BioNanoSci. (2014) 4:288–300 DOI 10.1007/s12668-014-0139-4
Carbon Nanotube Thin Film-Supported Fibroblast and Pluripotent Stem Cell Growth Zhimin Tao & Peizhe Wang & Fengzhi Zhang & Jie Na
Published online: 18 June 2014 # Springer Science+Business Media New York 2014
Abstract In this study, we functionalized superaligned carbon nanotube (CNT) thin film and performed detailed analysis to characterize the surface modifications. Our results demonstrated that chemical and biochemical modification of CNT thin films significantly altered the level of hydrophilicity. We also showed that the growth of mammalian fibroblasts and pluripotent stem cells on functionalized CNT thin films were better than that on untreated CNT films. Moreover, extracellular matrix protein-conjugated CNT thin film can support pluripotent stem cell growth without feeder cells. These results suggest that once properly functionalized, CNT thin film can be a suitable scaffolding material for a broad range of mammalian cell culture. Our work provided basis for future cell/tissue engineering with this material. Keywords Carbon nanotube . Tissue engineering . Primary fibroblasts . Stem cell culture
1 Introduction Due to their unique physical and chemical properties, novel materials at nanoscale are taking cutting edges and thereby becoming useful tools in biomedical research. Numerous nanoparticles have been fabricated with diverse chemical components and physical characters to induce diverse biological effects [1–7]. Researches on the applications of nanotechnology in stem cell biology have maintained their steadily rapid growth Z. Tao (*) Department of Physics, Tsinghua University, Beijing 100084, China e-mail:
[email protected] P. Wang : F. Zhang : J. Na (*) School of Medicine, Tsinghua Universitys, Beijing 100084, China e-mail:
[email protected]
in recent years, as scientists are making efforts to marry the advanced nanochemistry with regenerative medicine, in order to find new ways to solve conventional medical problems. Nano or submicron particles, in a dimension of 1–1,000 nm, have been used in stem cell research, including cell labeling and tracking, the delivery of drugs and biomolecules, and directed cell growth and differentiation [8–15]. One of the biggest challenges in current stem cell therapy is how to make injected stem cells integrate into the host tissues. Changes in the microenvironment during the transplantation may inhibit the proliferation or differentiation of the engrafted stem cells. Being layer-by-layer assembled thin films of various materials, nanostructured scaffolds provided a niche for the encapsulated stem cells to grow, expand, and differentiate as desired, thereby maximizing their survival and minimizing the immune response. To this end, chemical composition and surface topography are thought to be the major determinants to affect the stem cell attachment, growth, and differentiation. For instance, mouse mesenchymal stem cells (MSCs) showed a much faster growth rate on a rougher surface when the number of layers of TiO2 thin films was increased, with no apparent toxicity on the attached cells [16]. Carbon nanotube (CNT) thin films with enhanced biocompatibility selectively differentiated the mammalian embryonic stem cells into neural or bone cells while maintaining the cell viability and morphology [17–20]. With few data reported, how different nanostructured scaffolding materials affect stem cell functions is still poorly characterized. Cytotoxicity of CNTs has been systematically investigated in many mammalian cells or animal models, reaching a conclusion that the materials are non-toxic once tolerable doses and functionalized surface are achieved [4, 21–24]. Studies on the toxicity profiles of CNTs in stem cells suggested that the safe dosage of CNTs towards cell proliferation and differentiation varied, depending on the functional group on their outer surface [25]. Pilot studies on the differentiation behaviors of mouse neural stem cells (NSCs) on layer-by-layer assembled single-walled CNT
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composites have been carried out in Kotov group [26, 27]. Mouse NSCs were seeded on glass coverslips coated with six bilayers of single-walled CNT-poly(ethyleneimine) (PEI) thin films, and the cell viability was maintained up to 3 days. To demonstrate the differentiation of mouse NSCs on functionalized CNT thin films, authors performed immunostaining using cell type-specific antibodies to show that NSCs grown on the layerby-layer assembled single-walled CNT composites successfully differentiated into neurons, astrocytes, and oligodendrocytes [26]. Similar studies of NSCs were also carried out in single-walled CNT thin films, with connection of laminin between layers, a glycoprotein which had been widely used to enhance cell adhesion [27]. The annealed CNTlaminin substrate exhibited a nice biocompatibility to stem cells and offered a novel and convenient method to study differentiation behaviors of stem cells. Here, we report a CNT thin film matrix-based culture of human and mouse fibroblasts and feeder cell layersupported or feeder-free growth of mouse embryonic stem cells (mESCs) on functionalized CNT thin films. A unique superaligned CNT thin film has been recently developed with many physical applications [28–33]. Multi-walled CNTs were first grown perpendicularly on the silicon wafer in parallel and with high density, followed by being pulled out to form a continuous yarn as CNTs were connected end by end due to van der Waals force [28–33]. Through this method, vertically grown CNTs could be transformed into the horizontally superaligned CNT thin films with excellent conductivity and elasticity. One critical drawback of many nanostructured materials is their hydrophobicity and the associated toxicity, due to their Scheme 1 Various functionalizations of CNT thin films. As-made (untreated) CNT films underwent oxidization, PEGylation, and laminin conjugation in sequence
