Substrate Fluidity Regulates Cell Adhesion and Morphology on Poly(Îµ

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Substrate Fluidity Regulates Cell Adhesion and Morphology on Poly(#-caprolactone)-based Materials Koichiro Uto, Sharmy S Mano, Takao Aoyagi, and Mitsuhiro Ebara ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00058 • Publication Date (Web): 08 Feb 2016 Downloaded from http://pubs.acs.org on February 13, 2016

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ACS Biomaterials Science & Engineering

Substrate Fluidity Regulates Cell Adhesion and Morphology on Poly(ɛ-caprolactone)-based Materials Koichiro Uto†, Sharmy S. Mano, Takao Aoyagi and Mitsuhiro Ebara* Biomaterials Unit, International Center for Materials Nanoarchitectonics (WPI-MANA), National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba, Ibaraki, 305-0044, Japan.

ABSTRACT: Although mechano-structural signals from the surrounding matrix have been known to regulate cell functions, the effects of substrate fluidity are poorly understood. Here, we demonstrate that the adhesion and morphology of cells are regulated by the fluidity on widely used biodegradable polymer substrates, rather than the substrate elasticity. We have designed cell culture films with different elasticity and fluidity using poly(ɛ-caprolactone-co-D,L-lactide) (CL-DLLA). The elasticity was successfully controlled by adjusting the amorphous-crystal phase transition temperature (Tm) of CL-DLLA without changing the surface wettability; i.e., the CL-DLLA displays more viscous (liquid-like) behavior at 37oC with increasing DLLA contents. The fluidity was varied by chemically crosslinking the polymer networks. This CL-DLLA system was used to test the effect of variations in a substrate’s fluidity on cell behavior. Differences were observed in adhesion, spreading and morphology of NIH 3T3 fibroblasts. Increasing the fluidity decreased cell spread area but enhanced the formation of spheroids. Although direct comparison of the elastic modulus between crosslinked and non-crosslinked samples are difficult, it was found that the substrate stiffness produced little changes in cell spread area, indicating that cells sense more dynamic nature of their surrounding environment. These findings will serve as the basis for new development of tissue engineering scaffolds and engineered stem cell niche as well as investigation of dynamic effects of mechano-structural stimuli on cell fate.

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KEYWORDS: viscoelasticity, fluidity, semicrystalline polymer, temperature-responsive polymer, mechanobiology INTRODUCTION There is growing recognition that mechano-structural stimuli from the surrounding microenvironments of cells crucially influence cellular functions.1-5 Especially, recent reports have revealed that elasticity,6, 7 topography8-10 or geometry11, 12 of the matrix can direct stem cell fate. Substrate stiffness, for example, has been demonstrated to be a key control parameter in the mechanotransduction signaling pathways by assembling and reassembling focal adhesions, and up- and down-regulating cell adhesion molecules that are associated with cell-cell and cell-extracellular matrix (ECM) interactions.13-16 It was shown that cells seeded on synthetic hydrogels, the modulus of the substrate can influence their fate, even in the absence of soluble differentiation factors.7, 17 Although many studies have investigated the mechanisms that cells use to sense and respond to their mechanical environment, however, the role of the fluidity is less well known. For hydrogel systems, the elastic modulus has been generally controlled by varying the crosslinking density within the gel.18-20 The crosslinking density of hydrogels, however, influences not only rigidity but also the fluidity and swelling properties. Moreover, covalently crosslinked hydrogels may be able to regard as purely elastic materials since it only exhibit chemically stable bonds,21 but it is exactly viscoelastic materials being composed of both elastic and viscous natures. Cameron et al. have recently reported the effect of viscous modulus of polyacrylamide (PAAm) substrate on differentiation of mesenchymal stem cells (MSCs), because most of the researches have mainly focused on the effects of elastic aspect of substrate despite the most synthetic substrates are viscoelastic materials.22 They clearly demonstrated the viscous modulus of substrates also influences MSC differentiation, and that variations in the viscoelasticity of polydimethylsiloxane (PDMS) may also influence epithelial sheet movement.23 Trappmann et al. have investigated how substrate stiffness influences the differentiation of MSCs and human epidermal stem cells using a series of PDMS substrates of different stiffness with a covalently


