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Langmuir 2007, 23, 9104-9108
“On the Fly” Continuous Generation of Alginate Fibers Using a Microfluidic Device Su-Jung Shin,† Ji-Young Park,† Jin-Young Lee, Ho Park, Yong-Doo Park, Kyu-Back Lee, Chang-Mo Whang, and Sang-Hoon Lee* Department of Biomedical Engineering, Korea UniVersity, 126-1, Anam-dong 5ga, Soungbuk-gu, Seoul, Korea ReceiVed March 21, 2007. In Final Form: June 6, 2007 In this paper, we introduce a new continuous production technique of calcium alginate fibers with a microfluidic platform similar to a spider in nature. We have used a poly(dimethylsiloxane) (PDMS) microfluidic device embedded capillary glass pipet as the apparatus for fiber generation. As a sample flow, we introduced a sodium alginate solution, and, as a sheath flow, a CaCl2 solution was introduced. The coaxial flows were generated at the intersection of both flows, and the sodium alginate was solidified to calcium alginate by diffusion of the Ca2+ ions during traveling through the outlet pipet. The diameter changes in the sample and sheath flow variations were examined, and the size of alginate fibers was well regulated by changing both flow rates. In addition, we have measured the elasticity of dried fibers. We evaluated the potential use of alginate fibers as a cell carrier by loading human fibroblasts during the “on the fly” fabrication process. From the LIVE/DEAD assay, cells survived well during the fiber fabrication process. In addition, we evaluate the capability of loading the therapeutic materials onto the alginate fibers by immobilized bovine serum albumin-fluorescein isothiocyanate in the fibers.
Introduction For the past decade, many microfluidic devices have been broadly applied in the field of analytical chemistry, tissue engineering, microbiology, biotechnology, and drug discovery.1-5 Recently, one of the emerging microfluidic applications introduced was the “on the fly” fabrication of diverse curved microstructures via the photopolymerization of liquid monomers or polymers. Many microstructures have been produced by this method, such as microparticles,6-9 microfibers,10 microstrips,11 and microcapsules.12,13 This fabrication procedure is simple and cost-effective; it enables mass production of diverse microstructures with uniform distribution of size. The fabrication environment is comparatively safe, and many sensitive biological materials such as enzymes, proteins, drugs, and cells can be loaded without serious damage. Because of these advantages, the “on the fly” fabrication method via the microfluidic platform has been regarded as a new tool that can be used to create small * Corresponding author. Tel: +8229206457. Fax: +8229216818. Email:
[email protected]. URL: http://biomems.korea.ac.kr. † These authors contributed equally to this work. (1) El-Ali, J.; Sorger, P. K.; Jensen, K. F. Nature 2006, 442, 403-411. (2) Hong, J. W.; Studer, V.; Hang, G.; Anderson, W. F.; Quake, S. R. Nat. Biotechnol. 2004, 22, 435-439. (3) Wu, H. K.; Wheeler, A.; Zare, R. N. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 12809-12813. (4) Dittrich, P. S.; Manz, A. Nat. ReV. Drug DiscoVery 2006, 5, 210-218. (5) Tsang, V. L.; Bhatia, S. N. AdV. Drug DeliVery ReV. 2004, 56, 1635-1647. (6) Dendukuri, D.; Pregibon, D. C.; Collins, J.; Hatton, T. A.; Doyle, P. S. Nat. Mater. 2006, 5, 365-369. (7) Jeong, W. J.; Kim, J. Y.; Choo, J.; Lee, E. K.; Han, C. S.; Beebe, D. J.; Seong, G. H.; Lee, S. H. Langmuir 2005, 21, 3738-3741. (8) Xu, S.; Nie, Z.; Seo, M.; Lewis, P.; Kumacheva, E.; Stone, H. A.; Garstecki, P.; Weibel, D. B.; Gitlin, I.; Whitesides, G. M. Angew. Chem., Int. Ed. 2005, 117, 734-738. (9) Nie, Z.; Xu, S.; Seo, M.; Lewis, P. C.; Kumacheva, E. J. Am. Chem. Soc. 2005, 127, 8058-8063. (10) Jeong, W. J.; Kim, J. Y.; Kim, S. J.; Lee, S. H.; Mensing, G.; Beebe, D. J. Lab Chip 2004, 4, 576-580. (11) Kim, S. R.; Oh, H. J.; Baek, J. Y.; Kim, H. H.; Kim, W. S.; Lee, S. H. Lab Chip 2005, 5, 1168-1172. (12) Oh, H. J.; Kim, S. H.; Baek, J. Y.; Seong, G. H.; Lee, S. H. J. Micromech. Microeng. 2006, 16, 285-291. (13) Utada, A. S.; Lorenceau, E.; Link, D. R.; Kaplan, P. D.; Stone, H. A.; Weitz, D. A. Science 2005, 308, 537-541.