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chemical composition and physical profile [5–7]. This significantly limited their use in biomedical research and future clinical applications. Unmodified CNTs exhibited hydrophobic characteristics and were incompatible with mammalian cell culture. Thus, pristine CNT films need to be functionalized to improve their hydrophilicity and therefore biocompatibility. In previous work by other groups, surface functionalization of CNT films were typically done by two steps. First, dispersed CNTs were modified with chemical or biochemical ligands; second, modified CNTs were assembled on a solid surface to form a CNT film [25–27]. As such, thin films made from above method had random surface topography, which compromised their electrical and mechanical properties compared to suspended CNT particles. In our method, to preserve the unique elasticity and conductivity of superaligned CNT films for future applications in both experimental and clinical medicine scenarios, we adopted a facile chemistry to oxidize CNT films [34] and then conjugated synthetic polymers and functional protein ligands onto CNT films (Scheme 1). Our results demonstrated that this surface modification indeed significantly improved the biocompatibility of CNT films, and both human foreskin fibroblasts (HFFs) and mouse embryonic fibroblasts (MEFs) can attach, spread, and grow on these CNT films. We also showed that mouse embryonic stem cells (mESCs) can grow on CNT films covered with MEF feeder cells as well as on functionalized CNT films without cell feeder layers. Our research convincingly demonstrated that, once properly modified, CNT films can even support the self-renew growth of pluripotent stem cells which is known to be very sensitive. This research lays the foundation of using properly functionalized CNT films for stem cell research and tissue engineering.
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2 Materials and Methods 2.1 Functionalization of SACNT Films Superaligned CNT films were prepared as previously reported [28–33]. In brief, SACNT thin films were cross-placed on each top; that is, one latitudinal and another longitudinal in turn for a total of 30 layers, being untreated CNT. For oxidation, 30-layer untreated CNT films were placed over a pool of 65–68 % (wt%) nitric acid in a sealed Teflon-lined autoclave and heated at 120 °C for 6 h. Films were then soaked in dH2O, followed by the addition of 1 M NaOH drop-wise until the value of pH reaches 8 in the solution. The neutralized samples were kept in dH2O overnight and rinsed throughout before further use. For PEGylated CNT film preparation, 4×4 cm2 of oxidized SACNT films were first soaked in a 5-mL 1×PBS solution, followed by consequent additions of 4.8 mg of 1ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (i.e., EDC, final concentration=5 mM, Alfa Aesar, Tianjin, China) and 50 mg of 6-armed PEG-amine ((NH2)6-PEG, m.w.=5, 000, SunBio, Korea). After incubation at room temperature for 3–4 h, another 14.4 mg of EDC was added into the solution to reach a final concentration of 20 mM, left overnight. The pH value of the reaction solution was confirmed slightly over 7.0 (∼7.4). At the next day, the PEGylated CNT films were rinsed throughout by and immersed in 1× PBS solution for the next use. For laminin-conjugated CNT films, 4×4 cm2 of PEGylated CNT films were incubated with 10-μg/cm2 laminin proteins (from Engelbreth-Holm-Swarm murine sarcoma basement membrane, 1 mg/mL in Trisbuffered NaCl, Sigma-Aldrich), and 20 mM EDC in 1×PBS solution, kept overnight. The pH value of the reaction solution was confirmed ∼7.4. The laminin-conjugated CNT films were rinsed by PBS throughout before use.
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angle tests. Contact angle values were measured at room temperatures, using an optical contact angle measuring device (OCA-20, DataPhysics Instruments GmbH, Filderstadt, Germany). In each measurement, a water droplet of 2 μL was dispensed onto the surface of CNT films. Water contact angles of at least three different spots on one CNT film were acquired and averaged. Fourier transform infrared spectra (FT-IR) were next acquired for CNT films, collected on a Bruker IFS 66 v/s spectrometer at 2-cm−1 resolution. For XPS measurements, an area of 1×1 cm2 of various CNT film was individually placed onto silicon wafer, wetted by ethanol, and left completely dry. XPS measurements were then performed in a PHI-5300ESCA Perkin-Elmer spectrometer, using monochromatic Mg Kα (1253.6 eV) radiation at 14 kV and 250 W. Element analyses were conducted for C, O, and N elements. CNT films after the same preparation were subject to measurements. Finally, to estimate the surface roughness of all films, we instrumented a profilometer (Ambios, XP-1) and quantified the surface roughness by the value of Ra. Each film was measured at three random spots, scanned by stylus tip for 2.0 mm, and each datum was taken every 3.3 mm length. As a result, the arithmetical mean roughness (Ra, Eq. 1) was calculated to describe the topographic profile of the surface. Calculations were illustrated as follows. In each sample, three individual tests from three random spots were finally averaged for a statistical purpose.
