Synthesis,permeability and biocompatibility of tricomponent membranes containing polyethylene glycol,polydimethylsiloxane and polypentamethylcyclopentasiloxane domains

The synthesis of "smart" tricomponent amphiphilic membranes containing poly(ethylene glycol) (PEG), polydimethylsiloxane (PDMS) and polypentamethylcyclopentasiloxane (PD(5)) domains is described. Contact angle hysteresis indicates that in air, the surfaces of such PEG/PD(5)/PDMS membranes are enriched by the hydrophobic components, PDMS and PD(5), while in water, the surfaces are rich in the hydrophilic PEG. The oxygen permeability of a series of membranes with varying M(c,hydrophilic) (M(n,PEG)=4600, 10,000 and 20,000 g/mol) and varying PEG/PD(5)/PDMS compositions was studied. Oxygen permeability increased with the amount of PDMS in the membrane. The molecular weight cut-off (MWCO) ranges and permeability coefficients of insulin through a series of PEG/PD(5)/PDMS(=29/14/57) membranes with varying M(c,hydrophilic) were determined. Insulin permeability is directly related to M(c,hydrophilic) of the membrane. MWCO studies show that the membranes are semipermeable to, i.e., allow the transport of smaller proteins such as insulin (M(n)=5733 g/mol, R(s)=1.34 nm) and cytochrome c (M(n)=12,400 g/mol, R(s)=1.63 nm), but are barriers to larger proteins such as albumin (M(n)=66,000 g/mol, R(s)=3.62 nm). Implantation of representative membranes in rats showed them to be biocompatible. According to these studies, PEG/PD(5)/PDMS membranes may be suitable for biological applications, e.g., immunoisolation of cells.     

Preparation of polyimide/polydimethylsiloxane graft copolymer and its permeabilities for gases and liquids

Polyimide/polydimethylsiloxane (PI/PDMS) graft copolymers with various PDMS contents were prepared by polycondensation of PDMS macromonomer 1 with aromatic diamines and pyromellitic dianhydride followed by heating at 220°C. The PDMS content of the grafts copolymers was controlled by changing the average degree of polymerization of the PDMS macromonomer in the range between 4 and 21. Graft copolymer membranes were prepared by casting films in the poly(amic acid) stage 2 and subsequent imidation upon heating. PI/PDMS membranes thus obtained are insoluble. The permeability coefficients towards nitrogen, oxygen, hydrogen, methane and carbon dioxide were evaluated. In the region of a PDMS content above 50 wt.-%, the oxygen permeability coefficient of a PI/PDMS graft copolymer membrane is > 10^?8 cm³(STP) · cm · cm^?2 · s^?1 · cmHg^?1. In addition, the permeability coefficient of methane is remarkably enhanced in comparison with a PI membrane. Preferential permeation of ethanol was observed through the graft copolymer membrane in the case of pervaporation of aqueous ethanol solution. Furthermore, acetone, acetonitrile and tetrahydrofuran were efficiently separated from water at any solvent/water ratio in the mixture.     

Microfibrillated Cellulose Sheets Coating Oxygen-Permeable PDMS Membranes Induce Rat Hepatocytes 3D Aggregation into Stably-Attached 3D Hemispheroids

Here we report the use of natural, chemically-unmodified, microfibrillated cellulose (MFC) as a matrix for hepatocyte culture. We developed an original cell-culture design composed of a thin 3D-microstructured fibrous substrate consisting of a MFC sheet coating a highly O₂-permeable polydimethylsiloxane (PDMS) membrane. The MFC-coated PDMS membranes were obtained according to a simple process where cellulose fibres were deposited from an aqueous suspension on the PDMS surfaces and the films were dried under mild conditions. To enable oxygen diffusion through the membranes, they were assembled on bottomless frames ('O₂+' condition). Rat hepatocytes primary-cultured on such MFC-PDMS membranes quickly organized themselves into large hemispherical 3D aggregates which were tightly anchored to the MFC sheets. In contrast, hepatocytes cultured on smooth PDMS membranes in the O₂+ system (O₂+, PDMS) organized into unstable 2D monolayers which easily detached from the surfaces. Hepatocyte 3D cultures obtained on MFC-PDMS membranes exhibited higher liver-specific functions over a 2-week culture period, as assessed by both the higher albumin secretion and urea synthesis rate. The MFC-PDMS membranes appear suitable for obtaining stably-attached and functional hepatocyte 3D cultures and appear interesting for drug/chemical screenings in a microplate format, but also for microfluidic applications.     

