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## Creator

A. JEAN, [C. YOSHIKAWA](https://orcid.org/0000-0002-6589-387X)

## Rights

[In Copyright](http://rightsstatements.org/vocab/InC/1.0/)

## Other metadata

[SELF-AGGREGATION OF CELLS WITH CONCENTRATED POLYMER BRUSH-MODIFIED ELECTROSPUN FIBERS](https://mdr.nims.go.jp/datasets/790d742d-553d-4f54-8af8-ecffcad68492)

## Fulltext

Proceedings of Biomaterials International 2026 July 26 – 30, 2026 · Seoul, Korea SELF-AGGREGATION OF CELLS WITH CONCENTRATED POLYMER BRUSH-MODIFIED ELECTROSPUN FIBERS  A. JEAN1,2, and C. YOSHIKAWA1,2* 1Research Center for Macromolecules and Biomaterials, National Institute for Materials Science (NIMS), 1-2-1 Sengen, Tsukuba, Ibaraki 305-0047, Japan  – yoshikawa.chiaki@nims.go.jp 2 Graduate School of Life Science, Hokkaido University, Kita 10, Nishi 8, Sapporo 060-0808, Japan  Abstract – Electrospun fiber mat with an average diameter = 1.5 μm, having initiating groups for surface-initiated atom transfer radical polymerization (SI-ATRP) was prepared using a random copolymer of styrene (ST) and 4-vinylbenzyl 2-bromopropionate (VBP). Then, SI-ATRP of hydrophilic styrene sodium sulfonate (SSNa) was carried out on the fiber mat, yielding high-dense graft layer on the fiber (the graft density was about 0.2 chains/nm2). The short fiber with an average length = 30 - 150 μm was obtained by mechanical cutting. We cultured chicken embryonic keratocyte cells with the short fibers for prescribe time. SEM observation confirmed self-aggregation of the cells and the short fibers. Histological analysis revealed that cells homogenously existed inside the aggregates and the cell viability was high (~ 80%).   Keywords: electro spinning, concentrated polymer brush, flocculation, cell, self-aggregation  Introduction  Recently, we have successfully developed “short” electrospun (e-spun) fibers with a polymer brush of high graft density, i.e., concentrated polymer brush, by surface-initiated atom transfer radical polymerization (SI-ATRP), one of LRPs. [1] The core fiber and the outer polymer brush were composed of styrene based copolymer (hydrophobic) and poly(styrene sodiumsulfate) (PSSNa) (hydrophilic), respectively. Due to the rod-like structure (short length) and the hydrophilic PSSNa brush, the fibers showed good dispersiblity in water. Utilizing the shortened fibers with PSSNa brushes as a model material, in this work, we planned to fabricate a new type of tissue engineering scaffold. The aim of this work was to demonstrate a new scaffold based on a converse idea, namely, we cultivated cells not on the fibrous scaffolds, but with the “short” fibers. Since the short e-spun fibers can easily move, cells (as colloid particles) flocculated, namely self-aggregated with the short fibers (as flocculant) due to the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory that flocculation of colloid particles is caused by a balance between van der Waals and electrostatic forces.   Experimental  Synthesis of the random copolymer A solution of ST (133 mmol), VBP (7.6 mmol) and VR110 (0.009 mmol) was prepared in a Schlenk tube and air in the solution was removed by argon bubbling for 20 minutes. Then, the polymerization tube was heated to 110 °C for 11h. After the polymerization, poly(ST-r-VBP) was purified by reprecipitation with MeOH. Mn and Mw/Mn (PDI) of poly(ST-r-VBP) were determined by gel permeation chromatography (GPC) in N,N-dimethylformamide (DMF) with 10mM LiCl. The composition of poly(ST-r-VBP) was determined by nuclear magnetic resonance (1H-NMR). Fabrication of poly(ST-r-VBP) electrospun fiber mats A 30wt% solution of poly(ST-r-VBP) in DMF was electrospun to fabricate nanofibers at an applied potential of 20kVusing a needle inner of about 0.45 mm diameter and a solution feeding rate of 1mlh−1. The obtained fiber mats were observed with a scanning electron microscope (SEM). The average diameter was calculated from more than 60 fibers, imaged in 3 different areas on a nonwoven mat (1×1cm2). The surface area of the nonwoven mat, SBET, was determined by Brunauer–Emmett–Teller (BET) analysis of two different samples SI-ATRP of SSNA from fiber mats. The fiber mats were first degassed over two days prior to polymerization to degas any entrapped oxygen. Surface modification of the fiber mats was done via SI-ATRP: A methanol/water solution (10ml, 1/3 vol/vol) of Cu(I)Br (2.56 mg, 0.018 mmol), Cu(II)Br2 (1.71 mg, 0.0077 mmol), 2,2'-bipyridine (8.0 mg, 0.051 mmol), PEGBr (12.7 mg, 0.026 mmol), and SSNa (1.05g, 5.1 mmol) were prepared under N2, and the solution was added to a schlenk tube with poly(St-r-VBP) electrospun fibers (20 mg). The polymerization solution was mixed at 30 °C for 5h. After the polymerization, the solution was quenched with air until the solution color changed to blue. Mn and Mw/ Mn for free polymers were determined by standard PSSNa-calibrated