PDMS Sylgard 527-Based Freely Suspended Ultrathin Membranes

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Biological and Medical Applications of Materials and Interfaces

PDMS Sylgard 527 Based Freely Suspended Ultrathin Membranes Exhibiting Mechanistic Characteristics of Vascular Basement Membranes Mitesh L Rathod, Jungho Ahn, Biswajit Saha, Prashant Purwar, Yejin Lee, Noo Li Jeon, and Junghoon Lee ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b12309 • Publication Date (Web): 16 Oct 2018 Downloaded from http://pubs.acs.org on October 20, 2018

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ACS Applied Materials & Interfaces

PDMS Sylgard 527 Based Freely Suspended Ultrathin Membranes Exhibiting Mechanistic Characteristics of Vascular Basement Membranes Mitesh L. Rathod,† Jungho Ahn,†,§, Biswajit Saha,† Prashant Purwar,† Yejin Lee,† Noo Li Jeon,† and Junghoon Lee*,† †

School of Mechanical and Aerospace Engineering, Seoul National University, Seoul 151-744,

South Korea §

George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology,

Atlanta, GA 30332, USA KEYWORDS: PDMS Sylgard 527, ultrathin, membrane, vascular basement membrane, compliance, cell-substrate interaction, cell-cell interaction, monolayer

ACS Paragon Plus Environment

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ABSTRACT

In the past significant effort has been made to develop ultrathin membranes exhibiting physiologically relevant mechanical properties, such as thickness and elasticity, of native basement membranes. However, most of these fabricated membranes have relatively high elastic modulus, ~MPa - GPa, relevant only to retinal and epithelial basement membranes. Vascular basement membranes exhibiting relatively low elastic modulus, ~kPa, on the contrary, have seldom been mimicked. Membranes demonstrating high compliance, moduli ranging in ~kPa along with submicroscale thicknesses have rarely been reported, and would be ideal to mimic vascular basement membranes in vitro. To address this, we fabricate ultrathin membranes demonstrating the mechanistic features exhibited by their vascular biological counterparts. Salient features of the fabricated ultrathin membranes include free suspension, physiologically relevant thickness ~submicrometres, relatively low modulus ~kPa, and sufficiently large culture area ~20 mm 2. To fabricate such ultrathin membranes, undiluted PDMS Sylgard 527 was utilised as opposed to the conventional diluted polymer-solvent mixture approach. In addition, the necessity to have sacrificial layer for releasing membranes from the underlying substrates was also eliminated in our approach. The novelty of our work lies in achieving the distinct combination of membranes having thickness in sub-micrometres and the associated elasticity in kPa using undiluted polymer, which past approaches with dilution have not been able to accomplish. The ultrathin membranes with average thickness of 972 nm (thick) and 570 nm (thin) were estimated to have an elastic modulus of 45 kPa and 214 kPa respectively. Contact angle measurements revealed the ultrathin membranes exhibited hybrophobic characteristics in unpeeled state and transformed to hydrophilic behaviour when freely suspended. Human Umbilical Vein Endothelial Cells were cultured on the polymeric ultrathin membranes and the temporal cell response to change in local compliance of the


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membranes was studied by evaluating cell spread area, density, percentage area coverage and spread rate. After 24 hours, single cells, pairs and group of three to four cells were noticed on highly compliant thick membranes, having average thickness of 972 nm and modulus of 45 kPa. On the contrary, cell monolayer was noted on glass slide acting as a control. For the thin membranes, featuring average thickness of 570 nm and modulus of 214 kPa, the cells tend to exhibit similar response to that on control with initiation of monolayer formation. Our results indicate, the local compliance, in turn, the membrane thickness governs the cell behaviour and this can have vital implications during disease initiation and progression, wound healing and cancer cell metastasis.

