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. 2021 Jan 30;14(3):643.
doi: 10.3390/ma14030643.

Cytocompatibility of Graphene Monolayer and Its Impact on Focal Cell Adhesion, Mitochondrial Morphology and Activity in BALB/3T3 Fibroblasts

Iwona Lasocka  1 , Lidia Szulc-Dąbrowska  2 , Michał Skibniewski  3 , Ewa Skibniewska  1 , Karolina Gregorczyk-Zboroch  2 , Iwona Pasternak  4 , Marie Hubalek Kalbacova  5
Affiliations

Cytocompatibility of Graphene Monolayer and Its Impact on Focal Cell Adhesion, Mitochondrial Morphology and Activity in BALB/3T3 Fibroblasts

Iwona Lasocka et al. Materials (Basel). .

Abstract

This study investigates the effect of graphene scaffold on morphology, viability, cytoskeleton, focal contacts, mitochondrial network morphology and activity in BALB/3T3 fibroblasts and provides new data on biocompatibility of the "graphene-family nanomaterials". We used graphene monolayer applied onto glass cover slide by electrochemical delamination method and regular glass cover slide, as a reference. The morphology of fibroblasts growing on graphene was unaltered, and the cell viability was 95% compared to control cells on non-coated glass slide. There was no significant difference in the cell size (spreading) between both groups studied. Graphene platform significantly increased BALB/3T3 cell mitochondrial activity (WST-8 test) compared to glass substrate. To demonstrate the variability in focal contacts pattern, the effect of graphene on vinculin was examined, which revealed a significant increase in focal contact size comparing to control-glass slide. There was no disruption in mitochondrial network morphology, which was branched and well connected in relation to the control group. Evaluation of the JC-1 red/green fluorescence intensity ratio revealed similar levels of mitochondrial membrane potential in cells growing on graphene-coated and uncoated slides. These results indicate that graphene monolayer scaffold is cytocompatible with connective tissue cells examined and could be beneficial for tissue engineering therapy.

Keywords: cytocompatibility; fibroblast; focal contact; graphene; mitochondria.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
(a) Raman spectrum of graphene film on glass substrate. (b) Micro-Raman map of intensity ratio of the 2D to the G peaks. (c) histogram of the intensity ratio of the 2D to the G peaks.
Figure 2
Figure 2
Optical image of graphene layer transferred onto glass substrate. Scale bar is 10 µm.
Figure 3
Figure 3
Morphology of BALB 3T3 cells growing on glass and graphene substrate at different time of incubation 1, 3, 6, 12 and 24 h.
Figure 4
Figure 4
Spreading (cell area) of BALB/3T3 fibroblasts 12 and 24 h after seeding on glass and graphene platform. Data are represented as mean ± SD (standard deviation) of three independent experiments. Approximately 70 cells were evaluated in each experiment. Single cell-area was measured using ImageJ software. **-statistically significant differences.
Figure 5
Figure 5
Focal contacts morphology of BALB 3T3 cells and calculation. Vinculin staining of focal contact on control (A) and graphene substrate (B). Focal contact number and size area of BALB 3T3 cells (C,D). Representative vinculin staining of attachment points (focal contacts) localized in lammelum area. The arrow indicates the direction of cell movement (E). Cells were stained with phalloidin-FITC (F-actin, green fluorescence), anti-vinculin Abs (vinculin, red fluorescence) and Hoechst 33,342 (DNA, blue fluorescence); Magnification in squares presents focal contacts and interaction between vinculin and actin. For focal contact number per cell at least 50 cells of each experiment were counted, and for focal contacts size, at least 10 areas of focal contacts per cell were analyzed. Data from three independent experiments are presented as mean ± SD (standard deviation). **-statistically significant differences.
Figure 5
Figure 5
Focal contacts morphology of BALB 3T3 cells and calculation. Vinculin staining of focal contact on control (A) and graphene substrate (B). Focal contact number and size area of BALB 3T3 cells (C,D). Representative vinculin staining of attachment points (focal contacts) localized in lammelum area. The arrow indicates the direction of cell movement (E). Cells were stained with phalloidin-FITC (F-actin, green fluorescence), anti-vinculin Abs (vinculin, red fluorescence) and Hoechst 33,342 (DNA, blue fluorescence); Magnification in squares presents focal contacts and interaction between vinculin and actin. For focal contact number per cell at least 50 cells of each experiment were counted, and for focal contacts size, at least 10 areas of focal contacts per cell were analyzed. Data from three independent experiments are presented as mean ± SD (standard deviation). **-statistically significant differences.
Figure 6
Figure 6
Trypan blue and WST-8 assay results on the cell viability and mitochondrial activity of BALB 3T3 cells on the test material. Data from three independent experiments are presented as mean ± SD (standard deviation). **-statistically significant differences.
Figure 7
Figure 7
The morphology of mitochondrial network after staining with Mito Tracker Green FM and Mito Red Green fluorescence in living cells and red fluorescence in fixed cells. Magnification in squares presents different phenotypes of mitochondria: straight rods, twisted rods, branched rods and loops.
Figure 7
Figure 7
The morphology of mitochondrial network after staining with Mito Tracker Green FM and Mito Red Green fluorescence in living cells and red fluorescence in fixed cells. Magnification in squares presents different phenotypes of mitochondria: straight rods, twisted rods, branched rods and loops.
Figure 8
Figure 8
Evaluation of mitochondrial membrane potential using JC-1 dye. Cytograms of JC-1-stained cells; Red populations—cells with high membrane potential. Green populations—cells with low membrane potential. Blue populations—cells with high mitochondrial depolarization. Bar chart of red/green fluorescence intensity ratio of JC-1-stained cells. Data from three independent experiments are presented as mean ± SD (standard deviation) (n = 10,000 cells). ** - statistically significant differences.
Figure 9
Figure 9
Fluorescence microscopy showing mitochondrial network in cells growing on graphene and glass substrate. Staining cells with JC-1 demonstrated the influence of hydrogen peroxide on mitochondria.
See this image and copyright information in PMC

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