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Graphene and carbon nanotube–graphene hybrid nanomaterials for human embryonic stem cell culture
Materials Letters 92 (2013) 122–125
Contents lists available at SciVerse ScienceDirect
Materials Letters journal homepage: www.elsevier.com/locate/matlet
Graphene and carbon nanotube–graphene hybrid nanomaterials for human embryonic stem cell culture Meriam Sebaa a, Thanh Yen Nguyen a, Rajat K. Paul b, Ashok Mulchandani c, Huinan Liu a,d,e,n a
Department of Bioengineering, University of California at Riverside, CA 92521, United States Department of Mechanical Engineering, University of California at Riverside, CA 92521, United States c Department of Chemical and Environmental Engineering, University of California at Riverside, CA 92521, United States d Stem Cell Center, University of California at Riverside, CA 92521, United States e Materials Science and Engineering Program, University of California at Riverside, CA 92521, United States b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 September 2012 Accepted 9 October 2012 Available online 22 October 2012
The objective of this study was to explore the potential of graphene and multi-walled carbon nanotube (MWNT)–graphene hybrid nanomaterials for culturing delicate human embryonic stem cells (hESCs) and maintaining their viability. This first in vitro study reports the hESC viability and proliferation when cultured with graphene and MWNT–graphene nanomaterials. The results demonstrated that the hESCs remained viable and pluripotent, and they proliferated on graphene and MWNT–graphene hybrid during 9 days of culture with or without Geltrexs. With Geltrexs, the cells attached and proliferated on graphene-based nanomaterials during 9 days of culture. Without Geltrexs, the cells remained viable and formed cell aggregates suspended in media during 9 days of culture. & 2012 Elsevier B.V. All rights reserved.
Keywords: Graphene Multi-walled carbon nanotubes (MWNT) Multi-walled carbon nanotube–graphene hybrid Human embryonic stem cells Cell viability Cytocompatibility
1. Introduction Recent advances in graphene and carbon nanotubes have created a novel group of nanomaterials with superior electrical, mechanical and biological properties for cell and tissue engineering applications [1]. One advantage of graphene and carbon nanotubes is their stable conductivity in the aqueous physiological environment, thus making them attractive for cellular stimulation. Specifically, graphene showed an electrical conductivity of 1000–2300 S/m [2], and the electrical conductivity of aligned MWNTs was reported to be 1000 S/m along the longitudinal axis of a nanotube and 150 S/m perpendicular to the nanotube axis [3]. Moreover, graphene and carbon nanotubes are both strong and flexible, making them attractive for medical device and implant applications [4]. In addition, carbon nanotubes and graphene can boost neuronal cell activity by providing a shortcut for electrical coupling between somatic and dendritic neuronal compartments. Graphene was studied as a neural electrode for sensing neural cell and tissue activities and recording neural signals [5]. As graphene is an attractive building block for nanoscale electronic devices, graphene field-effect transistors (Gra-FET) were investigated for n Corresponding author at: Department of Bioengineering, University of California at Riverside, 900 University Avenue, Riverside, CA 92521, United States. Tel.: þ 1 951 827 2944; fax: þ 1 951 827 6416. E-mail address: [email protected] (H. Liu).
0167-577X/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.matlet.2012.10.035
their interfacing with embryonic chicken cardiomyocytes [6]. Gra-FET conductance signals recorded from spontaneous beating of cardiomyocytes have produced well-defined and robust extracellular signals with a signal-to-noise ratio routinely greater than 4, which exceeds typical values of other devices [6]. Coupling cells that can differentiate into neurons (such as embryonic stem cells) with conductive graphene and carbon nanotubes can potentially lead to more effective nerve regeneration. In this study, a three-dimensional (3D) MWNT–graphene hybrid nanomaterial and graphene itself were explored for their effects on the human embryonic stem cell (hESC) culture. As reported previously, in the MWNT–graphene hybrid nanomaterials, the roots of MWNTs had seamless crystalline contact with the graphene substrate consisting of a typical C–C covalent bond between the two carbon allotropes [7]. This crystalline contact between MWNTs and graphene would remarkably reduce the contact resistance to achieve maximum charge transfer through the MWNT–graphene hybrid system [7]. This freestanding highly conductive MWNT–graphene hybrid nanomaterial is promising for use as nerve conduit or neural electrode considering its desirable flexibility and strength. Moreover, the 3D nanotubular structure of MWNT on the graphene surface mimics the nanometer dimensions of physiological proteins that mediate cell functions. The biomimetic nano-features of carbon nanotubes were reported to improve stem cell differentiation into neurons and decrease the functions of astrocytes [8,9]. As the viability of
M. Sebaa et al. / Materials Letters 92 (2013) 122–125
embryonic stem cells is the first critical requirement for long-term cell function and tissue engineering, the objective of this first in vitro study was to investigate the viability of hESC when cultured with the novel MWNT–graphene hybrid nanomaterial and graphene itself for potential neural applications.
