Film Thickness Determines Cell Growth and Cell Sheet Detachment

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Film Thickness Determines Cell Growth and Cell Sheet Detachment from Spin Coated Poly(N-Isopropylacrylamide) Substrates. Nina A. Dzhoyashvili, Kerry Thompson, Alexander Vladimirovich Gorelov, and Yuri A. Rochev ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b09711 • Publication Date (Web): 23 Sep 2016 Downloaded from http://pubs.acs.org on September 27, 2016

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ACS Applied Materials & Interfaces

Film Thickness Determines Cell Growth and Cell Sheet Detachment from Spin Coated Poly(N-Isopropylacrylamide) Substrates.

Nina A. Dzhoyashvili 1, 2,*, Kerry Thompson3, Alexander V. Gorelov4, 5, Yuri A. Rochev1, 6

1

School of Chemistry, National University of Ireland Galway, H91 CF50,

Galway, Ireland, 2National Centre for Biomedical Engineering Sciences, National University of Ireland Galway, H91 CF50, Galway, Ireland 3

Center for Microscopy and Imaging, Anatomy, School of Medicine,

National University of Ireland Galway, H91 CF50, Galway, Ireland 4 School of Chemistry and Chemical Biology, University College Dublin, D04 R7R0, Belfield, Dublin 4, Ireland. 5Institute of Theoretical and Experimental Biophysics, Russian Academy of Science, 142290, Pushchino, Moscow region, Russia 6 Sechenov First Moscow State Medical University, Institute for Regenerative Medicine, 119991, Moscow, Russia *Corresponding author [email protected]

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ABSTRACT Poly(N-isopropylacrylamide) (pNIPAm) is widely used to fabricate thermoresponsive surfaces for cell sheet detachment. Many complex and expensive techniques have been employed to produce pNIPAm substrates for cell culture. The spin coating technique allows rapid fabrication of pNIPAm substrates with high reproducibility and uniformity. In this study, the dynamics of cell attachment, proliferation and function on non-crosslinked spin coated pNIPAm films of different thicknesses were investigated. The measurements of advancing contact angle revealed increasing contact angles with increasing film thickness. Results suggest that more hydrophilic 50 and 80nm thin pNIPAm films are more preferable for cell sheet fabrication while more hydrophobic 300 and 900 nm thick spin coated pNIPAm films impede cell attachment. These changes in cell behavior were correlated with changes in thickness and hydration of pNIPAm films. The control of pNIPAm film thickness using spin coating technique offers an effective tool for cell sheet-based tissue engineering. Key-words pNIPAm, spin coating, film thickness, wettability, protein adsorption, cell sheet engineering


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1. INTRODUCTION A short-term goal of regenerative medicine is the development of functional tissue substitutes created from a patient’s own autologous cells that can be applied in clinical practice 1. Cell sheet transplantation provides a promising therapeutic approach for tissue regeneration because it does not cause inflammation and enables improved integration and attachment to a host tissue

2, 3

.

Biomaterials play a significant role in the creation of synthetic cell culture substrates. Poly(Nisopropylacrylamide) (pNIPAm) is one of the most studied intelligent biomaterials widely used for cell sheet harvesting4-5. The substrates when coated with pNIPAm or its copolymers enable the detachment of cells from culture dishes by temperature reduction below the lower critical solution temperature (LCST) and without using digestive reagents which destroy extracellular matrix assembly, intercellular connections and focal adhesion complexes. The preserved ECM and developed cell-cell and cell-matrix interactions in cell sheets greatly supports cell adherence to natural or polymer matrices, or directly to tissue after in vivo transplantation. Despite pNIPAm’s biocompatibility, bulk pNIPAm coatings display resistivity to cell adhesion6. This fact can present a significant obstacle for cell sheet fabrication. The choice of deposition method can significantly affect the surface bioactivity. The methods which are used to fabricate thin pNIPAm films can be divided, according to preparation procedure, into two major categories: covalently polymerization bonding7-9 and polymer coating10-14. Several studies have shown the dependency of cell behavior on the thickness of covalently grafted pNIPAm films. In particular, the pNIPAm films with thickness higher than 20 nm fabricated by electron beam irradiation impede cell attachment, and the optimal thickness was found to be between 15 and 20nm8. It was found that atom transfer radical polymerization (ATRP)-fabricated pNIPAm brushes with thickness between 20 and 45 nm are the most suitable for HepG2 cell adhesion and detachment15.

