Controlled Fabrication of Bioactive Microfibers for Creating Tissue

Letter www.acsami.org

Controlled Fabrication of Bioactive Microfibers for Creating Tissue Constructs Using Microfluidic Techniques Yao Cheng, Yunru Yu, Fanfan Fu, Jie Wang, Luoran Shang, Zhongze Gu,* and Yuanjin Zhao* State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China S Supporting Information *

ABSTRACT: The fabrication of heterogeneous microstructures, which exert precise control over the distribution of different cell types within biocompatible constructs, is important for many tissue engineering applications. Here, bioactive microfibers with tunable morphologies, structures, and components are generated and employed for creating different tissue constructs. Multibarrel capillary microfluidics with multiple laminar flows are used for continuously spinning these microfibers. With an immediate gelation reaction of the cell dispersed alginate solutions, the cell-laden alginate microfibers with the tunable morphologies and structures as the designed multiple laminar flows can be generated. The performances of the microfibers in cell culture are improved by incorporating bioactive polymers, such as extracellular matrix (ECM) or methacrylated gelatin (GelMA), into the alginate. It is demonstrated that a series of complex three-dimensional (3D) architectural cellular buildings, including biomimic vessels and scaffolds, can be created using these bioactive microfibers. KEYWORDS: microfiber, tissue engineering, cell-encapsulation, microfluidics, hydrogel

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controlled heterogeneous cell-laden microstructures for mimicking biological features is still anticipated. Here, we present desired microfibers for creating different tissue constructs. These microfibers were continuously generated in multibarrel capillary microfluidics by employing multiple laminar flows. With an immediate gelation reaction of cell dispersed sodium alginate (Na-alginate) solutions, cellladen alginate microfibers with tunable morphologies and structures as the designed multiple laminar flows could be generated, shown in Figure 1a. This is different from current reported methods for constructing anisotropic or heterogeneous tissues in that by stacking monolayer cell sheets, packing spheroids, or assembling cell-laden hydrogel blocks,26,27 our proposed technology could be used to generate complex fibershaped cellular building blocks continuously in a one-step microfluidic spinning process. We have demonstrated that the performances of these microfibers in cell culture could be improved by incorporating bioactive polymers, such as extracellular matrix (ECM) or methacrylated gelatin (GelMA), into the alginate. Moreover, we have shown that a series of tissue constructs, including biomimic vessels and scaffolds (Figure 1b−d), could be created using these bioactive microfibers. We believe that these cell-laden microfibers will have important applications in tissue engineering.

trategies for culturing cells in three-dimensional (3D) environments have received growing attention in the fields of cell biology,1 tissue engineering,2 drug discovery,3 and so on.4,5 Compared with conventional two-dimensional (2D) cultures, the 3D approaches have been shown to induce cellular behavior that mimics natural tissue more closely.6 To engineer the desired 3D cellular processes, numerous biomimetic scaffolds that incorporate different biochemical, mechanical, or architectural cues have been developed for cell cultures.7 Among these scaffolds, microfibers are widely used because of their facile preparation, handling, and assembly. Various protocols have been developed to fabricate microfibers, such as macromolecule self-assembly, electrospinning, melt spinning, and template-assisted microfabrication.8−10 Ever since microfluidic technologies have been employed,11−16 the generation of biofunctional microfibers in particular, has shown great progress.17−20 On the basis of these technologies, a considerable number of cell-laden hydrogel microfibers with symmetrical or isotropic structures have been prepared and assembled for mimicking tubular, multilayer−tubular, or other fiber-shaped tissues.21−25 However, because of a lack of bioactive elements, most of these hydrogels are insufficient to reconstruct a biomimic microenvironment for cell growth. In addition, different from uniform cell distribution in these hydrogel microfibers, cell distribution in vivo is extremely complex, and spatially anisotropic or heterogeneous tissues are widespread, which is of great significance in maintaining the physiological functions of organisms. Thus, fabrication of novel bioactive microfibers with © 2016 American Chemical Society