2.2 Characterization of CNT Films
2.3 Fibroblasts Cell Isolation and Culture
For SEM images, CNT films were placed onto a 1×1-cm2 glass coverslide, pre-treated with 0.1 % (w/v) polyethylenimine (PEI). Samples were completely dried and characterized using a Hitachi S-3600N SEM (electron beam=10 kv). For analysis of Raman spectroscopy, an area of 2×2 cm2 of various CNT film was individually placed onto a square Teflon frame (outer length= 20 mm, inner length=16 mm, with a frame length of 2 mm) and dried completely. All samples were then investigated by Raman spectra, using a Renishaw spectrometer (1,800 gr/mm grating mode) with a laser excitation at 514.5 nm. Raman shift was scanned from 400 to 2,800 cm−1. The laser was focused to a 1–2-μm spot size on the samples, and the power was set to less than 2 mW. Three different spots on one film were randomly measured, and the characteristic peak intensities of D- and G-line were recorded to calculate D/G ratio, showing the quality of CNT films. The prepared samples were further used for contact
The use of experimental animal was reviewed and approved by the School of Medicine, Tsinghua University, China. To isolate MEF cells, a 13.5-day pregnant mouse was sacrificed by cervical dislocation. Abdomen was liberally covered with 70 % alcohol then cut through the skin and peritoneum to expose the uterine horns, followed by removal of the uterine horns. Skin tissues were placed in a Petri dish containing PBS (w/o Ca2+, Mg2+). Embryos were removed from the embryonic sac and then dissected out, and the placenta and membranes were discarded. The embryos were decapitated and eviscerated, and the remaining carcases were washed three times with PBS (w/o Ca2+, Mg2+). Carcasses were put in a clean Petri dish and then minced with a scalpel blade, followed by the addition of 2-mL trypsin/EDTA and incubation at 37 °C for 10–20 min. Five milliliter Dulbecco’s modified Eagle’s medium (DMEM)/fetal calf serum (FCS) were then
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added, and the solution was transferred to a 15-mL centrifuge tube, pipetted vigorously. After the large tissue chunks settled due to gravity, the supernatant was transferred to a T75 flask and added with another 15 mL of DMEM/FCS, kept in the 37 °C incubator over night. At the next day, fresh media were added to remove floating cellular debris. Cells were grown to 90 % confluence (counted as the first generation) and then stored at −80 °C. For MEF culturing, cells were maintained at 37 °C with 5 % CO2 in DMEM (with high glucose, 10 % FCS, 1 % glutaMAX, 0.5 % penicillin/ streptomycin, and 0.1 % gentamicin). Human Foreskin Fibroblast (HFF) cells were obtained from the foreskins of young male patients following informed consent. They were maintained in DMEM (Invitrogen) with 10 % FCS (Hyclone), 1× Pen/Strep (Invitrogen) and passaged with 0.25 % trypsin/EDTA. Cells were maintained under standard conditions in a humidified, 37 °C, 5 % CO2 atmosphere. 2.4 Mitomycin C Inactivation of MEFs MEF cells were inactivated with Mitomycin C to be feeder cells for mESCs. In brief, MEF cells were cultured in DMEM/FCS medium with 0.01 mg/mL of Mitomycin C (M-4287, Sigma-Aldrich) at 37 °C for 2–3 h. Afterwards, the Mitomycin C medium was removed, and MEF cells were washed three times with PBS and maintained in DMEM/FCS medium. Inactivated MEF cells were used within 1 week after Mitomycin C treatment. 2.5 Culturing mESCs The R1 mESCs were obtained from Life Technologies. We first prepare inactivated MEFs in T25 or 6-well plate and then thaw ESCs (vial contains ∼106 cells) in a 37 °C water bath and add to 10 mL of pre-warmed ESC medium. Mix well by swirling, spin 3 min at 1,000 rpm, aspirate off the supernatant, and resuspend cells in 5 mL of pre-warmed ESC media. For passaging mESCs, on the day cells grow to confluent, feed cells 2 h before passaging. Then, aspirate medium off and wash once with pre-warmed PBS. Cover cells with 0.05 % trypsin/EDTA solution and return to 37 °C incubator for 2 min or until cells are uniformly dispersed into small clumps. Medium 4.5 mL was added to inactivate the trypsin. Finally, 1 mL (∼106 cells) was added to new tissue culture flask with MEF. mESC culture media include: high-glucose DMEM (-pyruvate, -glutamine) (Invitrogen/Gibco 11960-044), 15 % FBS (Hyclone SH30070.03) or 15 % KO-serum replacement (Invitrogen/Gibco 10828-028), 1× glutaMAX™-I supplement (Dilute from Glutamax-I, Invitrogen/Gibco 35050061), 1× non-essential amino acid solution (dilute from