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attached collagen coating.24 Interestingly, cell spreading and differentiation were unaffected by PDMS stiffness but regulated by the elastic modulus of PAAm. This is because cells are more sensitive to how ECM molecules are tethered to a substrate; i.e. when the collagen is more loosely bound, it cannot provide the mechanical feedback that the integrin complex requires to cluster in focal adhesions and signal through extracellular-signal-related kinase (ERK)/mitogen-activated protein kinase (MAPK). These observations indicate that elastic modulus cannot fully account for the different cell responses to different substrates. While tissue engineering research have recently focused on the micromechanical properties of a scaffold and its effects on cells, it is yet unclear how the mechanical properties of widely used biodegradable polymers such as poly(ɛ -caprolactone) (PCL),25,

26

poly(glycolic acid) (PGA)27 and

poly(lactide-co-glycolic acid) (PLGA)28, 29 influence cell behavior. Since the need to tailor the surface chemistry of an implantable material has gained significant attention for decades,30, 31 the mechanical properties of an implantable material has to be also considered for the new development of an implantable material. From this regard, we have studied how viscoelastic properties of PCL-based scaffold influence cell spreading and morphology using planner films with different elasticity and fluidity but similar surface wettability. We first synthesized four-armed poly(ɛ-caprolactone-co-D,L-lactide) (P(CL-co-DLLA)) by ring-opening copolymerization of CL and DLLA. The obtained copolymers were then reacted with acryloyl chloride to introduce vinyl groups at the end chains. The elasticity was successfully controlled by adjusting the CL/DLLA composition. The fluidity was controlled by chemically crosslinking the functionalized end chains. This CL-DLLA system was used to test the effect of variations in a substrate’s fluidity on cell behavior (Figure 1). Figure 1. Schematic illustrations of cell adhesion behavior on crystalline and non-crystalline substrate with or without crosslinking.



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Elastic (crosslinked)

Fluidic (non-crosslinked)

Crystalline (below Tm)

Non-crystalline (above Tm)

Materials and Methods ɛ-Caprolactone (CL) was purchased from Tokyo Kasei (Tokyo, Japan), and purified by distillation over calcium hydride (Wako Pure Chemical Industries, Tokyo, Japan) under reduced pressure. Pentaerythritol and acryloyl chloride were purchased from Tokyo Kasei and used as received. D,L-lactide (DLLA) was kindly supplied by Musashino Chemical Laboratory (Tokyo, Japan) and recrystallized twice from ethyl acetate before use. Triethylamine was purchased from Wako Pure Chemical Industries, Ltd., and dehydrated by distillation over potassium hydroxide. Tin octanoate and other chemicals were also purchased from Wako Pure Chemical Industries, Ltd. Benzoyl peroxide (BPO) was purchased from Sigma (St. Louis, MO, USA) and used as received. The quartz crystal microbalance (QCM) electrode (frequency = 9 MHz, AT-cut) was purchased from SEIKO EG&G Co., Ltd (Tokyo, Japan). Synthesis of four-armed P(CL-co-DLLA) macromonomers Four-armed copolymers with different CL and DLLA compositions were synthesized by ring-opening copolymerization from terminal hydroxyl groups of pentaerythritol using tin octanoate as a catalyst according to our previous reports.32-35 Acryloyl chloride was then reacted with the end of the branched chains to introduce the crosslinkable functional group. Preparation of P(CL-co-DLLA) films The non-crosslinked P(CL-co-DLLA) films were prepared by a melting press method (Figure S1 in the supporting information). The powder of P(CL-co-DLLA) with hydroxyl end group was placed on a glass


