Royal Society Publishing

Time-lapse observation of cell alignment on nanogrooved patterns

Satoshi Fujita, Masahiro Ohshima, Hiroo Iwata

Abstract

Cells elongate on a surface with nanogrooved (NG) patterns and align along that pattern. Although various models have been proposed for how this occurs, much remains to be clarified. Studies with fixed cells do not lend themselves to answering some of these open questions. In this study, the dynamic behaviours of living mesenchymal stem cells on an NG substrate with a 200 nm groove depth, an 870 nm ridge width and a 670 nm groove width were observed using time-lapse microscopes. We found that filopodia moved as if they were probing the surroundings of the cell protrusion, and then some cell protrusions invaded the probed areas. Cell protrusions that extended perpendicular to the NG direction tended to retract more rapidly than those parallel to the grooves. From these facts, we think that the retracting phase of cell protrusions play a rule in cell alignment along the NG patterns.

1. Introduction

A large number of studies have examined the effects on cell behaviours of surface characteristics, such as wettability, electrostatic charges and functional groups (Witt et al. 2004; Navarro et al. 2006). These studies form the basis for the development of materials for artificial organs, disposable medical devices and cell culture substrates. In the last decade, the effects of surface micro- and nanotextures on cell adhesion and cell morphology have attracted much attention (Clark et al. 1990; Curtis & Wilkinson 1997; Flemming et al. 1999; Teixeira et al. 2006; Lim & Donahue 2007). Substrates with fine grooves induce various cell responses, such as cell alignment and migration along the grooves (Dalton et al. 2001; Diehl et al. 2005; Su et al. 2007), and, moreover, differentiation of stem cells (Charest et al. 2007). Most researchers have assessed cell alignments after cells have been fixed in formaldehyde, methanol or glutaraldehyde. Although those static observations are useful for visualizing intracellular protein fibre alignment in detail, they provide scarce information about dynamic cell behaviours on surfaces with nanopatterns. Studies are needed on dynamic cell behaviours in an acute phase and are expected to give more detailed information on the mechanism of cell alignment on nanogrooved (NG) patterns.

Epithelial cells and fibroblasts have been used to examine cell responses to substrate topography (Lim & Donahue 2007). In this study, we used mesenchymal stem cells (MSCs). MSCs are multipotent stem cells that can differentiate into osteoblasts, adipocytes, chondrocytes and other kinds of mesenchymal cells (Pittenger et al. 1999). It has been reported that the morphology of MSCs can determine their fates, such as proliferation or differentiation direction (McBeath et al. 2004; Engler et al. 2006). In addition, the morphology of MSCs is affected by some physicochemical properties and topography of cell culture substrates (Park et al. 2004; Zhu et al. 2005; Kurpinski et al. 2006; Dalby et al. 2007). Thus, we expected that the differentiation direction of MSCs can be controlled by the nanostructure of a substrate. It is meaningful to study the effect of topography of cell culture substrates on the morphology of MSCs.

In this research, NG patterns were printed on polycarbonate (PC) plates using supercritical CO2-assisted embossing, as previously reported (Fujita et al. 2008), and the dynamic behaviours of MSCs on NG patterns were observed under 5 per cent CO2 and at 37°C using time-lapse microscopes. Time dependence of cell alignment, cell protrusion movements and their remodelling were analysed to infer the mechanism of the topographical effect of the substrate on cell responses.

2. Material and methods

2.1 Fabrication of the nanogrooved substrate

An NG pattern (200 nm groove depth, 870 nm ridge width and 670 nm groove width; scheme 1a) was printed on PC plates using a supercritical CO2-assisted embossing machine, as reported previously (Fujita et al. 2008). In brief, the vessel of the embossing machine was separated into two chambers by a partition. An under pedestal could move between the two chambers through a partition, as a syringe. A disc-shaped PC plate (diameter=20 mm; thickness=3 mm; bisphenol-A type; Mw=58 000; Tg=153°C; Tsutsunaka Plastic Industry Co. Ltd., Osaka, Japan) was placed on top of the pedestal. A nickel mould (supplied by Hitachi Maxell, Ltd., Tokyo, Japan) was set on the ceiling of the upper chamber.