2. Materials and methods Synthesis of graphene and MWNT–graphene hybrid films using chemical vapor deposition (CVD): Copper (Cu) foil was first decorated with 1 nm thick iron nanoparticles on one side using an ebeam evaporator (Temescal, BJD-1800) and then used as growth substrate for one-step MWNT–graphene growth. The growth substrate was placed inside a fused silica tube (5 cm inner diameter by 100 cm long) and the temperature was ramped to 750 1C in a flowing Ar/H2 atmosphere. The flow rate was 100 sccm (standard cubic centimeter per min) for Ar and 50 sccm for H2. A 50 sccm flow of C2H2 was supplied into the tube for 10 min once the temperature stabilized at 750 1C. After the growth process, the mass flow controller (MFC) for C2H2 was turned off, and the furnace was cooled to room temperature in the flowing Ar/H2 atmosphere. The MWNT–graphene hybrid film formed on the side of iron decorated Cu foil, whereas only graphene film formed on the backside of the Cu foil. The graphene on the backside of the Cu foil was removed by O2 plasma, followed by etching the Cu foil in a 1 M aqueous FeCl3 solution to collect MWNT–graphene films grown on the other side with iron decoration. Subsequently, the films were cleaned with an aqueous HCl (5%) solution and deionized (D.I.) water several times. For graphene film growth, only Cu foil was used as a growth substrate without any iron nanoparticle decoration. All growth conditions were kept the same as for the MWNT–graphene growth. In this case, graphene formed on both sides of the Cu foil. To extract monolayer graphene film on any side, the graphene on the other side of the Cu foil was removed by O2 plasma before the Cu-etching process. Material characterization: Both the graphene and MWNT– graphene films were characterized using scanning electron microscopy (SEM; LEO 1550 or FEI XL30). SEM images were obtained to show the uniformity of the MWNTs on the graphene. The crystalline integrity of MWNT roots on the surface of the graphene film was previously confirmed by transmission electron microscopy (TEM; Philips CM300) [10]. Stem cell culture: The graphene and MWNT–graphene hybrid films were transferred onto a SiO2/Si substrate for the ease of handling during cell culture. The graphene and MWNT–graphene on the SiO2/Si substrates as well as the SiO2/Si control substrates were rinsed in 70% ethanol followed by UV radiation for 12 h on each side to disinfect the surface. All the samples were then placed into a 12-well polystyrene tissue culture plate (BD Falcon) for hESCs culture. All substrates were tested in triplicate either with or without Geltrexs (Invitrogen). Three blank tissue culture wells with hESCs only were used as references. Two 12-well plates with identical sample layout were prepared for the cell culture experiment. Geltrexs was used in only one plate, and the other plate had no Geltrexs. The H9 hESCs (WiCell) were stably transfected with an OCT4eGFP (octamer-binding transcription factor 4-enhanced green fluorescence protein) reporter plasmid as previously described [11]. OCT4 was a marker for undifferentiated cells. H9 hESCs were maintained as feeder-free cultures on Geltrexs in mTeSRs1 media (Stem Cell Technologies). One of the 12-well plates was coated with Geltrexs while the other plate was left uncoated. A confluent T25 flask of H9 OCT4 was enzymatically detached with Accutase (Invitrogen) and centrifuged at 800 rpm for 3 min
123
to form a pellet. The supernatant was removed, and the pellet was re-suspended in mTeSRs1 medium. Next, 0.5 mL of cells (500 cells/mL) in mTeSRs1 media was plated into each well of the two 12-well plates. The cells were incubated for 9 days under standard cell culture conditions. For the plate coated with Geltrexs, the media were replenished every 24 h for all wells. For the plate without Geltrexs, to prevent undesirable removal of hESCs in suspension, 500 mL of media was removed from the top of each well and replaced with fresh mTeSRs1 media every three days during the 9 days of culture. Stem cell imaging: A fluorescence microscope (Nikon Eclipse Ti) was used to image the hESCs after 9 days of culture. Two detection channels were used in this study: One was for imaging the fluorescence from the eGFP in hESCs and the other was for collecting bright field images. The eGFP fluorescence in the hESCs was recorded under auto exposure at 3 ms (excitation wavelength 490 nm and emission wavelength 525 nm). The imaging software (Nikon NIS elements) was used for merging the images. The eGFP expression provides an indicative reporter for OCT4 transcriptional activity in undifferentiated pluripotent hESCs.