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100× MEM non-essential amino acid solution, Invitrogen/ Gibco 11140-035), 0.1 mM β-mercaptoethanol (dilute from stock, 7 μl ß-ME in 10 mL of DMEM, 10 mM), 1× penicillin/ streptomycin (dilute from Pen 5,000 units/Strep 5,000 ug 100×, Invitrogen/Gibco 15070-063), 1,000 U/mL of ESGRO® (LIF, Millipore), and trypsin/EDTA 0.05 % trypsin (w/v) (Gibco; cat no.15090-046):5 mM EDTA (Sigma cat no E-5134). 2.6 Preparation for SEM Images of Cells For SEM analyses of cell growth on various substrates, cell media were carefully removed, and cells on the films were washed three times by PBS, followed by fixation of 2.5 % glutaraldehyde (25 % glutaraldehyde diluted 10 times in 1 × PBS) for 3 h. The films were then washed by PBS for three times and dehydrated in 30, 50, 70, 80, and 90 % (v/v) ethanol solutions, respectively, 5 min for each dehydration. Lastly, films were soaked in 100 % ethanol for three times, 5 min each time. When completely dried under air flow, films were coated by 10-nm gold (Hitachi E-1010 Ion Sputter), ready for SEM observation. 2.7 Fluorescence Staining and Imaging Calcein-AM to stain the calcium-rich cytoplasm (1:500, green) and Hoechst dyes to indicate the living cells (1:500, blue) were purchased from Dojindo Laboratory. Dyes were added into cell media and incubated for 30 min, individually. Samples were washed intensively before observation under fluorescence microscopy (Olympus Q-imaging RETIGA 2000R). SSEA1 mouse monoclonal antibody (1:50) and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG antibody (1:500) were applied to visualize the growth of mESCs (green). 3 Results and Discussion 3.1 Functionalization and Characterization of CNT Films First, for oxidization, in a sealed Teflon-lined autoclave, untreated CNT films were placed over a pool of 65–68 % (wt%) nitric acid and then heated at 120 °C for 6 h, leading to the formation of oxidized CNT film with carboxyl groups on the surfaces [34]. In this process, higher or lower temperatures were initially applied to adjust the oxidation, but with a failure owing to either damage to film integrity or minimal oxidization. Simultaneously, at 120 °C, extension of exposure time of CNT films to nitric acid steams, to 10 h or longer, caused the serious rupture of the film. Therefore, steaming of untreated CNT films at
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120 °C for 6 h was finally adopted in the following experiments to prepare oxidized superaligned CNT films [34]. After oxidization, samples were soaked in dH2O, followed by the addition of 1 M NaOH drop-wise until pH =8 in the solution. Then, neutralized samples were kept in dH2O overnight and rinsed throughout before the next use. Polyethylene glycols (PEGs) are water-soluble polymers, whose conjugation significantly enhances the hydrophilicity of CNT surfaces [35–38]. Laminin, an important extracellular matrix (ECM) protein, displays a crossshaped structure that comprises of four arms to execute their binding capability and has been recognized as a membrane glycoprotein to enhance cell attachment [39, 40]. PEGs and laminins were conjugated to the surface of CNT films to prepare PEGylated and laminin-conjugated CNT films following the procedures in the “Materials and Methods” section and were rinsed by PBS thoroughly before the next use. Further investigation of all films by scanning electron microscopy (SEM) (Fig. 1) revealed that superaligned structures of CNT films were well-reserved during various functionalizations, while multi-walled CNTs with diameters of dozens of nanometers in untreated film tended to form bundles with diameters of ∼100 nm in all modified films, suggesting that van der Waals forces increased when oxidation took place and thereby interactions between neighboring multi-walled CNTs, both horizontally
Fig. 1 SEM images of differently functionalized CNT films. Scale bar indicates a size of 2 μm
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in one layer and vertically between layers, strengthened. Under a high pressure in the sealed container, acid steams drove out the stored gas inside CNTs and simultaneously wetted oxidized CNTs; as a result, condensed SACNT films were produced after oxidation. All samples were then investigated by Raman spectra (Fig. 2a) to analyze their structural composition. CNT films were measured using a Renishaw spectrometer with a laser excitation at 514 nm, and the Raman shift was scanned from 400 to 2,800 cm−1. Three different spots on one film were randomly measured, and the characteristic peak intensities of D-line (signature for defective graphitic structures) and G-line (the tangential vibration of the carbon atoms) were recorded to calculate D/G values (ratio of D-line peak intensity to G-line one), showing the surface defection of various CNT films [34, 41]. Apparently, oxidation significantly increased D/G ratio from (0.64±0.05) to (1.01±0.03), confirming that excessive defects were formed on the surfaces of CNTs after acidic oxidation at a high temperature. Compared to the untreated one, D/G ratio for PEGylated and laminin-conjugated CNT films were also increased to 0.73±0.03 and 0.82±0.05, respectively. In parallel, infrared (IR) spectra of various CNT films were obtained and shown in Fig. 2b. Compared to those in untreated film, the peaks at ∼1,800 and ∼1,100 cm−1 resulted from vibrations of –C=O and –C–O bonds, respectively, in the spectra of all oxidized CNT films. In addition, a peak at ∼1,570 cm−1 in oxidized film was attributed to the asymmetric stretching