The mould and the PC plate were cleaned by spraying with fluorocarbon gas. The upper chamber was filled with CO2 gas to 10 MPa, and the chamber temperature was increased to 100°C to achieve the CO2 supercritical condition. The surface of the PC plate was plasticized by dissolution of CO2 under the supercritical condition for 5 min. Then, CO2 was released from the upper chamber, and immediately the pressure of the lower chamber was increased by the introduction of the CO2 gas to lift the pedestal. The PC plate was pressed to the nickel mould at 12 000–15 000 N for 30 s at 100°C. After stamping, CO2 was completely released from both chambers and the plate was detached from the mould.

Scheme 1

(a) Dimensions of the NG pattern. (b) Terminologies used in this study. (c) Schematic of accumulated distance and Euclidean distance of movements of the leading edge of the cell protrusion.

2.2 Cell culture

Human bone marrow-derived MSCs (Lonza, Basel, Switzerland) were maintained in Dulbecco's modified essential medium (DMEM; Invitrogen Corp., Carlsbad, CA, USA) supplemented with 10 per cent foetal bovine serum (FBS; BIOWEST, France), 100 U ml−1 penicillin and 100 μg ml−1 streptomycin (Invitrogen) at 37°C under 5 per cent CO2 in a humidified atmosphere, and subcultured at 5×103 cells per cm2 every 3–4 days. Cells at passages 3–8 were used for experiments.

To give cell adherent property to PC plates, they were treated with 10 μg ml−1 of fibronectin (Invitrogen) for 2 hours at 37°C (Keselowsky et al. 2004), and washed five times with Dulbecco's phosphate-buffered saline Ca2+ Mg2+ free (DPBS(−); Nissui Pharmaceutical Co. Ltd., Tokyo, Japan). Prior to cell seeding on the fibronectin-coated plates, uniformity of fibronectin coating was examined by immunostaining (electronic supplementary material, figure 1b). MSCs were treated with 10 μg ml−1 mitomycin C (Wako Pure Chemical Industries, Ltd., Osaka, Japan) for 2 hours to stop cell division, because it is hard to see cell morphology when cell division occurs during a long-term tracking. After the mitomycin C treatment, cells were washed thrice with DPBS(−). The MSCs were seeded at 5×103 cells per cm2 on fibronectin-coated plates and cultured for 4 days in the maintaining medium (DMEM/10% FBS).

Figure 1

Low-magnification time-lapse images of MSCs seeded on the NG pattern. The NG lines were parallel to the x-axis. Recording under the time-lapse microscope started from 20 min post-seeding. Scale bar, 200 μm. See also the electronic supplementary material, movie 1.

2.3 Time-lapse video microscopy

For time-lapse imaging, 1.5×104 cells of MSCs, which were treated with 10 μg ml−1 mitomycin C and suspended in 300 μl of medium, were seeded onto a fibronectin-treated plate, placed in a 35 mm culture dish and then statically incubated at 37°C under 5 per cent CO2 for 60 min for adhesion. Then, after the addition of 2 ml medium, the dish was stored in a gas-tight container with capnophilic powder, which consists mainly of ascorbic acid (CulturePal; Corefront Corp., Tokyo, Japan), to maintain 5 per cent CO2 during observation. For an analysis of cell orientation, cell images were captured every 2.5 min for 5 days using an inverted-phase contrast microscope (Cellwatcher; Corefront).

For visualization of cells under high magnification, a plate carrying MSCs was placed upside down with cells facing a culture dish on a silicone spacer (thickness, 0.5 mm). Cells could be observed under high magnification with an inverted microscope (Biostation IM; Nikon Corp., Tokyo, Japan). For visualization of the extension of cell protrusions, cell images were recorded every 2.5 min for 7 hours. For investigating filopodial movements, time-lapse images were captured every 4 s for 2 hours. Captured images were stacked by ImageJ (v. 1.39f, distributed by NIH) and converted to windows media video format.

