Preparation of Cell-Paved and -Incorporated Polysaccharide Hollow

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Preparation of Cell-Paved and -Incorporated Polysaccharide Hollow Fibers Using a Microfluidic Device Kazutoshi Iijima, Seiko Ichikawa, Shohei Ishikawa, Daisuke Matsukuma, Yusuke Yataka, Hidenori Otsuka, and Mineo Hashizume ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b01500 • Publication Date (Web): 27 Feb 2019 Downloaded from http://pubs.acs.org on March 10, 2019

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ACS Biomaterials Science & Engineering

Preparation of Cell-Paved and -Incorporated Polysaccharide Hollow Fibers Using a Microfluidic Device Kazutoshi Iijima,†,‡,1 Seiko Ichikawa,‡ Shohei Ishikawa,§ Daisuke Matsukuma, ¶ Yusuke Yataka, †,‡ Hidenori Otsuka,§,¶ Mineo Hashizume†,‡,* †Department

of Industrial Chemistry, Faculty of Engineering, Tokyo University of Science,

12-1 Ichigayafunagawara-machi, Shinjuku-ku, Tokyo 162-0826, Japan, [email protected] ‡Graduate

School of Engineering, Tokyo University of Science, 12-1 Ichigayafunagawara-

machi, Shinjuku-ku, Tokyo 162-0826, Japan §Graduate

School of Science, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku-ku,

Tokyo 162-8601, Japan

Present Address: Faculty of Engineering, Yokohama National University, 79-5 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan 1

ACS Paragon Plus Environment

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ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

¶Department

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of Applied Chemistry, Faculty of Science, Tokyo University of Science, 1-3

Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan KEYWORDS: Microfluidics, Polysaccharides, Fiber, Polyion complex, Cell scaffold

Abstract

Cellular constructs having hollow tubular structures are expected to be used as artificial blood vessels. We have recently demonstrated that water-insoluble polyion complexes (PICs) were formed from water-soluble polysaccharides with opposite charges at the interface of coaxial flows, which resulted in the formation of hollow fibers. In this study, both inside- and outsidecell-laden chondroitin sulfate C (CS)/chitosan (CHI) hollow fibers were prepared by utilizing a microfluidic device and modification with cell adhesive molecules. Loading of type I collagen (COL) and surface modification with fibronectin and gelatin using layer-by-layer assembly techniques improved the adhesion and spreading of fibroblast cells to/on the surface of CS/CHI hollow fibers. On the other hand, by suspending mesenchymal stem cells (MSCs) in the core flow solution, cells were successfully loaded in the walls of the hollow fibers. As the culture time extended, cells trapped in the PIC structures consisting the wall of the hollow fibers migrated to the interface between the hollow fibers and the medium: cells adhered to and stretched “on” the lumen surfaces in the COL-loaded fibers. In contrast, for the case of unmodified hollow fibers, cells were hard to adhere to the lumen surfaces. Therefore, cell aggregates were formed “in” the lumen. Results of live/dead assay and MTT assay clearly


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demonstrated that MSCs possessed certain levels of cell viability and proliferated for up to ten days, especially for the cases of COL-loaded hollow fibers. Based on these results, the utility of the present hollow fibers in the formation of cellular constructs corresponding to blood vessels is also discussed.

Introduction Tissue engineering has great potentials for not only regenerative medicine but also drug screening and alternative for toxicity assessment. Artificial tissues with ordered structures and improved functions have been constructed from cells combined with three-dimensional (3D) scaffolds. Among them, fibers have been used for regeneration of muscles1 and component units for the construction of more complex objects.2 Cellular constructs having hollow structures such as hollow fibers, in other words, tubes, are expected to be used as artificial blood vessels for vascular regeneration,3 construction of vascularized tissues4 and renal tubules.5 Moreover, they are applicable for drug absorption/transport model systems,6 and tumor metastasis models.7 These cellular constructs with hollow structures have been fabricated by using cell migratable scaffolds,8 rolling of cell sheets,9 shaping in a mold,10 3D printing,11,12 and microfluidic techniques.13 Among then, microfluidic technique offers single-step and continuous fabrication of cellular constructs having hollow fibrous (tubular)


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structures. 13 For example, solid and hollow fibers incorporating various kinds of cells were fabricated using extracellular matrix proteins combined with calcium alginate.2 Currently, synthetic polymers such as polyethylene terephthalate (PET) and expanded polytetrafluoroethylene (e-PTFE) are used for artificial blood vessels without cells. For preparation of cellular constructs, synthetic or natural polymers,14 or their composites, and decellularized vascular structures15 have been used as cell scaffolds. As synthetic polymers, biodegradable polymers such as polyglycolic acid and poly(ester urethane)urea8 were widely used. Natural polymers have been investigated for such purposes include proteins such as type I collagen (COL)10 and silk fibroin,16 and polysaccharides.17 In the use of polysaccharides, the formation of polyion complexes (PICs) of anionic and cationic polysaccharides is a promising approach because it makes water-soluble polysaccharides water-insoluble without the use of cross-linking agents or chemical modification. Composite fibers made of PICs of polysaccharides can be prepared by the interface spinning method,18 the spray spinning method,19 and microfluidics.20 We have succeeded in fabrication of water-insoluble composite hollow fibers made of PICs of chondroitin sulfate C (CS) and chitosan (CHI) by utilizing microfluidic techniques.20 Water-insoluble PIC were formed from water-soluble polysaccharides with opposite charges at the interface of coaxial flow which resulted in formation of hollow fibers. The diameters and thickness of the fibers were also controllable depending on the flow rate and concentration of polysaccharides, respectively.20 In addition, protein such as bovine serum


