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Publication Date (Web): March 15, 2018 ... A protein adsorption test indicates that affinity interaction between the proteins (cells) and CPB is criti...
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Cite This: ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

Flocculation of Cells by Cellulose Nanofibers Modified with Concentrated Polymer Brushes Chiaki Yoshikawa,*,† Takashi Hoshiba,†,‡ Keita Sakakibara,*,§ and Yoshinobu Tsujii§ †

International Center for Materials Nanoarchitectonics, National Institute for Materials Science, 1-2-1 Sengen, Tsukuba, Ibaraki 305-0047, Japan ‡ Frontier Center for Organic Materials, Yamagata University, 4-3-16 Jonan, Yonezawa, Yamagata 992-8510 Japan § Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan S Supporting Information *

ABSTRACT: A new type of three-dimensional cell culture using a cellulose nanofiber modified with a concentrated polymer brush (CNF-CPB) is described from the viewpoint of colloidal flocculation. By varying the CNF-CPB concentrations from 0.005 to 0.5 wt %, we demonstrate that hepatocyte cells self-assemble with the CNF-CPBs and change the floc size and shape (fully filled or hollow) in a concentration-dependent manner. A protein adsorption test indicates that affinity interaction between the proteins (cells) and CPB is critical for cellular flocculation. In addition, gene expression analysis confirms that the cellular flocs using CNF-CPBs enhance cell functions compared to CNFs with semidilute polymer brushes and pristine CNFs. KEYWORDS: cellulose nanofiber, living radical polymerization, concentrated polymer brush, flocculation, hepatocyte cell, scaffold 5 μm, are very similar to those of collagen fiber, a natural extracellular matrix. Thus, CNF would be suitable as a cell culture scaffold. In spite of their potential, only two groups have reported 3D cell cultures using pristine CNF gels so far.14,15 Native CNF spontaneously forms hydrogels in aqueous solutions at certain concentrations.14 Because cell adhesive proteins are difficult to adsorb on the nonmodified CNF surface, the cells barely or weakly interacted with the CNF surface, consequently forming cell spheroids through a strong cell−cell interaction. In this study, we demonstrate a novel CNF-based 3D scaffold inspired by colloidal flocculation, where cells are regarded as colloidal particles. Our method provides easy cell entrapment (homogeneous cell diffusion) and unique self-assemblies (flocs). The key to tuning interactions between the cells and CNF is to introduce concentrated polymer brushes (CPBs) on the CNF surface, which are prepared by living radical polymerization (LRP) and exhibit unique entropic properties, so-called “CPB effects”, including high repulsion (colloidal stabilization), superlubrication, and strict size-exclusion properties.16 Unlike native CNF, CPB-grafted CNFs (CNF-CPBs) well disperse in aqueous solutions without gel formation at any concentration. Here, an anionic CPB was grafted onto the CNF surface to enhance protein adsorption, namely, cell adhesion, affording electrostatic repulsion, which is another representative mechanism for colloidal stabilization and is summarized in the

T

he importance of three-dimensional (3D) cell cultures is rapidly increasing in various medical applications such as regenerative medicine, cell therapy, and drug screening/ designing.1,2 Because 3D cell culture better resembles the natural microenvironment within our body than two-dimensional (monolayer) cell culture, the cellular responses are more reflective of natural cells in vivo and the obtained information is more reliable.3,4 In particular, for regenerative medicine, in order to replace deficient or damaged tissues/organs, it is essential to culture cells in 3D using scaffolds5,6 formed from materials such as gels, sponges, particles, and fibers, which are shaped to mimic the anatomical defect size and shape. To date, thin sheet-type tissues such as cartilage sheets and epithelial cell sheets have been successfully regenerated.7−12 However, the regeneration of larger and more complicated tissues and organs has not been realized. Scaffolds are typically required for cell attachment, providing the appropriate space for cell migration, diffusion of nutrients/oxygen, waste release, biocompatibility, generation of a robust structure until cells form tissues/organs, and so on. For scaffold-based 3D cell culture, there are roughly two preparation methods: one is to seed cells on a preshaped scaffold such as a gel and a porous material, and the other is to entrap cells in the scaffold by cross-linking a mixture of cells and a gel precursor. Both approaches have been extensively studied; however, there are no scaffolds that satisfy all requirements for the regeneration of larger and more complicated tissues and organs. Recently, cellulose nanofiber (CNF) has attracted attention in life sciences because it is an inexpensive renewable resource with unique mechanical and biological properties.13 The scale ranges of CNF, i.e., diameters of 10−20 nm and lengths of over © XXXX American Chemical Society

Received: January 31, 2018 Accepted: March 15, 2018 Published: March 15, 2018 A

DOI: 10.1021/acsanm.8b00172 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

Letter

ACS Applied Nano Materials Scheme 1. Overview of the Studya

a (a) Affinity interaction between the cell and CNF-CPB. (b) Flocculation of cells and CNF-CPBs. The cell number is fixed (early stage). (c) Size and shape changes of the floc. The CNF concentration is fixed (later stage). Gray circles indicate the initial seeded cells, and yellow circles represent proliferating cells.

