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Bundle Gel Fibers with Tunable Microenvironment for In Vitro Neuron Cell Guiding Sayaka Tachizawa, Haruko Takahashi, Young-Jin Kim, Aoi Odawara, Joris Pauty, Yoshiho Ikeuchi, Ikuro Suzuki, Akihiko Kikuchi, and Yukiko T. Matsunaga ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b14585 • Publication Date (Web): 31 Oct 2017 Downloaded from http://pubs.acs.org on November 1, 2017

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ACS Applied Materials & Interfaces

Bundle Gel Fibers with Tunable Microenvironment for In Vitro Neuron Cell Guiding Sayaka Tachizawa,†, ‡,¶ Haruko Takahashi,†, ¶ Young-Jin Kim,† Aoi Odawara,§,∥ Joris Pauty,†,# Yoshiho Ikeuchi,† Ikuro Suzuki,§ Akihiko Kikuchi,‡ Yukiko T. Matsunaga†,#,* †

Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo

153-8505, Japan. ‡

Department of Materials Science and Technology, Graduate School of Industrial Science and

Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo 125-8585, Japan. §

Department of Electronics, Graduate School of Engineering, Tohoku Institute of Technology,

35-1 Yagiyama, Kasumicho, Taihaku-ku, Sendai, Miyagi 982-8577, Japan. ∥

Research Institute of Electrical Communication, Tohoku University, 2-1-1 Katahira, Aoba-

ku, Sendai, Miyagi 980-8577, Japan. #

LIMMS/CNRS-IIS UMI 2820; SMMiL-E project, Institute of Industrial Science, The

University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan. ¶

These authors contributed equally.

* To whom correspondence should be addressed: Yukiko T. Matsunaga E-mail: [email protected] Tel: +81-3-5452-6470, Fax: +81-3-5452-6471

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ABSTRACT

As scaffolds for neuron cell guiding in vitro, gel fibers with a bundle structure, comprising multiple microfibrils, were fabricated using a microfluidic device system by casting a phaseseparating polymer blend solution comprising hydroxypropyl cellulose (HPC) and sodium alginate (Na-Alg). The topology and stiffness of the obtained bundle gel fibers depended on the microstructure of them derived by the polymer blend ratio of HPC and Na-Alg. High concentrations of Na-Alg led to the formation of small microfibrils in a one-bundle gel fiber and stiff characteristics. These bundle gel fibers permitted for the elongation of the neuron cells along their axon orientation with the long axis of fibers. In addition, human-induced pluripotent-stem-cell-derived dopaminergic neuron progenitor cells were differentiated into neuronal cells on the bundle gels. The bundle gel fibers demonstrated an enormous potential as cell culture scaffold materials with an optimal microenvironment for guiding neuron cells.

KEYWORDS: bundle structure, phase separation, hydroxypropyl cellulose, microfluidics, neuron guiding.


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1. INTRODUCTION Neural tissues play roles in signal transduction, thereby controlling sense and motion throughout the human body. This neural control system is crucial for vital activities, and its dysfunction causes severe diseases, including motor paralysis, sensory paralysis, and respiratory failure.1-5 Meanwhile, the reconstruction of deficient neural tissues by guiding neuron cells on an appropriate scaffold satisfies the demands of recovery of neural networks and functions. Thus far, several materials have been developed for the insertion of a defect of a neural tissue in the body and for the development of a bridge between terminal parts, leading to nerve regeneration6-10. Approved implant materials available in the market mainly include biocompatible polymers such as polyglycolic acid with a hollow tubular structure.11, 12

Moreover, currently, an increasing number of studies have reported alternative functional

materials with fiber13, 14, sheet15, 16, and tubular structures17, 18. Nevertheless, because these approaches induce the regeneration of nerve tissues in the body, it typically takes several months for joining the nerve defects, and the recovery rate of signal transduction varies depending on the disease case. Recently, another new approach involving the building of physiologically functional nerve tissue units in vitro via tissue engineering has been reported for the implantation and direct bridging between defects.19 This physiological nerve tissue unit built in in vitro exhibits considerable merits because it serves as a “living” line, permitting the connection of the missing parts in the body. Furthermore, the matured nerve tissue unit may be usable as a connection wire between a brain–machine interface20,

21

which have been getting huge

attention as a next-gen motion support system in the future. Nerve tissues have a fiber structure comprising a bundle of small nerve fibers. The creation of fiber-structured scaffolds, such as silk and microtextured gel fibers, exhibits a particular advantage for guiding nerve cells.22-24 However, it is challenging to produce materials for scaffolds that mimic the bundled 3 ACS Paragon Plus Environment