Received: November 25, 2015 Accepted: January 7, 2016 Published: January 7, 2016 1080

DOI: 10.1021/acsami.5b11445 ACS Appl. Mater. Interfaces 2016, 8, 1080−1086

Letter

ACS Applied Materials & Interfaces

Figure 1. Schematic illustrations of (a) the capillary microfluidic device with a three-barrel injection capillary and three parallel inserted spindlier capillaries for the generation of microfibers with the tunable morphologies and structures, (b) the biomimic vessels by using the generated microfibers, and (c, d) scaffolds by (c) weaving or (d) stacking the microfibers.

Figure 2. (a−d) Digital photographs of a microfluidic device for generating Janus-hollow microfibers under different on−off state, and the correspondingly (e−h) longitudinal-sectional CLSM images, (i−l) cross-sectional CLSM images of microfibers with different hollow structures Scale bar indicates 200 μm.

a tapered multibarrel capillary and several single-barrel capillaries inserted in a given spatial configuration (Figure 1a). Alginate was chosen for the microfibers generation because it can be quickly

In a typical experiment, the microfluidic spinning devices were assembled by aligning scalable injection capillaries coaxially within a collection capillary. The injection capillaries consisted of 1081


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Figure 3. Schematic illustrations and CLSM images of the Janus-hollow microfibers with controllable channels: (a) Janus microfibers with four channels; (b) Janus microfibers with three channel; (c, d) Janus microfibers with two channel; (e) Janus microfibers with one channel. Scale bar indicates 200 μm.

fluorescent-tagged Na-alginate precursor middle phase and the CaCl2 outer phase were pumped through the above-mentioned channels. When three types of fluid were introduced into the device, an immediate gelation of the middle Na-alginate fluid occurred simultaneously at the tip of the injection capillary. The complex three-layered coaxial flow moved through the collection capillary, and the solidified Janus hollow microfiber was continuously generated and extruded, as shown in Figure 2a. It is worth mentioning that the microfibers could form three types of morphology under different flow rates: wavy microfibers, straight microfibers, and spiral microfibers (Figure S2). Among these morphologies, the straight microfibers were the desired product in this research. The diameter of the microfibers was proportional to the Na-alginate flow rate, increasing to about 200 μm as Na-alginate flow rate increased from 0.4 to 4 mL/h. Although the CaCl2 flow rate increased from 4 to 40 mL/h, the diameter decreased to 140 μm on average (Figure S3). In addition, the flow rate ratio and viscosity of two Na-alginate phases could also determine the internal architecture and the volume ratio of each compartment in the microfibers (Figure S4). It was worth noting that the microfibers in our system can be continuously fabricated in any length as long as the solution is enough; their diameters from several micrometers to several millimeters can also be achieved by using different microfluidic devices. Under the optimized conditions, the microfibers had a cylindrical structure and showed a very sharp interface between the two compartments, in which parallel, independent channels

created and shaped in microfluidic devices with an immediate gelation reaction. An active dispersed Na-alginate precursor solution was infused into the injection capillaries, while a CaCl2 solution was pumped along the same direction into the collection capillary. Because of hydrodynamic focusing effect, a 3D coaxial sheath flow stream around the flow of the precursor was formed at the merging point of both flows, and so, for a fast diffusioncontrolled ion cross-linking process, hydrogel microfibers could be generated in situ. If the precursor flow had a low Reynolds number, then a laminar flow would remain in the microfluidic channels and adjacent streams would only mix slowly and diffusively. Therefore, the resulting hydrogel microfibers would have the same heterogeneous structures and active distributions as the injection flows. A notable advantage of our design was that a series of microfibers with different morphologies and structures could be generated by using microfluidic spinning devices with the corresponding injection capillary configurations. To the best of our knowledge, this is the first report of such a design of microfibers using microfluidic spinning approach. To confirm the validity of our method, a microfluidic device with a theta-type injection capillary configuration (Figure 2 and Figure S1) was first employed for spinning microfibers. Two tapered cylindrical capillaries were inserted into the barrel of a theta-type capillary for fabricating a hollow tubular structure in the resulting microfibers, which is a very common form in organisms. The poly(vinyl alcohol) (PVA) inner phase was pumped through two tapered cylindrical capillaries. The different 1082