Derjaguin−Landau−Vervey−Overbeek (DLVO) theory.17 Thus, we planned to make the charged CNF-CPB cover cell surfaces via nonspecific protein adsorption, generating enough charges on the cell surface to cause flocculation and reducing unfavorable cell−cell interactions. We show herein that a combination of steric and electrostatic interactions, both repulsive and enthalpic (protein-mediated) attractive interactions, leads to reversible association of the cells. As a consequence, the assembly of cells with CNF-CPBs could vary in size and morphology even in active proliferation. In addition, different from the preshaped 3D scaffolds, it is advantageous for cells to homogeneously disperse inside the flocs. The CNFCPBs existing between cells would provide a proper gap for substance transportation. Scheme 1 shows the results supporting our proof-of-concept. The CNF-CPBs were prepared in a two-step reaction (Figure S1): (i) the introduction of initiating sites for atom-transfer radical polymerization (ATRP), one of the LRPs, and (ii) surface-initiated ATRP from the CNF surface. We also prepared a CNF with a semidilute polymer brush (SDPB), the graft density of which is much lower than that of CPB, to demonstrate the efficacy of CNF-CPB as a scaffold. Two CNFs with different amounts of initiating sites (for CPB and SDPB) were prepared by varying the esterification time for pinederived CNF (diameter ∼15 nm; Figure S2) and isolated via a mechanical grinder treatment.18 The initiator densities on the CNF surface were regulated at >6 and 0.04 groups/nm2 (calculation provided in Table S1), which are sufficient to prepare CPB and SDPB on the surface, respectively.

Subsequently, because charged polymers induce nonspecific protein adsorptions and, hence, cell adhesions, poly(sodium 4styrenesulfate) (PSSNa), an anionic polymer, was grafted on CNF (Scheme 1a). It is generally thought that cationic polymers (positive charges) would enhance cell adhesion more than anionic polymers because cells are negatively charged. However, our recent study confirmed that surface charges of polymers had little effect on the protein adsorption and cell adhesion under normal experimental conditions.19 In addition, it is known that some of the cationic polymers, such as poly(ethylenimine), have relatively high cytotoxicity.20 Thus, herein, we chose PSSNa, an anionic polymer. Surface-initiated ATRP of SSNa was performed with the CNF-Br according to our previous report.21 Table S2 summarizes the results of graft polymerization. The number-average molecular weights of the free polymers (Mn) were 9200 and 14400 for CNF-CPB and CNF-SDPB, respectively, which were prepared as a good index for the graft polymers. These values were almost identical with the theoretical ones (Mn,c) of 11100 and 10900 for the CPB and SDPB samples, respectively, and the polydispersity indexes were relatively narrow ( CFN-SDPB > pristine). In fact, in contrast to CNF-CPB, the cells with pristine CNF and CNFSDPB did not form strong assemblies and were easily disrupted upon collection. Thus, only the flocs of CNF-CPBs could be observed by confocal laser microscopy (Figure S6). Actin filaments and cell nuclei were stained in red and blue, respectively. The CNF-CPBs were also colored in blue, probably because of autofluorescence. The flocs of cells with the CNF-CPBs were observed in the range of the examined concentrations; however, the sizes of the flocs decreased with increasing CNF-CPB concentration. This was because excess CNF-CPBs prevented the cells from making a large floc (Scheme 1b). Images of the 24-multiwell plate in Figure S7 provide further evidence for this observation. The flocs were observed as white precipitates in the center of the wells. Excess CNFs, which appeared as white turbidity, sank to the bottom of the plate after 4 days for the 0.5 and 0.05 wt % samples. In addition, we observed the flocs by phase-contrast microscopy (Figure 1). Dark-brown round constructs show flocs of cells and CNF-CPBs. For example, in the 7-day images, part of the flocs are shown with dotted circles. The sizes of all of the flocs increased with incubation. As illustrated in Scheme 1c, this was because the cell population in the floc increased with time and the new cells (yellow circles in the scheme)