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albumin (BSA) can be incorporated in CS/CHI follow fibers by adding them into core flow of CHI solution.20 In our body, there are several types of blood vessels with different cellular components and structures.12 Arteries and veins with diameters larger than 600 m consists of endothelial cell (EC) layers and dense smooth muscle cell (SMC) layers, which are enclosed by fibroblasts (Figure 1a). On the other hand, arterioles/venules consist of EC layers surrounded by a small number of SMCs. Capillaries comprise an EC surrounded by pericytes. As mentioned above, artificial blood vessels with large diameters (several millimeters to several centimeters) made of PET and e-PTFE were clinically available. In addition, capillary networks with small diameters (several micrometers) were spontaneously formed from EC. In contrast, there are some difficulties in preparation of artificial blood vessels with middle diameters (submillimeters), for example, prevention of occlusion in fibers made of synthetic polymers and formability in cell-laden fibers. Therefore, the development of artificial blood vessels having submillimeter diameters has not progressed well, even though there are potential needs such as that for vascularization of the regenerated tissue constructs. In this study, we focused on the fibers with diameters of submillimeters. Microfluidic processes have advantageous for preparation of hollow fiber structures of these sizes. The purpose of this study is evaluation of the potential of the system to obtain polysaccharide hollow fibers using a microfluidic device20 for the development of artificial blood vessels having submillimeter diameters consisted of multilayered cellular hollow fibers


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(Figure 1). For this purpose, preparation of cell-paved and -incorporated polysaccharide hollow fibers was investigated in this study. As the first attempt, we utilized CS/CHI hollow fibers as the polysaccharide fibers because their preparative conditions have been optimized and their basic physical properties have been characterized. In addition, because glycosaminoglycans are major components of extracellular matrix and contribute to functionalization of cells, cell scaffolds of made of composites of glycosaminoglycans such as CS and CHI have been used for regeneration of wide range of tissues including vessels.21 Therefore, preparation of CS/CHI composite fibers using microfluidic devices great potentials for constructing of tissues with tubular structure. As shown in Figure 1, cell-laden CS/CHI hollow fibers were prepared by the following two ways; (1) seeding of the cells on the surface of fibers (Figure 1c), and (2) incorporation of the cells inside the fibers during fiber formation (Figure 1d). In (1), modification of the hollow fibers with proteins were also conducted to enhance cell attachment to the fiber surfaces.


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Figure 1. Conceptual illustrations of this study. (a) Multilayered cellular structures of blood vessels. (b) Artificial blood vessels (our target). In order to fabricate hollow fibers with multilayered cells mimicking blood vessels, both inside- and outside-cell-laden chondroitin sulfate C (CS)/chitosan (CHI) hollow fibers were prepared by utilizing a microfluidic device. (c) Cell-paved fibers were prepared by seeding fibroblast on the fibers. (d) Cell-incorporated fibers were prepared by incorporating mesenchymal stem cells in the fibers during fiber formation.

Materials and Methods Reagents CS (sodium salt, from shark cartilage, molecular weight (MW) ca 20,000), CHI (from crab shell, MW ≥ 100,000) and other chemicals were purchased from Nacalai Tesque, Inc. (Japan). Type I collagen (from calf skin) and rhodamine B isothiocyanate were obtained from Sigma Co., Ltd (MO, USA). Fibronectin (FN) and gelatin (GN) were purchased from Wako Pure Chemical Industries, Ltd. (Japan) and Bio-Rad laboratories (CA, USA). Reagents were used without further purification. The distilled water and ultrapure water (18.2 MΩ cm) used in experiments were prepared using Advantec RFD210TA and Advantec RFU414BA (Advantec Toyo Kaisha, Ltd., Japan), respectively.


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Cell culture Mouse embryonic fibroblast NIH3T3 cells (Health Science Research Resources Bank, Osaka, Japan) and human bone marrow-derived mesenchymal stem cells (MSCs) with an extended life span through retroviral transduction, UE7T-13 cells22 (Japanese Collection of Research Bioresources (JCRB) Cell Bank, National Institute of Biomedical Innovation) were maintained in standard Dulbecco’s modified Eagle’s medium (D-MEM, Sigma-Aldrich Co. LLC., MO, USA) supplemented with 10% (vol./vol.) fetal bovine serum (MP Biomedicals, LLC., CA, USA) and 2% penicillin-streptomycin (D-MEM(+)) at 37 ºC under a humidified 5% CO2 atmosphere.