3. Results Graphene characterization and properties: Fig. 1 shows scanning electron microscopy (SEM) images of graphene and MWNT–graphene hybrid films transferred onto SiO2/Si substrate. The graphene film formed a few wrinkles during the transfer process due to its ultrathin dimension. In Fig. 1(B), the MWNT–graphene hybrid film showed highly dense MWNTs on the graphene surface; MWNTs were directly grown on the entire graphene surface during the one-step CVD process. The average nanotube diameter was 30 nm. This unique 3D carbon nanotube architecture would provide high surface area for cell growth as well as 3D electrical conductivity. Stem cell viability and adhesion: Fig. 2 shows the merged images of H9 hESCs, which were seeded into the culture well with Geltrexs and cultured for 3 h. The expression of eGFP (green fluorescence) indicated cell viability and pluripotency. The hESC colonies were scattered and not confluent at 3 h. Fig. 2 shows the initial stage of hESC culture with lower hESC density, which served as an important reference point for the status of hESCs during the subsequent 9 days of culture.
Graphene
2 µm
2 µm
SiO2/Si
MWNT on Graphene
Fig. 1. Scanning electron micrographs of (A) graphene and (B) MWNT–graphene hybrid nanomaterials synthesized by chemical vapor deposition (CVD).
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Fig. 3 shows the merged images of the hESCs after they were cultured on the substrates of interest with or without Geltrexs for 9 days. Fig. 3(a–d) shows the cells that were grown in the wells without Geltrexs. Fig. 3(e–h) shows the cells that were grown in the wells coated with Geltrexs. After 9 days of incubation under the standard cell culture conditions, the hESCs remained viable and pluripotent, as indicated by the expression of eGFP (green fluorescence). Significant cell proliferation was observed in all the wells after 9 days of culture as compared with that at 3 h of culture. Without Geltrexs, the viable and pluripotent hESCs formed aggregates and became suspended in the media. As indicated by the presence of green fluorescence, the hESCs cultured with graphene, MWNT–graphene hybrid, and SiO2/Si substrates were as viable as the blank reference wells with hESCs only (Fig. 3a–d). This indicated that graphene and MWNT–graphene hybrid did not affect the viability of H9 hESCs, even though hESCs could not directly attach on any surface due to the absence of Geltrexs. With Geltrexs, the hESCs attached, maintained cell morphology, and proliferated to confluence after 9 days of culture in contrast to scattered hESC colonies at 3 h of culture. The hESCs cultured with graphene and MWNT–graphene hybrid films proliferated as confluent as the cells on the SiO2/Si
500 µm Fig. 2. The H9 hESC colonies that attached on Geltrexs after 3 h of culture served as an initial reference point. Original magnification was 4 . Scale bar is 500 mm.
Without Geltrex
Graphene
500 µm
With Geltrex
200µm
MWNT-Graphene
500 µm 200µm
control and blank reference (Fig. 3e–h). There was no significant difference between the wells containing graphene and MWNT– graphene films. The dark areas in Fig. 3(e–h) are the corners of the substrates (graphene, MWNT–graphene, SiO2/Si substrates). The cells grew very confluent in the presence of all substrates with no significant differences in viability and pluripotency.
4. Discussion This study demonstrated, for the first time, the viability and proliferation of hESCs when cultured with the novel MWNT– graphene hybrid and graphene nanomaterials. As expected, H9 hESCs did not adhere to any of the substrates without Geltrexs because the adhesion of hESCs requires either a feeder layer or Geltrexs. Geltrexs is a commercial product intended to eliminate the necessity of feeder layer for hESC culture, thus reducing the risks for future experiments in vivo. Moreover, Geltrexs is designed to decrease variability of the hESC cell culture experiments. The results suggested that MWNT–graphene hybrid and graphene nanomaterials are compatible with hESCs and suitable for hESCs culture. Although no significant differences in hESC viability were observed in this study when comparing MWNT– graphene hybrid and graphene alone, it is still important to demonstrate the cytocompatibility of MWNT–graphene because the advantageous electrical properties of MWNT–graphene hybrid have a great potential for future electrical stimulation of nerve regeneration. This in vitro study with hESCs was the first step toward developing graphene-based nanomaterials for neural applications, and more in-depth characterization is needed for their potential in coupling with hESCs to induce neural tissue regeneration. A few recent studies in the literature examined neural cell behavior on graphene and showed promising results for neural applications [12]. For example, Park et al. reported that graphene enhanced adhesion and neuronal differentiation of immortalized human neural stem cells (hNSC; ReNcell VM, Millipore) as compared with glass coverslips. To note, both the graphene and cover glass substrates were coated with laminin to help hNSC attachment. Moreover, more neurons (39%) than glial cells (23%) were observed on graphene substrates, while more glial cells (35%) than neurons (22%) were found on the glass coverslip substrates [12]. Glass coverslip substrates also had a smaller number of differentiated cells as compared with the graphene
SiO2 /Si
Control
500µm 200µm
500µm 200µm
Fig. 3. Fluorescence images of H9 hESCs cultured with the substrates of interest with and without Geltrexs for 9 days. (a–d) without Geltrexs; magnification at 4 . (e–h) with Geltrexs; magnification at 10 . H9 hESCs cultured with (a,e) graphene substrates, (b,f) MWNT–graphene hybrid substrates, (c,g) SiO2/Si control, and (d,h) blank (hESC only) reference.
