Pre-culture Sudan Black B treatment suppresses autofluorescence signals emitted from polymer tissue scaffolds
Pre-culture Sudan Black B treatment suppresses autofluorescence signals emitted from polymer tissue scaffolds
Erin K. Knapton
OPEN In tissue engineering, autofluorescence of polymer scaffolds often lowers the image contrast, making it difficult to examine cells and subcellular structures. Treating the scaffold materials with Sudan Black B (SBB) after cell fixation can effectively suppress autofluorescence, but this approach is not conducive to live cell imaging. Post-culture SBB treatment also disrupts intracellular structures and leads to reduced fluorescence intensity of the targets of interest. In this study, we introduce pre-culture SBB treatment to suppress autofluorescence, where SBB is applied to polymeric scaffold materials before cell seeding. The results show that the autofluorescence signals emitted from polycaprolactone (PCL) scaffolds in three commonly used fluorescence channels effectively decrease without diminishing the fluorescence signals emitted from the cells. The pre-culture SBB treatment does not significantly affect cell viability. The autofluorescence suppressive effect does not substantially diminish during the culturing period up to 28 days. The results also show that cell migration, proliferation, and myogenic differentiation in preculture SBB-treated groups do not exhibit statistical difference from the non-treated groups. As such, this approach greatly improves the fluorescence image quality for examining live cell behaviors and dynamics while the cells are cultured within autofluorescent polymer scaffolds.
Polymeric biomaterials are widely used as cell culturing scaffolds in tissue engineering. Cell interactions with
the scaffolds are critical for cell proliferation, differentiation, and tissue growth1?3. A variety of engineering
approaches has been developed to examine and quantify cell responses, including scanning electron microscopy
(SEM), flow cytometry, western blotting, and fluorescence labeling4?6. Among these, flow cytometry and
western blotting can quantitatively determine the expressions of proteins in cells. Unfortunately, these approaches
cannot reveal the morphology relationship between cells and scaffolds, making it difficult to study cell responses
to specific stimulations applied by the scaffolds or the environment. SEM provides a powerful tool to unveil cell
morphological responses in three-dimensional formats. The examination of protein expressions, however, is
difficult. This has been addressed by?using immunogold SEM, where gold nanoparticles are used to immunolabel the
targets of interest before the samples are prepared for SEM observation7. Nonetheless, SEM requires a fairly
complicated and lengthy sample preparation process. The fixed cells are often vulnerable to cracking and distortion. In
addition, SEM is only applicable to end-point examinations. In-situ observation of live cell responses is difficult.
In view of these limitations, fluorescence labeling is used as a primary approach to investigate the interactions
between cells and polymeric scaffolds. The fluorescence dyes are bounded to corresponding molecules in the cells
through chemical reactions or physical adsorption. This allows for the investigation of cell responses to scaffolds
and environment (e.g. cell morphology change, protein expression, and distribution) in live and fixed cells
without disrupting cell-scaffold morphology relationship. Nonetheless, the efficacy of fluorescence labeling is limited
by autofluorescence of scaffold materials, which emit fluorescence signals that are difficult to be distinguished
The optimal SBB concentration. The SBB concentration should be adjusted to the value that can suppress
the autofluorescence of polymer scaffolds while not substantially affecting cell viability. An experiment was
performed to determine the appropriate SBB concentration (Fig.?2a?h). 3T3 fibroblasts were used and the culture
lasted for 3 days before examination. The results showed the average live/dead cells ratio reached its maximum
at 0.3% SBB concentration. The ratio dropped as the SBB concentration kept increasing (Fig.?2i). The image
contrast increased with the increasing SBB concentration when the SBB concentration was below 0.1% (Fig.?2a?d).
There was no statistical difference in the image contrast when the SBB concentration was above 0.1%. The cells
on 1% SBB treated PCL slabs had relatively lower confluence than other groups. Some cells in this group showed
rounded shapes (Fig.?2h). This phenomenon was not observed in the groups with lower SBB concentrations.