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surface and incubated at 60 oC for 1 h. The molten P(CL-co-DLLA) on the glass plate was then covered with a teflon sheet and pressed with another glass plate for 15 min. The temperature was immediately cooled down to 10 oC (below crystallization temperature; Tc 20 oC) and maintained for another 30 minutes. The film having a 100 µm thick was obtained by peeling the teflon sheet off. To prepare crosslinked P(CL-co-DLLA) films, four-armed macromonomers (45 wt%) and BPO (1.5 wt%) were dissolved in xylene and cured between two glass plates with a 200 µm thick teflon spacer for 3 h at 80 oC. Cell culture on the films Prior to cell culture, the non-crosslinked and crosslinked P(CL-co-DLLA) films were immersed in PBS and equilibrated in a 37°C incubator for 2 h. For fibronectin coating, the surface of the films were coated with 10 µg mL-1 fibronectin (Sigma-Aldrich) and equilibrated in a 37°C incubator for 2 h. NIH 3T3 fibroblasts (ATCC, Manassas, VA, USA) were then seeded on the films at densities varying in a range from 1.0 x 104 cells cm-2 to 10 x 104 cells cm-2 and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma-Aldrich) in the presence of 10% fetal bovine serum (FBS) (EQUITECH-BIO. INC., Kerrville, TX, USA) and 1 % antibiotic-antimycotic (Gibco Invitrogen, Carlsbad, CA, USA) at 37°C. Characterizations The structures and molecular weights of synthesized P(CL-co-DLLA)s were estimated by 1H NMR spectroscopy (JEOL, Tokyo, Japan) and gel permeation chromatography (JASCO International, Tokyo, Japan) respectively. The thermal property of P(CL-co-DLLA)s were characterized by DSC (DSC6100, Seiko Instruments, Chiba, Japan). The measurements were conducted from 0 to 120 oC at the heating rate of 5 oC min-1. Viscoelastic properties of non-crosslinked samples were tested using a rheometer (MCR 301, Anton Paar, Tokyo, Japan) with parallel plate geometry (rotating top plate of 10 mm diameter). The viscoelastic spectrum (storage modulus, G’ and loss modulus, G’’) as a function of temperature and angular frequency were obtained. The mechanical properties of crosslinked or crystallized noncrosslinked samples (10 mm wide by 0.2 mm thick by 30 mm long, rectangular shaped) were characterized by a



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tensile tester (EZ-S 500N, Shimadzu, Kyoto, Japan) equipped with a thermo chamber (Chromato chamber M-600FN, TAITEC, Saitama, Japan). The tensile tests were carried out with the elongation rate of 10 mm min-1 at 37 oC, and the elastic modulus of samples was calculated from the slope of linear region of the stress-strain curve, which appears just after toe region. Contact angles on the samples were determined from the shapes of water droplets. Water droplet (5 µL) was mounted onto each substrate and measured the contact angle at 37 oC in air. To characterize the stability of non-crosslinked fluidic substrate in aqueous environment, quartz crystal microbalance (QCM) technique was conducted. The non-crosslinked P(CL-co-DLLA) was spin-coated on the QCM substrate and the frequency changes in PBS and acetone were monitored by frequency counter (QCA917, SEIKO EG&G Co., Ltd, Tokyo, Japan). The fluidity of non-crosslinked P(CL-co-DLLA) substrate was continuously monitored and imaged using a phase contrast microscope (Olympus IX71, Tokyo, Japan). To determine the cell morphology on the substrate, cells were fixed with 4 % paraformaldehyde (PFA) (Sigma-Aldrich) and treated with rhodaminephalloidin (Sigma-Aldrich) and 4’,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) to stain F-actin and nucleus respectively. To stain vinculin, the samples were incubated with primary antibody (vinculin produced in mouse; Sigma-Aldrich) for 1 h at room temperature, and stained with goat anti-mouse Alexa Fluor 488 secondary antibody (Invitrogen) for 1 h at room temperature. To visualize vinculin, confocal laser scanning microscopy (CLSM) was performed using a Zeiss LSM510 microscope (Carl Zeiss, Oberkochen, Germany). Alamar blue® assay was used to study the adhesion rate of fibroblast to crosslinked and non-crosslinked non-crosslinked P(CL-co-DLLA)s substrates with or without fibronectin coating. Fluorescence intensity (590 nm emission) was measured by an ARVO MX1420 mutilabel counter (Perkin Elmer, Waltham, Massachusetts, US). The images were analyzed by Image J software to quantify the adhesion area of NIH3T3 fibroblasts, the diameter and surface coverage of fibroblast spheroid. Results and Discussion Preparation of crosslinked and non-crosslinked P(CL-co-DLLA) films