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Fig. 2 Characterization of functionalized CNT films. a Raman spectra of all CNT films as indicated. Three individual measurements from randomselected spots on the film were conducted, and representative spectra were shown. D/G values show the intensity ratios of D- and G-line in a characteristic CNT film (average ± stand deviation). b Infrared spectra of CNT films, collected on a Bruker IFS 66 v/s spectrometer for untreated (black), oxidized (red), PEGylated (blue), and lamininconjugated (purple) CNT film, respectively. Each characteristic ligand after each functionalization was observed as indicated by arrows in the spectra
vibration of carboxylate anion, possibly due to the reaction of carboxylic group with residual metal catalysts used for CNT growth [34]. Signature vibrations of –C–N bonds in amides at ∼1,650 cm−1, –C–H bonds in long chain alkanes at ∼2,900 cm−1 (split at 2,860 and 2,920 cm−1, respectively), and –N–H bonds at ∼3,300 cm−1 proved a successful PEGylation in both PEGylated and lamininconjugated CNT films. Besides, vibrations of –C–N bond were found stronger in laminin-conjugated CNT films than that in PEGylated one, indicating the formation of additional peptide bonds there after protein conjugation. Fig. 3 Various CNT film was individually placed onto a square Teflon frame (outer length= 20 mm, inner length=16 mm, with a frame length of 2 mm), and dried completely. Contact angle values were measured at room temperatures, using an optical contact angle measuring device (OCA 20, DataPhysics Instruments GmbH, Filderstadt, Germany). Values of water contact angles on different surfaces were shown on the top of each image (average ± stand deviation)
Next, contact angle analysis of water droplet on different CNT substrates was conducted to estimate the wettability of all functionalized surfaces. Various CNT films were placed on a Teflon frame and kept dried completely. At room temperature, a water droplet of 2 μL was dispensed onto each surface of CNT film, and water contact angles measured from at least three spots on every film were then averaged. Results were shown in Fig. 3. Apparently, the surface of untreated CNT film was highly hydrophobic, as it has a water contact angle of (138.1± 2.7)0. On oxidized form of CNT film, the contact angle
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decreased significantly to (41.9±2.1)0, less than one-third of that on the untreated film. Moreover, water droplet contacted PEGylated CNT film at an angle of (21.9±0.9)0, indicating this functionalized film now has a highly hydrophilic surface. However, further conjugation of laminin increased the consequent water contact angle over twofolds of that on PEGylated surface, with a value of (46.8±2.8)0, suggesting that laminin conjugation to CNT film made the hydrophobic region of this protein uncovered. Indeed, domain of hydrophobic amino acids in laminin was believed responsible for some important biological activities of this protein, such as its enhancement in cell attachment and neurite growth [40]. Therefore, while the exposure of hydrophobic side chains in laminin to aqueous solvents increased the water contact angle, this conjugation might help to preserve the function of this protein when immobilized to CNT surfaces via covalent bond. Furthermore, surface roughness of various CNT films was estimated by a profilometer (Ambios, XP-1) and quantified by the value of R a (see Eq. 1). For untreated, oxidized, PEGylated, and laminin-conjugated surface of CNT films, Ra read (10,333±3,887), (948±106), (720±155), and (1,568 ±296), respectively, demonstrating that there is a significant decline of surface roughness from as-made CNT film to all oxidized films, whereas among all oxidized substrates, laminin conjugation made the surface rougher. Interestingly, this tendency in roughness after each modification was in a good agreement with that of water contact angle. Functionalization of CNT film was further verified by element analyses through X-ray photoelectron spectra (XPS). XPS data of all CNT films were analyzed and summarized in Table 1, where oxygen content jumped over threefolds after oxidization of untreated CNT films and increased further after PEGylation and laminin conjugation, whereas nitrogen content remained relatively low until laminin conjugation were complete. These results confirmed that polypeptides were immobilized onto the surface of CNT films successfully.
