**4.4. Carbon nanowall scaffold to control culturing of cervical cancer cells**

In recent years, cell culturing that uses carbon nanomaterials as scaffolds has been studied intensively [55-57]. The culturing rate is generally discussed with respect to the surface wettability of the scaffold. It has been reported that the cell-culturing rate would peak when the WCA on the scaffold surface is between 60˚ and 80˚ [58,59]. Moreover, many factors, including morphology, chemical termination, surface charge, scaffold surface stiffness, and the quantity of adsorbed protein are also essential for determining cell-culturing rates [60-63]. As mentioned in Section 4.2, the wide range control of surface wettability of CNWs was attained by postgrowth plasma treatments [52]. The unique features of CNWs and the variety of surface modification would give CNWs a high potential for scaffold application.

Here, the dependence of cell-culturing rate on the morphology and chemical termination of CNW scaffold was systematically investigated. Three types of CNW scaffolds with different densities (or different wall-to-wall distances) were prepared using RI-PECVD with CH4/H2 on quartz plates by changing the total pressure, CCP power, and growth period. The substrate temperature was 560°C. The flow rates of CH4 and H2 were fixed at 50 and 100 sccm, respec‐ tively. The total pressure was varied in the range of 1 to 5 Pa. The power applied to the SWP was 400 W and that to the CCP was changed in the range of 100 to 500 W. The growth period ranged from 8 to 80 min. Figures 24(a)-(c) show SEM images of the resulting CNW scaffolds with different densities. CNW scaffolds with average wall-to-wall distances of 95, 131, and 313 nm were obtained, which are denoted as "high-density [Figure 24(a)]", "medium-density [Figure 24(b)]", and "low-density [Figure 24(c)]" CNW scaffolds, respectively. The as-grown samples are denoted as H-terminated CNWs.

atmospheric pressure plasma treatment, which had slightly hydrophilic surface, weak oxidation and reduction peaks were observed in anode peak potential at 0.2 V and cathodic peak potential at -0.3 V, respectively. In the CV profile using the typical oxidized CNW electrode, on the other hand, a broad oxidation and a high peak reduction currents were observed in anode peak potential of 0.2 V and cathodic peak potential at -0.75 V, respectively. The surface of as-grown CNW electrode could be easily modified into superhydrophilic one by the surface oxidation using the Ar atmospheric pressure plasma. In the case of oxidized CNW electrode with low height, the CV profile exhibited small peak currents due to the small surface area. The results in Figure 23 indicate that superhydrophilic surfaces of CNWs with

**Figure 23.** CV profiles of as-grown (bare) CNWs (500 nm in height), oxidized CNWs (500 nm), and oxidized CNWs of

In recent years, cell culturing that uses carbon nanomaterials as scaffolds has been studied intensively [55-57]. The culturing rate is generally discussed with respect to the surface wettability of the scaffold. It has been reported that the cell-culturing rate would peak when the WCA on the scaffold surface is between 60˚ and 80˚ [58,59]. Moreover, many factors, including morphology, chemical termination, surface charge, scaffold surface stiffness, and the quantity of adsorbed protein are also essential for determining cell-culturing rates [60-63]. As mentioned in Section 4.2, the wide range control of surface wettability of CNWs was attained by postgrowth plasma treatments [52]. The unique features of CNWs and the variety

of surface modification would give CNWs a high potential for scaffold application.

**4.4. Carbon nanowall scaffold to control culturing of cervical cancer cells**

large surface areas were useful as electrodes for biosensor.

166 Graphene - New Trends and Developments

low height (350 nm) in PBS containing BSA. Scan rate: 100 mVs-1 [52].

**Figure 24.** SEM images of (a) high-density CNW, (b) medium-density CNW, and (c) low-density CNW scaffolds pre‐ pared using RI-PECVD with CH4/H2 [25].