The capability of autofluorescence suppression of SBB treatment was represented by the fluorescence
signal reduction compared to the non-treated PCL groups. The results showed that the autofluorescence of the
SBB-treated PCL slabs decreased with the increasing SBB concentration in all the three fluorescence channels
(TRITC, FITC, and DAPI) (Fig.?2j). The autofluorescence suppressive efficacy reached the maximum at 0.05%
SBB in FITC and TRITC channels, and at 0.1% in DAPI channel. When the SBB concentration kept increasing,
there were no statistical changes in the fluorescence intensity: p = 0.2438 between 0.05% and 0.1% groups in
TRITC channel; p = 0.6795 between 0.05% and 0.1% groups in FITC channel; and p = 0.4157 between 0.1% and
0.3% groups in DAPI channel. Therefore, the SBB concentration >0.1% is sufficient to maintain the maximal
autofluorescence suppressive effect for all the three fluorescence channels.
Considering both the cell viability and autofluorescence suppressive efficacy, the SBB concentration of 0.3%
is chosen in this study. Figure?2k provides a representative fluorescence image of a cell-cultured PCL slab with
0.3% SBB treatment. After 3 days of cell culturing, the SBB treatment effectively suppressed autofluorescence
signals emitted from PCL, leading to clear boundaries of subcellular structures. The image contrast sharply
increased comparing to the control groups without SBB treatments (Fig.?2l). The differences in the image
contrast between the SBB-treated slabs and the corresponding controls were highly statistically significant for all
the three fluorescence channels (p < 0.0001, p = 0.0024, and p = 0.0003 for FITC, TRITC, and DAPI channels,
respectively) (Fig.?2m). Among these, FITC channel exhibited the greatest difference in the image contrast. The
average image contrast of the SBB-treated PCL slabs (0.862 ? 0.057) was more than two times of the control group
(0.429 ? 0.185).
Cell viability tests. To investigate whether the SBB treatment with the selected concentration (0.3%) affects
cell viability, live/dead assays were performed using human vein umbilical endothelial cells (HUVECs, Fig.?3a and
b), skeletal myoblasts (C2C12, Fig.?3c and d), endodermal cells (PYS2, Fig.?3e and f) and primary cells from rat
hippocampi (Fig.?3g and h) after culturing the cells on SBB-treated PCL slabs for 3 days. The results showed that
there was no statistical difference in the ratios of live/dead cells between the SBB-treated and non-treated groups
(p = 0.0953, 0.8505, 0.1851, 0.5524 for the four cell types, respectively) (Fig.?3i).
Endurance test of autofluorescence suppression. The autofluorescence suppressive effect should
preferably span over the entire period of cell culturing. In tissue engineering, most examinations of cell responses
to the scaffolds are within the first few weeks of cell seeding29?33. During the period, polymer degradation may
change the dimensions and molecular structures of the scaffolds15, 34, 35, and in turn affects the autofluorescence
suppression capability of SBB treatments. 3T3 fibroblasts were cultured on PCL slabs. The autofluorescence
suppression was examined on Days 7, 14, 21, and 28 (Fig.?4a?d). For the SBB-treated groups, the image contrast
in DAPI channel did not show statistical changes over time (Fig.?4e). This suggested that SBB treatment has
long-term autofluorescence suppressive capability. Here, the F-actins in TRITC channel were not used for
calculating the image contrast due to the fact that the very?high cell confluence after long-term culturing left too?little
uncovered area for image contrast determination.
Effect of SBB treatment on cell behaviors. The effects of SBB treatments on cell behaviors were
investigated using 3T3 fibroblasts and C2C12 skeletal myoblasts.