to estimate the graft amount of PSSNa, enabling an estimation of the graft amounts for both CNF-CPB and CNF-SDPB from the S atom originating from PSSNa. The graft densities of CNF-CPB and CNF-SDPB were then calculated using the known Mn, surface area, and graft amount. The values of the CPBs obtained by FT-IR and elemental analysis were slightly higher than those of the typical CPBs prepared on flat substrates (σ < 1.0), which were within the experimental error range. Using the criteria of CPBs based on the scaling theory,16 we categorized the two brushes as CPB and SDPB, respectively. The morphology of CNF-CPB in a swollen state can be estimated by using a fully stretched core−shell model.22 This model is reasonable in the case for CPB-afforded nanoparticles as well as nanofibers with short polymer brushes, where the graft polymers are highly stretched to exhibit over 80% of the full length.23 The thickness of the swollen PSSNa shell is equal to the weight-average full (contour) length of the graft chain in the all-trans conformation, evaluated to be around 10 nm (see the Supporting Information). Thus, the total diameter of CNFCPB swollen under aqueous conditions was estimated to be 35 nm (length >150 μm), which is thin and long enough to cover the cell surfaces. As a human hepatocyte model, HepG2 cells were cultured with the obtained PSSNa-grafted CNFs and pristine CNFs as references. The CNF concentrations were varied from 0.005 to 0.5 wt %, while the cell concentration was fixed at 1 × 105 cells/ mL. The cell−CNF mixtures were placed in a 24-multiwell plate and incubated for 1, 4, and 7 days. After incubation for the stipulated time, the CNFs with cells were gently washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde. Then, the flocs were transferred to fresh dishes for confocal laser microscopy. Figure S4 shows the washed plate C

DOI: 10.1021/acsanm.8b00172 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

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ACS Applied Nano Materials

Because the flocs were difficult to distinguish from excess unbound CNF-CPBs for the day 1 and 4 samples, the flocs of the day 7 samples were estimated. When the flocs had oval shapes, the long axis was measured. The results indicate that the size of the flocs could be roughly controlled by adjusting the cell and CNF concentrations. These results were very similar to the behavior of colloidal flocs, such as a morphology change with time and a redispersion by agitation.17,24 Further, it should be noted that, after 7 days, flocs of 0.005 wt % CNF-CPB started to develop hollows. The cross sections of the confocal fluorescent images of the flocs are shown in Figure 2. Movies of two hollows are also provided in the Supporting Information. Cells and CNFs were homogeneously packed in the flocs of 0.5 and 0.05 wt % CNF-CPB, but almost nothing was present inside the 0.005 wt % floc. As mentioned above, a longer cell culture time increased the floc (cluster) size. At a critical point after 4 days, the volume fraction of the cells relative to the CNF would probably be high enough for the floc to form voids. In general, cells normally require adhesion to matrixes for survival. The CNF-CPB concentration (matrixes) is fixed in the cluster. Therefore, at certain concentrations, HepG2 migrated to the limited number of CNF-CPBs, which decreased the surface area required for adhesion, creating a hollow inside the floc (Scheme 1c). We also assessed the cell viability and function in the flocs after 4 days. Because the flocs with CNF-SDPB and pristine CNF are fragile, we directly performed live/dead cell staining on the flocs without any transfer after washing with PBS. Figure S9 shows the images of the stained cells, where living and dead cells are shown in green and red, respectively. Few dead cells were observed in the samples as well as the 24-multiwell plate surface (TCPS), indicating that surface interaction with CNFs did not affect the cell viability. Next, we evaluated the HepG2 function by gene expression analysis.25 The expressions of albumin (ALB) and hepatocyte nuclear factor 4α (HNF4A) were measured as indicators of liver-specific function. Figure 3 shows ALB and HNF4A expressions of HepG2 against CNFs and TCPS. Gene expression was normalized with that of a house-keeping gene (glyceraldehyde-3-phosphate dehydrogen-

Figure 2. Cross-sectional confocal laser microscopy images of the flocs of cells and CNF-CPBs. HepG2 = 1.0 × 105 cells/mL and CNF-CPB = 0.005 wt %; cultured for 1, 4, and 7 days; scale bar = 100 μm. Parts A−D indicate the positions of the cross-sectional images from the floc surface: (A) top surface; (C) middle.

expanded the floc. The floc growth of 0.005 wt % was most prominent, and the diameter was larger than 500 μm after 7 days. Figure S8 shows the average size of the flocs after 7 days.