After the preparation of CNWs, some of them were subjected to various plasma treatments to realize the chemical termination of the edges and surfaces of CNWs. For oxygen termination, the CNW film was exposed to the atmospheric pressure plasma employing O2 (50 sccm)/Ar (2000 sccm) at room temperature for 30 s (O-terminated CNWs). For fluorine termination, the CNW film was set in the VHF CCP employing CF4 (36 sccm)/Ar (10 sccm) in the RI-PECVD system without SWP at room temperature for 5 s (F-terminated CNWs). The applied CCP power was 200 W, and the pressure was at 107 Pa during the plasma treatment. For nitrogen termination, the CNW film was set in the VHF-CCP region of the RI-PECVD system employing N2 (12.5 sccm)/H2 (37.5 sccm) at 560°C for 30 s (N-terminated CNWs). The applied SWP and CCP powers were 400 and 300 W, respectively. The pressure was 1 Pa. Prepared CNW scaffolds with different densities and terminations were put in multiwell cell-culturing plates. Cervical cancer cells (HeLa cells) at a density of 1.0 × 104 cell/cm2 were seeded on each well. Incubation was conducted under a CO2 (5%) atmosphere at 37°C for 96 h with 2 ml/well of the medium culture. The cells were maintained in a medium of minimum essential medium (MEM) Eagle, which consisted of 5 ml of L-glutamine (200 mM), 50 ml of fetal bovine serum (FBS), 5 ml of nonessential amino acids for MEM Eagle, and 5 ml of penicillin streptomycin. The HeLa cells cultured on the CNW scaffolds were picked up by using trypsin (0.5 w/v%, 5.3 mmol/l). After 24 Fig. 25

25 Fig. 26

26 Fig. 27

culturing for 24 h, the numbers of the cells with each of two different shapes were counted. One is the nonspreading cell with the circular shape and the other is the spreading cell with the noncircular shape [25].

Figure 25 shows the WCAs on the CNWs after various plasma treatments. The wettability of the CNWs depended on the types of chemical termination, not on the densities of the CNWs. The wettability of CNWs was controllable by plasma treatments in the range from superhy‐ drophilic (WCA ≤ 10˚) to near superhydrophobic (WCA ≥ 150˚). The insets show the C 1s XPS profiles for the medium-density CNWs with (a) O-termination, (b) N-termination, (c) Htermination (as-grown), and (d) F-termination [52]. As shown in inset (a) of Figure 25, after the Ar/O2 atmospheric pressure plasma treatment, a small broad peak related to C-O single bonding was observed at around 286.5 eV [64]. The composition ratio O/C was 0.21. After the N2/H<sup>2</sup> plasma treatment, a broad peak tail was observed at around 285-287 eV, corresponding to nitrogen-related bondings [inset (b)] [65]. The composition ratio N/C was 0.08. In the case of as-grown CNWs, the composition ratio O/C was 0.05. A weak broad peak related to C-O single bonding was observed at around 286.5 eV [inset (c)]. Because of ex situ XPS measure‐ ments, CNW surface was oxidized when exposed to the atmosphere. Because of the slight existence of oxygen at the surface of CNWs, the as-grown CNWs exhibited slightly hydrophilic property. In contrast, after the CF4/Ar plasma treatment, sharp peaks related to C-CFX (X ≤ 3) bonding structures were evident as shown in inset (d). The composition ratio F/C was estimated to be approximately 0.49.

**Figure 25.** WCAs on the CNWs after various plasma treatments for different terminations. Inset shows C 1s XPS spec‐ tra of CNWs, in which the average wall-to-wall distance is 131 nm; (a) O-terminated, (b) N-terminated, (c) H-terminat‐ ed (as-grown), and (d) F-terminated CNWs [25].

Optical microscope images of HeLa cells cultured on the CNW scaffolds with different terminations and wall densities after incubation for 24 h are shown in Figure 26 [25]. The