M. Sebaa et al. / Materials Letters 92 (2013) 122–125
substrates. The hNCSs exhibited neurite outgrowths and elongated cell shapes on both substrates after 1 month of cell culture, leading to neural network formation. Li et al. investigated the neurite sprouting and outgrowth of mouse hippocampal neurons when cultured with graphene [13]. The results showed that the number and length of neurites increased on graphene substrates compared with tissue culture polystyrene plate control [13]. These results indicated that graphene provided a more favorable microenvironment for hNSC differentiation [12]. Although these recent studies provided promising evidences for the future biomedical applications of graphene-based nanomaterials, this first in vitro study of graphene and MWNT–graphene hybrid for hESCs culture provided critical information on the potential of coupling graphene-based nanomaterials with hESCs for neural stimulation and regeneration.
5. Conclusions This study demonstrated that MWNT–graphene hybrid and graphene nanomaterials are not toxic to hESCs. The H9 hESCs remained viable and pluripotent on graphene-based nanomaterials with or without Geltrexs. With Geltrexs, hESCs attached and proliferated on MWNT–graphene hybrid and graphene during 9 days of culture. Without Geltrexs, hESCs still remained viable and formed cell aggregates suspended in the media during 9 days of culture. This study, together with other recent cell studies on graphene, suggest that MWNT–graphene hybrid and graphene can be used for promoting and stimulating adhesion, proliferation, and differentiation of stem cells for neural tissue regenerative therapies. In addition, MWNT–graphene hybrid and graphene can be used for neural electrodes and prostheses considering their excellent electrical properties, mechanical properties, and cytocompatibilities. Therefore, MWNT–graphene hybrid and graphene nanomaterials should be further studied for neural and other biomedical applications.
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Acknowledgments The authors would like to thank the University of California at Riverside for financial support. We would also like to thank the Central Facility for Advanced Microscopy and Microanalysis (CFAMM) and the Stem Cell Center at the University of California at Riverside. References [1] Zhang Y, Nayak TR, Hong H, Cai WB. Graphene: a versatile nanoplatform for biomedical applications. Nanoscale 2012;4:3833–42. [2] Schniepp HC, Li JL, McAllister MJ, Sai H, Herrera-Alonso M, Adamson DH, et al. Functionalized single graphene sheets derived from splitting graphite oxide. J Phys Chem B 2006;110:8535–9. [3] De Heer WA, Bacsa WS, Chˆatelain A, Gerfin T, Humphrey-Baker R, Forro L, et al. Aligned carbon nanotube films: production and optical and electronic properties. Science 1995;268:845–7. [4] Yang L, Zhang LJ, Webster TJ. Carbon nanostructures for orthopedic medical applications. Nanomedicine-Uk 2011;6:1231–44. [5] Chen CTL CH, Chen JJ, Hsu WL, Chang YC, Yeh SR, Li LJ, et al. A graphenebased microelectrode for recording neural signals. Transducers 2011:1883–6. [6] Cohen-Karni T, Qing Q, Li Q, Fang Y, Lieber CM. Graphene and nanowire transistors for cellular interfaces and electrical recording. Nano Lett 2010;10:1098–102. [7] Paul RK. Synthesis and applications of large area graphene-based electrode systems. Riverside: University of California-Riverside; 2011. [8] Jan E, Kotov NA. Successful differentiation of mouse neural stem cells on layer-by-layer assembled single-walled carbon nanotube composite. Nano Lett 2007;7:1123–8. [9] Tran PA, Zhang LJ, Webster TJ. Carbon nanofibers and carbon nanotubes in regenerative medicine. Adv Drug Deliv Rev 2009;61:1097–114. [10] Paul RK, Ghazinejad M, Penchev M, Lin JA, Ozkan M, Ozkan CS. Synthesis of a pillared graphene nanostructure: a counterpart of three-dimensional carbon architectures. Small 2010;6:2309–13. [11] Chatterjee P, Cheung Y, Liew C. Transfecting and nucleofecting human induced pluripotent stem cells. J Vis Exp 2011. [12] Park SY, Park J, Sim SH, Sung MG, Kim KS, Hong BH, et al. Enhanced differentiation of human neural stem cells into neurons on graphene. Adv Mater 2011;23:H263–7. [13] Li N, Zhang XM, Song Q, Su RG, Zhang Q, Kong T, et al. The promotion of neurite sprouting and outgrowth of mouse hippocampal cells in culture by graphene substrates. Biomaterials 2011;32:9374–82.