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structure in nerve tissues. In addition, optimal stiffness, which is highly correlated to the original microenvironment, and biocompatibility are crucial elements for the effective guiding of nerve cells.25, 26 In this study, new hydrogel materials, referred to as bundle gels, were developed as cell culture scaffold materials for in vitro neuron cells guiding (Figure 1). Previously, our group has reported the formation of a bundle structure of gels via the integration of phase separation in a polymer blend solution, comprising a mixture of hydroxypropyl cellulose (HPC) and sodium alginate (Na-Alg), and a microfluidic device system.27 The obtained gel fibers consisted of multiple microfibrils, comprising biocompatible cellulose derivatives, and these gel fibers were capable of functionalization via the addition of a third material, e.g., carbon nanotubes (CNTs).28 Accordingly, we envision the bundle gel fiber is adequate as a neuron cell guiding scaffold to build the nerve tissue unit in vitro. However, thus far, knowledge about the parameters required for controlling the topology and stiffness of the bundle gel fibers is not investigated in previous studies, although these parameters essentially determine cellular adherence, growth, and organization25,

26, 29, 30

. To develop suitable

scaffolds for guiding nerve cells, the effects of the HPC/Na-Alg blend ratio and polymer blend solution pH on the phase-separating pattern and characteristics of the fabricated bundle gels (i.e., fibril diameter and stiffness) were investigated. Subsequently, the application of the bundle gels with different topologies and stiffness as neuron cell culture scaffolds and the behavior of neuron cells on the prepared gels were investigated. The functionality of the bundle gels for neuron axonal guidance and the differentiation of human-induced pluripotent stem cells (iPSCs) into neurons were estimated.


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Figure 1. Concept of generating bundle gel fibers for neuron cell guiding. (A) Schematic of a dynamic microfluidic gelation system for the fabrication of bundle gels and a representative phase-contrast image of a microfluidic channel. The phase-separated solution (inner flow) and its cross-linking agent CaCl2 solution (outer flow) were injected into a coflow microfluidic device to produce bundle gel fibers. (B and C) Chemical structures of hydroxypropyl cellulose (HPC) (B) and sodium alginate (Na-Alg) (C). (D) Proposed cell culture system to investigate the effect of the gel structure and diameter of fibril in gels with various stiffness values.

2. MATERIALS AND METHODS 2-1. Materials



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Na-Alg (Grade I–1G, M/G ratio: approximately 0.6−0.8) was provided by Kimica Corporation (Tokyo, Japan). HPC (average MW: 100 kDa), bovine serum albumin (BSA) and FITC-labeled poly-L-lysine (MW: 15—30 kDa) were purchased from Sigma Aldrich (MO, USA). Divinyl sulfone (DVS) was purchased from Tokyo Chemical Industry (TCI, Tokyo, Japan). Calcium chloride and sodium hydroxide were purchased from Kanto Chemicals (Tokyo, Japan). Citric acid was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Polydimethylsiloxane (PDMS, SILPOT 184) and its catalyst SILPOT were purchased from Dow Corning Toray (Tokyo, Japan). Poly-L-lysine (20 kDa) grafted with poly(ethylene glycol) (PEG) and RGD (-Gly-Tyr-Gly-Arg-Gly-Asp-Ser-Pro-Gly-NH2) functionalized PEG (PLL-g-PEG-RGD) was purchased from SuSoS (Dübendorf, Switzerland). Nerve growth factor (NGF) was purchased from Cosmo Bio (Tokyo, Japan). Laminin-511 solution (iMatrix-511) was purchased from Nippi (Tokyo, Japan). Rat pheochromocytoma PC-12 cells were purchased from RIKEN BRC (Ibaraki, Japan). D-MEM (high glucose), 100× penicillin– streptomycin solution (PS), and a 4% paraformaldehyde solution were purchased from Wako Pure Chemical Industries (Osaka, Japan) in liquid form. Fetal bovine serum (FBS) was purchased from Japan Bio Serum (Hiroshima, Japan). Horse serum (HS) was purchased from Gibco (CA, USA). Dorsal root ganglion (DRG) neuron was obtained from 10-week-old male Wistar rats by dissection according to a previous study31. MACS Neuro Medium and MACS Neuro Brew-21 were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). iPSCderived dopaminergic neuron progenitor, neural expansion-XF medium, and neural maintenance-XF medium were purchased from Axol Bioscience (Cambridge, UK). Mouse anti-β-tubulin III antibody was purchased from BioLegend (CA, USA) and Sigma Aldrich (St. Louis, MO, USA). Alexa Fluor 488-conjugated goat anti-mouse IgG, Alexa Fluor 488conjugated donkey anti-rabbit IgG, Alexa Fluor 546-conjugated goat anti-mouse IgG, and Hoechst 33342 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Anti-


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human nestin (N1602) rabbit IgG affinity purify was purchased from Immuno-Biological Laboratories (Gunma, Japan). Hoechst 33258 was purchased from Dojindo (Kumamoto, Japan). Pure water from a Milli-Q system (Merck Millipore, Darmstadt, Germany) was used for all experiments.