3

3

numbers and morphologies of cultured HeLa cells somewhat depended on the chemical terminations as well as on the wall densities of CNW scaffolds. Figure 27 shows the numbers of HeLa cells cultured on the CNW scaffolds after incubation for 96 h as a function of WCA. For comparison, HeLa cells were also cultured on commercial glass plates, and the maximum number of cells was achieved at a WCA of 60˚, as previously reported [66]. Similarly, on the medium density CNW scaffolds, the maximum number of cells was obtained at a WCA of 55˚. In the case of low-density and high-density CNW scaffolds, however, the number of cells decreased and increased, respectively, with increasing WCAs. Hence, superhydrophilic surface is suitable for cell culturing on low-density CNW scaffolds, while superhydrophobic surface is suitable for high-density CNW scaffolds. As was shown in Figure 25, the surface wettability was nearly independent of the wall density. On the other hand, the cell-culturing rate was strongly dependent on the wall density of CNW scaffolds. These results suggest that the surface wettability is not dominant factor for determining the cell-culturing rate, while it is useful as approximate index of the expected cell-culturing rate. These experimental results indicate that the density of CNWs is the most essential factor for cell culturing rather than the surface wettability and types of chemical termination of CNW scaffolds. However, detailed mechanisms of cell/scaffold interactions in cell culturing on CNW scaffolds have not yet been clarified.

culturing for 24 h, the numbers of the cells with each of two different shapes were counted. One is the nonspreading cell with the circular shape and the other is the spreading cell with

24 Fig.

25 Fig.

26 Fig.

27

26

25

Figure 25 shows the WCAs on the CNWs after various plasma treatments. The wettability of the CNWs depended on the types of chemical termination, not on the densities of the CNWs. The wettability of CNWs was controllable by plasma treatments in the range from superhy‐ drophilic (WCA ≤ 10˚) to near superhydrophobic (WCA ≥ 150˚). The insets show the C 1s XPS profiles for the medium-density CNWs with (a) O-termination, (b) N-termination, (c) Htermination (as-grown), and (d) F-termination [52]. As shown in inset (a) of Figure 25, after the Ar/O2 atmospheric pressure plasma treatment, a small broad peak related to C-O single bonding was observed at around 286.5 eV [64]. The composition ratio O/C was 0.21. After the N2/H<sup>2</sup> plasma treatment, a broad peak tail was observed at around 285-287 eV, corresponding to nitrogen-related bondings [inset (b)] [65]. The composition ratio N/C was 0.08. In the case of as-grown CNWs, the composition ratio O/C was 0.05. A weak broad peak related to C-O single bonding was observed at around 286.5 eV [inset (c)]. Because of ex situ XPS measure‐ ments, CNW surface was oxidized when exposed to the atmosphere. Because of the slight existence of oxygen at the surface of CNWs, the as-grown CNWs exhibited slightly hydrophilic property. In contrast, after the CF4/Ar plasma treatment, sharp peaks related to C-CFX (X ≤ 3) bonding structures were evident as shown in inset (d). The composition ratio F/C was

**Figure 25.** WCAs on the CNWs after various plasma treatments for different terminations. Inset shows C 1s XPS spec‐ tra of CNWs, in which the average wall-to-wall distance is 131 nm; (a) O-terminated, (b) N-terminated, (c) H-terminat‐

Optical microscope images of HeLa cells cultured on the CNW scaffolds with different terminations and wall densities after incubation for 24 h are shown in Figure 26 [25]. The

3

the noncircular shape [25].

168 Graphene - New Trends and Developments

estimated to be approximately 0.49.

ed (as-grown), and (d) F-terminated CNWs [25].

24 Fig. 25

25 Fig. 26

26 Fig. 27


**Figure 26.** Optical microscope images of HeLa cells cultured on CNW scaffolds with different chemical terminations and wall densities after incubation for 24 h [25].

As a result of systematic investigation on the cell-culturing rates and morphological changes of HeLa cells on CNW scaffolds with respect to the wall densities and wettability of CNWs, it was found that the cell-culturing rates were significantly dependent on the CNW densities but seemed to be independent of the surface wettability of the CNW scaffolds. These results enable us to understand the detailed mechanisms of cell culturing on such scaffolds. Moreover, findings in the present study should also contribute to realize various nano-bioapplications using carbon nanomaterials.

24 Fig. 25

25 Fig. 26

26 Fig. 27

**Figure 27.** Number of HeLa cells cultured after incubation for 96 h as a function of the water contact angle. For com‐ parison, data for a commercial glass substrate are also presented [25].