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Figure 4. Schematic illustrations and the cross-sectional CLSM images of the three-compartment-hollow microfibers, with (a) three hollow channels; (b) two hollow channels; (c) one hollow channel; (d) no hollow channel. Inserts are the schematics of their correspondingly crosssectional microfluidic injection channels. Scale bar indicates 200 μm.

capillary for pumping the inner fluid (Figure S6), Janusstructured microfibers with multiple hollow core channels in each compartment could be spun out from the microfluidic device continually (Figure 3). In addition, by replacing the theta-type injection capillary with a three-barrel injection capillary (Figure S7), three compartment hollow alginate microfibers could also be fabricated, as shown in Figure 4. On the basis of the same “on−off” tuning approach of the inner PVA phase, a series of index microfibers with different structures could be generated in the same spinning device. It is worth mentioning that more complex microfibers with the features of multiple channels and multiple compartments could be created by using multibarrel capillaries to construct much more complex laminar flow microfluidic devices. To confirm the value of these anisotropic or heterogeneous microfibers in biomedical engineering, we employed them as controllable cell encapsulations and in coculture research, and showed their significance in constructing biomimic hepatic lobules and blood vessels. For the hepatic lobules mimicking, HepG2 cells and NIH 3T3 cells were dispersed in a Na-alginate solution and the mixture was pumped into several microfluidic devices to fabricate cell-laden microfibers with different structures and cell distributions, as shown in Figure 5a−d. It can be seen that there was a distinct interface between the HepG2 and NIH 3T3 cells in both the four-compartmental

separated by a thin wall were observed (Figure 2e and Figure S1c). When the inner phase fluid was turned off, the ambient Na-alginate fluid occupied the previous inner phase space and immediately formed a filling structure. If the inner phase fluid was reopened, it would turn into a nonreactive stream inside Na-alginate fluid and transformed into a hollow tubular structure. Thus, four types of Janus microfibers with different structures could be generated in the same spinning device by simply turning the inner PVA phase on and off, as can be seen in Figure 2e−h. To discern the internal architecture of these microfibers,we took cross-sectional slices and observed then using confocal laser scanning microscopy (CLSM), as shown in Figure 2i−l and Figure S5. It was found that the internal architecture of the microfibers also showed a clear boundary at the compartmental interface, in which the hollow microstructures were also confirmed to correspond to their injection flows. As there was minimal mass transfer between the different compartments, each compartment of the microfibers could have its own chemical composition, which was determined by the chemical composition of the initial injecting solution. A noticeable advantage of our microfluidic spinning method was that both the number of hollow core channels and compartments in the anisotropic or heterogeneous microfibers could be expanded. By inserting more spindle capillaries orientated parallel to each barrel of the theta-type injection 1083


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Figure 5. (a−d) Fluorescent images and optical images of two types of cell-laden microfibers with different structure and cell distribution: (a, b) four-compartmental core−shell microfibers and cells encapsulated in each compartment; (c, d) two-compartmental microfibers encapsulated with different cells in each side; the NIH 3T3 cells were stained with a green fluorescent dye, and HepG2 cells were stained with a red fluorescent dye. Scale bar indicates 200 μm. (e) Albumin secretion and (f) urea synthesis of the cells in the microfibers was measured at different time points during a 10 day culture. All date were measured at the same initial cell number and were expressed as mean ± s.d. (n = 4).