The key advantage of a spin-coating technique is the combination of

reproducibility and versatility. The method allows very fast, gentle and non-deforming drying of polymer films with uniform surface which characteristics can be controlled very carefully16. The technique provides a more inexpensive and convenient way to prepare thermoresponsive cell culture surface and can be deployed in many research labs. The adhesion properties of pNIPAm films are closely related to protein adsorption. Considering the surface characteristics and conditions of surrounding environment, surface hydration, roughness, surface chemistry, pH and temperature can be important for the adsorption of proteins and in the evaluation of material’s cell adhesion properties17-20. While the cell behaviors on



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covalently grafted pNIPAm brushes is well understood, there are a limited number of studies which systematically investigated cellular adhesiveness of spin coated pNIPAm films. In the present study, we tested cell behavior on non-crosslinked pNIPAm films of different thicknesses. Cell response to pNIPAm films were analyzed in terms of mouse stromal (MS-5) cell attachment, growth, and detachment, metabolic activity, viability and proliferation rate, morphology and cytoskeleton organization. The stromal cells including MS-5 cells have been commonly used for cytotoxicity evaluation of biomaterials21-23. Moreover, they provide a supportive environment for stem cells24, 25, including human hematopoietic stem/progenitor cells (HSPCs) 26, 27. The thickness of pNIPAm films was measured optically by viewing optical profilometry and employing a laser ablation technique. To analyze pNIPAm film morphology and wettability, the films of different thicknesses were characterized using atomic force microscopy (AFM) and contact angle measurements, respectively. Immunofluorescence staining of F-actin and paxillin was used to examine the cells grown on pNIPAm films. The actin cytoskeleton is connected to the extracellular matrix via macromolecular complexes, such as focal adhesions. Paxillin, the major focal adhesion adaptor protein, binds several proteins that contribute to the organization of actin cytoskeleton and thus plays a crucial role in signaling network that can regulate cell adhesion and spreading28. We observed that the attachment, growth and detachment as well as metabolic activity of MS-5 cells depend on the thickness of pNIPAm films. The thick pNIPAm films showed a lower cell attachment when compared to a Thermanox control and to thin pNIPAm films. The possibility to detach the cells in the form of a contiguous sheet was shown using thin pNIPAm films by lowering the temperature. No relevant detachment was observed on Thermanox control or thick pNIPAm films. These spin coated thin pNIPAm films can find application as intelligent surfaces for use in research, medical devices and regenerative medicine. The results of present study may be important for chemists improving polymer properties, engineers optimizing the polymer production processes and biomedical scientists specializing in cell sheet based tissue engineering.