1. INTRODUCTION Endothelial cells (ECs), lining the lumen of all blood vessels, perform several important functions; such as presenting a permeability barrier, trafficking of biochemical cues and relaying information about fluid shear stress to the underlying tissues and cells in the arterial walls.1,2 ECs attach to and interact with the underlying stromal elements via a specialization of extracellular matrix, the basement membrane (BM). BM essentially consists of a network involving fibres and pores at nanometre and submicrometre scales.3-5 The inherent topography and the local compliance of the BM play a critical role in providing the biophysical cues to the overlying ECs. These signals later regulate ECs migration, adhesion, proliferation, and differentiation.6-9 Over the years a well-established relation has been developed between the alterations in mechanical properties of vessels and progression of the disease state.10,11 Numerous vascular pathologies have resulted on account of modifying the micromechanical environment, including the underlying BM, available to the ECs.12-16 Cardiovascular pathological state such as atherosclerosis has been identified with increased basement membrane thickness and stiffness in


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some vessels.17,18 Modifying the mechanistic characteristics of BM thus indicate that the mechanical properties of BM may be involved directly or indirectly in the initiation and progression of cardiovascular disease. Such evidences clearly demonstrate the detrimental impacts of altering the biophysical microenvironment, comprised primarily of BM, and have started receiving attention lately. Recent technological advances have facilitated guided tissue regeneration by developing the much needed vascular scaffolds. However, such prosthetic designs lack the essential in vivo like micromechanical environment, namely compliance and topography of native vessel. Nonexistence of such biophysical features, in past, may be a crucial factor for prosthetic vessel failures.19,20 Enormous efforts have been applied over the past decade to mimic basement membranes in vitro. These involved from utilising sophisticated technologies such as conventional nano and microfabrication techniques, replica moulding, soft lithography, layer by layer approach, micro contact printing to approaches as simple as spin coating based fabrication. Numerous materials such as poly (l-Lactic acid) PLLA, polyelectrolyte, photoresist (SU 8, 1002F), polystyrene, hydrogel, silicon nitride, paralyene HT, glass, silicon dioxide were explored by these techniques to develop membranes with thicknesses ranging from nano to micrometre scales.21-32 Commercial polycarbonate and polyester membranes as thick as 10 µm have also been integrated with microfluidic platforms to mimic the physiological micromechanical environment in a more realistic manner.33,34 Some techniques and materials mentioned before could be applied to fabricate membranes with nanoscale thicknesses. However, such membranes featured significantly high elastic modulus (MPa - GPa), thereby making it physiologically irrelevant. The membranes exhibiting elasticity in tens of MPa could only mimic retinal and epithelial basement membranes, as these are characterized by relatively high elastic modulus (0.95-20 MPa).35-38 In contrast,


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depending on the vessel type, vascular basement membranes (VBM) featuring relative low modulus (8-70 kPa)38-40 and sub-micrometre scale thickness (120-900 nm)5,41 could not be mimicked by past approaches. In this regard, ultrathin membranes exhibiting elasticity in ~kPa would be ideal to mimic VBM in vitro. Polydimethylsiloxane (PDMS) based ultrathin films/membranes are other promising candidates mimicking the BM. PDMS has been widely applied for diverse applications such as sensors, microfluidic devices and transducers, to name a few.42-45 PDMS, with its optical clarity and relatively lower modulus and biocompatibility, belongs to an interesting class of polymer that is utilised for fabricating ultrathin films. The most common and simplest technique to generate PDMS membrane is by spin coating. Spin coating based approach using pure PDMS Sylgard 184 typically was used to develop membranes with thickness in micrometre range.46-48 To achieve submicrometre and nanometre scale thicknesses, the dilution of pure PDMS using solvent such as hexane is needed. By such technique sub-micron and nanoscale membranes are routinely generated. 38,46,49 However, hexane results in swelling of cured PDMS. Such shortcoming can be overcome by using tert-butyl alcohol as a solvent for diluting the PDMS or increasing the spin coating duration. With such approach, however, the reported membrane thickness still has been in micrometre range.47 In addition, irrespective of the thickness of such membranes, the associated modulus has always been estimated in ~MPa range. Such solvent based dilution approach fall short in developing ultrathin membranes with modulus ranging in ten-hundreds of kPa. In this study we describe the fabrication of freely suspended ultrathin membrane (FSUM) having physiologically relevant thickness, relatively low elastic modulus similar to the VBM and sufficiently large area (~ 20 mm2) for cell culturing. FSUMs were developed using pure PDMS unlike the previously described PDMS solvent mixture based approach. Moreover, our approach