Fibroblast migration is important in wound healing and pathological fibrosis. The scratching experiment
using 3T3 fibroblasts (Fig.?5a?h) showed that the cell migration progress was ~56% after 12 hours, ~78% after
24 hours, and ~100% after 48 hours on both the SBB-treated and non-treated surfaces (Fig.?5i). There was no
statistical? difference in the cell migration progress between SBB-treated and non-treated groups at each time point,
indicating that the SBB treatment does not significantly affect 3T3 migration capability.
Skeletal myoblasts have the capability of forming muscle tissues through myogenesis process. Periodic
microgrooves were used as the contact guidance to facilitate linear myotube formation (Fig.?6a). After 7 days
following the addition of the differentiation medium, most myotubes aligned along the longitudinal direction of the
microgrooves in both the SBB-treated and non-treated groups (Fig.?6b and c). A large amount of long myotubes
(>1 mm) was observed in both groups. ELISA analysis showed that the MHC expression/cell was not statistically
different between the SBB-treated and non-treated groups (p = 0.2922), indicating that SBB treatment does not
significantly affect the myogenic differentiation. The effect of SBB treatment on cell proliferation rate was
examined by culturing C2C12 on planar PCL slabs and in the growth medium up to 7 days. The results showed that
there was no statistical difference in DNA content between SBB-treated and non-treated groups of the same day
(p = 0.0752 on Day 1, p = 0.3927 on Day 3, p = 0.8300 on Day 5, and p = 0.2018 on Day 7) (Fig.?6e).
Image contrast improvement for cells cultured on 3D porous PCL scaffolds. PCL particulate struc
tures were fabricated and served as 3D porous scaffolds. C2C12 myoblasts were cultured on the scaffolds, and
stained by live/dead assay on Day 3. A representative SEM micrograph of such a scaffold is shown in Fig.?7a (~80 ?m
in width and ~100 ?m in height). Similar to the results from planar PCL slabs, SBB-treated 3D porous PCL scaffolds
(Fig.?7b) exhibited statistically lower autofluorescence signals than the non-treated groups (Fig.?7c) in FITC channel.
Quantitative analysis showed that the image contrast decreased from 0.616 ? 0.066 in SBB-treated 3D scaffolds to
0.372 ? 0.100 in non-treated 3D porous scaffolds (Fig.?7d). The change was statistically significant (p= 0.0242).
Comparison with pre- and post-permeabilization SBB treatments. In our study, PCL scaffolds
were treated with SBB prior to cell seeding, while the previous protocols treated the substrates with SBB after
cell fixation, either prior to or after cell membrane permeabilization. To compare the impacts of different SBB
treatment protocols on autofluorescence suppression, planar PCL slabs were treated using all the three protocols
and cultured with C2C12 myoblasts. The fluorescence images acquired after 3 days culturing showed that all
the protocols were able to suppress autofluorescence signals emitted?from the PCL slabs in DAPI and TRITC
channels (Fig.?8). The intensities of the fluorescence images in the pre-culture group were 158.171? 37.428
(DAPI) and 222.117 ? 7.749 (TRITC), comparing to 94.222 ? 18.877 (DAPI) and 188.867 ? 16.591
(TRITC) in the pre-permeabilization group and 59.567 ? 7.480 (DAPI) and 48.667 ? 18.180 (TRITC) in the
post-permeabilization group. Statistical analysis showed that the pre-culture SBB treatments exhibited
statistically higher intensity than the pre-permeabilization group (p = 0.0443 for DAPI and p = 0.0347 for TRITC) and
the post-permeabilization group (p = 0.0071 for DAPI and p = 0.0013 for TRITC). In addition, a number of dark
particles were observed in the pre-permeabilization and the post-permeabilization groups: some particles laid on
the top of cell bodies and disrupted fluorescence images, as indicated by the arrows in Fig.?8d and f. Such
particles were not observed in the pre-culture SBB group (Fig.?8b). The exposure time, analog gain, and lookup tables
(LUTs) setting at 40? (Fig.?8b,d and f) were adjusted to obtain the best brightness and contrast.