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PCL is an important class of the biocompatible and biodegradable synthetic polymers, which has been approved for biomedical applications by the US Food and Drug Administration (FDA). PCL is a semicrystalline polymer that has a melting temperature (Tm) over which the mobility of polymer chains changes dramatically. Therefore, the mechanical property can be tuned by changing the temperature. However, the relatively high Tm of PCL (~60 oC) limits the potential use of PCL in biological conditions. To overcome this shortcoming, we have previously reported two different approaches to controlling the Tm of PCL by tailoring the branched numbers10, 36 or incorporating non-crystalline segments such as DLLA.32-35 In this study, P(CL-co-DLLA) copolymers were used because it shows an elastic modulus in hundreds kPa range above its Tm. Four-armed P(CL-co-DLLA) copolymers with various CL/DLLA ratios were synthesized by ring- opening polymerization by pentaerythritol. The copolymer properties are summarized in Table 1. The DLLA contents of the copolymers were determined by 1H-NMR as 29 and 39 mol% DLLA when the feed concentrations were 30 and 40 mol%, respectively. These copolymers were abbreviated as CL-DLLA 71/29 and 61/39, where X and Y in CL-DLLA X/Y are mol% of CL and DLLA, respectively. The molecular weights of these copolymers determined by GPC were almost consisted with the target molecular weight of 100 monomer units in each arm. They possess relatively narrow molecular weight distribution (Mw/Mn = 1.34 and 1.57 for CL-DLLA 71/29 and CL-DLLA 61/39, respectively). These results indicate that copolymers with the desired branch number, molecular weights, and compositions of CL and DLLA have been successfully obtained. Table 1. Characterization and thermal properties of four-branched P(CL-co-DLLA) derivatives.



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The obtained copolymers were then reacted with acryloyl chloride to introduce vinyl groups at the end chains. The end-functionalized macromonomers were finally crosslinked between glass plates to obtain planner films. Figure S2 (supporting information) compares the thermal properties of CL-DLLA 71/29 and 61/39 before and after crosslinking. The Tm of CL-DLLA 71/29 was found at 48 ± 2 oC, whereas that of CL-DLLA 61/39 was at 40 ± 1 oC. The melting enthalpy (∆H) also decreased when DLLA content increased. This trend was also observed in the macromonomer and crosslinked films. The Tm was shifted to lower temperature for CL-DLLA 71/29 (38 ± 1 oC) or disappeared for CL-DLLA 61/39 after crosslinking. This is because crosslinking can prevent polymer segments from crystallization. Figure 2 shows the photographs of films at 37 oC. The CL-DLLA 71/29 is opaque since it is in crystalline state at 37 oC. But, of particular interest is that non-crystalline CL-DLLA 71/29 at 37 oC can be also prepared by thermal annealing because the crystallization temperature (Tc) was lower than 20 oC. The annealed CLDLLA 71/29 showed highly transparent. Figure 2. Photographs of (left) crosslinked and (right) non-crosslinked P(CL-co-DLLA) films with different CL/DLLA compositions. The amorphous CL-DLLA 71/29 is also obtained by annealing crosslinked CL-DLLA 71/29 at 42 oC.


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Mechanical properties of crosslinked and non-crosslinked P(CL-co-DLLA) films To provide insight into the mechanical properties of the non-crosslinked films, rheological characterization was carried out. Figure 3 shows the storage modulus (G’), loss modulus (G”), and tanδ (G”/G’) curves for non-crosslinked CL-DLLA 71/29 and 61/39 as a function of temperature. For noncrosslinked CL-DLLA 71/29, both G’ and G” were nearly constant below the Tm and declined sharply around 43°C. (Figure 3A) G’ is higher than G” at 37 oC and the crossover of the G’ and G” was observed around 47 oC. For non-crosslinked CL-DLLA 61/39, on the other hand, both G’ and G” were lower than those of CL-DLLA 71/29 and the crossover of the G’ and G” was observed around 32 oC for CL-DLLA 61/39 (Figure 3B). In other words, the G” of the CL-DLLA 61/39 is higher than the G’ at 37 oC, indicating that it is viscous liquid at 37 oC. To further investigate the viscoelastic properties of noncrosslinked substrates under the cell culture condition of 37 oC, angular frequency test was performed on the non-crosslinked CL-DLLA 71/29 and 61/39, where the frequency was increased from 0.1 rad sec-1 to 100 rad sec-1 at a strain of 0.1%. As expected, the viscoelastic behavior against frequency sweeps at 37 oC



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was significantly different between two samples. CL-DLLA 71/29 shows no angular frequency dependency in both of G’ and G” that the G’ is always higher than the G”, while those of CL-DLLA 61/39 are increased with increasing angular frequency that G” is higher than the G’ (Figure 4). These results strongly supported that the viscoelastic property can be tuned by the CL/DLLA composition, resulting in ‘solid (G’ > G’’)’ or ‘viscous liquid (G’ < G’’)’ like behavior at 37 oC.