Contents lists available at SciVerse ScienceDirect
Materials Letters journal homepage: www.elsevier.com/locate/matlet
Graphene and carbon nanotube–graphene hybrid nanomaterials for human embryonic stem cell culture Meriam Sebaa a, Thanh Yen Nguyen a, Rajat K. Paul b, Ashok Mulchandani c, Huinan Liu a,d,e,n a
Department of Bioengineering, University of California at Riverside, CA 92521, United States Department of Mechanical Engineering, University of California at Riverside, CA 92521, United States c Department of Chemical and Environmental Engineering, University of California at Riverside, CA 92521, United States d Stem Cell Center, University of California at Riverside, CA 92521, United States e Materials Science and Engineering Program, University of California at Riverside, CA 92521, United States b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 September 2012 Accepted 9 October 2012 Available online 22 October 2012
The objective of this study was to explore the potential of graphene and multi-walled carbon nanotube (MWNT)–graphene hybrid nanomaterials for culturing delicate human embryonic stem cells (hESCs) and maintaining their viability. This first in vitro study reports the hESC viability and proliferation when cultured with graphene and MWNT–graphene nanomaterials. The results demonstrated that the hESCs remained viable and pluripotent, and they proliferated on graphene and MWNT–graphene hybrid during 9 days of culture with or without Geltrexs. With Geltrexs, the cells attached and proliferated on graphene-based nanomaterials during 9 days of culture. Without Geltrexs, the cells remained viable and formed cell aggregates suspended in media during 9 days of culture. & 2012 Elsevier B.V. All rights reserved.
Keywords: Graphene Multi-walled carbon nanotubes (MWNT) Multi-walled carbon nanotube–graphene hybrid Human embryonic stem cells Cell viability Cytocompatibility
1. Introduction Recent advances in graphene and carbon nanotubes have created a novel group of nanomaterials with superior electrical, mechanical and biological properties for cell and tissue engineering applications [1]. One advantage of graphene and carbon nanotubes is their stable conductivity in the aqueous physiological environment, thus making them attractive for cellular stimulation. Specifically, graphene showed an electrical conductivity of 1000–2300 S/m [2], and the electrical conductivity of aligned MWNTs was reported to be 1000 S/m along the longitudinal axis of a nanotube and 150 S/m perpendicular to the nanotube axis [3]. Moreover, graphene and carbon nanotubes are both strong and flexible, making them attractive for medical device and implant applications [4]. In addition, carbon nanotubes and graphene can boost neuronal cell activity by providing a shortcut for electrical coupling between somatic and dendritic neuronal compartments. Graphene was studied as a neural electrode for sensing neural cell and tissue activities and recording neural signals [5]. As graphene is an attractive building block for nanoscale electronic devices, graphene field-effect transistors (Gra-FET) were investigated for n Corresponding author at: Department of Bioengineering, University of California at Riverside, 900 University Avenue, Riverside, CA 92521, United States. Tel.: þ 1 951 827 2944; fax: þ 1 951 827 6416. E-mail address: [email protected] (H. Liu).
0167-577X/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.matlet.2012.10.035
their interfacing with embryonic chicken cardiomyocytes [6]. Gra-FET conductance signals recorded from spontaneous beating of cardiomyocytes have produced well-defined and robust extracellular signals with a signal-to-noise ratio routinely greater than 4, which exceeds typical values of other devices [6]. Coupling cells that can differentiate into neurons (such as embryonic stem cells) with conductive graphene and carbon nanotubes can potentially lead to more effective nerve regeneration. In this study, a three-dimensional (3D) MWNT–graphene hybrid nanomaterial and graphene itself were explored for their effects on the human embryonic stem cell (hESC) culture. As reported previously, in the MWNT–graphene hybrid nanomaterials, the roots of MWNTs had seamless crystalline contact with the graphene substrate consisting of a typical C–C covalent bond between the two carbon allotropes [7]. This crystalline contact between MWNTs and graphene would remarkably reduce the contact resistance to achieve maximum charge transfer through the MWNT–graphene hybrid system [7]. This freestanding highly conductive MWNT–graphene hybrid nanomaterial is promising for use as nerve conduit or neural electrode considering its desirable flexibility and strength. Moreover, the 3D nanotubular structure of MWNT on the graphene surface mimics the nanometer dimensions of physiological proteins that mediate cell functions. The biomimetic nano-features of carbon nanotubes were reported to improve stem cell differentiation into neurons and decrease the functions of astrocytes [8,9]. As the viability of
M. Sebaa et al. / Materials Letters 92 (2013) 122–125
embryonic stem cells is the first critical requirement for long-term cell function and tissue engineering, the objective of this first in vitro study was to investigate the viability of hESC when cultured with the novel MWNT–graphene hybrid nanomaterial and graphene itself for potential neural applications.