Figure 3. HepG2 cell functions against CNFs and TCPS: (a) ALB and (b) HNF4A expressions. Data represent the means ± standard deviation (n = 3). *P < 0.05 versus TCPS. HepG2 = 1.0 × 105 cells/mL and CNFs = 0.005 wt % in 1 mL of a medium, cultured for 4 days. D

DOI: 10.1021/acsanm.8b00172 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

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ACS Applied Nano Materials

Discovery and Cell-Based Biosensors. Assay Drug Dev. Technol. 2014, 12, 207−218. (2) Langhans, S. A. Three-dimensional In Vitro Cell Culture Models in Drug Discovery and Drug Repositioning. Front. Pharmacol. 2018, 9, 1−14. (3) Laschke, M. W.; Menger, M. Life is 3D: Boosting Spheroid Function for Tissue Engineering. Trends Biotechnol. 2017, 35, 133− 144. (4) Petrenko, Y.; Sykova, E.; Kubinova, S. The Therapeutic Potential of Three-Dimensional Multipotent Mesenchymal Stromal Cell Spheroids. Stem Cell Res. Ther. 2017, 8, 1−9. (5) Lee, K. Y.; Mooney, D. Hydrogels for Tissue Engineering. Chem. Rev. 2001, 101, 1869−1880. (6) Knight, E.; Przyborski, S. Advances in 3D Cell Culture Technologies Enabling Tissue-Like Structures to Be Created In Vitro. J. Anat. 2015, 227, 746−756. (7) Niklason, L. E.; Gao, J.; Abbott, W. M.; Hirschi, K. K.; Houser, S.; Marini, R.; Langer, R. Functional Arteries Grown In Vitro. Science 1999, 284, 489−493. (8) Oberpenning, F.; Meng, J.; Yoo, J. J.; Atala, A. De Novo Reconstitution of A Functional Mammalian Urinary Bladder by Tissue Engineering. Nat. Biotechnol. 1999, 17, 149−155. (9) Pomahac, B.; Svensjo, T.; Yao, F.; Brown, H.; Eriksson, E. Tissue Engineering of Skin. Crit. Rev. Oral Biol. Med. 1998, 9, 333−344. (10) Ma, P. X.; Langer, R. Morphology and Mechanical Function of Long-Term In Vitro Engineered Cartilage. J. Biomed. Mater. Res. 1999, 44, 217−221. (11) Service, R. F. Tissue Engineers Build New Bone. Science 2000, 289, 1498−1500. (12) Lin, V. S.; Lee, M. C.; O’Neal, S.; McKean, J.; Sung, K. L. P. Ligament Tissue Engineering Using Synthetic Biodegradable Fiber Scaffolds. Tissue Eng. 1999, 5, 443−452. (13) Lin, N.; Dufresne, A. Nanocellulose in Biomedicine: Current Status and Future. Eur. Polym. J. 2014, 59, 302−325. (14) Bhattacharya, M.; Malinen, M. M.; Lauren, P.; Lou, Y.; Kuisma, S. W.; Kanninen, L.; Lille, M.; Corlu, A.; GuGuen-Guillouzo, C.; Ikkala, O.; Laukkanen, A.; Urtti, A.; Yliperttula, M. Nanofibrillar Cellulose Hydrogel Promotes Three-Dimensional Liver Cell Culture. J. Controlled Release 2012, 164, 291−298. (15) Kidoaki, S.; Tuji, Y.; Hayashi, H.; Iwama, T.; Horikawa, M. Material for Undifferentiated State-Maintaining Culture. Patent WO2015111734A1. (16) Tsujii, Y.; Ohno, K.; Yamamoto, S.; Goto, A.; Fukuda, T. Structure and Properties of High-Density Polymer Brushes Prepared by Surface-initiated Living Radical Polymerization. Adv. Polym. Sci. 2006, 197, 1−45. (17) Dickinson, E.; Eriksson, L. Particle Flocculation by Adsorbing Polymers. Adv. Colloid Interface Sci. 1991, 34, 1−29. (18) Abe, K.; Iwamoto, S.; Yano, H. Obtaining Cellulose Nanofibers with A Uniform Width of 15 nm from Wood. Biomacromolecules 2007, 8, 3276−3278. (19) Hoshiba, T.; Yoshikawa, C.; Sakakibara, K. Characterization of Initial Cell Adhesion on Charged Polymer Substrates in SerumContaining and Serum-Free Media. Langmuir, 201810.1021/acs.langmuir.8b00233 (20) Cherng, J. Y.; van de Wetering, P.; Talsma, H.; Crommelin, D. J. A.; Hennink, W. E. Effect of Size and Serum Proteins on Transfection Efficiency of Poly((2-dimethylamino)ethyl methacrylate)-Plasmid Nanoparticles. Pharm. Res. 1996, 13, 1038−1042. (21) Yoshikawa, C.; Zhang, K.; Zawadzak, E.; Kobayashi, H. A Novel Shortened Electrospun Nanofiber Modified with a ‘Concentrated’ Polymer Brush. Sci. Technol. Adv. Mater. 2011, 12, 015003-1−0150037. (22) Ohno, K.; Morinaga, T.; Takeno, S.; Tsujii, Y.; Fukuda, T. Suspensions of Silica Particles Grafted with Concentrated Polymer Brush: Effects of Graft Chain Length on Brush Layer Thickness and Colloidal Crystallization. Macromolecules 2007, 40, 9143−9150. (23) Yamamoto, S.; Ejaz, M.; Tsujii, Y.; Fukuda, T. Surface Interaction Forces of Well-Defined, High-Density Polymer Brushes