quantities of diverse functional polymeric microstructures. The potential applications of this technology include the following: (1) a carrier for various therapeutic materials such as chemical drugs, cells, and recombinant proteins for the treatment of disease, (2) a microscaffold for regenerative medicine and (3) an actuating or sensing element that can be integrated into the microfluidic device.14 However, the “on the fly” fabrication process also has limitations that include (1) the safety of exposure of sensitive materials or cells to UV light has not been fully proven even with very short exposure times and 2) the photopolymerizable hydrogels used in this process have not been proven to be biocompatible and biodegradable. Therefore, these problems should be resolved before the application of the ‘on the fly’ fabrication process to biomedical fields. Calcium alginate has been used as a stable material in biomedical fields. Recently, calcium alginate has been extensively used in tissue engineering,15-18 cell culture,19,20 and drug delivery.21-24 It has many attractive properties such as low cytotoxicity, nonimmunogenicity, biodegradability, and the ability to be molded under mild conditions. Some investigators have started to use calcium alginate for the “on the fly” fabrication (14) Park, J. Y.; Oh, H.; Kim, D. J.; Baek, J. Y.; Lee, S. H. J. Micromech. Microeng. 2006, 16, 656-663. (15) Talei Franzesi, G.; Ni, B.; Ling, Y.; Khademhosseini, A. J. Am. Chem. Soc. 2006, 128 (47), 15064-15065. (16) Takai, T.; Sakai, S.; Yokonuma, T.; Ijima, H.; Kawakami, K. Biotechnol. Prog. 2007, 23 (1), 182-186. (17) Bouhadir, K. H.; Lee, K. Y.; Alsberg, E.; Damm, K. L.; Anderson, K. W.; Mooney, D. J. Biotechnol. Prog. 2001, 17 (5), 945-950. (18) Augst, A. D.; Kong, H. J.; Mooney, D. J. Macromol. Biosci. 2006, 6 (8), 623-633. (19) Li, X.; Liu, T.; Song, K.; Yao, L.; Ge, D.; Bao, C.; Ma, X.; Cui, Z. Biotechnol. Prog. 2006, 22 (6), 1683-1689. (20) Shapiro, L.; Cohen, S. Biomaterials 1997, 18 (8), 583-590. (21) Kim, Y. J.; Park, H. G.; Yang, Y. L.; Yoon, Y.; Kim, S.; Oh, E. Biol. Pharm. Bull. 2005, 28 (2), 394-397. (22) Laurienzo, P.; Malinconico, M.; Mattia, G.; Russo, R.; La Rotonda, M. I.; Quaglia, F.; Capitani, D.; Mannina, L. J. Biomed. Mater. Res. A 2006, 1, 78 (3), 523-531. (23) Giunchedi, P.; Gavini, E.; Moretti, M. D.; Pirisino, G. AAPS PharmSciTech 2000, 2, 1 (3), E19. (24) Qurrat-ul-Ain; Sharma, S.; Khuller, G. K.; Garg, S. K. J. Antimicrob. Chemother. 2003, 51 (4), 931-938.