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2. EXPERIMENTAL SECTION 2.1. Materials. The polymer pNIPAm (poly-N-isopropylacrylamide) with Mn =20000-40000 and anhydrous ethanol (200 proof, >99.5% assay) were purchased from Sigma Aldrich (St. Louis, MO, USA), Thermanox™ plastic 25mm discs from NUNC™ (Naperville, IL, USA), the high quality fused silica glass discs 20mm in diameter from UQG Optics Ltd (Cambridge, UK), borosilicate cover glass slides 25mm in diameter from VWR (Radnor, PA, USA). Plastic consumables were purchased from Sarstedt (Nümbrecht, Germany). Dulbecco’s Phosphate Buffered Saline (DPBS), Hanks Balanced Salt Solution (HBSS) and Dulbecco’s Modified Eagles Medium (DMEM) were purchased from Lonza (Switzerland), antibiotics (penicillin -streptomycin) and fetal bovine serum (FBS) from HyClone (Logan, UT, USA). The alamarBlue® reagent, Live/Dead® viability/cytotoxicity kit and Quant-iTTM PicoGreen® dsDNA assay kit were purchased from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA). Rabbit monoclonal anti-paxillin antibody (ab32084) and Alexa Fluor® 488 goat anti-rabbit secondary antibody (ab150077) were purchased from Abcam (Cambridge, UK). Phalloidin eFluor660 (50-6559-05) was purchased from Affymetrix (CA, USA). UltraCruzTM mounting medium was purchased from Santa Cruz Biotechnology (CA, USA). Heating dry bath from Torrey Pines Scientific (Carlsbad, CA, USA) was used for careful temperature control. 2.2. pNIPAm Film Preparation and Deposition. Spin-coated pNIPAm films were fabricated by initially depositing a 150-μl aliquot of a 5, 10, 20,40 and 80 mg/ml ethanol pNIPAm solution onto a slowly spinning, (150 RPM), substrate (Thermanox™ plastic discs or glass discs) for 9s followed by rapid acceleration to 4000 RPM for 30s, on a Laurell Technologies WS-400B-256 . The coated samples were slowly dried overnight in an ethanol saturated atmosphere and then left in a vacuum oven at 40 °C and 600 mBar for a minimum of 4 h to eliminate any residual solvent. Thermanox coverslips were used as the underlayer substrates for advancing contact angle measurements, cell morphology, qualitative cell assays (alamarBlue and PicoGreen) and cell detachment analysis. Clean and optically flat borosilicate cover glass slides were used for atomic force microscopy and fluorescence microscopy. The fused silica glass substrates were used for laser ablation and optical profilometry analysis.



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2.3. Evaluation of pNIPAm Film Thickness. An ArF excimer laser (ATL Atlex®, Wermelskirchen, Germany) was used to ablate the selected areas on the thin pNIPAm film deposited on fused silica glass discs, 20mm in diameter from UQG optics. The excimer laser operates at a wavelength of 193 nm with a pulse length of a few ns. Laser parameters included a pulse repetition frequency of 200Hz at a fluence of 66mJ/cm2. A standard mask projection machining approach was used to shape the laser beam. An optical demagnification of 5X was employed to produce 9 periodically arranged ablated areas of approximately 400μm×400μm. Optical profilometry analysis was used to assess the thickness of pNIPAm films. All measurements were made in air. The thickness of pNIPAm films was measured using white light interferometry (Zygo New View 100) with an accuracy of 0.1nm. The z-height distance between the remaining polymer and the underlying substrate was measured to accurate assess the thickness. Statistically relevant data was obtained by repeating all measurements three times, and scans of 5 randomly selected ablated windows were recorded on 3 different samples. The objective used for all measurements was a 20X Mirau, with zoom set at 0.5X. 2.4. Contact Angle Measurements. Advancing contact angle measurements were performed on a home-built goniometer assembled on an optical rail from Newport Optics with opto-mechanical components from Newport Optics and Edmund Optics. DROPimage software marketed by Rame Hart and developed by F.K. Hansen was applied to determine contact angles29. pNIPAm -coated samples were placed in a temperature-controlled environmental chamber mounted on an adjustable tilt stage. Contact angles were taken at 40°C, i.e. above the LCST of the pNIPAm polymer, and controlled using a thermocouple attached to the stage surface. A drop of ultrapure water was deposited on the surface with an initial radius of about 3mm. For the advancing contact angle experiments, a thin stainless steel needle (gauge 22) was inserted in the center of the drop from above. The volume of a drop was increased by pumping liquid into the drop using a syringe pump .The pumping speed was maintained at the rate of advancing to below 0.5mm/min. The values of advancing contact angles were taken and averaged between approximately 450 and 500 seconds. The values of contact angles were measured in two different positions for each coverslip. Contact angle was also measured after thermal annealing at 120°C for 2h in a vacuum oven.