Figure 3. Temperature dependent viscoelastic property of non-crosslinked P(CL-co-DLLA)s: Storage modulus (G’, closed circles), loss modulus (G”, open circles) and tanδ (G”/G’, open triangles) as a function of temperature for (A) CL-DLLA 71/29 and (B) CL-DLLA 61/39.


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Figure 4. Angular frequency dependence of storage modulus (G’, closed circles), loss modulus (G”, open circles) and tanδ (G”/G’, open triangles) at 37 oC for (A) non-crosslinked CL-DLLA 71/29 and (B) CL-DLLA 61/39.



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Because living cells are bathed in an aqueous medium, the stability and fluidic nature of noncrosslinked CL-DLLA 61/39 were investigated under the aqueous condition. First, we observed the dynamic fluidic nature in PBS at 37 oC by optical microscopy (Figure S3 in the supporting information). The scratch totally disappeared after 10 min, suggesting that non-crosslinked CL-DLLA 61/39 behave as liquid-like substrate in cell culture conditions. Next, the stability of non-crosslinked CL/DLLA 61/39 in the aqueous solution was investigated to confirm whether non-crosslinked polymers could elute into the solution. To analyze the weight loss preciously, the QCM measurement was conducted because the


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accuracy is in nano-gram scale. The results are summarized in Table S1 (supporting information). The polymer dissolved immediately in acetone which is a good solvent for CL-DLLA. But, the polymer showed good stability in PBS for 24 h (> 94 %) regardless of the thickness. These results suggested that the non-crosslinked CL-DLLA 61/39 is stable in water, even though it dynamically flows in aqueous environment imitating the cell culture conditions. The mechanical property of crosslinked CL-DLLA was also characterized by tensile test. In tensile test, we observed the small toe region at early stage of stress-strain curve (within around 10% of strain), suggesting the crosslinked CL-DLLA was not perfectly elastic but was viscoelastic. Therefore, Young’s modulus was determined from the slope of a linear region of stress-strain curve appeared after small toe region. The Young’s modulus (E) for each sample at 37 oC was summarized in Table 2. The crosslinked CL-DLLA 71/29 and 61/39 show the E values around 9,000 kPa and 260 kPa at 37 oC, respectively. The E of annealed CL-DLLA 71/29 was very similar to that of CL-DLLA 61/39 because it became amorphous by annealing. Interestingly, the non-crosslinked 71/29 exhibits the highest E value of 38500 kPa. This can be explained by the highest ∆H value among the samples tested as shown in Table 1. That is, the noncrosslinked 71/29 possesses more crystalline regions. Although direct comparison of the E between solid and liquid samples is difficult, we have also calculated the E values of non-crosslinked CL-DLLA 61/39 from the equation as follow;22

where

is a Poisson’s ratio and G’ and G” represents storage modulus and loss modulus (we adopted the

values at frequency of 0.1 rad sec-1), respectively. Since the Poisson ratio of PCL-based materials has been reported to range between 0.3-0.47,37-39 the E values of non-crosslinked CL-DLLA 61/39 can be approximately 1.5 kPa.



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Table 2. Mechanical properties of crosslinked and non-crosslinked P(CL-co-DLLA) substrates.