In this study, PCL slabs were stained by SBB prior to cell seeding and culturing. The result showed that this pre-culture
SBB treatment is capable of suppressing autofluorescence signals emitted?from the PCL slabs and enhancing the
contrast of fluorescence images. The cytotoxicity of the treatment was examined using live/dead assays with four cell lines
and one group of primary cells. All the experiments showed that there was no statistical difference in the ratios of
live/dead cells between the experimental and the control groups. Quantitative measurements of representative cell
behaviors, including the migration progress of fibroblasts, and the myogenic differentiation and proliferation
capability of skeletal myoblasts, confirmed that the pre-culture SBB treatment has little effects on changing cell behaviors.
Pre-culture SBB treatment with significantly reduced background autofluorescence can thus provide a powerful tool for
examining cell responses to the scaffolds in live cells with better image contrasts. The comparison of the mechanisms
and performance of this treatment and of other SBB treatment schemes are as below.
Mechanism of autofluorescence suppression by SBB. SBB has poor solubility in aqueous solution but
soluble in several organic solvents. Due to the hydrophobic effect, SBB is more affinitive to hydrophobic surfaces36,
37. SBB staining is, therefore, more of a physical adsorption by the hydrophobic regions of the subjects, rather than
bonding through chemical reactions. The autofluorescence suppressive effect of SBB is believed mainly attributed
to its superior light absorption capability, which masks autofluorescence (i.e. absorbing autofluorescence and/or
scattering light that is generated from histological samples38?40 and polymeric scaffolds22). This is different from
some other fluorescence suppressive methods, such as photobleaching where the fluorophores are consumed
through chemical reactions. Jaafar and colleagues examined the effect of the post-permeabilization SBB treatment
for several polymers through ATR-FTIR and UV-vis spectroscopy. They concluded that SBB has minor chemical
interactions with the polymers, but showed a significant light absorption for all examined polymers21. Although
PCL was not tested, the result of PLCL, a copolymer of PCL and PLA (polylactic acid), is expected similar to that
of PCL. Therefore, it is plausible to deduce that the mechanism underlying the image contrast improvement of the
pre-culture SBB treatment is similar with post-culture SBB treatments: by absorption of autofluorescence signals
emitted from the polymers.
Preparation of planar polymer slabs. PCL (Mn ~ 45,000; Sigma-Aldrich, MO, USA) was dissolved in
acetone (Fisher Scientific, PA, USA) at a concentration of 10% (w/v), and molded by solvent casting. The polymer
solution was dispensed into 2 cm2 cell culture plates and evaporated to cast a 3 mm thick slab. SBB solutions with
the concentrations of 0.0005% to 1% (saturated) (w/v) were prepared by dissolving SBB powder (Sigma-Aldrich)
in 70% (v/v) ethanol, and syringe-filtered (0.2 ?m). The PCL slabs were then immersed in the SBB solution
overnight at 4 ?C. After staining, the PCL slabs were rinsed in 1? phosphate-buffered saline (PBS; Fisher Scientific)
three times and exposed to UV light overnight for sterilization. Before cell seeding, all PCL slabs were coated with
fibronectin (Sigma-Aldrich) at a concentration of 50 ?g/ml overnight at 4 ?C to enhance cell adhesion.
Fabrication of grooved topographical PCL substrates. PCL slabs with microgrooved topography
(linear microgrooves with the period of 30 ?m and the amplitude of ~2.8 ?m) were fabricated by solvent casting
in a pre-molded polydimethylsiloxane (PDMS) template. The PDMS template was replica molded by dispensing
and curing PDMS pre-polymer (Sylgard@184, Dow Corning, MI) on a pre-fabricated silicon wafer at 65 ?C. The
silicon wafer, with the grooved geometry with 30 ?m in period, was fabricated by standard photolithography.