2-2. Preparation of polymer blend solutions Gel fibers were fabricated using a polymer blend solution consisting of HPC and NaAlg at a pH of 7 or 13. HPC (5.0 wt%) and 1.0, 1.5, or 2.0 wt% of Na-Alg were dissolved in Milli-Q pure water and stirred for 17 h at 25°C (Note: The polymer blend solution consisting of X wt% of HPC and Y wt% of Na-Alg was referred to as HXAY. For example, 5.0 wt% HPC and 1.0 wt% of the Na-Alg mixture solution was referred to as H5A1.0). NaOH aq. (5.0 mol/L) was then added to the H5AY solution for adjusting the pH to 13 (referred to as H5AYpH13) and for salting out. To prepare pH 7 solutions (H5AY-pH7), Milli-Q pure water was added. The polymer blend solutions were filtered using a membrane filter (pore size: 0.2 µm, Sartorius, Göttingen, Germany) and maintained at 4°C.

2-3. Fabrication of gel fibers using a dynamic microfluidic gelation system The co-flow microfluidic device was fabricated by a method reported previously by Kim et al.27, 28 The polymer blend solution and 100 mmol/L CaCl2 aq. were injected into the inner channel and outer channel of the device, respectively (inner flow rate: 300 µL/min and outer flow rate: 2500 µL/min), to fabricate gel fibers. The gels were dipped in 2 wt% DVS aq. for 24 h for the cross-linking of hydroxyl groups in the HPC and alginate molecules and subsequently in citric acid aq. for 3 h to remove residual alginate. Then the final gel fibers were obtained by washout with pure water. The obtained bundle gel fibers were immersed in pure water and split into disassembled fibrils using tweezers. The disassembled fibrils were



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observed by phase-contrast microscopy (Axio Observer D1, Carl Zeiss, Oberkochen, Germany) to determine the diameter using AxioVision application (Rel. 4. 8. 2., Carl Zeiss).

2-4. Observation of gel fibers by scanning electron microscopy (SEM) The gel fibers were frozen in a freezer at −30°C and then lyophilized (FD-1000, Tokyo Rikakikai Co., Ltd., Saitama, Japan). The surfaces of the freeze-dried fibers were coated using a magnetron sputter coater (MSP-1S, Vacuum Device Inc., Ibaraki, Japan), and samples were observed by scanning electron microscopy (SEM) (acceleration voltage: 2.0 kV, SU8000, Hitachi High-Technologies Corp., Tokyo, Japan).

2-5. Tensile test of gel fibers Tensile tests were carried out to investigate the mechanical properties of gel fibers. First, the gel fiber was cut into a length of approximately 40 mm and attached to a tensile testing machine (load: 0–100 kPa, gripping distance: 20 mm, and tension speed: 10 mm/min, EZ-SX, Shimadzu Corp., Kyoto, Japan), and stress–strain curves were recorded. Young’s moduli of the gels were calculated from the stress–strain curve. Stress σ [Pa], strain ε [mm/mm], and Young’s moduli E [Pa] are expressed in equations (1)–(3), respectively: ி

ߪ=஺ ߝ=

∆ఌబ ఌబ

߃=

ఙ ఌ

–(1) –(2) –(3)

Here, F is the force exerted on an object under tension, A is the cross-sectional area of the fiber, ε0 is the original length of the fiber, and ∆ε is the amount by which the length of the object changes.

2-6. Transmittance measurement of the polymer blend solution 8 ACS Paragon Plus Environment

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The turbidity of the prepared polymer blend solutions in the quartz cell was measured using a UV–visible spectrometer (V-630 BIO UV–vis spectrophotometer, JASCO Co., Tokyo, Japan). The solution in the quartz cell was maintained at 4°C for 10 min to achieve stabilization before measurement. After incubation for 10 min, the transmittance of the solution between 4°C and 50°C was recorded (a temperature elevation rate of 0.5°C/min and a wavelength of 500 nm).