10.1021/la700818q CCC: $37.00 © 2007 American Chemical Society Published on Web 07/18/2007
Continuous Generation of Alginate Fibers
of microparticles. Zhang et al. used a microfluidic device to produce calcium alginate capsules by emulsifying an aqueous alginate solution in an organic phase containing a dissolved crosslinking agent.25 Liu et al. reported a novel method of manufacturing shape-controlled calcium alginate gel microparticles (e.g., microspheres, disks, and plugs) in a microfluidic device.26 Both methods focused on the generation of separated microparticles by minimization of the interfacial energy of liquid flow. In this paper, we introduce a new continuous production technique of calcium alginate fibers with a microfluidic platform similar to a spider in nature. To date, many methods have been reported to fabricate alginate fibers using diverse apparatus.27-30 However, a continuous generation of calcium alginate fibers, based on the microfluidic chip approach, has not been previously reported. Such microfluidic generation of continuous polymer fiber has many advantages such as easy control of size, simple fabrication process of microscale fibers, and easy loading of biological materials. However, it is still a challenge because the flow control in a fluidic channel is difficult because of high viscosity and the need for the solidified fibers to be extruded easily without clogging. In addition, the solidification time, by diffusion-controlled ionic cross-linking, should be short enough to be completed within the microfluidic channel. We used a poly(dimethylsiloxane) (PDMS) microfluidic device embedded capillary glass pipet as the fiber generation apparatus. As a sample flow, we introduced a sodium alginate solution, and, as a sheath flow, a CaCl2 solution was introduced. The sheath flow plays two roles: (1) it solidifies the sodium alginate by diffusion of the Ca2+ ions and (2) it serves as a lubricant to extrude the solidified fibers without clogging. The long (3 cm) outlet pipet was employed to provide sufficient reaction time for the sodium alginate and Ca2+ ions. The diameter changes in the sample and sheath flow variations were examined; in addition, we also measured the elasticity of dried fibers. We evaluated the potential use of alginate fibers as a cell carrier by loading human fibroblasts during the “on the fly” fabrication process. In addition, we evaluate the capability of loading the therapeutic materials onto the alginate fibers by immobilized bovine serum albumin-fluorescein isothiocyanate (BSA-FITC) in the fibers. Materials and Methods Materials. Sodium alginate powder was obtained from Sigma Aldrich, and 2 g of the powder was dissolved in 98 g of deionized water solution using a mechanical stirrer at 500 rpm for 2 h. Calcium chloride powder was obtained from Sigma Chemicals, and a 784 mM CaCl2 solution (100 g) was prepared for the reaction with sodium alginate solution. The borosilicate capillary micropipets, which were used to focus flow, were purchased from Sutter Instruments, and the PDMS was obtained from Dow Corning. BSA-FITC was obtained from Sigma Aldrich. Continuous Production of Calcium Alginate Microfibers. Figure 1a illustrates the schematics of the microfiber generation apparatus. The microfluidic device used for fiber production was fabricated by combining the PDMS platform and the pulled borosilicate glass pipet. The procedure for the construction of the microfluidic device has been reported previously.7 The inner diameter (25) Zhang, H.; Tumarkin, E.; Peerani, R.; Nie, Z.; Sullan, R. M. A.; Walker, G. C.; Kumacheva, E. J. Am. Chem. Soc. 2006, 128 (37), 12205-12210. (26) Liu, K.; Wu, Y.; Henderson, F.; McCoy, D. M.; Salome, R. G.; McGowan, S. E.; Mallampalli, R. K. Langmuir 2006, 22 (22), 9453-9457. (27) Bhattarai, N.; Li, Z.; Edmondson, D.; Zhang, M. AdV. Mater. 2006, 18, 1463-1467. (28) Wan, A. C. A.; Yim, E. K. F.; Liao, I. C.; Visage, C. L.; Leong, K. W. J. Biomed. Mater. 2004, A11, 586-595. (29) Takei, T.; Sakai, S.; Ijima, H.; Kawakami, K. Biotechnol. J. 2006, 1, 1014-1017. (30) Fan, L.; Du, Y.; Huang, R.; Wang, Q.; Wang, X.; Zhang, L. J. Appl. Polym. Sci. 2005, 96, 1625-1629.