2. Materials and methods Synthesis of graphene and MWNT–graphene hybrid films using chemical vapor deposition (CVD): Copper (Cu) foil was first decorated with 1 nm thick iron nanoparticles on one side using an ebeam evaporator (Temescal, BJD-1800) and then used as growth substrate for one-step MWNT–graphene growth. The growth substrate was placed inside a fused silica tube (5 cm inner diameter by 100 cm long) and the temperature was ramped to 750 1C in a flowing Ar/H2 atmosphere. The flow rate was 100 sccm (standard cubic centimeter per min) for Ar and 50 sccm for H2. A 50 sccm flow of C2H2 was supplied into the tube for 10 min once the temperature stabilized at 750 1C. After the growth process, the mass flow controller (MFC) for C2H2 was turned off, and the furnace was cooled to room temperature in the flowing Ar/H2 atmosphere. The MWNT–graphene hybrid film formed on the side of iron decorated Cu foil, whereas only graphene film formed on the backside of the Cu foil. The graphene on the backside of the Cu foil was removed by O2 plasma, followed by etching the Cu foil in a 1 M aqueous FeCl3 solution to collect MWNT–graphene films grown on the other side with iron decoration. Subsequently, the films were cleaned with an aqueous HCl (5%) solution and deionized (D.I.) water several times. For graphene film growth, only Cu foil was used as a growth substrate without any iron nanoparticle decoration. All growth conditions were kept the same as for the MWNT–graphene growth. In this case, graphene formed on both sides of the Cu foil. To extract monolayer graphene film on any side, the graphene on the other side of the Cu foil was removed by O2 plasma before the Cu-etching process. Material characterization: Both the graphene and MWNT– graphene films were characterized using scanning electron microscopy (SEM; LEO 1550 or FEI XL30). SEM images were obtained to show the uniformity of the MWNTs on the graphene. The crystalline integrity of MWNT roots on the surface of the graphene film was previously confirmed by transmission electron microscopy (TEM; Philips CM300) [10]. Stem cell culture: The graphene and MWNT–graphene hybrid films were transferred onto a SiO2/Si substrate for the ease of handling during cell culture. The graphene and MWNT–graphene on the SiO2/Si substrates as well as the SiO2/Si control substrates were rinsed in 70% ethanol followed by UV radiation for 12 h on each side to disinfect the surface. All the samples were then placed into a 12-well polystyrene tissue culture plate (BD Falcon) for hESCs culture. All substrates were tested in triplicate either with or without Geltrexs (Invitrogen). Three blank tissue culture wells with hESCs only were used as references. Two 12-well plates with identical sample layout were prepared for the cell culture experiment. Geltrexs was used in only one plate, and the other plate had no Geltrexs. The H9 hESCs (WiCell) were stably transfected with an OCT4eGFP (octamer-binding transcription factor 4-enhanced green fluorescence protein) reporter plasmid as previously described [11]. OCT4 was a marker for undifferentiated cells. H9 hESCs were maintained as feeder-free cultures on Geltrexs in mTeSRs1 media (Stem Cell Technologies). One of the 12-well plates was coated with Geltrexs while the other plate was left uncoated. A confluent T25 flask of H9 OCT4 was enzymatically detached with Accutase (Invitrogen) and centrifuged at 800 rpm for 3 min
123
to form a pellet. The supernatant was removed, and the pellet was re-suspended in mTeSRs1 medium. Next, 0.5 mL of cells (500 cells/mL) in mTeSRs1 media was plated into each well of the two 12-well plates. The cells were incubated for 9 days under standard cell culture conditions. For the plate coated with Geltrexs, the media were replenished every 24 h for all wells. For the plate without Geltrexs, to prevent undesirable removal of hESCs in suspension, 500 mL of media was removed from the top of each well and replaced with fresh mTeSRs1 media every three days during the 9 days of culture. Stem cell imaging: A fluorescence microscope (Nikon Eclipse Ti) was used to image the hESCs after 9 days of culture. Two detection channels were used in this study: One was for imaging the fluorescence from the eGFP in hESCs and the other was for collecting bright field images. The eGFP fluorescence in the hESCs was recorded under auto exposure at 3 ms (excitation wavelength 490 nm and emission wavelength 525 nm). The imaging software (Nikon NIS elements) was used for merging the images. The eGFP expression provides an indicative reporter for OCT4 transcriptional activity in undifferentiated pluripotent hESCs.
