank Dr. Teru Okitsu (Kyoto University Hospital) and Dr. Satoru Kobayashi (3D Matrix Inc.) for their valuable comments and discussions. We also thank 3D Matrix Inc. (Tokyo, Japan) for providing the PuraMatrix. Supporting Information Available: Internal gelation method to produce cell-encapsulating SAP microgel beads, microscopic views of beads, and confocal microscopy of hierarchically cocultured cells using SAP microgel beads. This material is available free of charge via the Internet at http://pubs.acs.org.

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