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Figure 1. Schematic of microfiber generation apparatus and principle of gelation (dotted circle). of the glass tip was 35 µm, and the end of the tip was cut using a microforge (MF-900, Narishige). A detailed view of the microfluidic device, to create the coaxial flow, is demonstrated in the dotted circle (Figure 1). Two fluids (2% (w/w) sodium alginate solution and 784 mM CaCl2 solution) were introduced into the sample and the sheath inlet ports, respectively, and two syringe pumps were employed for the injection. A three-dimensional (3D) coaxial sheath flow stream, around the sample flow, was formed at the point where the two flows merged. At the interface of both fluids, the sodium alginate solutions met with the polycation (Ca2+) material, and gelation of the cylindrical fibers was achieved by diffusion-controlled ionic cross-linking. We employed a long outlet pipet to increase the reaction time between the sodium alginate and the Ca2+ ions. During the time that the alginate flow travels inside of the outlet pipet, it forms spiral curls, and such solidifying alginate sample flow causes clogging of the outlet pipet. To eliminate this problem, we made both the sample and the sheath fluids move downward (i.e., gravitational direction). For the precise regulation of both of these flows, the viscosity is important; we measured the viscosities of all solutions using a viscometer (DV-II+, Brookfield). Measurement of the Diameter According to the Flow and Elasticity of Dried Fibers. The polymerized calcium alginate fibers, which were fabricated under the various flow conditions, were collected in a Petri dish and characterized. We used a digital camera to take microscopic photographs of the calcium alginate fibers and measured their diameters using Photoshop 7.0. The elasticity of the dried calcium alginate fibers was evaluated. For the elasticity measurements, we fixed one end of several of the fibers (mean length: 12.9 mm; diameter: 19 µm) on a glass plate using adhesive tape, and then we applied stress at the other end by hanging the adhesive mass step by step; the percentage elongation of the fiber to the tensile stress was then measured. All of the experiments were carried out under a stereoscope. Cell Preparation. To demonstrate the applicability of continuous alginate fibers as cell-carriers for tissue engineering, we encapsulated human fibroblast cells in the microfibers. Human fibroblast cells (L929) were cultured in Dulbecco’s modified Eagle medium (DMEM, GIBCO) with 10% fetal bovine serum (FBS, GIBCO) and 1% antibiotics containing 10 000 units (GIBCO) of penicillin and streptomycin at 37 °C under 5% CO2 and 95% atmospheric air. Cells were detached with a solution of 0.25% trypsin EDTA 1 × (GIBCO) for 2-3 min at 37 °C. For fluorescent examination of the cells inside of the alginate fibers, we stained human fibroblast cells with cell tracker (Red CMTPX, Invitrogen). For the viability test of the encapsulated cells, we incubated the cell-loaded fibers in a CO2 incubator for 24 h and treated the incubated alginate fibers with LIVE/DEAD assay reagents (LIVE/DEAD Viability/Cytotoxicity Kit, Molecular Probe) for 10 min at room temperature. The labeled cells were evaluated under fluorescence microscopy, and their viability was measured. Cell and Protein Encapsulation. For the encapsulation of cells, we prepared a sample solution by mixing cell suspensions (1 × 106 cells/mL) with sodium alginate solution (50% (w/w)). By introducing alginate solution including cells and 100 mM CaCl2 solution into
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Figure 2. (a) Calcium alginate fibers generated by using microfluidic system. (b-d) Formation of spiral curl of alginate solution in the outlet pipet according to the flow changes: (b) sheath flow rate: 20 mL/h, sample flow rate: 1 µL/min; (c) sheath flow rate: 20 mL/h, sample flow rate: 3 µL/min; (d) sheath flow rate: 20 mL/h, sample flow rate: 5 µL/min. the sample and sheath inlets, respectively, we created calcium alginate fibers with human fibroblast cells. To demonstrate the capability of loading the therapeutic materials onto the alginate fibers, we immobilized a fluorescently labeled bovine serum albumin (BSAFITC) in the fibers. BSA-FITC (3.5 mg) was dissolved in deionized water (700 µL) and added to the sodium alginate solution (700 µL); we fabricated the protein-loaded fiber using the microfluidic device.