GPC. The graft amount on the fiber (g/g) was determined by a fourier transform infrared spectroscopy according to our previous report [1]. Cutting nanofibers with a homogenizer The electrospun fibers with PSSNa brushes were cut with a homogenizer (IKA, ULTRA-TURRAX T25) in water for prescribed time. After the cutting, the shortened fibers were dried and observed by SEM. The average length was estimated with three different SEM images. Cell culture with the short fibers Proceedings of Biomaterials International 2026 July 26 – 30, 2026 · Seoul, Korea Suspension of chicken embryonic keratocyte primary cells (6.0104 cells/ml) and the short fibers (0.2 wt%) were placed in a 24- multiwell plate (1 ml / well). The cells with the short fibers were cultured in DMEM with 10% FBS at 37 oC in a humidified atmosphere of air containing 5% CO2. After the prescribed time, the aggregations of cells and fibers were washed with PBS and fixed by by 4 % paraformaldehyde.  Results and Discussion We first synthesized a random copolymer of ST and VBP by conventional radical polymerization as shown in Scheme 1.  1H-NMR measurement confirmed that the VPB content in the copolymer was about 6 mol%, almost equal to monomer feeding ratio. We then carried out the SI-ATRP of SSNa from the fiber mat. In the polymerization system, we added a free initiator to control polymerization. In addition, it is known that Mn and PDI of the free polymers can be good indices for the graft polymers. After 3h of polymerization, Mn of the free polymer was well agreed with the theoretical Mn calculated with conversion and PDI was narrow, indicating the polymerization proceeded in living manner. The graft density of PSSNa brush (0.2 chains/ nm2) was calculated using the values of SBET, graft amount, density of PolySSNa and Mn. This graft density is high enough to be categorized as concentrated polymers. Then, the fiber mat with PSSNa brushes was cut by a homogenizer (Figure 1a). By varying the cutting time, we obtained short fibers with various length from 30 – 150 μm.  Recently, we reported that cellulose nanofibers (CNFs) modified with charged concentrated polymer brushes flocculated (self-aggregated) with various cell lines such as HepG2 and hMSC [2-4]. The floc size and the cell functions were dependent on surface charge, the CNF concentration and length. The results indicate that the cellular flocculation can be explained by the DLVO theory. Therefore, we hypothesized that when the short e-spun fibers have appropriate length and charge for cells, flocculation would also occur according to the DLVO theory. To verify this, we co-cultured cells and the short fibers with PSSNa brushes. Herein, we used the short fiber with the length of 30 μm and chicken embryonic keratocyte primary cells as a model fibroblast cell. It should be noted that the short fiber is much longer than CNF (0.2- 1.8 μm) which we used in previous work [2-4]. After 24 hours, the cells self-assembled to form aggregates with the short fibers. SEM confirmed that the floc sizes were larger than 200 μm. In addition, hematoxylin-eosin staining showed that cells homogenously existed in the aggregates. TUNEL staining confirmed that almost cells in the aggregate were alive (~ 80%). To further investigate the self-aggregate, we varied the fiber concentrations, cell types and culture period. As we expected, independent of cell types, cellular self-aggregates were observed. Importantly, the floc size increased with decreasing fiber concentration and increasing cell culture period. This was consistent with the results on CNF with polymer brushes. The details will be discussed.  Conclusions  We successfully demonstrated a new type of cell scaffold based on cellular self-assembly with e-spun fibers. This resulted from easy movement of “short” e-spun fiber and charge interaction of cells and the brushes (DLVO theory). Different from the conventional e-spun (uncut) fiber mats or sponges, the cells homogenously existed inside the cell-fiber aggregates and showed high viability. Therefore, our self-assembly system using short e-spun fibers would be a new route for a tissue engineering scaffold.   Acknowledgements This work was supported by the JSPS KAKENHI Grant Number 19KK0368, 22H02133 and 24KF0166.   References 1. Yoshikawa, C.; Zhang, K.; Zawadzak, E.; Kobayashi, H. Sci. Technol. Adv. Mate., 2011, 12(1),015003-1-7. 2. Yoshikawa, C.; Sakakibara, K.; Nonsuwan, P.; Shobo, M.; Xida, Y.; Matsumura, K. Biomacromolecules 2022, 23, 3186-3197.  3. Nonsuwan, P.; Nishijima, N.; Sakakibara, K. Nakaji-Hirabayashi, T.; Yoshikawa, C. J. Mater. Chem. B 2022, 10(14), 2444-2453. 4. Xida; Y.; Nounsuwan, P.; Shobo, M.; Rojan, R.; Yamazaki, T.; Sakakibara, K.; Matsumura, K.; Yoshikawa, C. Biomacromolecules 2022, 23(3), 1101-1111.   Scheme 1. Random copolymerization of ST and VBP.   Figure 1. The fiber cutting by a homogenizer.