Construction of microfluidic device The microfluidic device was fabricated according to our previous report.20 Schematic illustration of the device was shown in Figure 2. A stainless-steel 28G needle (inner diameter: 150 m, outer diameter: 350 m, length: 28 mm, Kyowa Interface Science, Co. Ltd.) was fixed temporarily inside a glass capillary tube (inner diameter: 800 m, outer diameter: 1300 m, length: 55 mm, Sansyo, Co. Ltd., Japan) using a brass tube (inner diameter: 400 m, outer diameter: 800 m) and the root of the capillary tube was coated with silicon grease to make a cavity for connection with the flow paths. These were set in a mold (20 mm × 75 mm × 5 mm) and filled with epoxy resin (Nissin resin, Co. Ltd., Japan).


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After removing the brass tube, flow paths from both sides of the device to the cavity of the root of glass capillary at an angle of 45° were generated using a drill. Stainless-steel 18G needles (Terumo Corp., Japan) were inserted in the paths and fixed with epoxy resin. Silicon grease in a cavity was flushed out.

Preparation of polysaccharide composite hollow fibers 2 wt% CHI solutions in 2 wt% aqueous acetic acid and 4 wt% CS solutions in ultrapure water were continuously infused into the core channel at 50 L min-1 and the sheath channels at 1000 L min-1, respectively using syringe pumps (KDS Legato 110, KD Scientific Inc., MA, USA). Fabricated fibers were injected into sheath solution in a glass beaker. For fluorescence microscopic observation, rhodamine B-labeled CHI synthesized according to our previous report20 were blended in the core solution at 1/100 (w/w), and fibers were fabricated in a same manner.

Preparation of COL-loaded hollow fibers COL-loaded hollow fibers were fabricated by blending COL in the core flow solution. Indicated amount of COL were dissolved in 2 wt% aqueous acetic acid with CHI. Hollow fibers were then fabricated by following the same procedure. Amounts of COL incorporated


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in the hollow fibers were estimated by quantification of hydroxyproline with the chloramine-T/Ehrlich’s reagent assay.23 Briefly, fibers were hydrolyzed in 5 N KOH at 110 ºC for 24 h. 200 L PBS, 200 mg sodium chloride and 100 L 0.5 M borate buffer (pH 8.2) were then added and pH was adjusted to pH 7-8 with 5 N HCl. 100 L of 0.2 M chloramine T solution in 2-methoxyethanol was added and incubated for an hour at room temperature followed by addition of 100 l of 3.6 M sodium thiosulfate solution. After heating at 100 ºC for 15 min and cooled for 15 min. 300 L toluene was added and mixed vigorously. Then samples were centrifugated at 1500 rpm for 5 min. 200 L of organic fraction were mixed with 80 L of Ehrlich’s reagent (p-dimethylaminobenzaldehyde in ethanol with sulfuric acid) and incubated for 15 min at room temperature. Absorbance at 560 nm was measured using a microplate reader (Varioskan Flash, Thermo Fisher Scientific, Inc., MA, USA) and quantified by using the standard curve generated with L-hydroxyproline.

Surface coating of the hollow fibers using layer-by-layer (LbL) assembly Layer-by-layer (LbL) assembly with FN and GN was applied to the surface of the hollow fibers in order to improve adhesin of fibroblast. LbL conditions such as concentration of protein and incubation time were set based on the previous report about LbL modification of cells with FN and GN.24 40 g mL-1 FN solution in PBS was added to each well of 24-well polystyrene (PS) plates (AGC Techno Glass, Co., Ltd., Japan) with hollow fibers and


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incubated for one minute. After removing FN solution, 40 g mL-1 GN solution was added and incubated for one minute. The set of treatments were performed 20 times. The treated fibers were washed with 50 mM Tris-HCl buffer (pH 7.4) three times.

Preparation of cell-paved hollow fibers The CS/CHI hollow fibers and COL-loaded CS/CHI hollow fibers were set into each well of a 24-well non-treated PS plate (AGC Techno Glass, Co., Ltd.) with PBS and sterilized by UV irradiation (254 nm, 9 W, 2 h). After removing PBS from the well, 1 × 104 NIH3T3 cells dispersed in 500 L D-MEM(+) were seeded into each well with the fibers. The cells were cultured at 37 ºC for up to 96 h. For the fibers modified with LbL assembly, the sterilized fibers were treated with FN and GN in the clean bench. 1 × 106 NIH3T3 cells dispersed in 1 mL D-MEM(+) were then seeded into each well with the fibers. The cells were cultured at 37 ºC for up to 24 h.