2-7. Observation of the phase-separating pattern of polymer blend solutions To observe the phase-separated pattern, two cover slips (size: 22 mm × 24 mm and thickness: 0.12–0.17 mm, Matsunami Glass Co. Ltd., Osaka, Japan) were used for observation. The polymer blend solution was placed between cover slips so that the thickness between the cover slips was 300 µm. The cell was placed on a thermo plate for microscopic observation (Tokai Hit, Shizuoka, Japan) at 28°C for 20 min to stabilize the phase-separating pattern and to heat up the plate to visualize the HPC- and solvent-rich phases. The asobtained patterns were recorded using a digital camera. The area of the HPC-rich phase was analyzed using Image J software (version 1.50i, downloaded from NIH, USA).

2-8. Cell culture on the gels

Pretreatment of gels. The gel fibers were coated with PLL-g-PEG-RGD to enhance the adhesion of cells on the gels. The freeze-dried fibers were then dipped into 1 mg/mL of PLL-

g-PEG-RGD solution in phosphate-buffered saline (PBS, pH 7.2) for 1 h. After washing the gel fibers with PBS, the fibers were placed on a glass-bottom dish (φ = 14 mm, Matsunami Glass Co. Ltd., Osaka, Japan) and dried and sterilized under UV for 1 h. The fibers were kept overnight at 4°C. To estimate coating by PLL derivatives, FITC-coated PLL with similar molecular weight (approximate MW: 20 kDa) to PLL-g-PEG-RGD were used and coated



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fibers were observed by fluorescence microscope (Axio Observer Z1, Carl Zeiss, Oberkochen, Germany). For culturing DRGs and iPSCs, the gel fibers were also immersed in laminin-511 (2.5 µg/mL) in PBS for 1 h at 37°C with 5% CO2 and used in cell culture experiments.

PC-12 cells. PC-12 cells (3.0 × 105 cells) in high-glucose D-MEM supplemented with 10% of FBS, 10% of HS, and 1% of PS were seeded on the fibers placed on a glass-bottom dish and incubated overnight at 37°C with 5% CO2. After the adhesion of cells on the gel fiber, the medium was changed to the differentiation medium (high-glucose D-MEM supplemented with 0.5% of FBS, 1% of HS, and 1% of PS) containing 100 ng/mL of NGF, and PC-12 cells were incubated for 8 days at 37°C with 5% CO2.

DRGs. DRG neuron cells were harvested from 10-week-old male Wistar rats and cultured according to a protocol described by Malin et al.31 Animal experiments were carried out in accordance with the “Guidelines of Tohoku Institute of Technology on Animal Use” and the “Guidelines of the University of Tokyo on Animal Use.” DRGs (200 µL, 1.0 × 104 cells/mL) in the media consisting of the MACS Neuro Medium and MACS Neuro Brew-21 added with 50 µg/mL of NGF, 8.1 µg/mL of L-glutamine, 10% FBS, and 1% PS were seeded on the fibers placed in a microtube, followed by incubation for 30 min at 37°C with 5% CO2. The gel fibers were then placed in the glass-bottom dish to maintain the culture at 37°C with 5% CO2.

Human iPSCs. iPSCs (200 µL, 1.0 × 106 cells/mL) in the neural expansion-XF medium with 20 ng/mL of fibroblast growth factor-2 (FGF2) and epidermal growth factor (EGF) were seeded on the fibers placed in a microtube, followed by incubation for 1 h at 37°C with 5% CO2. The gel fibers were then placed in the glass-bottom dish to maintain the culture at 37°C with 5% CO2. After 1-day culture, half of the volume of the medium was changed using neural maintenance-XF medium. The medium was changed every 4 days.


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The cells cultured on the gel fibers were observed by phase-contrast microscopy (Axio Observer D1, Carl Zeiss, Oberkochen, Germany).

2-9. Immunohistochemistry Cells used for immunohistochemical analysis were fixed with 4% paraformaldehyde in PBS for 10 min, permeabilized with 0.5% Triton X-100 in PBS for 10 min, and blocked overnight with 5% BSA in PBS at 25°C. Immunostaining for each sample was performed as described below.

PC-12 cells. Cells were treated overnight with mouse anti-β-tubulin III antibody (1:2000; initial concentration, 1.0 mg/mL, BioLegend) at 4°C. After rinsing with PBS, the cells were exposed to Alexa Fluor 488-conjugated goat anti-mouse IgG (1:500; initial concentration, 5 mg/mL) at 25°C for 2 h, followed by Hoechst 33342 (1:1000; initial concentration, 10 mg/mL) at 25°C for 10 min.