Table 1 Various CNT films were individually placed onto silicon wafer, wetted by ethanol, and left completely dry. XPS measurements were then performed in a PHI-5300ESCA Perkin-Elmer spectrometer, using monochromatic Mg Kα (1253.6 eV) radiation at 14 kV and 250 W. Element analyses were conducted for C, O, and N elements. O/C and N/C stand for mole ratios of oxygen and nitrogen to carbon, respectively Samples
C1s (%)
O1s (%)
N1s (%)
O/C
N/C
Untreated Oxidized PEGylated Laminin-conjugated
93.2 78.6 72.9 68.1
5.8 19.2 21.9 23.3
1.0 2.1 5.2 8.5
0.06 0.24 0.30 0.34
0.01 0.03 0.07 0.12
3.2 Growth of Human Foreskin Fibroblasts on Differently Functionalized CNT Films We first tested the utility of differently functionalized CNT films using HFFs. 1×105/mL HFFs were placed on differently functionalized CNT films and let to grow for 3 days. Calcein-AM (green) was added to visualize the cytoplasm of cells and Hoechst (blue) to stain the nuclei of live cells. Samples were then washed extensively before observation using fluorescence microscopy (Olympus Q-imaging RETIGA 2000R). Results are shown in Fig. 4a. Few HFFs can be seen on untreated CNT films, indicating that unmodified CNT films are not suitable for cell attachment and thereby growth or proliferation, most likely due to their highly hydrophobic and rough surfaces. In contrast, HFF grew to much higher density on the oxidized, PEGylated, and laminin-conjugated surfaces of CNT films. To further quantify cell density on different substrates, we randomly selected 10 areas of the same size from each film and counted the number of cells within the defined view area (1.1×1.1 mm2) using ImagePro. Data are presented in the form of mean cell number ± standard deviation of 10 areas and shown in Fig. 4b. Our quantitative analysis demonstrated that there were 82±9 cells per area (cpa) on oxidized surface, over fourfolds higher than the average cell count 19±6 cpa on pristine films. Cell counts were 41±3 cpa and 60 ± 4 cpa on PEGylated and laminin-conjugated CNT films, respectively, all significantly higher than that on pristine films (p≤0.05, Student t test). Our quantification is consistent with cell images shown in Fig. 4a. As oxidized CNT films supported better HFF attachment and growth compared to PEGylated and laminin modified surfaces, thus in this case, the physical property of the substrate, such as hydrophilicity, is the most crucial factor determining cell adhesion, while the conjugation of chemical polymer PEG and extracellular matrix protein laminin onto oxidized CNT films may be required by other types of cells for their enhanced adhesion. Based on this result, we can speculate that (1) an optimized degree of hydrophilicity is favored for cell attachment and growth with specific origin; (2) hydrophobicity on a defined surface is a crucial but not unique factor to affect HFF cell attachment and growth; (3) biological factors, such proteinprotein interaction to enhance cell receptor binding, need to be taken into account when a good biocompatibility of material surface is desired, whereas an optimized substrate for cells to grow should be primarily equipped with proper physical or chemical characters. The positive influence of substrate matrix on cell attachment and growth is determined by many physicochemical profiles of growth substrate, including hydrophilicity, surface roughness/flexibility, the type and density of functional groups on surface, and other mechanical (or electrical)
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Fig. 4 HFF cell growth on different CNT films. a Fluorescent images of HFF cells when grown on different substrates as indicated. Cells were stained with Hoechst to visualize the nuclei (blue) and Calcein-AM to show the cytoplasm (green) in living cells. Scale bar shows a size of 100 μm. b Relative cell density on various conditions. Cell density on different substrates was quantified by first randomly choosing 10 areas with the same area from each film and then counting the number of cells within this view field (1.1× 1.1 mm2) individually using ImagePro. Data are presented as a bar graph. Each bar represents mean cpa ± standard deviation on indicated substrates (n=10). Cell counts on all groups of treated CNT films were significantly higher than that on untreated CNT film, p ≤0.05 (calculated using Student t test on each indicated condition)
properties, where an optimal combination can offer a favored cell behavior, such as adhesion, growth, and proliferation [42–46]. In addition, those biocompatible characters of scaffolding materials could be cell type-dependent, as cells of different origins may require different extracellular matrix environment when cultured in vitro. Combinatorial factors as mentioned above from both material and cellular aspects contribute to exact adhesion energy when cells are seeded onto a specific substrate, decisively guiding their attachment and further proliferation [47]. 3.3 Growth of Mouse Embryonic Fibroblasts on Differently Functionalized CNT Films Next, we tested if freshly isolated MEFs could be grown on CNT films. MEF cells were prepared from mouse embryos and cultured as described in the “Materials and Methods” section. 1×105/mL MEF cells were grown on different substrates for 3 days. At the end of the experiment, we added Hoechst dyes to stain nuclei of live cells and took fluorescence images (Fig. 5a, blue color indicates cell nucleus). We also performed cell number quantification using the same method