High-grade optical polydimethylsiloxane for microfluidic applications

Commercially available polydimethylsiloxane (PDMS) elastomers, such as Sylgard 184® are widely used in soft lithography and for microfluidic applications. These PDMS elastomers contain fillers to enhance their mechanical stability. The reinforcing fillers, often sub-micrometer small SiO₂ particles, tend to aggregate, swell with water, and thereby become cognoscible in a way that can strongly interfere with the visualization of micro-scale events taking place next to PDMS structures. As PDMS microfluidics are often used for studying cells and micro-/nanoparticles and for creating/handling nanodroplets, it has become highly desirable to employ a PDMS having high optical quality and that allows microscopy observation without artifacts. Here, we present a PDMS formulation that is free of fillers and has sufficiently low viscosity to perform a filtration step of the mixed prepolymers before curing. By molding a bi-layer microfluidic network (MFN), composed of a thin filler-free PDMS layer and a thicker Sylgard 184® backing layer, PDMS MFNs featuring both high optical quality and mechanical stability, can be fabricated.     

Practical fabrication of microfluidic platforms for live-cell microscopy

We describe a simple fabrication technique – targeted towards non-specialists – that allows for the production of leak-proof polydimethylsiloxane (PDMS) microfluidic devices that are compatible with live-cell microscopy. Thin PDMS base membranes were spin-coated onto a glass-bottom cell culture dish and then partially cured via microwave irradiation. PDMS chips were generated using a replica molding technique, and then sealed to the PDMS base membrane by microwave irradiation. Once a mold was generated, devices could be rapidly fabricated within hours. Fibronectin pre-treatment of the PDMS improved cell attachment. Coupling the device to programmable pumps allowed application of precise fluid flow rates through the channels. The transparency and minimal thickness of the device enabled compatibility with inverted light microscopy techniques (e.g. phase-contrast, fluorescence imaging, etc.). The key benefits of this technique are the use of standard laboratory equipment during fabrication and ease of implementation, helping to extend applications in live-cell microfluidics for scientists outside the engineering and core microdevice communities.     

An ultra-thin PDMS membrane as a bio/micro–nano interface: fabrication and characterization

We report a method for making ultra-thin PDMS membrane devices. Freely suspended membranes as thin as 70 nm have been fabricated. Bulging tests were performed with a custom built fluidic cell to characterize large circular membranes. The fluidic cell allows the media (such as air or water) to wet one side of the membrane while maintaining the other side dry. Pressure was applied to the membrane via a liquid manometer through the fluidic cell. The resulting load-deflection curves show membranes that are extremely flexible, and they can be reproducibly loaded and unloaded. Such devices may potentially be used as mechanical and chemical sensors, and as a bio-nano/micro interface to study cellular mechanics in both static and dynamic environments.     

Membrane-activated microfluidic rotary devices for pumping and mixing

Microfluidic devices are operated at a low-Reynolds-number flow regime such that the transportation and mixing of fluids are naturally challenging. There is still a great need to integrate fluid control systems such as pumps, valves and mixers with other functional microfluidic devices to form a micro-total-analysis-system. This study presents a new pneumatic microfluidic rotary device capable of transporting and mixing two different kinds of samples in an annular microchannel by using MEMS (Micro-electro-mechanical-systems) technology. Pumping and mixing can be achieved using a single device with different operation modes. The micropump has four membranes with an annular layout and is compact in size. The new device has a maximum pumping rate of 165.7 μL/min at a driving frequency of 17 Hz and an air pressure of 30 psi. Experimental data show that the pumping rate increases as higher air pressure and driving frequency are applied. In addition, not only can the microfluidic rotary device work as a peristaltic pumping device, but it also is an effective mixing device. The performance of the micromixer is extensively characterized. Experimental data indicate that a mixing index as high as 96.3% can be achieved. The developed microfluidic rotary device can be easily integrated with other microfluidic devices due to its simple and reliable PDMS fabrication process. The development of the microfluidic rotary device can be promising for micro-total-analysis-systems.     

Engineering micropatterned surfaces for the coculture of hepatocytes and Kupffer cells

Bioartificial liver (BAL) devices are used for applications ranging from pharmaceutical testing to temporary liver replacement. The capabilities of these devices can be improved by optimizing the range of hepatocyte functions that the BAL is able to perform. One means of achieving this is to design the BAL such that it establishes communication between hepatocytes and nonparenchymal cells. To understand how these heterotypic interactions can be favorably utilized in BAL design, it is first necessary to establish a culture environment that permits the controlled interactions of multiple cell types. This is the goal of the current study, which focuses on micropatterned cocultures of hepatocytes with Kupffer cells. The micropatterning technique relies on a polydimethylsiloxane (PDMS) membrane to achieve various two-dimensional configurations of the ECM prior to seeding the cell populations. The easy and inexpensive method of making the PDMS membranes differs from that reported in the literature and is detailed in the current study. To demonstrate the success of the method, surface characterization of the resultant micropatterns, as well as morphological and functional results are also presented.     