DRG cells and iPSCs. Cells were treated overnight with anti-human nestin (N1602) rabbit IgG affinity purify (IBL, 18741; 1:1000; initial concentration, 0.1 mg/mL) and monoclonal anti-β-tubulin III antibody produced in mouse (1:1000; initial concentration, 1 mg/mL, Sigma) at 4°C. After rinsing the cells with PBS, the cells were exposed to Alexa Fluor 488conjugated donkey anti-rabbit IgG (1:1000; initial concentration, 2 mg/mL), Alexa Flour 546conjugated goat anti-mouse IgG (1:1000; initial concentration, 2 mg/mL), and Hoechst 33258 (1:1000; initial concentration, 1 mg/mL) at 25°C for 1 h. Finally, the immunostained samples were washed three times with PBS and observed by laser scanning confocal microscopy (LSM 700, Carl Zeiss) with objective lenses of 20× for PC-12 and 40× for DRGs and iPSCs. The axon length of PC-12 cells (10 cells) was recorded using ZEN software version 8.1 (Carl Zeiss) and shown as average length ± S.D.



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3. RESULTS AND DISCUSSION 3-1. Fabrication of bundle gel fibers To fabricate the bundle gel fibers, the co-flow microfluidic device enabling dynamic gelation was utilized (Figure 1A). The phase-separating polymer blend solution, comprising HPC (Figure 1B) and Na-Alg (Figure 1C), with a bicontinuous structure, was introduced into the microchannel, allowing a multiple aligned fibril structure by the generated shear stress in laminar flow. To facilitate phase-separated pattern, the system and blend solution were maintained at 28°C, and the obtained structures were preserved through the use of immediate cross-linking of Na-Alg by the CaCl2 solution from the outer channel, allowing long bundle gel fibers (Figure 1D). These structures were made permanent by first soaking the gels in DVS solution to covalently couple DVS sulfone to hydroxyl groups in the HPC and alginate molecules. Then, the unreacted alginate molecules were solubilized and removed by soaking in citric acid. Finally, the bundle gel fibers were obtained by washout with pure water. Six polymer blend solutions with three Na-Alg concentrations (5.0 wt% of HPC and 1.0, 1.5, and 2.0 wt% of Na-Alg) at pH 7 or 13 were introduced into the dynamic gelation system, affording gel fibers.

3-2. Characterization of the bundle gel fibers In a HPC/Na-Alg blend solution, the phase separation depends on the pH of blend solutions; briefly, phase separation was observed only at high pH because of the salting-out effect32, 33. Figure 2A shows the as-obtained gels fabricated from the polymer blend with a homogenous solution at pH 7 or a phase-separating solution at pH 13 by the variation in the Na-Alg concentration. The gels fabricated by the H5AY-pH7 solutions were transparent and their surfaces were very smooth. On the other hand, the H5AY-pH13 gels showed 12 ACS Paragon Plus Environment

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microstructure by packing of multiple small fibrils under the phase-contrast microscope observation, producing bundle structure. These assemble of small fibrils in a gel fiber were more clearly observed using freeze-dried gel fiber owing to increasing phase contrast (Figure S1 in the Supporting Information (SI)). The bundle gel structure permitted the splitting of the bundle gel fibers by tweezers (Figure 2B and Movie S1 in the SI). The gels were easily split into multiple gels; however, it was difficult to obtain single microfibrils because each of the microfibrils was connected at some point within the gel, generating a net structure. This phenomenon was correlated to the morphology of the bicontinuous phase-separating structure of the polymer blend solution. The SEM observation was also carried out to obtain the surface and cross-sectional images of the gel fibers (Figure 2A, bottom). The H5AY-pH7 gels showed smooth surfaces in the side views and single collapsed and shrunken fibers in the cross-sectional views. The shrinkage of fibers was due to drying and dehydration process in the sample preparation for SEM. In contrast, the H5AY-pH13 gels showed bumpy surface caused by small fibrils in the side views and multiple collapsed fibers, which are corresponding to small fibrils, in the cross-sectional views. The SEM cross-sectional images revealed that opening pores caused by space between microfibrils existing within the bundle gels increases with the increasing concentration of Na-Alg in the blend ratio, suggesting that the number of microfibrils in one gel fiber increased. Also, the diameter of fibrils in a bundle gel fibers in water decreased with the increase in the concentration of Na-Alg in the polymer blend solution; H5A1.0-pH13, H5A1.5-pH13, and H5A2.0-pH13 exhibited diameters of 11.4 ± 0.6, 6.9 ± 0.5, and 3.2 ± 0.4 µm, respectively (Figure 2C). Tensile tests indicated that bundle gels are stiffer than non-bundle gels at all of the HPC/Na-Alg ratios (Figure 2D). For example, from the comparison among the H5A1.0 gels, the Young’s modulus of H5A1.0-pH7 was 46.4 ± 11.0 kPa, whereas that of H5A1.0-pH13