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also eliminates the need for sacrificial layer as demanded by past fabrication methods for releasing the membrane from the substrate. To fabricate the ultrathin membranes using pure PDMS we use Sylgard 527 as compared to commonly used Sylgard 184. Viscosity of Sylgard 527 as per manufacturer data is 0.5 Pa-sec and was exploited to achieve membranes with sub-micron scale thicknesses. In addition, the bulk modulus of Sylgard 527 is reported to be 1.5-5 kPa,50,51 thereby making it an ideal candidate for developing ultrathin membranes exhibiting elasticity comparable with the local compliance of the VBM. Next, we cultured Human Umbilical Vein Endothelial Cells (HUVEC) on the FSUM, evaluated the cell viability and studied the cell response with respect to change in membrane thickness and local compliance.

2. EXPERIMENTAL SECTION 2.1 Ultrathin membrane fabrication. Schematic details for fabricating FSUM are illustrated in Figure 1. The process begins with cleaning a polystyrene petri dish (diameter 60 mm) with air gun. PDMS Sylgard 527 was prepared by mixing part A and part B in mass ratio of 1:1. The bubbles formed during rigours mixing were removed by degassing the mixture. Next, the freshly prepared PDMS Sylgard 527 was poured over the air cleaned petri dish and spin coated to form an ultrathin layer over the dish. The spin coating speed was varied to fabricate membranes with different thicknesses; however, the coating duration was fixed at 90 seconds. Once coated the samples were cured in a convection oven at 60 °C for 16 hours. Next, a PDMS Sylgard 184 frame structure having circular opening of 5 mm diameter and thickness of ~ 3-4 mm was prepared. Briefly, the preparation involved, mixing Sylgard 184 in a mass ratio of 10:1 (base:curing agent), followed by degassing, and allowing it to cure in convection oven in a petri dish. Later, an autopsy punch (diameter 5mm) was used to create circular


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Figure 1. Illustration of the process flow for fabricating PDMS Sylgard 527 based freely suspended ultrathin membranes. PDMS Sylgard 527 was spin coated at high speeds to generate ultrathin membranes. The membranes were next freely suspended on a PDMS Sylgard 184 frame structure. openings in the cured PDMS Sylgard 184. The PDMS was then cut into square shaped frame having circular opening of 5 mm. The Sylgard 184 frame structure was next bonded to the previously spin coated and cured PDMS Sylgard 527 samples using freshly prepared Sylgard 184. The complete assembly was then allowed to cure overnight in a convection oven at 60 °C. Once cured the samples were carefully peeled from the petri dish resulting in PDMS Sylgard 527 ultrathin membranes freely suspended on PDMS Sylgard 184 support structure. The PDMS frame structure provides the necessary stability to the freely suspended membranes and aids during the peeling process and handling of the ultrathin membranes.