Preparation of cell-incorporated hollow fibers The microfluidic device, CHI solution, and CS solution were sterilized by UV irradiation ((254 nm, 9 W, 2 h)). In preparation of cell-incorporated hollow fibers, aqueous acetic acid (2 wt%, pH 3.8) and acetate buffer (0.5 M, pH 5.3) with and without 0.2 wt% COL were used as


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solvents of CHI. MSCs were dispersed in indicated solvents containing CHI at a density of 1 × 107 cells mL-1 and hollow fibers were then fabricated by following the same procedure. The cell-incorporated fibers were cut into 2 cm and cultured in each well of a 24-well nontreated PS plate at 37 °C for the indicated period. Cell culture media were changed every three days.

Optical microscopic observation Morphologies of the obtained fibers were observed using an optical microscope ECLIPSE LV100 (Nikon Corp.) and Observer.D1 (Carl Zeiss AG, Oberkochen, Germany). The fibers were observed in the sheath solution. Diameter of at least three independent fibers were measured and shown as mean ± standard deviation.

Florescent microscopic and confocal-laser scanning microscopic (CLSM) observation Cells were treated with 5 g mL–1 Hoechst 33342 (Dojindo laboratories, Japan) in D-MEM(+) for an hour under dark condition. Then, cells were washed three times with PBS and fixed with 4 wt% paraformaldehyde solutions in PBS for 20 min followed by permeation with 0.5% TritonX-100 solution for 2 min. Cells were then treated with 1% Alexa Fluor 594 phalloidin (Life Technologies, Corp., CA, USA) for 2 h. After washing with PBS three times,


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cells were observed by a fluorescent microscope (Observer.D1) and a confocal-laser scanning microscope (CLSM, LSM-710, Carl Zeiss).

Live/dead assay Cell incorporated fibers were treated with PBS containing 2 M calcein-AM and 4 M ethidium homodimer-1 (Life technologies) for 30 min. After washing with PBS three times, cells were observed by CLSM. Live and dead cells were counted by using Image J software (NIH).25 Survival rate was calculated as the ratio of live cells to total number of cells.

MTT assay 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay was performed according to the method described elsewhere.26 Briefly, after days of culture in 24-well nontreated PS plates, MSCs-loaded fibers were transferred to 1.5 mL microtube and incubated in D-MEM with 0.5 mg/mL MTT (Wako Pure Chemical Industries, Ltd.) at 37°C and 5% CO2 for 4 h. After centrifugation, the solutions were removed. Then 150 L of isopropanol containing 40 mM HCl was added and incubated 24 h. Finally, the absorbance of the resulting solution was measured at 570 nm using a microplate reader (Varioskan Flash,


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Thermo Fisher Scientific) and quantified by using the standard curve. Three parallels were averaged for each specimen.

Results Loading of COL in the CS/CHI composite hollow fibers A microfluidic device used for preparation of PIC hollow fibers of CS and CHI was fabricated according to our previous report (Figure 2).20 COL was loaded in the hollow fibers by blending COL in the core flow solution. When COL was dissolved in 2 wt% aqueous acetic acid at 0.1 wt% and 0.2 wt% with CHI, polysaccharide composite hollow fibers were formed and there were no significant differences in morphology and diameter among the resultant fibers (Figure 3a-c). The amounts of COL loaded in the CS/CHI hollow fibers were estimated by quantification of hydroxyproline with the chloramine-T/Ehrlich’s reagent assay.23 As shown in Figure 3d, approximately 40 to 50% of charged COL molecules estimated from the used flow volumes were loaded in the follow fibers.


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Figure 2. The microfluidics device and polysaccharides used in this study. (a) Schematic illustration of the microfluidic device and preparation of fibers, (b) sectioned illustration of the flow exit, and (c) the chemical structures of polysaccharides.

Figure 3. Preparation of COL-loaded CS/CHI hollow fibers. (a-c) Microscopic images of fibers prepared from CHI solutions without (a) and with 0.1 wt% (b) and 0.2 wt% COL (c). Scale bars: 200 m. (d) Amount of COL loaded in the CS/CHI fibers made in 2 min. “Charged”


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means amount of COL estimated from the used flow volumes in core solution within 2 min. Diameters are mean ± standard deviation (S. D.) (n>3) (a-c) and values are mean ± S. D. (n>3) (d).

Preparation of cell-paved hollow fibers using COL-loaded fibers Mouse embryonic fibroblast NIH3T3 cells were seeded on the CS/CHI and COL-loaded CS/CHI hollow fibers and their adhesions and proliferation were evaluated. After 24 hculture, aggregates of cells stained with nucleus and actin were observed on the unmodified fibers (Figure 4a). Although cells stained with nucleus and actin were observed on the COLloaded fibers, the number of adhered cells seem to be larger than that on the unmodified fibers (Figure 4b and c). Figure 4d-f show the morphologies of cells on composite fibers after a 72-h culture. There were aggregates of cells stained with nucleus and actin (Figure 4d). On the COL-loaded fibers, larger numbers of cells were observed and part of them spread on the surface of the fibers (Figure 4e and f).