2.4 Quantification of orientation angle

Cell orientation angles were quantified by the analysis of immunostained images or low-magnification time-lapse phase contrast images using ImageJ. The orientation angle of an individual cell was determined as the angle between the groove direction and the direction of the longer axis of the approximated ellipse, which approximates to the cell shape (scheme 1b). The distribution of cell orientation was estimated by a wrapped normal distribution, as previously reported (Dakin et al. 2005; Bashur et al. 2006; Agostinelli 2007). Briefly, the probability distribution function was adapted from Fisher (1993) for a periodicity of π radians as follows:Embedded Image(2.1)Embedded Image(2.2)andEmbedded Image(2.3)where ρ is the mean resultant length and μ is the mean angle. These parameters were determined from a set of n measured cell orientation angles, θi. The angular standard deviation (s.d.), σ, for the distribution was determined by the following equation:Embedded Image(2.4)

2.5 Tracking of cell protrusions

The leading edges of cell protrusions were tracked through all of the captured images using the MTrackJ plug-in (v. 1.2.0) and Chemotaxis and Migration Tool plug-in (v. 1.01, distributed by ibidi GmbH, München, Germany) for ImageJ. The position of each leading edge was plotted in the coordinate axis, where the origin was the position where each edge first appeared and the x-axis was parallel to the grooves (scheme 1b). Tracking was terminated when the tip of the cell protrusion disappeared or recording ended. The distribution of protrusions was quantitatively compared by the s.d. of the angle between the vector of the endpoint of cell protrusion and x-axis calculated from equation (2.4). From the start point to the endpoint of each leading edge, an accumulated distance and the Euclidean distance were calculated as defined in scheme 1c. The velocity of a leading edge of a cell protrusion was defined as an accumulated distance of each edge divided by time (scheme 1c). The directionality of a leading edge of a cell protrusion was defined as given in the following equation:Embedded Image(2.5)

2.6 Immunostaining

Cells were stained with F-actin and vinculin. Cultures were fixed with 4 per cent paraformaldehyde (Nacalai Tesque, Inc., Kyoto, Japan) for 15 min, permeabilized with 0.2 per cent Triton-X (Wako) for 3 min, blocked with 2 per cent skimmed milk (Nacalai) for 1 hour at room temperature and incubated with anti-mouse vinculin (Chemicon, CA, USA) (1 : 200 dilution) overnight at 4°C. Then, they were washed with 0.05 per cent polyoxyethylene sorbitan monolaurate (Tween 20, Wako, Osaka, Japan) for 15 min thrice at room temperature and treated with Alexa-594-conjugated phalloidin (Invitrogen) (1 : 40 dilution) for F-actin staining, Hoechst 33342 (Dojindo, Kumamoto, Japan) (1 : 1000 dilution) for nucleus staining and Alexa-488-conjugated mouse anti-IgG (Invitrogen) (1 : 500 dilution) for vinculin staining, for 30 min at room temperature. Stained cultures were mounted on slides with a light anti-fade reagent (Vectashield, Vector Laboratories, Burlingame, CA, USA) and observed using a fluorescence inverted microscope (IX71, Olympus, Tokyo, Japan).

2.7 Statistical analysis

Comparisons between two groups were made using Student's t-tests. p<0.05 was considered statistically significant. All statistical calculations were performed using the software JMP v. 5.1.1.

3. Results

3.1 Cell alignment

In a previous study, we observed contact guidance of MSC alignment on NG substrates when the NG depth was more than 90 nm (Fujita et al. 2008). In this study, NG plates with 200 nm groove depth, 870 nm ridge width and 670 nm groove width were employed (scheme 1a and the electronic supplementary material, figure 1a). Dynamic cell behaviours on the NG plate were observed using time-lapse microscopes. Cells on the NG plate started to align along the NG patterns just after cell seeding, as shown in figure 1, and the dynamic features of this alignment can also be viewed in the electronic supplementary material, movie 1. Cells extended in parallel with the NG pattern and aligned with each other during the subsequent 24 hours of culture. Cell alignment was maintained for at least 4 days following this period.

To express cell alignment quantitatively, we determined the orientation angles of individual cells (n>140 for each time point) against the groove direction (scheme 1b), and calculated the s.d., σ, of the distribution of the orientation angles at each time point using equation (2.4). No specific orientation of cells in a specific direction was observed at 20 min after cell seeding, as shown in figure 1. Figure 2a shows cell orientation more quantitatively by histograms and the angular s.d. (σ). It was σ=58.4 at 20 min. After 1 hour of culture, a clear peak at 0° was observed in the histogram of the orientation angle distribution. This tendency became much clearer and the angular s.d. decreased rapidly with time afterwards. Figure 2b shows the time course of the values of the s.d., σ. The s.d. value sharply decreased with time during the initial several hours and remained at approximately 20 afterwards, indicating maintenance of cell alignment along the grooves.

Figure 2

Cell orientation angle against the NG lines and the angular s.d. (a) Histograms of cell orientation angle against NG lines at (i) 20 min (s.d.=58.4), (ii) 1 hour (s.d.=38.1), (iii) 6 hours (s.d.=28.9) and (iv) 24 hours (s.d.=19.4) post-seeding. Each figure includes the angular s.d. calculated from the wrapped normal distribution (see equation (2.4) in the text). (b) Time course of the angular s.d.

3.2 Cell protrusions

To elucidate the mechanism of cell morphological changes, we focused attention on cell protrusions (scheme 1a). A cell protrusion is defined as a thicker protrusive structure in amoeboid cells, carrying lamellipodia, pseudopodia, filopodia (microspikes) and microvilli (Adams 2002; DeMali & Burridge 2003). Figure 3a and the electronic supplementary material, movie 2, show that cells extended their cell protrusions in all directions on a flat substrate. Lifetimes of the cell protrusions differed from one another, but no clear dependence of lifetime on direction was seen on the flat substrate. Figure 3b and the electronic supplementary material, movie 3, however, show that cells extended cell protrusions in all directions equally on the NG plate, as seen on the flat substrate, but cell protrusions that extended in the groove direction remained longer than those that were perpendicular to the groove direction. Consequently, cells elongated and aligned along the groove direction.

Figure 3

Comparison of time-lapse images of MSCs seeded on (a) a non-treated (NT) flat substrate and (b) an NG substrate. Cultures were incubated for 60 min after cell seeding at 37°C, 5% CO2 and followed by recording under a time-lapse microscope every 2.5 min. Images of every 20 min are shown. In NG images, the NG lines were parallel to the x-axis. Scale bar, 20 μm. See also the electronic supplementary material, movies 2 and 3.

To evaluate the extension length and the direction of cell protrusions more quantitatively, the leading edges of the cell protrusions were tracked every 2.5 min from 1 to 7 hours after cell seeding. Movements of the cell protrusions from the cells shown in figure 3a,b are presented in the electronic supplementary material, movies 2 and 3. The trajectories of the leading edges of cell protrusions are plotted in figure 4a. The s.d. of the angles of the cell protrusions against the groove direction were 32.5 on the NG plate but 81.5 on the flat plate. Thus, the leading edges of the cell protrusions extended along the grooves on the NG plate. Additionally, the numbers of protrusion appearances per cell in an hour were 1.17±0.37 for cells on the NG and 0.98±0.25 for cells on the flat plate. No significant difference was observed between these. On the other hand, their lifespans (i.e. time from appearance to retraction) were 265±91 and 154±50 min for the cells on the NG and on the flat plates, respectively. Protrusions existed longer on the NG plate than the flat plate. These results indicate that cells tend to elongate along the NG patterns, although cell protrusions emerge at the same frequency.

Figure 4

Movements of leading edges of cell protrusions. (a) Tracks of leading edges of protrusions of cells on (i) NT flat substrate (number of tracks=62; s.d.=81.5) and (ii) the NG substrate (number of tracks=80; s.d.=32.5). Start point of a leading edge of each cell protrusion was set at the origin of the coordinate axes, its location every 2.5 min was plotted and a filled circle indicates its endpoint after observation. The values inserted represent the s.d. of angles between the lines from the origin to filled circles and the x-axis. (b) Average velocity of leading edges. The velocity of each leading edge was determined by dividing the contour length by observed time. Values were given as mean ±s.d. for n=62 protrusions for the flat substrate and 80 for the NG substrate, of 10 randomly selected cells, respectively. (c) Directionality of leading edge movement, which was defined as the ratio of the Euclidean distance and accumulated distance. Asterisks represent a significant difference (p<0.05) between two groups.

The averaged velocities and the directionality of edge movement of cell protrusions were determined as described in §2. Significant differences were observed in the velocities and the directions of movements of cell protrusions between cells cultured on non-treated (NT) plates and NG plates, as shown in figure 4b,c. Moreover, directionality was higher on the NG plate than on the NT plate. This outcome implies that the movement of the edge of cell protrusions on the NG plate would confine the movements of cell protrusions along the grooves because higher directionality indicates linear movement of cell protrusions.

3.3 Focal adhesion points

To give some insights into the anisotropic movements of cell protrusions, focal adhesion points under a cell protrusion onto the NG pattern were examined in detail by immunohistochemical staining of actin filaments and vinculin found at focal adhesion points (figure 5 and the electronic supplementary material, figure 2). Vinculin was found only on the ridges of the NG pitches as seen for vinculin staining in figure 5. When the cell protrusions extended parallel to the groove direction, vinculin aligned parallel to the NG direction and actin filaments aligned parallel to vinculin and their terminations attached to vinculin as shown in figure 5a. A long area of focal adhesion is expected to be able to resist the contraction force generated by actin filaments. On the other hand, when cell protrusions extended perpendicular to the groove direction, vinculin aligned parallel to the NG direction, but fragmented, and actin filament terminations attached to vinculin, but were not following the vinculin lines and were not aligning to a specific direction as seen in figure 5b and the electronic supplementary material, figure 2, and illustrated in scheme 2a. The cell protrusions will be easily retracted owing to fragmented focal adhesion. These results suggest that the retraction phase of the cell protrusions plays a role in the formation of cell contact guidance. Biggs et al. (2008a,b) focused on adhesion points and found that nanotopography affected adhesion formation. Our results are consistent with their observations of fixed cells using a scanning electron microscope.

Figure 5

Immunohistochemical staining of vinculin and actin filaments of MSCs after 4 days of culture on the NG substrate. A cell protrusion extended (a) parallel and (b) perpendicular to the NG lines. (i) Vinculin was stained with Alexa-488-conjugated antibody (green) and (ii) F-actin of the cytoskeleton was stained with Alexa-594-conjugated phalloidin (red) and (iii) merged images are shown. Arrows indicate leading edges of cell protrusions. Scale bar, 10 μm.

Scheme 2

Model for cell alignment on the NG substrate. (a) Actin filaments parallel to the grooves form wide focal adhesions at filament terminations. On the other hand, termination of perpendicular filaments is fragmented because focal adhesion is formed only on the ridge. (b) Filopodia movements are isotropic, i.e. no specific direction was observed for their extension and retraction against the NG structure. This finding suggests that filopodia probing does not play a major role in cell alignment. Cell protrusions isotropically extended, but some of them that were perpendicular to the NG pattern more rapidly retracted than those parallel to the NG pattern. These cell protrusion dynamics force a cell to elongate and align along the NG pattern.

3.4 Filopodial probing

It has been reported that filopodia play a sensory or exploratory role when a cell migrates and extends (Faix & Rottner 2006). The diameters of filopodia are 250–400 nm (McClay 1999), smaller than the width of the ridge and the groove of our NG plate. Filopodia were expected to attach to the ridges and to reach the bottom of the grooves without difficulty. Filopodia movements were followed by a time-lapse microscope to visualize their role in cell alignment along the NG plate.

A representative cell protrusion was observed under high magnification (80×objective lens) on a time-lapse microscope to see the dynamic features of the filopodia. Figure 6b and the electronic supplementary material, movies 4 and 5, show the representative movements of filopodia. Filopodia indicated by arrows in the figure moved as if they were probing the surroundings of the cell protrusion. Then, some cell protrusions invaded the probed areas, as indicated by arrowheads. Cell protrusions that extended perpendicular to the NG direction retracted more rapidly than those parallel to the grooves (arrow, R), while, on a flat substrate, this repeated probing and retraction of the cell protrusion was rarely observed (electronic supplementary material, movie 6). In these two cases, however, no difference in filopodia movements was observed. These facts suggest that filopodia cannot distinguish topological differences or that filopodia can do so but that the retracting phase of cell protrusions is a major factor for cell alignment along the NG patterns.

Figure 6

Observation of filopodia under high magnification. (a) Schematic of the observation area. Electronic supplementary material, movie 4, shows the rectangular area (A) and movie 5a shows the area (B). Pictures were captured with time-lapse microscopy every 4 s. (b) The extension and retraction of the leading edge of the cell protrusion and precession movements of representative filopodia on the NG substrate. Arrows in the figure indicate (F) filopodial probing, (E) extending cell protrusion and (R) retracting cell protrusion. Scale bar, 5 μm.

4. Discussion

A large number of studies have examined the effects of surface topography on cell behaviours or contact guidance. Dalby et al. (2004) reported that filopodia of human fibroblasts can sense topography down to a pitch of 35 nm in diameter and 50 nm in depth and that filopodial probing acts as an initial trigger for cell alignment in response to the topography of substrates. Teixeira et al. (2003) employed human corneal epithelial cells. In their study, they found that filopodia aligned along the grooves and that cells attached to the ridge and aligned along the NG pattern. From these findings, they speculated that filopodia sense differences in surface topography and induce cell alignment. In their other report, however, filopodia aligned perpendicular to the grooves (Teixeira et al. 2006). They mentioned that these two contradictory results reflected a difference in culture media. Wójciak-Stothard et al. (1996) reported that filopodia extending perpendicular to the groove direction were more frequently observed than those along the NG pattern, even though they were in cells that had aligned along the NG pattern. The role of filopodial probing remains controversial. Most researchers assessed this relationship of filopodial movements and cell alignments after cells were fixed with formaldehyde, methanol or glutaraldehyde.

We expected that the examination by time-lapse microscopes of the kinetics of cell alignment and dynamic features of cell protrusions and filopodia would give more detailed insights into cell responses to surface topography and the roles of cell protrusions and filopodia in contact guidance of cells. As shown in figure 6 and the electronic supplementary material, movie 6, filopodia moved as if they were probing the surroundings of the cell protrusion, and then some cell protrusions invaded the probed areas. Cell protrusions that extended perpendicular to the NG direction tended to retract more rapidly than those parallel to the grooves. From these facts, we think that the retracting phase of cell protrusions is a more important factor for cell alignment along the NG patterns (scheme 2) than filopodial sensing. On the other hand, it has been reported that filopodia can distinguish topological differences and cell protrusions follow filopodia movements, resulting in cell alignment (Teixeira et al. 2003, 2006; Dalby et al. 2004). At this point, it is difficult to identify which is the major mechanism in cell alignment on the NG pattern. We should carefully carry out additional experiments, such as the observation of the extension of filopodia under much shorter interval than this experiment, every 4 s, and clear visualization of filopodia movements employing fluorescent labelling.

Cell protrusions extended around cells. Some of those perpendicular to the NG pattern tended to rapidly retract compared to those parallel to the NG pattern. As shown in figure 5, when cell protrusions extended parallel to NG lines, both vinculin and actin filaments also aligned parallel to the NG lines; but when cell protrusions extended perpendicular to the NG lines, vinculin staining was fragmented, and no clear alignment of actin filaments was observed. The latter focal adhesion—the fragmented vinculin expression area—is expected to be a weaker adhesion than the longer vinculin expression area, resulting in easy retract of cell protrusions perpendicular to the NG.

MSCs are multipotent stem cells that can differentiate into osteoblasts, adipocytes, chondrocytes and other kinds of mesenchymal cells (Pittenger et al. 1999). The morphology of MSCs can determine their fates, such as proliferation or differentiation direction (McBeath et al. 2004; Engler et al. 2006). As shown in figure 1, the morphology of MSCs is affected by the nanostructure of cell culture substrates. Thus, we expected that the differentiation direction of MSCs can be controlled by the substrate topography. In future studies, we will carefully examine the effect of NG patterns on MSC differentiation.

5. Conclusion

We observed living cells on an NG pattern using time-lapse microscopes to clearly demonstrate the dynamic features of cell alignment along the NG pattern. Cell protrusions perpendicular to the NG pattern retracted more rapidly than those parallel to it. The anisotropic retraction rate of cell protrusions would induce cell elongation and alignment along the NG pattern. Filopodial roles in cell alignment are controversial, and detailed dynamic observations are required to elucidate them in future.

Acknowledgements

This study was partly supported by the Knowledge-Based Cluster Creation Project, MEXT and Leading Project: Development of Artificial Organs Utilizing Nanotechnology and Materials Science.

Footnotes

  • One contribution of 10 to a Theme Supplement ‘Japanese biomaterials’.

  • Received August 30, 2008.
  • Accepted January 6, 2009.

References

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