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was 121 ± 16.3 kPa. More importantly, gel bundles prepared from high Na-Alg concentration exhibited increased stiffness. The Young’s moduli of the H5A1.0-pH13, H5A1.5-pH13, and H5A2.0-pH13 bundle gels were 121 ± 16.3, 140 ± 20.0, and 188 ± 19.9 kPa, respectively. The results revealed that the diameter of the microfibril in bundle gels is successfully controlled by the variation in the Na-Alg concentration: High Na-Alg concentration led to the formation of small microfibrils and stiff characteristics.

Figure 2. Fabrication of gel fibers using polymer blend solutions at natural or alkaline pH. (A) Microscopic views of non-bundle (H5AY-pH7) and bundle gel (H5AY-pH13) fibers observed by phase-contrast microscopy and scanning electron microscopy (SEM). Bars: 100 µm in the phase-contrast mode and 5 and 50 µm in side and cross-sectional views by SEM, respectively. (B) Images showing the splitting process of the bundle gel (H5A1.0-pH13). The


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bundle gel was (i) immersed in water, (ii) pinched by a pair of tweezers, and (iii) split along the long axis using tweezers. Bars: 500 µm. (C) Diameter of fibrils in the H5AY-pH13 bundle gel (n=5, ave. ± S.D.). (D) The Young’s moduli of non-bundle (H5AX-pH7, white) and bundle (H5AY-pH13, gray) gels (n=5, ave. ± S.D.).

3-3. Characterization of phase-separated solutions To gain in-depth knowledge about the parameters of the polymer blend solution that affect the formation and characteristics of the gel, the physicochemical properties of the polymer blend solution were investigated at different Na-Alg concentrations, pH, and temperature. UV–Vis spectroscopy was employed to characterize the polymer blend solutions with increasing temperature from 4°C to 50°C. The polymer blend solution exhibited discontinuous transmittance changes within a distinctly narrow temperature range (Figure 3A). As has been described previously,27 H5AY-pH7 solutions exhibited a single decreased transmittance point around 37°C, whereas H5AY-pH13 solutions exhibited a two-step decreased transmittance point at ~20°C–25°C and ~37°C, respectively, which can be explained as follows: the first transmittance change in the phase-separated blend solutions corresponded to the initial phase separation (=Tphase separation) to polymer- and solvent-rich domains because of the decreased solubility of the polymers that accelerate phase separation. The second transmittance change corresponded to a coil–globule transition (=Tcoil-globule) of HPC in polymer-rich domains, which is a characteristic of temperature-responsive HPCs34. The Tphase

separation

values for H5A1.0-pH13, H51.5-pH13, and H52.0-pH13 were 25.1°C,

23.2°C, and 21.1°C, respectively. With the increase in the Na-Alg concentration, Tphase separation was shifted to low temperature because the dissociated salt ions from Na-Alg decreased the water solubility of the HPC polymer chains via the salting-out effect32,

33

, consequently



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shifting the phase-separation point to a low temperature. This salting-out effect also affected the variation of the phase-separated form of the polymer blend solutions. The polymer blends of H5AY-pH13 exhibited three domains depending on temperature, i.e., phase I (single phase), phase II (liquid–liquid phase separation), and phase III (liquid–solid phase separation), respectively (Figure 3A). For instance, the blend solution of H5AX1.0-pH13 exhibited the three phases at 4°C (phase I), 28°C (phase II), and 45°C (phase III; Figure 3B, upper). The fabricated gels in phase I were homogenous and transparent, whereas those prepared in phase II exhibited a fibrous or bundle structure (Figure 3B, bottom). The gels fabricated in phase III exhibited HPC aggregates. From these results, the essential condition for the H5AY-pH13 polymer blend solutions, which enable the fabrication of bundle gels, includes the temperature in the range of phase II that represents liquid–liquid phase separation. To understand the manner in which the concentration of Na-Alg in the polymer blend affects the bicontinuous structures of phase-separating polymer blend solutions, the phaseseparating solution was observed at 28°C. The H5AY-pH13 solutions were dropped on culture dishes and observed under the microscope. With increasing Na-Alg concentration, the phase-separated pattern became obvious (Figure 3C, upper). To clearly observe these differences, the phase-separating pattern of the polymer blend solutions was observed by the introduction of the solution within 300-µm-thick spaced glass cells (Figure 3C, bottom). The HPC- and solvent-rich phases were distinguished by rapid heating to the transition temperature of HPC (>37.7°C) because the HPC-rich phase, not the solvent-rich phase, became cloudy because of HPC aggregation. The percentages of the area corresponding to the HPC-rich phase in H5A1.0-pH13, H5A1.5-pH13, and H5A2.0-pH13 were 82.8% ± 1.5%, 51.8% ± 3.6%, and 32.0% ± 0.7%, respectively. With increasing Na-Alg concentration, the percentage of the HPC-rich phase area decreased. From the results observed in Figures 3A


