Name: thin film composite silicon elastomers for cell culture and skin applications: manufacturing and characterization
Uploaded: 2018-07-03T13:00:00
Duration: 8 min 2 s
Description: Watch this Scientific Journal Video about Thin Film Composite Silicon Elastomers for Cell Culture and Skin Applications: Manufacturing and Characterization at JoVE.com

Martin Danner is acknowledged for his assistance in preparing samples and establishment of cell culture procedures. The authors would like to thank Biesterfeld Spezialchemie GmbH (Hamburg, Germany), especially Robert Radsziwill for continuous support and discussions. The research leading to these results has received funding from the European Research Council under the European Union&#39;s Seventh Framework Programme (FP/2007-2013) /ERC Grant Agreement n. 340929.

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The authors have nothing to disclose.

The design of composite structures enables the simple adjustment of material properties, such as Young&#39;s modulus or the thickness of the samples. The Young&#39;s modulus of PDMS can be effectively changed in a wide range by either altering the mixing ratio between the two components or manufacturing of blends using a different silicone elastomer30,31. The described methods are not limited to the PDMS&#39;s used in the current investigation, but especially the adhesive performance depends strongly on the specific type used. A critical step within this protocol is the manufacturing process of the composite films (Figure 1). It was shown that thickness of the films affects significantly the adhesion behavior of the films on different substrates, including skin (Figure 5 and Figure 6). In addition to the film thickness, time and temperature during the curing process affects the material properties32. Therefore, parameters as the thickness of the polymeric layers have to be carefully adapted and verified.
Analysis of the adhesive properties of the thin films was performed with normal force adhesion measurements using two glass substrates with different surface roughness up to Ra = 0.338 &#181;m (Figure 3). In general, roughness impacts significantly the adhesion of surfaces, especially of elastic materials33,34. The roughness of glass can be easily varied by grinding with sandpaper of different asperity sizes, therefore allowing the fabrication of substrates exhibiting higher roughness values21. In addition, other materials, for example epoxy resin can be used for the production of substrates15,35. This might be an important modification strategy of the presented protocol. For example, if substrates exhibiting different surface free energies are needed or specific topographies are required. Here, pull-off stress and work of separation of the thin films manufactured of PDMS and SSA were analyzed with a custom-built setup (macroscopic adhesion measurement device (MAD, Figure 4)).36 Optical alignment of substrate and indenter is a critical step for the analysis of the measurement results. Therefore, adjustment of the tilt angle needs to be performed with the goniometer, as precise as possible. This can be achieved with sufficient precision by manually bringing the substrate into contact with the film surface until a horizontal contact is achieved.
In the current protocol the hold time was kept constant at one second (Figure 5 and Figure 7). Especially for the investigation of the adhesive performance of an elastic film to a rough substrate surface, an extension of the hold time provides additional information. For example, an increase in pull-off stress with increasing hold time has been reported8. In addition to the measurements performed in the current protocol, other methods, for example peel tests could be performed, allowing a more comprehensive investigation of adhesion performance37.
The adhesive properties of composite films exhibiting different film thicknesses of the soft skin adhesive were determined (Figure 7). Our results are in line with published data, showing that a decrease of film thickness lead to an increase of the pull-off stress as the confinement, i.e., the ratio between substrate diameter and film thickness, increases38,39. Based on these results and the data depicted in Figure 7, we conclude that composite films with a total thickness of approx. 100 &#181;m (a thickness of the SSA layer of approx. 60 &#181;m applied to a PDMS film with a thickness of approx. 40 &#181;m) exhibit favorable adhesion properties on rough surfaces.
Next, experiments related to the biological characterization have been performed on pristine composite films and plasma treated composite films (Figure 8). Plasma treatment of silicon elastomers is an often applied, versatile technique to increase the hydrophilic properties of surfaces and promoting cellular attachment and cellular spreading40,41. Silicones are well known for their low toxicity and high biostability but may contain residual monomers or catalysts which might influence physiological processes, leading also to cytotoxicity42,43. In the conducted experiments we have observed less than 5% cytotoxicity using LDH release as an indicator and a Trypan Blue exclusion assay. In the presented protocol, the entire cellular population, including cellular aggregates detached form the surface has been analyzed for proliferation analysis (Figure 9B). A modification of the protocol could produce more differentiated results. For each sample, the supernatant containing detached cellular aggregates could be transferred to a separate reaction tube and not combined with the cells enzymatically removed from the polymer surface. This would allow the exact assessment of cells attached to the surface and eventually reveal a more detailed determination of the influence of the polymers on the cellular adhesion process. In addition to the immunocytochemical methods presented here, cells might be harvested for investigation with immunoblot methods, allowing a detailed quantitative assessment of protein expression.
In summary, we have established manufacturing conditions for the production of thin elastomeric composite films for applications in advanced cell culture research. Additionally, these thin films possess high adaptability to skin roughness, enabling sophisticated design of skin adhesives.