Fabrication of three-dimensional (3D) PCL scaffolds. 3D porous PCL scaffolds were fabricated using
our previously described method43. Briefly, 10% (w/w) PCL/acetone solution was electrosprayed towards a
collecting substrate that has been patterned with interdigitated microelectrodes. By connecting one group of
microelectrodes at ground and keeping another group floated, PCL microparticles were attracted to the ground
microstructures and accumulated into 3D particulate scaffolds. After detaching the scaffolds from the collecting
substrate, the 3D porous structures were treated by SBB and fibronectin using the same procedure as for planar
Cell culturing. NIH/3T3 fibroblasts, C2C12 cells, PYS-2 endodermal cells (ATCC, VA, USA) were cultivated
in Dulbecco?s modified Eagle?s medium (DMEM) with 10% fetal bovine serum and 1% penicillin/streptomycin
(P/S) (all from Sigma-Aldrich). HUVECs (Lonza, MD, USA) were cultivated in Endothelial Basal Medium-2
(EBM-2) (Lonza) supplemented with growth factor kits. The four types of cells were seeded on planar PCL slabs
at the density of 5 ? 104 cells/cm2. C2C12 cells were also seeded on grooved PCL substrates and 3D PCL scaffolds
at the density of 5 ? 104 cells/cm2. The cells were then incubated at 37?C with 5% CO2 to allow cell attachment
and spreading. C2C12 differentiation was induced by changing to the differentiation medium (DMEM+2%
horse serum+1% P/S) and keeping the cells in the medium for 7 days. For all the above culturing conditions,
the media were changed every other day. The hippocampi of embryonic day 18 (E18) rat embryos were used to
generate the primary neuron culture following an established protocol44. The dissociated primary cells (mostly
hippocampal neurons with some glial cells) were cultured on the SBB-treated and non-treated PCL slabs at the
seeding density of 5 ? 104 cells/cm2 with plating medium (DMEM+10% FBS+1% P/S, with 0.45% glucose, 1 mM
sodium pyruvate, 25 mM glutamine, all from Thermo-Fisher). After 4hours of plating, the medium was replaced
by pre-warmed maintenance medium (neurobasal medium (Thermo-Fisher) with 2% of 50? B-27 supplement
(Thermo-Fisher), 0.5mM glutamine and 1% P/S). A half volume of the medium was changed with fresh
maintenance medium every other day. All animal experiments were conducted in accordance with ethics guidelines
stipulated by the Ohio State University animal ethics committee and with the NIH Animal Use Guidelines.
Fluorescence labeling. The cytoplasm was labeled by incubating cells in PBS with 2 mM calcein AM
(Life Technologies, CA) for 30 min. Afterward, the cells were fixed by incubation in PBS with 4%
paraformaldehyde (Sigma-Aldrich) for 30 min at room temperature. The cell membranes were permeabilized by
incubation with 0.1% Triton X-100 for 30 min. F-actins were stained by incubation with rhodamine-phalloidin
(Life Technologies) for 30 min at room temperature (25 ?C). The cell nuclei were labeled by incubation with
4?,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) for 30 min at room temperature.
Live/dead cells assay. Cell viability was examined by labeling live and dead cells using Live/Dead? kits
(Life Technologies). Briefly, cells were incubated in PBS with ethidium homodimer-1 (EthD-1, 4 ?M) and calcein
AM (2 ?M) for 30 min. Live cells were labeled with calcein AM (green) and dead cells were labeled with ethidium
Cell migration assay. The migration progress of fibroblasts was evaluated using scratching assay27. NIH/3T3
fibroblasts were cultured on SBB-treated and non-treated PCL slabs at the seeding density of 5 ? 104 cells/cm2 and
incubated for 3 days to allow cell confluence. On Day 3, the culture surface was scratched using a 200 ?L pipette
tip, resulting in a cell-free area with the width of ~500 ?m. The cell migration was monitored by labeling the cells
with calcein AM. The cell migration distance was determined by the distance of individual cells in the leading
edges of the scratch. The migration progress was normalized by dividing the migration distance by the initial
width of each scratch.