microfibers was better than in other microfibers. This was ascribed to the ECM microenvironment and complex microstructure of the cells in the microfibers, which provided biomimic 3D cellular interactions and a larger contact surface for the cells. The exhibition of the microfibers in constructing vessels was also investigated. To this point, GelMA hydrogel microfibers were successfully generated by using GelMA as bioactive additives in alginate. Primary normal human umbilical vein endothelial cells (HUVECs) were encapsulated in the cores of the microfibers during the generation. Althouth UV light which was used to polymerize GelMA was harmful for encapsulated cells, the cell viability was higher than 85% in our method. After 5 days, a tubular structure was formed with a monolayer of HUVECs along the direction of the microfiber (Figure 6a, i and ii). From the CLSM image of the cross-section microfiber, it can be seen that the HUVECs densely spread on the inner surface of the hollow microfiber and formed a monolayer cell tube (Figure 6a, iii and iv). In addition to the microfibers with single-channel cell tube, microfibers with two- and three-channel HUVECs tubes can also be successfully fabricated by adopting the above multicomponent microfibers (Figure 6b). These microfibers showed physical structures similar to blood vessels in the human body. Thus, they may have potential application in the replication of the complex fiber tissues. As the microfibers were thin and flexible, they have great potential in creating functional 3D tissue constructs by folding, bundling, reeling, and weaving.1,22,28 To demonstrate these concepts, we have fabricated a complex 3D gridding architecture by crisscross weaving multicomponent microfibers

core−shell microfibers and the two-compartmental microfibers. The encapsulated cells gradually proliferated into cellular spheroids in the microfibers (Figure S8). However, although with good biocompatibility, alginate hydrogels are not components of natural ECMs and thus are insufficient to reconstruct a microenvironment that are typical of living tissues for cell−cell connections and 3D cellular interactions. To address this problem, we dispersed bioactive ECM into alginate solution for cell encapsulations. To investigate the effects of ECM additives, cell distributions, and coculture on the biological function of the biomimic hepatic lobules, we used a Rat Albumin ELISA Quantitation kit and a Urea Nitrogen kit to quantify albumin secretion and urea synthesis, because these are two primary bioactive indices of hepatic cells. The results are shown in Figure 5e, 5f and Figure S9. It was found that during 10 days of culture, the albumin secreted by HepG2 cells were increased from 65.5 ± 2.3% to 114.3 ± 5.1% ng/mL in Janus microfibers, from 64.1 ± 3.7% to 128.2 ± 5.3% ng/mL in core−shell microfibers, and from 79.6 ± 2.8% to 145.2 ± 6.1% ng/mL in core−shell ECM microfibers, respectively. The urea synthesized by HepG2 cells were also increased from 27.1 ± 0.83 to 35.4 ± 0.75% mg/dL in Janus microfibers, from 26.2 ± 0.91% to 37.5 ± 0.68% mg/ dL in core−shell microfibers, and from 29.5 ± 0.67% to 43.6 ± 1.1% mg/dL in core−shell ECM microfibers, respectively. The reason for this may be from the formation of cellular spheroids, which are believed to be capable of reflecting the in vivo physiology of tumors more realistically than two-dimensional cell cultures. Importantly, it was found that the maintenance of HepG2 function in ECM microfibers was better than in pure alginate microfibers, and in the four-compartmental core−shell 1084


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Figure 6. (a) Biomimic vessel by culturing HUVECs in the hollow core of a GelMA microfiber in (i) optical image, (ii) fluorescent image, and (iii, iv) CLSM images in different viewpoints. (b) CLSM images of the biomimic vessels with different numbers of cell tubes in different viewpoints: (i, ii) is the microfiber with two cell tube; (iii, iv) is the microfibers with three cell tubes. (c) Gridding architecture by crisscross weaving multicomponent microfibers. (d) Layer-by-layer architecture by stacking hollow microfibers. All microstructures in c and d were fabricated by manually weaving and stacking microfibers under microscope. All scale bars indicate 200 μm.