3. Results Graphene characterization and properties: Fig. 1 shows scanning electron microscopy (SEM) images of graphene and MWNT–graphene hybrid films transferred onto SiO2/Si substrate. The graphene film formed a few wrinkles during the transfer process due to its ultrathin dimension. In Fig. 1(B), the MWNT–graphene hybrid film showed highly dense MWNTs on the graphene surface; MWNTs were directly grown on the entire graphene surface during the one-step CVD process. The average nanotube diameter was 30 nm. This unique 3D carbon nanotube architecture would provide high surface area for cell growth as well as 3D electrical conductivity. Stem cell viability and adhesion: Fig. 2 shows the merged images of H9 hESCs, which were seeded into the culture well with Geltrexs and cultured for 3 h. The expression of eGFP (green fluorescence) indicated cell viability and pluripotency. The hESC colonies were scattered and not confluent at 3 h. Fig. 2 shows the initial stage of hESC culture with lower hESC density, which served as an important reference point for the status of hESCs during the subsequent 9 days of culture.
Graphene
2 µm
2 µm
SiO2/Si
MWNT on Graphene
Fig. 1. Scanning electron micrographs of (A) graphene and (B) MWNT–graphene hybrid nanomaterials synthesized by chemical vapor deposition (CVD).
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M. Sebaa et al. / Materials Letters 92 (2013) 122–125
Fig. 3 shows the merged images of the hESCs after they were cultured on the substrates of interest with or without Geltrexs for 9 days. Fig. 3(a–d) shows the cells that were grown in the wells without Geltrexs. Fig. 3(e–h) shows the cells that were grown in the wells coated with Geltrexs. After 9 days of incubation under the standard cell culture conditions, the hESCs remained viable and pluripotent, as indicated by the expression of eGFP (green fluorescence). Significant cell proliferation was observed in all the wells after 9 days of culture as compared with that at 3 h of culture. Without Geltrexs, the viable and pluripotent hESCs formed aggregates and became suspended in the media. As indicated by the presence of green fluorescence, the hESCs cultured with graphene, MWNT–graphene hybrid, and SiO2/Si substrates were as viable as the blank reference wells with hESCs only (Fig. 3a–d). This indicated that graphene and MWNT–graphene hybrid did not affect the viability of H9 hESCs, even though hESCs could not directly attach on any surface due to the absence of Geltrexs. With Geltrexs, the hESCs attached, maintained cell morphology, and proliferated to confluence after 9 days of culture in contrast to scattered hESC colonies at 3 h of culture. The hESCs cultured with graphene and MWNT–graphene hybrid films proliferated as confluent as the cells on the SiO2/Si
500 µm Fig. 2. The H9 hESC colonies that attached on Geltrexs after 3 h of culture served as an initial reference point. Original magnification was 4 . Scale bar is 500 mm.
Without Geltrex
Graphene
500 µm
With Geltrex
200µm
MWNT-Graphene
500 µm 200µm
control and blank reference (Fig. 3e–h). There was no significant difference between the wells containing graphene and MWNT– graphene films. The dark areas in Fig. 3(e–h) are the corners of the substrates (graphene, MWNT–graphene, SiO2/Si substrates). The cells grew very confluent in the presence of all substrates with no significant differences in viability and pluripotency.
4. Discussion This study demonstrated, for the first time, the viability and proliferation of hESCs when cultured with the novel MWNT– graphene hybrid and graphene nanomaterials. As expected, H9 hESCs did not adhere to any of the substrates without Geltrexs because the adhesion of hESCs requires either a feeder layer or Geltrexs. Geltrexs is a commercial product intended to eliminate the necessity of feeder layer for hESC culture, thus reducing the risks for future experiments in vivo. Moreover, Geltrexs is designed to decrease variability of the hESC cell culture experiments. The results suggested that MWNT–graphene hybrid and graphene nanomaterials are compatible with hESCs and suitable for hESCs culture. Although no significant differences in hESC viability were observed in this study when comparing MWNT– graphene hybrid and graphene alone, it is still important to demonstrate the cytocompatibility of MWNT–graphene because the advantageous electrical properties of MWNT–graphene hybrid have a great potential for future electrical stimulation of nerve regeneration. This in vitro study with hESCs was the first step toward developing graphene-based nanomaterials for neural applications, and more in-depth characterization is needed for their potential in coupling with hESCs to induce neural tissue regeneration. A few recent studies in the literature examined neural cell behavior on graphene and showed promising results for neural applications [12]. For example, Park et al. reported that graphene enhanced adhesion and neuronal differentiation of immortalized human neural stem cells (hNSC; ReNcell VM, Millipore) as compared with glass coverslips. To note, both the graphene and cover glass substrates were coated with laminin to help hNSC attachment. Moreover, more neurons (39%) than glial cells (23%) were observed on graphene substrates, while more glial cells (35%) than neurons (22%) were found on the glass coverslip substrates [12]. Glass coverslip substrates also had a smaller number of differentiated cells as compared with the graphene
SiO2 /Si
Control
500µm 200µm
500µm 200µm
Fig. 3. Fluorescence images of H9 hESCs cultured with the substrates of interest with and without Geltrexs for 9 days. (a–d) without Geltrexs; magnification at 4 . (e–h) with Geltrexs; magnification at 10 . H9 hESCs cultured with (a,e) graphene substrates, (b,f) MWNT–graphene hybrid substrates, (c,g) SiO2/Si control, and (d,h) blank (hESC only) reference.