ase, GAPDH). Interestingly, the expressions of ALB and HNF4A were higher in cells with CNF-CPB, whereas no difference was observed in the remaining three samples. This was probably due to the formation of stable flocs of CNFCPBs, in which cell−CNF and cell−cell adhesions were significantly high. Unlike CNF-SDPB and pristine CNF, CNF-CPB showed the formation of stable flocs, unique 3D morphology, and enhancement of tissue-specific gene expression. In summary, we demonstrated that CNF-CPB flocs with HepG2 and significantly increased liver-specific gene expression compared well to the CNF-SDPBs and pristine CNFs. In addition, we proposed a new idea of cellular flocculation using the charged CNF-CPBs, confirming that the flocs changed their sizes and shapes (fully filled and hollow), in a concentrationdependent manner. The unprecedented hollow structure may improve the transportation of substances or enable new applications. With preliminary tests, we confirmed that fibroblasts such as L929 cells also self-assembled with CNFCPBs. Further investigation is now underway to validate the cellular flocculation. Because LRP techniques can easily introduce biofunctional groups such as a growth factor in/on the CPB layer, utilization of the capabilities of LRPs and the unique self-assembly system of the CNF-CPB would open new avenues for the development of novel 3D scaffolds that regenerate larger and more complicated tissue and organs.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsanm.8b00172. Experimental details, Figures S1−S9, and Tables S1 and S2 (PDF) Movie S1 (AVI) Movie S2 (AVI)

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AUTHOR INFORMATION

Corresponding Authors

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

Chiaki Yoshikawa: 0000-0002-6589-387X Takashi Hoshiba: 0000-0003-2070-9888 Keita Sakakibara: 0000-0002-6013-695X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was performed, in part, at the NIMS Molecules & Materials Synthesis Platform. This work was partly supported by a Grant-in-Aid for Young Scientists (A) (Grant 26702016 to T.H.) and a Grant-in-Aid for Young Scientists (B) (Grant 24750218 to K.S.) from MEXT, Japan, and by the Collaborative Research Program of Institute for Chemical Research, Kyoto University (Grant 2013-26 to C.Y.). We also thank Dr. K. Abe (RISH, Kyoto University) for supplying pinederived CNF.

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REFERENCES

(1) Edmondson, R.; Broglie, J. J.; Adcock, A. F.; Yang, L. Threedimensional Cell Culture Systems and Their Applications in Drug E

DOI: 10.1021/acsanm.8b00172 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

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ACS Applied Nano Materials Studied by Atomic Force Microscopy. 2. Effect of Graft Density. Macromolecules 2000, 33, 5608−5612. (24) Aoki, K.; Adachi, A. Kinetics of Polyelectrolyte Adsorption onto Polystyrene Latex Particle Studied Using Electrophoresis: Effects of Molecular Weight and Ionic Strength. J. Colloid Interface Sci. 2006, 300, 69−77. (25) Hoshiba, T.; Otaki, T.; Nemoto, E.; Maruyama, H.; Tanaka, M. Blood-Compatible Polymer for Hepatocyte Culture with High Hepatocyte-Specific Functions toward Bioartificial Liver Development. ACS Appl. Mater. Interfaces 2015, 7, 18096−18103.

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DOI: 10.1021/acsanm.8b00172 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX