Controlling retraction fibres during fibroblast
cell division via aspect ratio modulation of
nanoimprinted PLGA grooves
Yi-Hsuan Su,†,¶, ‡ Po-Chieh Chiang,†,‡ Li-Jing Cheng,†,♮ Chau-Hwang Lee,§,¥ Nathan
S. Swami,*,¶ and Chia-Fu Chou*,†,§,#
SUPPORTING INFORMATION
Figure S1 Representative fluorescence images of phalloidin in cells on various PLGA
substrates: (A) non-patterned. (B)-(E) 200 nm wide grooves of various depths: (B) 50 nm, (C)
100 nm, (D) 200 nm, and (E) 400 nm. (F) Angular distributions of the cell orientation on
non-patterned and 200/200 nm (depth/width) nanogrooves. Scale bar, 50 m.
Figure S2 Alignment and elongation of pre-mitotic protrusions on grooves of various widths
and aspect ratios. The values of standard deviations represent the degree of alignment of
pre-mitotic protrusions on grooves of various widths: (A) 200 nm; (C) 400 nm and (E) 800
nm. Each data is from the measurements of 750 cells. The percentage of cells represenst an
elongation factor larger than that of the cells cultured on non-patterned (N-p) substrate (mean
± SD) on grooves of various widths: (B) 200 nm; (D) 400 nm and (F) 800 nm. The elongation
factor is defined by the ratio a/b of the longest axis (a) of a cell to the maximum
perpendicular width (b). Each data is from the measurement of 300 cells.
Figure S3 Organization of actin filaments (green) (A-C), vinculin (red) (D-E) and nucleus
(blue) on various substrates. (A) The cells on the non-patterned substrate exhibit long and
prominent actin filaments across the entire cell body, and (D) prominent and numerous
vinculin spots are distributed broadly at the edges. The cells on grooves with an A.R. of 1.0
substrates: 800/800 (depth/width) nm (B) and on grooves with an A.R. of 2.0 substrates
(1600/800) (C) exhibit fewer and shorter actin filaments at both ends, and no long filament
across the cell bodies. The cells on grooves with an A.R. of 1.0 substrates (E) exhibit only few
vinculin spots at the cell edges but the nucleus morphology remains similar to that of the cells
on the non-patterned substrate. The cells on grooves with an A.R. of 2.0 substrates (F) also
exhibit few vinculin spots at the cell edges but the nucleus morphology is elongated. The
presence of vinculin-positive focal adhesion is confirmed by fluorescence intensity levels
greater than a threshold level, as set by the mean fluorescence intensity of each image plus
two standard deviations. For instance, the representative cell on the non-patterned substrate
(D) has 46 noticeable vinculin-positive focal adhesions; whereas the representative cells on
the groove with an A.R. of 1.0 (E) and 2.0 (F) show the vinculin-positive focal adhesion
numbers of 11 and 6, respectively. Scale bar, 20 m.
Figure S4 Cell migration trajectories on (A) non-patterned and (B) the grooves with an A.R.
of 1: 200/200 (depth/width) nm, axis scale in m. (C) Average cell migration speeds
measured in 6 hours (n = 30 for each patterns). (D) Vinculin positive focal adhesion numbers.
*, p ＜ 0.001 in comparison to the value on the non-patterned (N-p) substrate.
The cell migration speed on the non-patterned versus grooves with the A.R. of 1.0 (200 nm
depth/200 nm width and 800 nm depth/800 nm width), and A.R. of 2.0 (1600 nm depth/800
nm width). The speed is calculated as the total length of a migration track divided by the
duration of the recording. As shown in Figure R3, the average migration speed on the
non-patterned substrate is 30±8 m/h averaged over 30 cells. On the grooves with an A.R. of
1, the cells migrate twice as fast as those on the non-patterned substrate, with an average
speed around 60±28 m/h. However, the cell migration speed is lowered on sub-micron scale
grooves with an A.R. of 2.0, wherein the highest degree of cell guidance was observed. In
order to characterize the influence of the deeper trenches of high A.R. sub-micron scale
grooves on cell migration, we examine the number and depth distribution of the focal
adhesions as shown in Figure 4. As per Figure 4 C versus D, and Figure S4D, where the
vinculin-positive focal adhesion number of the cells is similar on patterned grooves at both,
A.R. 2.0 and A.R. 1.0 even though the migration speed is lower in the former case. We infer
the deeper extension of filopodia into grooves with an A.R. of 2.0 versus A.R. of 1.0, thereby
lowering migration speeds at the higher A.R. groove, whereas the mechanism for lowering of
the migration speeds on the non-patterned substrate is due to the greater focal adhesion
numbers.
All movies are time-lapse recording with 10 minutes interval with time stamp in hr:min.
Movie S1: Cells on 200 nm depth/200 nm width PLGA grooves undergo mitosis. Video
speed: 2 frame per second (fps). Scale bar applies to Movie S2 as well.
Movie S2: Cells on 1600 nm depth/800 nm width PLGA grooves undergo mitosis. Video
speed: 2 fps
Movie S3: Cells on 200 nm depth/800 nm width PLGA grooves undergo mitosis (Indicated
by black arrows). Video speed: 5 fps.