Effects of elasticity and fluidity on cell behavior To investigate the response of NIH3T3 fibroblasts to the substrate elasticity and fluidity, cells were cultured on CL-DLLA films at 37 oC for 24 h. All samples have similar surface wettability regardless of the compositions or crystallinity (Figure S4 in the supporting information). Although cell can attach and proliferate on the surface without fibronectin coating because the CL-DLLA films are relatively hydrophobic, we pre-coated the surface with fibronectin to improve an initial cell attachment. This treatment also improved cell spreading. We have compared the effect of fibronectin coating on the cell attachment (Figure S5 in the supporting information). Cell adhesion was significantly improved with fibronectin coating. Figure 5 shows immunostained images of adhered cells on CL-DLLA 71/29 and 61/39 with and without crosslinking. Cells adhered and spread well to the surface of crosslinked CLDLLA 71/29 (E =9100 kPa) (Figure 5A). Cells also spread well to the annealed CL-DLLA 71/29 even though the E value is 277 kPa (Figure. 5B). Cell morphologies on crosslinked CL-DLLA 61/39 were also very similar to those on the annealed CL-DLLA 71/29 (E =261 kPa) (Figure 5D). On the other hand, the average projected area of cells on non-crosslinked CL-DLLA 71/29 was slightly smaller than that on the crosslinked one (Figure 5C), nevertheless of the highest E values among the samples tested. Since it is widely accepted that the projected cell area is generally increased as substrate rigidity increased,40, 41 this result is dissimilar to previously observed dependency. In general, molecular motion and mobility at the


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liquid-solid interface of polymeric materials are higher than those in the bulk region.42 Such an enhanced mobility of polymer at the interface has been observed in the scale of approximately 10 nm down from the outermost surface. Therefore, it is plausible that the mobility of polymer chains at the outermost surface of the non-crosslinked CL-DLLA 71/29 can be also enhanced nevertheless of the highest E values. Significant difference in average cell spread area was observed between crosslinked and noncrosslinked CL-DLLA 61/39. Although cells attached to the non-crosslinked substrate, they remained round on the fluidic surface (Figure 5E). This is because the traction force by cells was dissipated due to the liquid nature of the non-crosslinked CL-DLLA 61/39. Figure 5. Fluorescent microscope images (scale bar = 100 µm) of NIH3T3 fibroblasts after 24 h culturing on the CL-DLLA 71/29 (A, B, and C) and 61/39 (D and E). (A and D) crosslinked, (B) annealed, and (C and E) non-crosslinked.

To further understand the difference of adhesion morphology depended on the substrate property observed in NIH3T3 fibroblasts, actin stress fibers as well as focal adhesion proteins were visualized



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using CLSM (Figure 6). Cells on crosslinked substrate were observed both stably formed focal adhesions and developed actin stress fiber formation (Figure 6A). Cells on non-crosslinked substrate hardly recruited vinculin to adhesion sites with increasing liquid nature of substrates (Figure 6B-C). Therefore, the vinculin recruitment followed by stress fiber formation is regulated by viscoelastic property of substrates, and the cells could not form the stable focal adhesion and stress fibers on it. Figure 7 plots the average projected cell area on each sample against the elastic modulus. This result does not correlate with previously observed results which demonstrated the increased in cell spread area as substrate rigidity increased,40, 41 suggesting that the cells are significantly more sensitive to fluidic motion of a substrate than elastic modulus, especially for relatively stiff biodegradable scaffolds. To our best knowledge, this study describes for the first time how viscoelastic properties of widely used biodegradable scaffold influence cell spreading and morphology. We believe that our finding highlights the importance of considering matrix fluidity as a fundamental property of the ECM that is critical to understand the basics of cell-ECM interactions. Figure 6. Confocal laser scanning microscope images (scale bar 50 µm) of NIH3T3 fibroblasts after 24 h culturing on (A) crosslinked CL-DLLA 61/39, (B) non-crosslinked CL-DLLA 71/29 and (C) 61/39. The cells were stained for F-actin (red), vinculin (green), and nuclei (blue).


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Figure 7. Relationship between elastic modulus of substrates and projected cell areas on crosslinked (closed) and non-crosslinked (open) CL-DLLA 71/29 (triangles) and 61/39 (circles) after 24 h of cultivation.