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(Figure 6c). Also, we have created a layer-by-layer 3D microstructure by stacking hollow microfibers (Figure 6d). It was found that the multicomponent or hollow microstructures of the microfibers were well preserved in their 3D architectures. This indicated that the cell-laden fabric-like spatially patterned structures could also be constructed by the methods, and the resulting 3D cell architectures would be useful in biomimicking microfiber-like network tissues for biological research. In this work, we have presented a microfluidic method for continuously fabricating bioactive microfibers for creating different tissue constructs. By employing a multiple laminar flow and an immediate alginate gelation reaction, our method could create a series of anisotropic or heterogeneous cell-laden hydrogel microfibers with tunable morphological and structural features from designed multiple injection flows. The performances of these microfibers in cell culture have been improved by incorporating bioactive polymers, including ECM protein and GelMA hydrogel, into the alginate. We have demonstrated that a series of complex three-dimensional (3D) architectural cellular buildings, such as biomimic vessels and scaffolds, could be created using these bioactive microfibers. We anticipate that the these cell-laden microfibers could be used as biological models in organisms, and the proposed microfluidic spinning method will lead to a host of novel applications in tissue engineering.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.5b11445. Schematic illustration and digital photograph of the capillary microfluidic devices; relationships between the microfiber diameters and flow rates; optical images and fluorescent images of cell-laden microfibers (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Science Foundation of China (Grants 21473029, 91227124, and 51522302), the NASF Foundation of China (Grant U1530260), the National Science Foundation of Jiangsu (Grant BK20140028), the research Fund for the Doctoral Program of Higher Education of China (20120092130006), the Program for Changjiang Scholars and 1085


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(21) Cheng, Y.; Zheng, F. Y.; Lu, J.; Shang, L. R.; Xie, Z. Y.; Zhao, Y. J.; Chen, Y. P.; Gu, Z. Z. Bioinspired Multicompartmental Microfibers from Microfluidics. Adv. Mater. 2014, 26, 5184−5190. (22) Onoe, H.; Okitsu, T.; Itou, A.; Kato-Negishi, M.; Gojo, R.; Kiriya, D.; Sato, K.; Miura, S.; Iwanaga, S.; Kuribayashi-Shigetomi, K.; Matsunaga, Y. T.; Shimoyama, Y.; Takeuchi, S. Metre-long Cell-laden Microfibres Exhibit Tissue Morphologies and Functions. Nat. Mater. 2013, 12, 584−590. (23) Kang, E.; Jeong, G. S.; Choi, Y. Y.; Lee, K. H.; Khademhosseini, A.; Lee, S. H. Digitally Tunable Physicochemical Coding of Material Composition and Topography in Continuous Microfibres. Nat. Mater. 2011, 10, 877−883. (24) Yu, Y.; Wen, H.; Ma, J.; Lykkemark, S.; Xu, H.; Qin, J. H. Flexible Fabrication of Biomimetic Bamboo- Like Hybrid Microfibers. Adv. Mater. 2014, 26, 2494−2499. (25) Matsunaga, Y. T.; Morimoto, Y.; Takeuchi, S. Molding Cell Beads for Rapid Construction of Macroscopic 3D Tissue Architecture. Adv. Mater. 2011, 23, H90−H94. (26) Matsuda, N.; Shimizu, T.; Yamato, M.; Okano, T. Tissue Engineering Based on Cell Sheet Technology. Adv. Mater. 2007, 19, 3089−3099. (27) Du, Y. N.; Lo, E.; Ali, S.; Khademhosseini, A. Directed Assembly of Cell-laden Microgels for Fabrication of 3D Tissue Constructs. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 9522−9527. (28) Hosseini, V.; Kollmannsberger, P.; Ahadian, S.; Ostrovidov, S.; Kaji, H.; Vogel, V.; Khademhosseini, A. Fiber-Assisted Molding (FAM) of Surfaces with Tunable Curvature to Guide Cell Alignment and Complex Tissue Architecture. Small 2014, 10, 4851−4857.

Innovative Research Team in University (IRT1222), and the Program for New Century Excellent Talents in University.

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Controlled Fabrication of Bioactive Microfibers for Creating Tissue

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