Results and Discussion Gelation of Sodium Alginate Fluids in the Microfluidic Channel. Alginate fibers were successfully generated as shown in Figure 2 a. During fabrication of the calcium alginate fibers, we discovered that the shape of the unsolidified sodium alginate flow, in the outlet pipet, varied according to the sample and sheath flow rates, even though they were in the laminar flow phase. For a clear visualization of alginate flow in the outlet channel, we mixed red dyes (Papicel Red IJ-F3B, Eastwell) with the sodium alginate solution, and Figure 2b-d shows the colored alginate flow. We fixed the sheath flow rate at 20 mL/h and changed the sample flow rate to 1, 3, and 5 µL/min, respectively. At low sample flow rates, the sample flow always formed straight lines. As the flow rate increased, spiral curls were formed, and their waves increased in density (a video of the curled alginate flow is provided in the Supporting Information). We suggest that the formation of the spiral curls was due to the following three factors: (1) a low-speed sheath flow that prevented fast movement of sample flow due to high friction at the interface of both flows; this caused the sample flow to be bent toward the direction of low flow resistance, and this generated the spiral curl; (2) as a
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result of high viscosity differences between sodium alginate (109 cP) and CaCl2 (1.3 cP) flows, the sample flow was bent toward the direction of low flow resistance; and (3) alginate was solidified, as it moved through the outlet pipet, and the sheath flow prevented fast movement of solidified alginate fibers, which have higher friction with sheath flow than the liquid alginate. Therefore, the curls were formed as a result of the presence of stagnation of the solidified fibers, which prevented fast flow of the oncoming alginate solutions. To determine the cause of the curls, we carried out two additional experiments by changing the sample and the sheath solution. First, we used a sample solution with red dye in the water and the sheath solution with only water; both solutions had almost the same viscosity (approximately 1 cP). We introduced both of the solutions through the microfluidic channels with the same flow speed (sheath: 20 mL/hour, sample flow: 1, 3, and 5 µL/min). In all cases, the shape of the sample flow rate was straight. Therefore, the first assumption, stated above, that the velocity difference generates a spiral curl was not supported by the results of this experiment. Next, we used a sample flow with 2% (w/w) dyed alginate solution (viscosity: 121 cP) and a sheath flow with water (viscosity: 1 cP); solidification of the alginate by the diffusion of Ca2+ ions did not occur in the outlet pipet. We performed the same experiment with changes in the flow rate of both fluids; however, the spiral curl from the sample flow was not formed in the outlet pipet. Therefore, the second assumption, which stated that a large difference in viscosity generated the spiral curl, was not supported by the experimental results. Another possible explanation for the formation of the spiral curls is that the stagnation of solidified fibers, caused by the high friction between the CaCl2 flow and solidifying calcium alginate flow, prevents the fast movement of sodium alginate solution and makes the oncoming liquid sodium alginate flow form the spiral curl. Usually, the gelatin formation of calcium alginate occurs by the diffusion of Ca2+ ions accompanied by an instantaneous chemical reaction, that is, binding of Ca2+ ions to the carboxylic groups on the sodium alginate. The gelatin formation process by Ca2+ ions may be approximated as follows:31