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and 3C, with increasing Na-Alg concentration, Tphase separation was shifted to low temperature, and the area ratio of the HPC-rich phase decreased. With the addition of Na-Alg, the HPCrich phase was speculated to exhibit more aggregation because Na-Alg exhibited a higher hydration force compared to HPC; therefore, water molecules in the HPC-rich phase move to the solvent-rich phase (Figure 3D). In addition, the aggregation of HPC contributed to the fibril diameter and stiffness. These characteristics of polymer blend solutions can be transformed to a bundle structure and controlled topology and stiffness of fabricate the gels.

Figure 3. Characteristics of the polymer blend solutions. (A) Transmittance of the polymer blend solutions from 10°C to 50°C. The polymer blend solutions exhibited three phases in the aqueous solution: (I) single phase, (II) liquid–liquid phase separation, and (III) liquid–solid phase separation. (B) Appearance of the H5A1.0-pH13 polymer blend solution at 4°C, 28°C, and 45°C (upper) and phase-contrast images of the fabricated bundle gel fibers (bottom) at 4°C, 28°C, and 45°C. (C) Microscopic (upper) and macroscopic (lower) images 17 ACS Paragon Plus Environment


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of the phase-separating pattern of the H5AY-pH13 polymer blend solutions. In the lower panel, white and gray colors indicate the HPC- and solvent-rich phases, respectively. (D) Schematic of the proposed mechanism showing the changes in the phase-separated pattern depending on the blend ratio of HPC and Na-Alg.

3-4. Bundle gel fibers as nerve cell guiding scaffolds To investigate whether the as-prepared bundle gels with varied topologies and stiffness can be used as neuron cell culture scaffolds and guiding materials, several types of nerve cells were cultured on the gel fibers. Before cell culture, all gel fibers were treated by PLL-g-PEG-RGD35, in which cationic poly-L-lysine is anchoring segment to adsorb to anionic HPC and alginate surface and grafted PEG-RGD is cell adherence segment by exposure of RGD motif, to enhance cell attachment. To confirm adsorption of PLL derivatives, FITC-labeled PLL was reacted, indicating that all surfaces of gel fibers were coated and covered by PLL polymers with their own topology (Figure S2 in SI). Rat pheochromocytoma (PC-12) cells were cultured on gel fibers for 8 days as a nerve model cell. PC-12 cells were attached and elongated on all gel fibers (Figure 4A). From the standpoint of using them as guiding materials, the axon of the PC-12 cells on the H5AY-pH13 bundle gels was successfully extended to the long axis. On the other hand, the axon of PC-12 on the H5A2.0-pH7 non-bundle gel was extended to the random axis. In addition, the axon lengths of the PC-12 cells on H5A1.0-pH13, H5A1.5-pH13, and H5A2.0-pH13 were 34.5 ± 18.1, 46.2 ± 12.2, and 165 ± 74.8 µm, respectively (Figure 4B), indicating that the H5A2.0pH13 bundle gel with a smaller diameter microfibril and stiffer mechanical property allowed longer axon of PC-12 cells. From the results obtained for the axon orientation along the long axis, the bundle structure constructed by multiple thin microfibrils contributed to guiding of