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Figure 4. Adhesion and proliferation of NIH3T3 cells on CS/CHI hollow fibers and COLloaded CS/CHI hollow fibers. (a-f) Fluorescent microscopic images of NIH3T3 cells stained with Hoechest 33342 (blue) and Alexa Fluor 594 phalloidin (red) on the hollow fibers prepared from CHI solution without (a, d) and with 0.1 wt% (b, e) and 0.2 wt% (c, f) COL after 24 h (a-c) and 72 h (d-f). Scale bars: 50 m.

Surface coating of the hollow fibers with FN and GN using LbL assembly LbL assembly with FN and GN was applied to the surface of the hollow fibers in order to improve the adhesion of fibroblast. There was no significant difference in morphology between unmodified fibers and fibers modified with FN and GN (Figure S1). Then the adhesion of NIH3T3 cells to these fibers was evaluated. After 24 h-seeding, the number of cells on the hollow fibers modified with FN and GN was much higher than that on unmodified ones (Figure 5b, d).


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Figure 5. Adhesion of NIH3T3 cells on the fibers modified with FN and GN by using LbL assembly. (a-d) optical (a, c) and fluorescent (b, d) microscopic images of NIH3T3 cells stained with Hoechest 33342 on hollow fibers without (a, b) and with modification of FN and GN (c, d). Scale bars: 50 m.

Preparation of cell-incorporated polysaccharide hollow fibers Cell-incorporated CS/CHI hollow fibers were prepared by mixing the cells in the core flow solutions. First, the effects of pH and osmotic pressure of the suspension on cell viability was investigated using acetate buffer and sucrose. CS/CHI hollow fibers were also obtainable in the condition of using acetate buffer (pH 5.3) and in the presence of 10% sucrose (Figure S2). There were no significant effects of solvent (acetic acid or acetate buffer) on morphology and diameter of the resultant fibers between the fibers (Figure S2a, b). Addition of sucrose


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slightly increased diameter of the resultant fibers (Figure S2c, d). Figure 6 shows the results of live/dead assay of MSCs incorporated in CS/CHI hollow fibers. Low pH condition and low osmotic pressure of acetic acid solution were harmful to the cells (Figure 6a). The use of acetate buffer as the solvent of CHI and the addition of sucrose markedly increased the survival rate of the cells (Figure 6b-d). About 70% of the cells were alive under the condition of acetate buffer with sucrose (Figure 6e).


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Figure 6. Viability of MSCs loaded in CS/CHI hollow fibers. (a-d) Fluorescence microscopic images of cells in the hollow fibers prepared from core flow of acetic acid (pH 3.8) (a, b) and acetate buffer (pH 5.3) (c, d) with (b, d) and without 10 % (vol./vol.) sucrose stained by 2 M calcein-AM (green; live) and 4 M ethidium homodimer-1 (red; dead). Scale bars: 200 m. (e) Cell viabilities obtained from fluorescent microscopic images.

Subsequently, the distribution of the cells in the CS/CHI hollow fibers was investigated. Most of the cells were incorporated in the walls of PIC (indicated as a yellow arrowhead in Figure 7b) but some of them were inside the hollow fibers (indicated as a white arrowhead in Figure 7b). After 10 days-culture, there were cell aggregates inside the unmodified CS/CHI hollow fibers (Figure 8a, b), whereas cells were spread inside the walls of COL-loaded hollow fibers (Figure 8c, d).


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Figure 7. Confocal-laser scanning microscope (CLSM) images showing distribution of MSCs incorporated in CS/CHI hollow fibers. (a) Single optical slice and (b) stack profile of CLSM images. Cellular nuclei were stained with Hoechest 33342 (blue). Red indicated CHI labeled with rhodamine isothiocyanate. Scale bars: 50 m.


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Figure 8. CLSM images of MSCs loaded in CS/CHI hollow fibers after 10 days culture. MSCs in CS/CHI hollow fibers (a, b), and COL-incorporated CS/CHI hollow fibers (c, d) with low magnification (a, c) and high magnification (b, d). Cellular nuclei and skeletons were stained with Hoechest 33342 (blue) and Alexa Fluor 594 phalloidin (red), respectively. Inset illustrations in (b) and (d) indicate observed planes. Scale bars: 100 m.

To assess viability and proliferation of the cells in fibers, we performed a live/dead assay and an MTT assay. Figure 9a-f show fluorescent images of cells stained with the live/dead reagent. The viability of cells incorporated in the COL-loaded CS/CHI fibers maintained considerable high, whereas that on the unmodified fibers decreased (Figure 9g). Growth rate


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of the cells in COL-loaded fibers was higher than that of cells in the unmodified CS/CHI fibers (Figure 9h).

Figure 9. Viability of MSCs incorporated in CS/CHI hollow fibers. (a-f) Fluorescence microscopic images of cells stained by 2 M calcein-AM (green; live) and 4 M ethidium homodimer-1 (red; dead) in the CS/CHI hollow fibers (a-c) and COL-loaded CS/CHI hollow fibers (d-f) at Day 0(a, d), Day 1 (b, e) and Day 10 (c, f). Scale bars: 100 m. (g and h) Cell viability (g) and number of cells (h) calculated from MTT assay.