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2.2 Elastic modulus estimation of ultrathin membrane. The complete assembly involving PDMS Sylgard 527 membrane suspended on PDMS Sylgard 184 frame structure was rested on two vertical columns made using PDMS Sylgard 184. Next, known amount of silicone oil (Alfa Aesar) was pipetted onto the freely suspended ultrathin membranes and the corresponding membrane extensions were recorded using a CCD camera (Basler scout scA640-70gc, Navitar 160123 zoom lens). The membrane extensions were next quantified using NIH ImageJ software for estimating the elastic modulus. 2.3 Contact angle measurement. Contact angle measurements (CAM) were performed for PDMS Sylgard 527 membranes in unpeeled and freely suspended states to assess the wettability. Membranes with different thicknesses, 570 nm, 972 nm and 1.8 µm were utilized for the measurements. In addition, CAM for substrates such as petri dish (Polystyrene, electron beam sterilized), glass slide (Marienfeld), PDMS Sylgard 527 and PDMS Sylgard 184 were also performed. Images of water droplets resting on different substrates were captured using a CCD camera (Basler scout scA640-70gc, Navitar 1-60123 zoom lens) and were later used for estimating the contact angles by utilizing NIH ImajeJ software. 2.4 Cell culture. Prior to cell seeding, the membranes were washed with phosphate buffered saline (PBS) and later UV sterilised for 5 minutes. Fibronectin (Sigma-Aldrich) 20 µg mL-1 was next coated on the sterilised membranes for 1 hour at 37 °C. After 1 hour, the membranes were rinsed with PBS to remove the excess fibronectin. HUVECs (Lonza), passage 3 to 6, cultured in EGM-2 culture medium (Lonza) were used for the experiments. The membranes were next seeded with HUVECs having a concentration of 0.5 million cells mL-1 and incubated in a 5% CO2 humidified incubator at 37 °C. The cells were finally fixed with 4% paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100 at the end of 3, 15 and 24 hours. Alexa 488 Phalloidin


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(Molecular Probes) and Hoechst 33342 (Molecular Probes) were used for staining actin and nuclei for immunofluorescence studies. Confocal images were acquired using Olympus FV 1000 microscope (20X dry objective). NIH ImajeJ software was utilised for quantification of cell area, percentage area coverage, density and spread rate measurements. 2.5 Live/dead assay protocol. The determination of live and dead cells was performed using the Live/Dead Viability/Cytotoxicity Assay (Invitrogen). After aspirating media from a reservoir in each sample, Live/Dead Assay solution was prepared approximately by mixing 4 μM of ethidium homodimer-1 (Etdh-1, detecting dead cells) and 2 μM of calcein-AM (detecting live cells) with working solution in Endothelial Growth Medium (EGM-2, Lonza). Next, samples were incubated at 37 ℃ in a 5% CO2 humidified incubator for 45 minutes. Before imaging, samples were washed 2-3 times using EGM-2. The fluorescence images were obtained using confocal microscope (Olympus FV 1000) and percent of surviving cells were analysed by counting the ratio of dead (red) and live (green) fluorescence at a given focal plane using Imaris (Bitplane). 2.6 Statistical Analysis. All data are presented as mean ± standard deviation. Statistical significance was evaluated using student’s t-test and one-way ANOVA followed by Tukey’s test. Significance was considered for p < 0.05.

3. RESULTS AND DISCUSSION To develop basement membranes with physiological relevant compliance and thickness appropriate to VBM we utilized PDMS Sylgard 527. Owing to its low modulus (1.5-5 kPa) and low viscosity (0.5 Pa-sec) as compared to Sylgard 184, PDMS Sylgard 527 seldom find its application in making micro and nano structures. Recent efforts have shown that even with such a low elastic modulus Sylgard 527 can be used to generate micro structures.51 Moreover, mixing