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2.5. AFM Analysis of pNIPAm Film Surface Roughness. AFM images were obtained in tapping mode in air using a Dimension 3,100 AFM (Digital Instruments, Santa Barbara, CA, USA) and Veeco 1–10 Ohm-cm phosphorus (n) doped Si tips and a matrix of 512×512 data points along the x-y plane were analyzed in a single scan. Four 10μm×10μm scans were recorded at a scan rate of 0.5Hz on each pNIPAm film to ensure statistical accuracy. Atomic force microscopy (AFM) was used to assess the roughness of the deposited pNIPAm coatings using 10μm×10μm scans. The roughness of the films was reported as root mean square (RMS) roughness values. 2.6. Cell Culture. For cell culture experiments, pNIPAm films were sterilized under mild UV light for 2 h. The mouse (MS-5) stromal cell line were cultivated in DMEM, supplemented with 10% FBS, 1% penicillin streptomycin antibiotics and maintained in a humidified incubator at 37°C and 5% CO2. 2.7. Cell Activity Assays. For experimentation, MS-5 cells were seeded in triplicate at a density of 40000 cells/cm2 on pNIPAm-coated and Thermanox bare tissue culture coverslips. The metabolic activity of MS-5 cells grown on pNIPAm films and Thermanox controls was assessed using an alamarBlue® assay 48 h after cell seeding. Total DNA content of MS-5 cells attached and grown on pNIPAm films and Thermanox controls was quantified at 24 h and 48h by the Quant-iTTM PicoGreen® dsDNA assay kit. Both assays were performed according to the manufacturer’s instructions. Cell numbers were obtained by calculation based on the cell DNA, using a calibration curve of total cell DNA versus known numbers of cells. AlamarBlue and PicoGreen fluorescence was measured using a Thermo Scientific Varioscan Flash Multimode plate reader. A Live/Dead cell viability assay was used for cell viability analysis. Titration was performed for both calcein-AM and ethidium homodimer-1 to define optimal dye concentration according to the manufacture protocol. A solution of 1 µM Calcein-AM and 2μM ethidium homodimer-1 in HBSS was mixed thoroughly, and then added to the cells for 20 min at 37°C. The fluorescent staining was observed using an Olympus IX81 fluorescence microscope (Olympus) and images were captured using a DP72 CCD camera (Olympus) linked to CellSens Dimension software (Olympus). This experiment was repeated three times.



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2.8. Immunofluorescent Staining. The MS-5 cells grown on pNIPAm films and control glass cover slides were fixed at 37°C with 4% paraformaldehyde in DPBS for 10 min. Afterward they were washed three times in DPBS and then incubated with 3% goat normal serum for 60 min at 37°C to block non-specific binding. MS-5 cells were stained to detect actin cytoskeleton and the focal adhesion protein paxillin. Actin stress fibers were detected using Phalloidin eFluor660 at 1:500 dilution for 30 min at 37°C. Immunostaining of paxillin was performed using rabbit monoclonal anti-paxillin antibody at 1:250 dilution for 1h at 37°C followed by incubation for 1 h with a 1: 1000 dilution of Alexa Fluor® 488nm goat anti-rabbit secondary antibody at 37°C. After rinsing with DPBS, samples were mounted with UltraCruz TM mounting medium containing 1.5 µg/ml DAPI for nuclear counter staining. All stained slides were observed under an Olympus IX81 fluorescence microscope (Olympus). Double-stained images were superimposed with Image J software. At least five images were taken from each sample for analysis. The number of focal adhesions per cell was assessed using immunofluorescence images of paxillin and Image J software30. The micrographs were magnified to the same size and a total of 10 cells were analyzed in each group. The absence of nonspecific binding was confirmed by the use of appropriate primary and secondary antibody controls. 2.9. Cell Detachment. MS-5 growth and detachment was microscopically observed using a Leica inverted-microscope (Leica, Solms, Germany) on either pNIPAm-coated or bare Themanox controls. The cells were rinsed with pre-warmed HBSS to remove any traces of serum. Cold serum-free DMEM was added to cells and the samples were left on a digitally controlled thermal/cooling plate set to 8°C. Micrographs of cells were taken every 10 min on a phase contrast microscope to monitor cell detachment. 2.10. Statistical Analysis. Statistical analysis was performed using Statistica 8.0 software. Values are expressed as mean ± standard error of the mean (SEM). Student t-test were conducted to compare independent groups. Multiple comparisons were made using one-way ANOVA test. Statistical significance was defined as p-value

Film Thickness Determines Cell Growth and Cell Sheet Detachment

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