Quantification of myogenic differentiation. After 7 days of myogenic differentiation, the expression
of myosin heavy chain (MHC) was determined using a commercial mouse MHC ELISA kit (cat#MBS756241,
MyBioSource, San Diago, CA). The expression of MHC was determined by measuring light absorbance by
examined samples at 450 nm. The cell number of each sample was determined by total DNA assay as described in the
section below. The myogenic differentiation of C2C12 was determined by the MHC expression per cell.
Total DNA assay. The DNA content of C2C12 was quantified on Days 1, 3, 5, and 7 using a CyQUANT?
Cell Proliferation Assay kit (Thermo-Fisher, MA, USA). Briefly, cells were lysed in 200?L of 1? cell lysis buffer
and centrifuged to collect the supernatants containing DNAs. A standard curve of DNA content was obtained
using serially diluted bacteriophage ?DNA with the concentration ranging from 50 to 1000 ng/mL. All the DNA
samples (50 ?L) were then transferred into a 96-well plate. CyQuANT? GR working solution (50 ?L) was added
to each well. After incubating the samples at room temperature for 5 min, the fluorescence intensity of the
samples was measured using a fluorescence microplate reader (BioTek, VT, USA) with a filter with 485nm excitation
wavelength and 535 nm emission wavelength.
Fluorescence imaging and analysis. Fluorescence microscopy was used to examine cells on planar PCL
slabs and on 3D porous PCL scaffolds using Nikon fluorescence microscope Eclipse 80i with 4? to 40? objective
lenses. Three fluorescence filters, namely DAPI (393nm), FITC (483 nm), and TRITC (555 nm) were used. The
intensities of the targets of interest and of the background were measured using Nikon NIS-Elements AR
software. The image contrast (D) is calculated as:
where Ic and Ib denote the intensity of targets of interest (e.g. cytoplasm, F-actins, cell nuclei), and background
fluorescence intensity, respectively. A high D indicates a high signal emitted?from the targets of interest and a low
background signal, and vice versa. To avoid overexposure, the intensity of the excitation light is modulated to
achieve the best imaging quality of FITC channel, since it has the highest fluorescence intensity.
Statistical analysis. Student t-test was used to examine whether there are statistical differences in the
intensities of fluorescence images between different SBB concentrations; whether there are statistical differences in
the image contrast of fluorescence images; the ratio of live/dead cells, the migration progress, MHC expression/
cell, and the total DNA amount between SBB-treated and non-treated groups; and whether there are
statistical differences in the intensities of fluorescence images between different SBB treatment protocols (pre-culture,
pre-permeabilization, and post-permeabilization). For the image contrast and fluorescence intensity analyses,
each group consists of four PCL samples (i.e. planar slabs or 3D porous scaffold). There are three images taken
at the same magnification in each sample, resulting in a total of 12 images in each group. For the live/dead cells
ratio and the migration progress evaluation, each group consists of three PCL slabs, and three images are acquired
from each slab (total 9 samples). For MHC expression/cell and the total DNA amount evaluations, each group
consisted of three PCL slabs, and two samples were collected from each PCL slab (total 6 samples). Statistical
analysis was performed using JMP 11.0 (SAS?, NC, USA). (*)p < 0.05 is considered significant and (**)p < 0.01 is
considered highly significant.
The authors acknowledge the support from National Science Foundation through a NSF CAREER award
DBI0954013, and a facility grant from Institute for Materials Research (IMR) at the Ohio State University.
L.Q. and Y.Z. conceived the study and designed the experiment. L.Q. performed the primary experiment. E.K.K
assisted the cell culture. X.Z. prepared the 3D scaffolds. T.Z. and C.G. dissociated tissues from rat hippocampi.
L.Q. analyzed the data. L.Q. and Y.Z. wrote the paper.
Competing Interests: The authors declare that they have no competing interests.
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