Spheroid formation on the fluidic substrate Platforms for 3D cell culture systems has recently attracted much attention because 3D in vitro models are more advantageous for various biological or medical assays including drug screenings for cancer chemotherapy.43 Among them, culturing cells into dense aggregations, so called spheroids, has been reported in several types of cells using a variety of substrates in specified conditions.44, 45 Spheroids show not only morphological but also functional similarities to tissues and organs, unlike the conventional 2D culture of cells. While the need to tailor the surface chemistry for the spheroid formation has been given significant attention for decades,46, 47 we utilized the fluidic substrate to induce the formation of spheroid without use of surface modifications. Figure 8 shows the immunostained images of adhered NIH3T3 fibroblasts on the non-crosslinked CL-DLLA 61/39 after 3 days of cultivation at various seeding density. When cells were seeded at density of 1.0 x 104 cells cm-2, the smaller aggregates were observed (Figure 8A). The diameter of cell aggregates increased with increase of the seeding density (Figure 8B-C). As shown in Fig.8D, there is a linear relationship between initial seeding density and spheroid size. The



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average size for the spheroid at day 3 for 10,000, 50,000, 100,000 cells/cm2 were 25.6 µm, 53.8 µm, and 78.4 µm, respectively. The % of confluency obtained by Image J were 5.6%, 13.0%, and 17.0%, respectively (Figure S6 in the supporting information). These data strongly suggest that the size and surface coverage of spheroids can be easily controlled by the seeding density. As shown in Fig.S7 in the supporting information, cells did not aggregate after 1 day of seeding at lower density. However, cells formed spheroid after 1 day when they are seeded at higher density. This result strongly suggests that spheroid formation depends on initial cell seeding density. The formation of fibroblast spheroids is mediated by a balance between two competing forces, that is, the interactions of cell-substrate and cellcell.48 When cell-substrate interaction is sufficiently strong compared to cell-cell interaction, the cells would stably attach and well spread on the substrate. In contrast, in the case that cell-cell interaction exceeds cell-substrate interaction, cells would aggregate into spheroids. Therefore, surface modification with hydrophilic polymers has been extensively explored.45,46 The proposed system, however, does not require the surface modification or patterning to modulate the adhesive ability between the cells and substrate. We have achieved the spheroid formation by the mechanical properties of a substrate, especially viscous fluidity in this study. Figure 8. Spheroid formation of NIH3T3 fibroblasts on non-crosslinked CL-DLLA 61/39 (scale bar = 100 µm). The fibroblasts were seeded on the fluidic substrates at the densities of (A) 1.0 x 104, (B) 5.0 x 104 and (C) 10.0 x104 cells cm-2 and cultured for 3 days at 37 oC. (D) The relationship between diameter of formed spheroids and seeding densities (**p < 0.01, t-test). Each point on the graph is a mean ± SD of 20-30 different aggregations.


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CONCLUSIONS This study elucidates the effects of substrate fluidity on cell responses using widely used biocompatible P(CL-co-DLLA) substrates with tunable mechanical properties. We have prepared crosslinked and noncrosslinked P(CL-co-DLLA) substrates by thermal polymerization of crosslinkable moiety at end groups and a melt press method, respectively. The non-crosslinked substrate with 39% of DLLA showed the liquid-like behavior at 37 oC. No significant effects of substrate elasticity on cell spreading have been observed on the samples tested. Increasing the fluidity, however, decreased cell spread area but enhanced the formation of spheroids. We believe that our finding will have a major impact on the design of synthetic cell culture substrates for the fields of not only tissue engineering but also basic cell biology.

ASSOCIATED CONTENT Supporting Information. The following files are available free of charge. Filename: brief description Filename: brief description

AUTHOR INFORMATION



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Corresponding Author * Dr. Mitsuhiro. Ebara, Fax: +81-29-860-4708; Tel: +81-29-860-4179; E-mail: [email protected] Present Addresses † Department of Bioengineering, University of Washington, 3720 15th Ave NE, Seattle, WA 98195, United States

ACKNOWLEDGMENT This research is partially granted by the Japan Society for the Promotion of Science (JSPS) through the “Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program),” initiated by the Council for Science and Technology Policy (CSTP).

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Table of Contents (TOC) Graphic. Substrate Fluidity Regulates Cell Adhesion and Morphology on Poly(e-caprolactone)-based Materials Koichiro Uto, Sharmy S. Mano, Takao Aoyagi, Mitsuhiro Ebara


Substrate Fluidity Regulates Cell Adhesion and Morphology on Poly(Îµ

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