Discussion


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In the present study, cell-paved and -incorporated polysaccharide hollow fibers were fabricated by using a microfluidic device. Usually, preparation of hollow fibers using microfluidics sometimes requires the use of a complex device generating double-coaxial laminar flows2,13 or additional process such as removing core region using chelate agents.4 In our system, hollow fibers are readily obtained using a simpler, standard device generating coaxial flow by selective PIC formation at the interface. It should be noted that solid fibers could be also fabricated in the same device by changing preparative conditions.20 First, paving of cells on the surface of the hollow fibers were examined using fibroblast. Fibroblast is one of the cells consisted outermost layers of blood vessels.27 The matrix proteins produced by fibroblasts are required for angiogenesis.28 They also contribute to the maintenance of vascular integrity and act as a mediator of vascular remodeling.29 Fibroblast adhered to the surface of the unmodified hollow fibers. Aggregates of small number of cells stained with nucleus and actin were observed (Figure 4a). However, fibroblast was not spread on the unmodified fibers and number of adhered cells were small. This indicated low adsorption of cell adhesion molecules contained in the culture media and that produced by cells on the surface of the fibers. We have previously demonstrated that NIH3T3 cells adhered but not spread on the CS/CHI composite films prepared by hot-pressing PIC gels and modification of the films with FN improved cellular adhesion.30 In order to improve the spreading of cells, loading of COL in the hollow fibers were examined. Cells adhere to COL through 11 and 21 integrins.31 We have found that


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CS/CHI hollow fibers containing BSA were obtainable by adding them into the core flow of CHI solutions.20 Basically the same strategy was used for loading of COL. COL was soluble in acidic solution like CHI. Although total charge of COL (isoelectric point (pI): 7.832) is positively charged in acidic solutions, there are some cationic and anionic side chains in COL: therefore, COL can electrostatically interact with CS and CHI. Quantification of hydroxyproline with the chloramine-T/Ehrlich’s reagent assay22 indicated that COL was successfully loaded in the hollow fibers depending on the charged amount of COL (Figure 3d). Incorporation of COL in the hollow fibers did not cause clear influence on cell morphology, whereas it seemed to increase the number of adhered cells. The results of MTT assay after 24h-culture also supported the latter point (data not shown). The effect of contents of COL in cell number was not significant. The present results indicated that there were certain effects of COL, but they were not significant as we expected. One possibility is that degree of COL molecules properly presented on the surface was not high under the present conditions because COL was incorporated in the core flow. Modification of the hollow fibers with FN and GN using LbL assembly was then examined as a more reliable method to present adhesive protein layers on the fiber surfaces. FN includes two heparin-binding domains33 and have affinity to CS.34 In the present system, firstly FN bound to CS on the surface of the fibers, followed by binding of GN to FN. Sequential treatments with GN and FN resulted in the formation of composite nanolayers of GN and FN. Although the thicknesses of the FN-GN layers were not determined at this


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moment, the thickness of 10 layers of FN-GN nanofilms formed onto single cell surfaces prepared using protein solutions of 40 g mL-1 was estimated as about 10 nm in the previous report.24 These nanofilms probably did not affect the morphology and mechanical properties of the parent substances. In fact, the LbL assembly of FN and GN did not affect morphology of the fibers under microscopic observation (Figure S1). On the other hand, number of the cells adhered to the surface of the fibers clearly increased (Figure 5), which indicated that FN and GN on the fibers acted as cell adhesion molecules. It was suggested that LbL coating of the fibers was better than loading of COL. However, the cells did not cover the fiber surface with a considerable density. There are technical difficulties in induction of cell adhesions to the outside surface of fibers. It is necessary to increase contact opportunity between cells and fibers. If the cells prefer the bottom surfaces of cell culture plates where the fibers are placed on, cells will adhere to and spread on the cell culture plate. The other reason for low cell adhesion is due to lower ability of CS/CHI for spreading of NIH3T3 cells, as we evaluated in a previous study.30 In this study, we used wellcharacterized CS/CHI PIC as the first attempt. Optimization of the seeding process and selection polysaccharides effective for cell adhesion will enable cells to coat the surface of the fibers at a higher density. Next, cell-incorporated CS/CHI hollow fibers were prepared by mixing the cells in the core flow solutions. The feature of this system is that formation of a hollow structure and incorporation of cells in walls can be achieved simultaneously. Here we used MSCs, UE7T-13