In this protocol, detailed methods for manufacturing of thin elastomer films are presented. The widely available doctor blade technique has been used for the production of thin composite films. The manufacturing technique has been performed on polyethylenterephtalate (PET) foils, enabling subsequent production of these films in large scale. Emphasis of this protocol is the assessment of reproducibility, precise manufacturing of the different layers of the composite films and determination of the biological and adhesion properties of the final composite patch. The silicone elastomer poly-(dimethylsiloxane) (PDMS) is extensively used in biomedical technology, including production of skin adhesives, microfluidics applications and additional research fields1,2,3,4. Recently, another subclass of PDMS, so called Soft Skin Adhesives (SSAs) have been introduced, particular for gentle skin bonding and de-bonding.
Silicone SSAs are vinyl functionalized elastomers, differing from analogous polymers by the absence of reinforcing silica5. Similar to other PDMS, SSA&#39;s Young&#39;s modulus can be adapted in a wide range by modulating cross-linker concentration or curing time6,7,8. This change in Young&#39;s modulus of silicone elastomers affects the adhesive properties of the material significantly and has also profound consequences on prokaryotic and eukaryotic cells cultured on the surface9,10,11. On the cellular biological level, it was shown, that eukaryotic cells respond on the signal transduction level to a modulation of the matrix elasticity or thickness of the surface9,10,12,13,14. Therefore, a broad interest in cell culture applications of polymers with tunable mechanical properties exist. Importantly, the intrinsically low surface energy of silicone based elastomers does not provide optimal conditions for cell culture of eukaryotic cells. Oxygen plasma treatment is a widely used technique to increase PDMS low surface energy temporarily, leading to an enhancement of its pull-off strength, decreased surface adsorption of molecules, while in parallel promoting attachment, spreading and proliferation of eukaryotic cells15,16,17,18.
In addition to the materials properties, the surface topography significantly affects cellular adhesion and the adhesive interaction between two materials19,20,21,22. Surface roughness has several effects on the contact formation between two surfaces: reduction of the contact area, high stored elastic energy surrounding asperities as well as influence on the crack propagation can alter the adhesive strength23,24. Adhesion of self-adhesive films to human skin is an emerging application field, e.g., wound dressings, fixation of ECG electrodes or other wearable electronic devices25,26,27,28. To measure the adhesive performance of self-adhesives in relation to surface topography, glass substrates with varying degrees of roughness can be used in normal adhesion measurements8,21. Here, two glass substrates have been selected to investigate the adhesive properties of the polymer films. First, composite films with a PDMS backing layer in a mixing ratio of 10 to 1 weight parts covered by PDMS with different mixing ratio were characterized. In a second step a adhesive SSA layer was prepared with equal weight amounts of both components and with varying film thickness on top of a supporting PDMS film.