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neuron cells. Moreover, with the increase in the concentration of Na-Alg in the gels, the axon length depended on the gel stiffness. In addition, rat DRG neurons were cultured to investigate whether the bundle gels can be used to regenerate peripheral nerves. Interestingly, 5-day cultured DRG cells stained with the neuronal marker β-tubulin III exhibited a bundle nerve tissue structure on the gels not only being aligned to the long axis (Figure 4C). In addition, the expression of the transient receptor potential vanilloid-1 (TRPV1) receptors, which are commonly found in DRG neurons and sense nociceptive temperature and pain36, was observed. These results indicated that the bundle gels provide a microenvironment for DRG neurons to form functional nerve tissue structures with the expression of indispensable ion channels. Toward the regeneration of neural tissues, one of the largest interests to examine the potential of bundle gel fibers involves the culture and differentiation of iPSCs. Human iPSCderived dopaminergic neuron progenitor cells were cultured on bundle gels (Figure 4D). The gel fibers were stable in the medium and kept their bundle structure during culturing iPSCs for long time (28 days). On the 28th day, the iPSCs on the bundle gels successfully differentiated into neuron cells, which was determined using the β-tubulin III, and the expression of nestin, which is known as a neural stem or progenitor cell marker37, was observed in a small area. In addition, cells on the bundle gels extended to the long axis as well as seen in other cell types (Figure 4D). These results indicated that the bundle structure is a powerful tool for reconstructing neuron tissues from iPSCs. Cell culture studies revealed that the topography caused by diameter of microfibrils and stiffness of bundle gels fabricated using different Na-Alg concentrations control the length and orientation of the axon and differentiation of iPSCs.



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Figure 4. Neuron cells cultured on bundle gels. (A) Confocal laser scanning microscopy (CLSM) images of PC-12 cells cultured on the bundle H5AY-pH13 gel fibers. β-tubulin III (green) and nuclei (blue) were stained. Images were merged with fluorescence and brightfield images. Bars: 50 µm. (B) Axon length of the PC-12 cells cultured on the bundled H5AY-pH13 gel fibers (n=10, ave. ± S.D.). (C) CLSM images of rat dorsal root ganglion (DRG) cells cultured on the bundle H5A2.0-pH13 gels. Immunofluorescence of the neuronal marker β-tubulin III (red), transient receptor potential vanilloid-1 (TRPV1) channel (green) and nuclei (blue). Bars: 50 µm. (D) CLSM images of human iPSC-derived neuron progenitor cells cultured on the bundle H5A2.0-pH13 gels. Immunofluorescence of the neuronal marker β-tubulin III (red), nestin (green), and nuclei (blue). Bars: 50 µm.

4. CONCLUSIONS


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In this study, gel fibers with a bundle structure, comprising multiple microfibrils, were developed by the integration of a phase-separating polymer blend solution and a microfluidic device system, and the potential of the obtained bundle gel fibers was investigated as scaffolds for in vitro neuron cells guiding. The topology and stiffness of the bundle gel fibers were controlled by the variation in the polymer blend ratio of HPC and Na-Alg: high Na-Alg concentration permitted the formation of microfibrils in a one-bundle gel fiber and stiff characteristics. These physicochemical features of the bundle gel fibers were related to the phase-separating properties of the polymer blend solution. With the liquid–liquid separation of the polymer blend solution because of the salting-out effect, the Na-Alg-induced dehydration of the HPC polymer chain was observed, leading to aggregation, followed by chemical cross-linking. The topology due to constitutive microfibrils and stiffness of the bundle gels aided in guiding neuron cells. The bundle gel fibers fabricated using the polymer blend solution with a high Na-Alg ratio permitted the elongation of the neuron cells along their axon orientation along with the long axis of fibers. In addition, human iPSC-derived dopaminergic neuron progenitor cells were successfully differentiated into the neuron cells on the stiff bundle gels. The gel fabrication method via the application of a phase-separating polymer blend solution and a microfluidic device system demonstrates a new paradigm for cell culture scaffold materials with an optimal microenvironment to built the functional nerve tissue unit in in vitro.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publication website at DOI: XXXXXXXXXX. The Supporting Information includes images of freeze-dried gel 21 ACS Paragon Plus Environment


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fibers and PLL-FITC coated gel fibers and a movie to show splitting gels by tweezers (Movie S1).

AUTHOR INFORMATION Corresponding Author *Email: [email protected] Present Addresses Y-J. K.: Bioengineering Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was partly supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI (Grant Numbers 25706010, 2503353, 16J02472, and 2656024) and JSPS Coreto-Core Program. J.P. is an International Research Fellow of the JSPS (P15767). The authors would like to thank Mr. Gustavo Garcia (Stanford University, USA) and Ms. Eri Otsuka (The University of Tokyo) for their technical assistance in the cell culture experiment and Dr. Haruka Oda (The University of Tokyo) for her technical assistance in SEM observation.


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