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International Journal of Bioprinting Curved cell-guided structures printed by FDM

migration in the curved channels [36,37] . The M-22 cells were (Leica, Germany) at 10× magnification. All the cells, nearly
cultured in DMEM/F12 (1:1) supplemented with 10% fetal 20 cells per group, in the channels were recorded and
bovine serum and 1% penicillin/streptomycin (culture measured by Fiji/ImageJ (National Institute of Health, US).
medium, Gibco, US) and incubated in a humidified cell The morphology of cells was characterized by the aspect
incubator (Thermo Fisher Scientific, US) at 37°C and ratio, which was defined as the ratio of the length to width
5% CO . of the fitting ellipse of cells. The orientation angle of the cell
2
The cells were uniformly seeded with 10,000 and 80,000 was defined as the acute angle between the ellipse’s major
cells per well to observe the effect of pattern curvatures axis that fits the cells’ shape and the tangent line on the
on cell proliferation and morphology, respectively pendant foot of the nucleus to the channel.
(Figure 1D). 2.6. Characterization of cell migration
For the observation of cell migration, the cells were The front-end speed of collective cells and the migration
firstly seeded in the center away from the entrance of the speed of single cells were quantified as mentioned below.
migration channels with 400,000 cells/mL (100 μL); after The front-end speed of collective cells was calculated by
6 h of cell adhesion, the frontier of the cell was slicked to the ratio of the displacement of the front end of the cell
a straight line to ensure that cells migrated from the same population to the observation time without eliminating
starting line, and then 2 mL culture medium was added the influence of cell proliferation. Bright-field images
to each well for continuously culturing the cells. A time- were taken one day after the cells entered the channels
lapse microscope was used to observe the migration of to measure the displacement of the front end of the cell
cells (Figure 1E). population; the time interval was 12 h.
2.3. Cell proliferation assay The single-cell migration speed was defined as the
The proliferation of M-22 cells was evaluated on days 1, 2, average ratio of the migration distance of the cells to
and 4 after culturing using a cell counting kit-8 (CCK-8) the observation time. The single-cell migration speed
(Beyotime Biotechnology Co., Ltd, China), and there were was recorded by a time-lapse microscope CytoSMART
four independent replications per group. Briefly, the CCK-8 Lux2 (CytoSMART Technologies, Netherlands) after
reagent was diluted (1:10) in a full culture medium as a the cells entered the curved channels. Photographs were
working solution and added to each well; after 30 min, taken at 5-min intervals for 12 h. The microscopy images
200 μL supernatant was extracted and added to 96-well were imported into the Fiji/ImageJ software, the manual
plates, this was repeated four times, and the optical density tracking function was used to track the displacement of
(OD) value at 450 nm was measured by a multifunction 30–40 cells per channel, and the average migration speed
microplate reader (Thermo Fisher Scientific, US). The cell of cells was calculated accordingly.
metabolism was quantified by the ratio of the OD value on 2.7. Computational model of cell migration
day n to the average OD value on day one. The cell migration was modeled and simulated by

[35]
2.4. Fluorescence staining modifying the methods based on Odde’s model . The
The morphology and orientation of the cells were analyzed migration simulation was based on the force equilibrium
by fluorescence staining and microscopic observation between the membrane tension, cell–cell, and cell–
after 48 h of incubation. Briefly, the samples were fixed in substrate interaction force. The boundaries were set as
4% paraformaldehyde (Solarbio, China) for 30 min and curved, which was consistent with our experiment. The
permeabilized with 0.5% Triton X-100 (Solarbio, China) detailed model description, parameter settings, and
for 20 min at room temperature. Next, the F-actin of cells calculation programs were described in the supporting
was stained with 1:200 phalloidin-FITC (Solarbio, China) information. The calculation programs were solved by
solution and incubated at room temperature in the dark for MATLAB (Mathworks, US).
2 h. Then, the cell nuclei were stained with 4’,6-diamidino-
2-phenylindole (DAPI) (Solarbio, China) for 10 min at 2.8. Statistical analysis
room temperature in the dark. Finally, all the samples were The data from the experiments are expressed in mean
rinsed three times with phosphate-buffered saline (PBS) ± standard deviation, and the error bar represented the
and fixed on a microscope for imaging. standard deviation of the mean value. The data were analyzed
by OriginPro 2021 learning edition (OriginLab, US). The
2.5. Characterization of cell morphology and statistical significance was determined using a one-way
orientation statistical analysis of variance (ANOVA) followed by the
The staining samples were observed and photographed Tukey’s post-hoc test for multiple comparisons. The statistical
under an inverted phase-contrast fluorescence microscope significance was defined as * P < 0.05 and ** P < 0.01.

Volume 9 Issue 3 (2023) 41 https://doi.org/10.18063/ijb.681