as described in the above section, and the results were shown in Fig. 5b. Similar to the results from HFF cells, untreated CNT film was the least favored surface for MEF cell attachment and growth as there were only about 59±11 cpa. After oxidization, CNT film surface can grow MEF cells very well where cell density reached 111±5 cpa, significantly higher than that on pristine CNT (p≤0.05, Student t test). Different from HFFs, MEFs can grow on PEGylated and laminin-conjugated CNT films in a density of 108±11 cpa and 103±10 cpa, respectively. Results above indicated that hydrophobicity seems to be the most important factor affecting MEFs’ attachment and growth on CNT films, as further chemical or protein conjugation did not significantly change cell growth. To better characterize the cell morphology on CNT films, we performed SEM analysis of MEF cells grown on various surfaces. As shown in Fig. 5c, cells were in spindle shape, and they sent out both large flat protrusions (lamellipodia) and fine protrusions (filapodia), while some protrusions were entangled with the CNT nanobundles underneath. Thus, fibroblast cells on CNT films appeared thinner and more stretched compared to those on the glass substrates. This is
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Fig. 5 MEF cell growth on different CNT films. a Fluorescent images of MEF cells when grown on different substrates as indicated. Cells were stained with Hoechst to visualize the nuclei (blue) in living cells. Scale bar shows a size of 100 μm. b Relative cell density on various conditions. Cell density on different substrates was obtained by first randomly choosing 10 spots with the same area from each film and then counting the number of cells within this view field (1.1×1.1 mm2) individually using ImagePro. Data are presented as a bar graph. Each bar represents mean cpa ± standard deviation (n=10). Cell counts on untreated CNT films were significantly lower than that on treated CNT films, p ≤0.05 (calculated using Student t test). But cell counts on differentially treated CNT films were similar. c SEM images of MEF cells when grown on different substrates as indicated
likely due to the unique physical connection between cell protrusions and CNT bundles. Our observation of MEF morphology on superaligned CNT films was in agreement with a previous report that significant decrease in cell size (10–15 %) was observed when
human skin fibroblasts were grown on a CNT sheet [48]. This morphological alteration could be associated with altered expression of cytoskeletal proteins, mainly actin and vinculin in focal adhesions, when fibroblasts were relocated onto carbon nanomaterial-coated substrates (e.g., CNT or graphene
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oxide) and had been shown to have no deleterious effects towards cells [49]. In our experiments, freshly isolated MEF cells could attach well to either oxidized, PEGylated, or laminin-conjugated CNT film, which is different from HFF cells. This may due to that MEF cells are a population of cells from mixed origins including fibroblast cells as well as some mesenchyme cells, which may prefer PEGylated or laminin-conjugated surfaces. In contrast, HFF cells were isolated from adult human foreskin and thus consisted of mostly one type of cells. Our results here suggested that surface modification with suitable macromolecules could be used to selectively enhance the adhesion of one particular type of cells on CNT films. 3.4 Growth of mESCs on Different Surfaces Pluripotent ESCs can self-renew and differentiate to all types of tissues in the body; therefore, they are promising cell source for regenerative medicine. However, they are particularly sensitive to external stimulus and easy to undergo differentiation. Traditionally, ESCs were maintained on inactivated MEF feeder cells. As we could grow MEF cells on CNT, we asked if CNT films covered with MEF cells could support the self-renewal of mESC. To answer this question, 1×105/mL of inactivated MEF cells were first plated on different CNT surfaces overnight, and then 1×105/mL of mESCs were added on top and let to grow for another 24 h. To test whether cells have undergone differentiation, we performed immunostaining of undifferentiated mESC surface marker, stage-specific embryonic antigen 1 (SSEA1). SSEA1 is a sensitive marker for pluripotent stem cells, and its expression would be downregulated upon differentiation [50, 51]. Since MEF cells do not express SSEA1, therefore, they will not be stained by this antibody. Fluorescence images of mESCs on MEF feeders grown on various substrates were shown in Fig. 6a. Very few ESC colonies can be found on untreated CNT films. As expected, there were much more ESCs colonies on all functionalized CNT films. On laminin-conjugated CNT films, ESC colonies that expressed SSEA1 appeared larger, indicating that extracellular matrix protein laminin may be favored by mESC for their attachment. SEM images of mESC on MEF feeder cells on various conditions were shown in Fig. 5b. We found that although most mESCs grew on top of MEF feeders as clumps, direct physical contact between mESCs and the substrates occurred as well. This suggested that specific cell-binding factors, such as laminin, could directly contribute to mESC attachment and growth. We next tried to grow mESCs on various substrates without MEF feeder cells. 1×105/mL mES cells were directly placed on different substrates and grown for 2 days, when SSEA1 mAb was used to visualize undifferentiated cells (green) and Hoechst dyes to stain the nuclei (blue). mESCs failed to grow on most substrates except for laminin-conjugated CNT films