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substrates. The hNCSs exhibited neurite outgrowths and elongated cell shapes on both substrates after 1 month of cell culture, leading to neural network formation. Li et al. investigated the neurite sprouting and outgrowth of mouse hippocampal neurons when cultured with graphene [13]. The results showed that the number and length of neurites increased on graphene substrates compared with tissue culture polystyrene plate control [13]. These results indicated that graphene provided a more favorable microenvironment for hNSC differentiation [12]. Although these recent studies provided promising evidences for the future biomedical applications of graphene-based nanomaterials, this first in vitro study of graphene and MWNT–graphene hybrid for hESCs culture provided critical information on the potential of coupling graphene-based nanomaterials with hESCs for neural stimulation and regeneration.
5. Conclusions This study demonstrated that MWNT–graphene hybrid and graphene nanomaterials are not toxic to hESCs. The H9 hESCs remained viable and pluripotent on graphene-based nanomaterials with or without Geltrexs. With Geltrexs, hESCs attached and proliferated on MWNT–graphene hybrid and graphene during 9 days of culture. Without Geltrexs, hESCs still remained viable and formed cell aggregates suspended in the media during 9 days of culture. This study, together with other recent cell studies on graphene, suggest that MWNT–graphene hybrid and graphene can be used for promoting and stimulating adhesion, proliferation, and differentiation of stem cells for neural tissue regenerative therapies. In addition, MWNT–graphene hybrid and graphene can be used for neural electrodes and prostheses considering their excellent electrical properties, mechanical properties, and cytocompatibilities. Therefore, MWNT–graphene hybrid and graphene nanomaterials should be further studied for neural and other biomedical applications.
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Acknowledgments The authors would like to thank the University of California at Riverside for financial support. We would also like to thank the Central Facility for Advanced Microscopy and Microanalysis (CFAMM) and the Stem Cell Center at the University of California at Riverside. References [1] Zhang Y, Nayak TR, Hong H, Cai WB. Graphene: a versatile nanoplatform for biomedical applications. Nanoscale 2012;4:3833–42. [2] Schniepp HC, Li JL, McAllister MJ, Sai H, Herrera-Alonso M, Adamson DH, et al. Functionalized single graphene sheets derived from splitting graphite oxide. J Phys Chem B 2006;110:8535–9. [3] De Heer WA, Bacsa WS, Chˆatelain A, Gerfin T, Humphrey-Baker R, Forro L, et al. Aligned carbon nanotube films: production and optical and electronic properties. Science 1995;268:845–7. [4] Yang L, Zhang LJ, Webster TJ. Carbon nanostructures for orthopedic medical applications. Nanomedicine-Uk 2011;6:1231–44. [5] Chen CTL CH, Chen JJ, Hsu WL, Chang YC, Yeh SR, Li LJ, et al. A graphenebased microelectrode for recording neural signals. Transducers 2011:1883–6. [6] Cohen-Karni T, Qing Q, Li Q, Fang Y, Lieber CM. Graphene and nanowire transistors for cellular interfaces and electrical recording. Nano Lett 2010;10:1098–102. [7] Paul RK. Synthesis and applications of large area graphene-based electrode systems. Riverside: University of California-Riverside; 2011. [8] Jan E, Kotov NA. Successful differentiation of mouse neural stem cells on layer-by-layer assembled single-walled carbon nanotube composite. Nano Lett 2007;7:1123–8. [9] Tran PA, Zhang LJ, Webster TJ. Carbon nanofibers and carbon nanotubes in regenerative medicine. Adv Drug Deliv Rev 2009;61:1097–114. [10] Paul RK, Ghazinejad M, Penchev M, Lin JA, Ozkan M, Ozkan CS. Synthesis of a pillared graphene nanostructure: a counterpart of three-dimensional carbon architectures. Small 2010;6:2309–13. [11] Chatterjee P, Cheung Y, Liew C. Transfecting and nucleofecting human induced pluripotent stem cells. J Vis Exp 2011. [12] Park SY, Park J, Sim SH, Sung MG, Kim KS, Hong BH, et al. Enhanced differentiation of human neural stem cells into neurons on graphene. Adv Mater 2011;23:H263–7. [13] Li N, Zhang XM, Song Q, Su RG, Zhang Q, Kong T, et al. The promotion of neurite sprouting and outgrowth of mouse hippocampal cells in culture by graphene substrates. Biomaterials 2011;32:9374–82.