<table>
	<tbody>
		<tr>
			<td>2-Propanol, 97%</td>
			<td>Stockmeier Chemie</td>
			<td>1000452610000</td>
			<td>Isopropanol</td>
		</tr>
		<tr>
			<td>Abrasive diamnod hand pad</td>
			<td>Bohle</td>
			<td>MO 5007522</td>
			<td>Grit: 220</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Capricorn Scientific</td>
			<td>ACC-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin Fraktion V</td>
			<td>Roth</td>
			<td>0163.2</td>
			<td>BSA</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Phalloidin</td>
			<td>ThermoFischer Scientific</td>
			<td>A12379</td>
			<td>highly toxic</td>
		</tr>
		<tr>
			<td>Aquamount</td>
			<td>Polysciences</td>
			<td>18606-20</td>
			<td>water soluble mounting medium</td>
		</tr>
		<tr>
			<td>CytoTox-ONE Homogeneous Membrane Integrity Assay</td>
			<td>Promega</td>
			<td>G7890</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, without Ca2+, Mg2+</td>
			<td>ThermoFischer Scientific</td>
			<td>14190094</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum gold</td>
			<td>GE Health Care Life Science</td>
			<td>A15-151</td>
			<td>FBS</td>
		</tr>
		<tr>
			<td>Goniometer OCA35</td>
			<td>Dataphysics</td>
			<td></td>
			<td>for the determination of the static water contact angle</td>
		</tr>
		<tr>
			<td>Hoechst Dye 33342</td>
			<td>Sigma-Aldrich</td>
			<td>B1155-100MG</td>
			<td>bisBenzimide H 33342 trihydrochloride, highly toxic</td>
		</tr>
		<tr>
			<td>Microscope Axiovert 25</td>
			<td>Zeiss</td>
			<td></td>
			<td>Microscope used for cell culture documentation</td>
		</tr>
		<tr>
			<td>Microscope Eclipse LV100ND</td>
			<td>Nikon</td>
			<td></td>
			<td>Microscope used for film thickness determination</td>
		</tr>
		<tr>
			<td>Paraformaldehyde, aqueous solution 16%</td>
			<td>Electron Microscopy Sciences</td>
			<td>RT 15710</td>
			<td>electron microscopy grade</td>
		</tr>
		<tr>
			<td>penicillin und streptomycin solution</td>
			<td>Sigma-Aldrich</td>
			<td>P4333-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenom XL Scanning Electron Microscope (SEM)</td>
			<td>Phenom</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-(vinyl alcohol) 4-88, MW 31000</td>
			<td>Sigma-Aldrich</td>
			<td>81381-1KG</td>
			<td>Mowiol 4-88</td>
		</tr>
		<tr>
			<td>Poly-dimethyl siloxanes, Sylgard 184</td>
			<td>Dow Corning</td>
			<td>(400)000108351397</td>
			<td>PDMS</td>
		</tr>
		<tr>
			<td>RPMI 1640 basal medium</td>
			<td>ThermoFischer Scientific</td>
			<td>21875034</td>
			<td></td>
		</tr>
		<tr>
			<td>soft skin adhesive (SSA)</td>
			<td>Dow Corning</td>
			<td>(400)000108251792</td>
			<td>MG 7-9800 Soft Skin Adhesive (SSA)</td>
		</tr>
		<tr>
			<td>speed mixer DAC 600.2 VAC-P</td>
			<td>Hauschild</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>stylus profilomter</td>
			<td>Zeiss</td>
			<td></td>
			<td>Model: SURFCOM 1500SD3</td>
		</tr>
		<tr>
			<td>Tecan Infinite M200 pro</td>
			<td>Tecan</td>
			<td></td>
			<td>fluorescence plate reader</td>
		</tr>
		<tr>
			<td>Triton X 100</td>
			<td>Calbiochem</td>
			<td>648466</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue solution</td>
			<td>Sigma-Aldrich</td>
			<td>T8154-100ML</td>
			<td>highly toxic</td>
		</tr>
		<tr>
			<td>Trypsin/EDTA solution</td>
			<td>PAN-Biotech</td>
			<td>P10-023500</td>
			<td>0.05% Trypsin, 0.02% EDTA in PBS</td>
		</tr>
		<tr>
			<td>UV glue</td>
			<td>Bohle</td>
			<td>BO MV76002</td>
			<td>medium viscosity</td>
		</tr>
	</tbody>
</table>

thin film composite silicon elastomers for cell culture and skin applications: manufacturing and characterization

In this protocol, we present methods to fabricate thin elastomer composite films for advanced cell culture applications and for the development of skin adhesives. Two different poly-(dimethyl siloxanes) (PDMS and soft skin adhesive (SSA)), have been used for in depth investigation of biological effects and adhesive characteristics. The composite films consist of a flexible backing layer and an adhesive top coating. Both layers have been manufactured by doctor blade application technique. In the present investigation, the adhesive behavior of the composite films has been investigated as a function of the layer thickness or a variation of the Young&#39;s modulus of the top layer. The Young&#39;s modulus of PDMS has been changed by varying the base to crosslinker mixing ratio. In addition, the thickness of SSA films has been varied from approx. 16 &#181;m to approx. 320 &#181;m. Scanning electron microscopy (SEM) and optical microscopy have been used for thickness measurements. The adhesive properties of elastomer films depend strongly on the film thickness, the Young&#39;s modulus of the polymers and surface characteristics. Therefore, normal adhesion of these films on glass substrates exhibiting smooth and rough surfaces has been investigated. Pull-off stress and work of separation are dependent on the mixing ratio of silicone elastomers.
Additionally, the thickness of the soft skin adhesive placed on top of a supportive backing layer has been varied in order to produce patches for skin applications. Cytotoxicity, proliferation and cellular adhesion of L929 murine fibroblasts on PDMS films (mixing ratio 10:1) and SSA films (mixing ratio 50:50) have been conducted. We have shown here, for the first time, the side by side comparison of thin composite films manufactured of both polymers and present the investigation of their biological- and adhesive properties.

Watch this Scientific Journal Video about Thin Film Composite Silicon Elastomers for Cell Culture and Skin Applications: Manufacturing and Characterization at JoVE.com

Thin Film Composite Silicon Elastomers for Cell Culture and Skin Applications: Manufacturing and Characterization

A protocol for the manufacturing process of polymeric thin film composite structures possessing either different Young&#39;s moduli or thicknesses is presented. Films are produced for advanced cell culture studies or as skin adhesives.

In this protocol, we present methods to fabricate thin elastomer composite films for advanced cell culture applications and for the development of skin ...

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