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(Fig. 6c), where typical mESC colonies could be seen albeit less than on feeder-covered surfaces. Our results here are in a line with other recent research findings, where laminin or other ECM protein-conjugated CNT or polymer matrices could promote the stem cell growth and/or direct a wellcontrolled stem cell differentiation [27, 52–55]. Maintenance of a homogenous growth of undifferentiated stem cells in a feeder-free condition is highly desired, particularly in cases of drug discovery and toxicity assessment, as feeder cells would complicate the circumstance. ESCs require specific attachment factors and the activation of cell surface receptors such as integrins to self-renew and differentiate. Our work here demonstrated that upon modification of CNT with ESC-preferred substrate protein laminin, they were able to grow without feeders and maintain undifferentiated state. This result opens up several new possibilities to use functionalized CNT films in stem cell research and tissue engineering. First, one can maintain feeder-free culture of pluripotent stem cells on CNT-based substrate while manipulating stem cell fate by taking advantage of unique properties of superaligned CNT films. For example, superaligned CNT films have excellent elasticity and conductivity inherited from CNT nanomaterials. Thus, the substrate stiffness and elasticity can be controlled to direct stem cell self-renewal or differentiation. Moreover, as superaligned CNT is conductive for electricity, thus, one can use this property to electro-transfect cells normally not amenable for chemical transfection as we have demonstrated in a separate study [34]. Second, it is possible to fabricate different surface patterns using well-functionalized CNT thin films to mimic the stem cell niche in the body and use it to directly test factors that can promote tissue regeneration.
4 Conclusions In this study, we first prepared superaligned CNT films and conducted various modifications of films to optimize the surface profiles for a better biocompatibility. Compared to conventional surface functionalization of CNT films, namely, functionalization of suspended CNT particles and the ensuing assembly of disperse CNTs to form thin films, we here adopted a facile chemistry to directly oxidize the superaligned CNT film, followed by sequential functionalization, including PEGylation and laminin conjugation. We proved that this process preserved the structural alignment and physical integrity of CNT thin films. For their biological applications as cell culturing substrates, both human and mouse fibroblasts were grown on differently functionalized CNT films. Untreated CNT films exhibited a hydrophobic character, incompatible with cell growth, whereas oxidized CNT films provided a hydrophilic surface, allowing good cell adhesion and proliferation. Both PEGylation (with the highest hydrophilicity as
298 Fig. 6 Growth of mESCs on different surfaces. a Fluorescent images of mESCs when seeded on MEF feeder cells, grown on different substrates as indicated. mESCs were stained with SSEA1 (green). b SEM images of mESCs on MEF feeder cells when grown on different substrates as indicated. Black arrows showed stem cell colonies grown on top of MEF feeders, while white arrows pointed to those directly in touch with CNT films (on lamininconjugated CNT films). c Fluorescent images of mESCs when directly seeded on lamininconjugated CNT films. They were stained with SSEA1 (green) and Hoechst (blue) in the nuclei
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well as the lowest surface roughness) and laminin conjugation (a well-known ECM protein in promoting cell attachment) did not significantly improve the cell adhesion, proving that a favored surface property is required for specific fibroblast growth, comprising of many physicochemical features more than just hydrophilicity, surface roughness, or chemical nature. Furthermore, we showed that mESCs can be grown on either MEF covered or laminin-conjugated CNT surfaces while staying undifferentiated. Our research developed a new methodology to modify superaligned CNT films and provided valuable information about the utility of this material in biomedical research settings. It will help broaden the application of CNT-based nanomaterials in stem cell research and tissue engineering. Acknowledgments This work is supported by the National Natural Science Foundation of China Grant 31171381 (to J.N.), the National Basic Research Program of China, 973 Program, 2012CB966701 (to J.N.), the core facility of the Tsinghua-Peking Center for Life Sciences, and financial supports from Tsinghua Research Funds (#100401007) (to Z.T.). We thank the Tsinghua-Foxconn Nanotechnology Research Center for their technical support of superaligned CNT films.
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