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cells. MSCs have ability to differentiate toward vascular lineages such as SMCs35 and EC.36 Cell viability through loading process is important for these applications. Protonation of the amino groups of CHI is suitable for efficient fiber production. Because the pKa value of the corresponding ammonium group of CHI is 6.5,37 it is preferred that the pH of the celldispersed solution containing CHI is lower than that value. Since the fiber formation itself can be finished within a short period, the use of acidic solutions as the core flow might possess the viability of the cells therein: however, results showed that the use of acetic acid aqueous solutions (pH 3.8) that usually used for fiber preparation caused serious damage to the cells (Figure 6a, b, e). By changing the solvent from acetic acid solution to the acetate buffer of higher pH (5.8) where CHI still can be protonated and dissolved and formed the fiber stably, and by addition of sucrose to the cell dispersions to optimize the osmotic pressure, the survival rate could be increased to about 70% (Figure 6d, e). Cell viability should be increased by shortening the total time from preparation of the cell-containing core flow dispersion to the end of the fiber preparation. As shown in Figure 7, most cells were incorporated in the walls of PIC (indicated as a yellow arrowhead) but some cells were inside the hollow fibers (indicated as a white arrowhead). Just after preparation of the hollow fibers, cells seemed to be trapped in the PIC networks because of their relatively quick formation. After 10 days culture, there were cell aggregates inside the unmodified CS/CHI hollow fibers (Figure 8a, b), whereas cells stretched inside the walls of COL-loaded hollow fibers (Figure 8c, d). It was speculated that cells


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trapped in the PIC structure consisting the wall of the hollow fibers migrated to the interface between the hollow fibers and the medium seeking for the environment where cells can be spread. In the COL-loaded fibers, cells adhered to and stretched “on” the lumen surfaces using COL molecules as footing. In contrast, for the case of unmodified hollow fibers, cells were hard to adhere to the lumen, therefore, cell aggregates were formed “in” the lumen. These results indicated that the cells embedded in the hollow fiber walls migrated to fiber inner surfaces to seek environments suitable for cell adhesion and spreading. For COL-loaded fibers, cells were able to adhere to the inner surfaces with the help of COL and then spread on the surfaces. For the case of fibers without COL, cells were difficult to adhere to the inner surfaces, which resulted in formation of cell aggregates in the lumen. There is a possibility that cells migrated to opposite direction: actually, cells migrated to the outside of the fibers were also observed. Although the detailed mechanisms for these cell behaviors are unclear at this moment, the present results clearly demonstrated that MSCs possessed certain levels of cell viability and proliferated for up to ten days, especially for the cases of COL-loaded hollow fibers (Figure 9g, h). In addition, we may say that cell aggregates in the hollow fibers applicable for cellular reactors and arrays can be prepared by using the system without COL. Another method for loading cells in on the lumen surface in the fiber is to inject the cell suspension into the lumen of a preformed fibers. It seems to be relatively easy method in a hollow fiber with a large inner diameter.38 Although we attempted the injection of cells into


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the lumen of the present hollow fibers using a micro syringe, it was failed due to technical reason (data not shown).

Conclusions By utilizing a microfluidic device and modification with cell adhesive molecules, both insideand outside-cell-laden CS/CHI hollow fibers can be prepared. Cell adhesion and spreading ability of the fibers can be improved by using other anionic polysaccharides known as useful for such purposes. We can also use polysaccharides having specific bioactivities. In the case of incorporation of the cells in the fibers, cells were initially incorporated in the walls made of PICs: therefore, if we find a way for retaining the cells inside the walls, the hollow fibers consist of three cellular layers –on the outer surface, inside the wall part, and on the lumen surface– can be prepared in future. Such a vessel-like cellular construct is applicable for a blood vessel for vascularization of the constructed tissues. High-flow and large-diameter artificial vessels for bypass can be prepared by changing the flow condition or diameter of the device. It exactly suggests utility of these hollow fibers in the formation of cellular constructs corresponding to blood vessels. Formation of cell aggregates in the lumen for the cases of unmodified hollow fibers is an unexpected finding. The study along this direction is also expected to utilize this system for various applications and to understand cell behaviors under specific spatial environments.


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ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: XXX. Figure S1 Optical microscopic images of the CS/CHI fibers modified with FN and GN by LbL assembly. Figure S2 Optical microscopic images of the CS/CHI fibers prepared using acetate buffer and in the presence of sucrose. AUTHOR INFORMATION Mineo Hashizume (Corresponding Author); [email protected], FAX: +81-3-52614631. Phone: +81-3-5228-8319 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources


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This work was supported by the Grants-in-Aid for Scientific Research (C) from JSPS KAKENHI Grant Number JP16K05799 (M. H.).