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Figure 2. Details of freely suspended ultrathin membranes. (A) Sylgard 527 membrane freely suspended on Sylgard 184 frame structure. Scale bar: 4 mm. Inset shows Newton’s rings developed on the FSUM. (B) 20 µl ink droplet resting on the freely suspended ultrathin membrane. Scale bar: 3mm. (C) Highly stretchable relatively thick, micrometre range, Sylgard 527 membrane fabricated at spin speed of 500 rpm. Scale bar: 7 mm. (D) Thickness vs spin speed curve for PDMS Sylgard 527 based freely suspended membranes. Sub-10 µm range membranes obtained at (E) 8000 rpm resulting in average thickness of 1.8 µm, (F) 5000 rpm having average thickness of 3.4 µm and (G) 3000 rpm having average thickness of 8.6 µm. Scale bar: 2 µm. (H) Membrane obtained at 10,000 rpm resulting in average thickness of 972 nm. (I) Membrane obtained at 12,000 rpm resulting in average thickness of 570 nm. both the PDMS variants results in a hybrid polymer that can be utilized to develop more complex microfluidic platforms.45 We hypothesized that low viscosity of Sylgard 527 can be effectively utilized to fabricate membranes with sub-micron scale thicknesses without any additional demand for polymer dilution with toxic solvents. To test the hypothesis, Sylgard 527 was spin coated at high speeds to form ultrathin membranes. These ultrathin membranes were later freely suspended on PDMS 184 frame structure. One of such freely suspended ultrathin membrane having diameter of 5mm and an ink droplet resting on


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such an ultrathin membrane is shown in Figure 2a and b respectively. To demonstrate the high elasticity of membranes developed using PDMS Sylgard 527 we fabricated a relatively thick membrane in micrometer range. As shown in Figure 2c, the micrometer thick membrane exhibits high stretching ability when peeled from the underlying substrate (petri dish). Demonstration of high pliability, mechanical strength and elasticity can be seen in Video S1 (Supporting Information). Even though the elastic modulus of PDMS Sylgard 527 is relatively low as compared to other PDMS variants, the mechanical strength and pliability displayed can be effectively exploited for numerous applications. The thickness vs spin speed curve is illustrated in Figure 2d. In the past membranes with thickness in sub-10 µm range using just PDMS Sylgard 184 have seldom been achieved. With our approach we demonstrate membranes in sub-10 µm range can be fabricated with relative ease. Scanning electron microscopy (SEM) images of PDMS Sylgard 527 membranes in sub-10 µm range are shown in Figure 2e, f and g. As seen from Figure 2d, by utilizing just Sylgard 527 membranes with average thickness of 570 nm can be achieved. To fabricate membranes with similar thickness past approaches required dilution of PDMS Sylgard 184 with various solvents.38,49,52 The low viscosity of Sylgard 527 eliminated the necessity of diluting the polymer with hazardous solvents. Moreover, the requirement of sacrificial layer to release the ultrathin membranes from the underlying substrates was also eliminated. With our approach, FSUM with 5mm diameter, resulting in sufficiently large area (~ 20 mm2) can be routinely generated for thickness as low as 570 nm. SEM images of the ultrathin membranes obtained at 10,000 and 12,000 rpm are shown in Figure 2h and i respectively. Hereinafter, membranes with average thickness of 972 nm and 570 nm will be referred to as thick and thin membranes.


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Figure 3. Evaluating the elastic modulus of FSUMs. The ultrathin membranes were loaded with known amount of fluid volume and the corresponding membrane extensions were used for estimating the elastic modulus. Thick membrane (972 nm) extension for silicone oil volume of (A) 80 µL, (B) 100 µL and (C) 120 µL. Scale bar: 1 mm. Thin membrane (570 nm) extension for (D) 40 µL (E) 60 µL, (F) 80 µL of silicone oil. Scale bar:1 mm. (G) Stretching of the ultrathin membrane in upward direction using a paper tissue after fluid loading test to confirm the intactness of the ultrathin membrane. (H) Intentional rupture of the ultrathin membrane using a paper tissue to demonstrate the difference between an intact and a ruptured membrane. (I) Elastic modulus estimation for thick (972 nm) and thin (570 nm) freely suspended ultrathin membranes. Inset illustrates the setup for estimating FSUM elastic modulus. ##p

PDMS Sylgard 527-Based Freely Suspended Ultrathin Membranes

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