∂C ∂2C ∂S )D 2 ∂t ∂t ∂x
(1)
where D is the diffusion coefficient of Ca2+, C is the concentration of Ca2+ ions in CaCl2 solution, and S is the concentration of cross-linked Ca2+ ions. The penetration of Ca2+ ions was governed by the concentration of Ca2+ ions, time of diffusion, and kinetics of cross-linking. Therefore, the concentration of CaCl2 and the time that the alginate solution resides in the microfluidic channel are important. The length of the outlet pipet and the flow rate determine the time that the alginate solution resided in the microfluidic channel. As the flow rate increases, the residence time in the microfluidic channel decreases, and the gelatin formation process, by the diffusion of Ca2+ ions, may be terminated before formation of the fibers. However, we did not observe termination of gelled fibers. The explanation appears to be that the spiral curl extended the residence time of the alginate fibers in the outlet pipet (length: 3 cm). The vertical positioning of the microfluidic channel is very important for the stable generation of fibers. When such curls move through the outlet pipet, located in the horizontal direction, they touch the inner surface of the outlet pipet, and this touch causes alginate clogging (31) Crank, J. The Mathematics of Diffusion, 2nd ed.; Clarendon Press: Oxford, 1975.
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Figure 3. Diameter change of alginate fibers according to the sample and sheath flow rate. Figure 5. SEM image of dried calcium alginate fiber (Sheath flow rate: 10 mL/hour, Sample flow rate: 5 µL/min).
Figure 4. Percent elongation to the stress of dried alginate fiber (diameter of fiber: 19 µm).
in the outlet pipet. By placing the microfluidic device in the vertical direction, we greatly reduced the clogging of the alginate fibers. Diameter According to the Flow Change and Elasticity of Alginate Fibers. We have measured the diameter of calcium alginate fibers according to the flow rate change of the sample and sheath flow, and the results are plotted in Figure 3. At a fixed sheath flow rate (10 mL/h), we changed the sample flow rates to 1, 2, 3, and 4 µL/min and then measured the diameters of the fibers under optical microscopy. On the basis of the increase of sample flow rates, the diameters of the fiber increased almost linearly. Similarly, we increased the sheath flow rate to 20 mL/h and changed the sample flow with the same flow rates. As expected, the diameter of the alginate fibers was regulated by both the sample and sheath flow changes. The sample flow more dominantly affected the determination of the fiber size. For the evaluation of the mechanical characteristics of the calcium alginate fiber, we measured the elasticity; the results are shown in Figure 4. In cases with wet calcium alginate fibers, it was impossible to measure the elasticity because of difficulties in handling the sample. With a dried alginate fiber, we measured the elongation of the fiber under a stereoscope. The fiber increased almost linearly with an increase in stress. When the length of the fiber increased more than 13%, most of the fibers would break. Compared to a 4-hydrobutylacrylate fiber,10 the dried calcium alginate fiber was comparatively rigid, and the tensile strength was weak. We evaluated the dried alginate fiber using a scanning electron microscope (Figure 5); many cracks were observed in the dried alginate fibers, and these cracks appeared to cause deterioration of the mechanical properties of the alginate fibers.
Figure 6. (a) Human fibroblast cell-laden calcium alginate fiber. (b) Corresponding fluorescent image of cells, which were stained with cell tracker (sheath flow rate: 20 mL/h, sample flow rate: 3 µL/min).