ABBREVIATIONS 3D, three-dimensional; CHI, chitosan; CLSM, confocal-laser scanning microscopy; COL, type I collagen; CS, chondroitin sulfate C; D-MEM, Dulbecco’s modified Eagle’s medium; EC, endothelial cell; e-PTFE, expanded polytetrafluoroethylene; FN, fibronectin; GN, gelatin; LbL, layer-by-layer; MSCs, mesenchymal stem cells; MTT, 3-(4,5-di-methylthiazol-2-yl)-2,5diphenyltetrazolium bromide; PBS, phosphate buffered saline; PET, polyethylene terephthalate; PIC, polyion complex; PS, polystyrene; SMC, smooth muscle cell;

Tris,

tris(hydroxymethyl)aminomethane

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For Table of Contents Use Only Preparation of Cell-Paved and -Incorporated Polysaccharide Hollow Fibers Using a Microfluidic Device Kazutoshi Iijima, Seiko Ichikawa, Shohei Ishikawa, Daisuke Matsukuma, Yusuke Yataka, Hidenori Otsuka, Mineo Hashizume*


39


Conceptual illustrations of this study. (a) Multilayered cellular structures of blood vessels. (b) Artificial blood vessels (our target). In order to fabricate hollow fibers with multilayered cells mimicking blood vessels, both inside- and outside-cell-laden chondroitin sulfate C (CS)/chitosan (CHI) hollow fibers were prepared by utilizing a microfluidic device. (c) Cell-paved fibers were prepared by seeding fibroblast on the fibers. (d) Cell-incorporated fibers were prepared by incorporating mesenchymal stem cells in the fibers during fiber formation. 299x260mm (300 x 300 DPI)


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The microfluidics device and polysaccharides used in this study. (a) Schematic illustration of the microfluidic device and preparation of fibers, (b) sectioned illustration of the flow exit, and (c) the chemical structures of polysaccharides. 262x201mm (300 x 300 DPI)



Preparation of COL-loaded CS/CHI hollow fibers. (a-c) Microscopic images of fibers prepared from CHI solutions without (a) and with 0.1 wt% (b) and 0.2 wt% COL (c). Scale bars: 200 μm. (d) Amount of COL loaded in the CS/CHI fibers made in 2 min. “Charged” means amount of COL estimated from the used flow volumes in core solution within 2 min. Diameters are mean ± standard deviation (S. D.) (n>3) (a-c) and values are mean ± S. D. (n>3) (d). 144x108mm (208 x 208 DPI)


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Adhesion and proliferation of NIH3T3 cells on CS/CHI hollow fibers and COL-loaded CS/CHI hollow fibers. (af) Fluorescent microscopic images of NIH3T3 cells stained with Hoechest 33342 (blue) and Alexa Fluor 594 phalloidin (red) on the hollow fibers prepared from CHI solution without (a, d) and with 0.1 wt% (b, e) and 0.2 wt% (c, f) COL after 24 h (a-c) and 72 h (d-f). Scale bars: 50 μm. 173x115mm (208 x 208 DPI)



Adhesion of NIH3T3 cells on the fibers modified with FN and GN by using LbL assembly. (a-d) optical (a, c) and fluorescent (b, d) microscopic images of NIH3T3 cells stained with Hoechest 33342 on hollow fibers without (a, b) and with modification of FN and GN (c, d). Scale bars: 50 μm. 109x82mm (208 x 208 DPI)


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Viability of MSCs loaded in CS/CHI hollow fibers. (a-d) Fluorescence microscopic images of cells in the hollow fibers prepared from core flow of acetic acid (pH 3.8) (a, b) and acetate buffer (pH 5.3) (c, d) with (b, d) and without 10 % (vol./vol.) sucrose stained by 2 μM calcein-AM (green; live) and 4 μM ethidium homodimer-1 (red; dead). Scale bars: 200 μm. (e) Cell viabilities obtained from fluorescent microscopic images. 108x166mm (208 x 208 DPI)



Confocal-laser scanning microscope (CLSM) images showing distribution of MSCs incorporated in CS/CHI hollow fibers. (a) Single optical slice and (b) stack profile of CLSM images. Cellular nuclei were stained with Hoechest 33342 (blue). Red indicated CHI labeled with rhodamine isothiocyanate. Scale bars: 50 μm. 108x118mm (300 x 300 DPI)


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CLSM images of MSCs loaded in CS/CHI hollow fibers after 10 days culture. MSCs in CS/CHI hollow fibers (a, b), and COL-incorporated CS/CHI hollow fibers (c, d) with low magnification (a, c) and high magnification (b, d). Cellular nuclei and skeletons were stained with Hoechest 33342 (blue) and Alexa Fluor 594 phalloidin (red), respectively. Inset illustrations in (b) and (d) indicate observed planes. Scale bars: 100 μm. 93x93mm (208 x 208 DPI)



Viability of MSCs incorporated in CS/CHI hollow fibers. (a-f) Fluorescence microscopic images of cells stained by 2 μM calcein-AM (green; live) and 4 μM ethidium homodimer-1 (red; dead) in the CS/CHI hollow fibers (a-c) and COL-loaded CS/CHI hollow fibers (d-f) at Day 0(a, d), Day 1 (b, e) and Day 10 (c, f). Scale bars: 100 μm. (g and h) Cell viability (g) and number of cells (h) calculated from MTT assay. 136x151mm (208 x 208 DPI)


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Preparation of Cell-Paved and -Incorporated Polysaccharide Hollow

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