Cell and Protein Encapsulation and Viability Testing. We encapsulated human fibroblast cells in the calcium alginate fibers and investigated their morphology and viability. Figure 6a illustrates a micrograph of human fibroblast cells loaded onto alginate microfibers taken just 1 h after encapsulation. The cellencapsulated fibers can be handled with a tweezer in the cell culture media without damaging the cells; the calcium alginate capsule protects the embedded cells. Figure 6b shows the corresponding fluorescent micrograph of the cell tracker. All the human fibroblast cells were encapsulated and loaded well onto the fiber and the features of the cells maintained stable. We maintained cell-loaded fibers for 7 days in the CO2 incubator, and the features of the embedded cells remained stable. After cultures of the fiber-containing cells were kept in a CO2 incubator for 24 h, we performed a viability test by evaluating the LIVE/
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Figure 8. Fluorescent image of BSA-FITC-laden calcium alginate fiber.
show that the calcium alginate protected and preserved the embedded cells and therefore could be used as cell-carriers for cell therapy or tissue regeneration. We fabricated calcium alginate microfibers containing BSA-FITC; Figure 8 illustrates the findings by fluorescent micrograph. The BSA-FITC was loaded stably and uniformly in the fibers by the “on the fly” fabrication process. These results suggest that other therapeutic materials can be easily loaded during the alginate fabrication process and that these fibers can be utilized for the delivery of drugs or other therapeutic materials.
Conclusion
Figure 7. (a) Micrograph of alginate fiber containing fibroblast cells. (b) Corresponding fluorescent image of cells that were stained with LIVE/DEAD assay reagent. (c) Micrograph of cells that were cultured for 24 h in the fiber (calcium alginate was completely dissolved by the EDTA in the LIVE/DEAD assay reagent); dotted lines represent the locus of calcium alginate fiber. (d) Corresponding fluorescent image of cells that were stained with LIVE/DEAD assay reagent.
DEAD staining (green/red, respectively). Figure 7a illustrates the micrograph of alginate fiber containing fibroblast cells, and Figure 7b shows the corresponding fluorescent image of cells, which were stained with LIVE/DEAD assay reagent (red: dead cells; green: live cells). Cells survived (green color) well during the “on the fly” fabrication process. After the 24 h harvesting, the viability was over 80%; this indicates that cells were not seriously damaged during the encapsulation process and were preserved well inside of the alginate fibers. We treated the cellloaded fibers for 30 min with the LIVE/DEAD assay reagents in the CO2 incubator. Then the calcium alginates were dissolved completely, and only the embedded cells remained in the dish. The micrograph of the remaining cells and their corresponding fluorescent micrograph are demonstrated in Figure 7c,d and there were many loci of alginate fibers (e.g., dotted line) in the optical micrograph. We measured the viability of remaining cells, and they were over 80%, which indicates that cells survived well during the dissolving process of calcium alginate. These results
In this paper, we have demonstrated that calcium alginate fibers can be produced continuously using microfluidic devices. In the outlet pipettes (length: 3 cm), an instantaneous chemical reaction (binding of Ca2+ ions to the carboxylic groups on sodium alginate) is produced, and the solidified calcium alginate fibers are extruded successfully without clogging when the microchannel was set to the gravitational direction. The flow characteristics of solidifying calcium alginate, according to the sample flow changes, were described, and the diameter of the fiber could be controlled by adjusting the flow rates of both fluids. We have shown that human fibroblast cells and BSA-FITC can be easily loaded during the fiber fabrication process without serious damage. Therefore, this technology can be used as a vehicle for cells or therapeutic molecules for the regeneration of damaged tissues or used as a 3D-line scaffold to fabricate nerve or muscle fibers. In addition, the alginate fibers, including cells or proteins, can be produced safely and in large quantities by using the microfluidic chip. A large quantity of fibers can also be molded into a 3D shaped scaffold for organ regenerations. Acknowledgment. This study was supported by a grant of the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (0405-ER01-0304-0001). Supporting Information Available: A video image of curl generation in the outlet pipet (sample flow rate: 3 µL/min; sheath flow rate: 20 mL/hour). This material is available free of charge via the Internet at http://pubs.acs.org. LA700818Q