In vitro effect on cancer cells: synthesis and preparation of polyurethane membranes for controlled delivery of curcumin

Urethane polymers (PU) have been prepared from low-molecular weight polylactic acid (PLA) and hexamethylene diisocyanate (HMDI) using polydimethylsiloxane (PDMS) as a chain extender. These formed the supporting polymeric matrix of curcumin-containing PU membranes which were prepared using a solvent evaporation technique. FTIR and XRD data indicated the molecular-level dispersion and random distribution of curcumin in the polymer matrix, and data were consistent with observations from tensile-strength measurements and from AFM imaging. Determination of water vapor permeability and moisture uptake measurements have indicated that the PU membrane were appropriate for use on human skin. Skin permeation studies of curcumin were consistent with zero order (R2 = 0.9874) and with Korsmeyer-Peppas (R2 = 0.9978) kinetics-analytical data pointed to permeation by a combination of diffusion and erosion processes, with the latter dominating. The biocompatibility of these PU membranes was indicated by in vitro cytotoxicity studies using 3T3-L1-murine fibroblast cell. The in vitro therapeutic potential of the patches was demonstrated against A549 human lung cancer cells.     

Quantitative analysis of spherical microbubble cavity array formation in thermally cured polydimethylsiloxane for use in cell sorting applications

Microbubbles are spherical cavities formed in thermally cured polydimethylsiloxane (PDMS) using the gas expansion molding technique. Microbubble cavity arrays are generated by casting PDMS over a silicon wafer mold containing arrays of deep etched pits. To be useful in various high throughput cell culture and sorting applications it is imperative that uniform micron-sized cavities can be formed over large areas (in²). This paper provides an in-depth quantitative analysis of the fabrication parameters that effect the microbubble cavity formation efficiency and size. These include (1) the hydrophobic coating of the mold, (2) the mold pit dimensions, (3) the spatial arrangement of the pit openings, (4) the curing temperature of PDMS pre-polymer, (5) PDMS thickness, and (6) the presence and composition of residual gas in the PDMS pre-polymer mixture. Results suggest that the principles of heterogeneous nucleation and gas diffusion govern microbubble cavity formation, and that surface tension prevents detachment of the vapor bubble that forms in the PDMS over the pit. Paramerters are defined that enable the fabrication of large format arrays with uniform cavity size over 6 in² with a coefficient-of-variation <10 %. The architecture of the microbubble cavity is uniquely advantageous for cell culture. Large format arrays provide a highly versatile system that can be adapted for use in various high-throughput cell sorting applications. Herein, we demonstrate the use of microbubble cavity arrays to dissect the cellular heterogeneity that exists in a tumorigenic cutaneous squamous cell carcinoma cell line at the single cell level.     

The covalent attachment of adhesion molecules to silicone membranes for cell stretching applications

Strain devices with expandable polydimethylsiloxane (PDMS) culture membranes are frequently used to stretch cells in vitro, mimicking mechanically dynamic tissue environments. To immobilize cell-adhesive molecules to the otherwise non-adhesive PDMS substrate, hydrophobic, electrostatic and covalent surface coating procedures have been developed. The efficacy of different coating strategies to transmit stretches to cells however is poorly documented and has not been compared. We describe a novel and simple procedure to covalently bind extracellular matrix proteins to the surface of stretchable PDMS membranes. The method comprises PDMS oxygenation, silanization, and covalent protein cross-linking to the silane. We demonstrate improved attachment (～2-fold), spreading (～2.5-fold) and proliferation (～1.2-fold) of fibroblasts to our new coating over established coating procedures. Further, we compared the efficiency of different PDMS coating techniques to transmit stretches. After 15% stretch, the number of maximally (15 ± 5%) stretched cells on our PDMS surface coating was ～7-fold higher compared with alternative coating protocols. Hence, covalent linkage of adhesive molecules is superior to non-covalent methods in providing a coating that resists large deformations and that fully transmit this stretch to cultured cells.     

Measurement of cell traction force with a thin film PDMS cantilever

Adherent cells produce cellular traction force (CTF) on a substrate to maintain their physical morphologies, sense external environment, and perform essential cellular functions. Precise characterization of the CTF can expand our knowledge of various cellular processes as well as lead to the development of novel mechanical biomarkers. However, current methods that measure CTF require special substrates and fluorescent microscopy, rendering them less suitable in a clinical setting. Here, we demonstrate a rapid and direct approach to measure the combined CTF of a large cell population using thin polydimethylsiloxane (PDMS) cantilevers. Cells attached to the top surface of the PDMS cantilever produce CTF, which causes the cantilever to bend. The side view of the cantilever was imaged with a low-cost camera to extract the CTF. We characterized the CTF of fibroblasts and breast cancer cells. In addition, we were able to directly measure the contractile force of a suspended cell sheet, which is similar to the CTF of the confluent cell layer before detachment. The demonstrated technique can provide rapid and real-time measurement of the CTF of a large cell population and can directly characterize its temporal dynamics. The developed thin film PDMS cantilever can be fabricated affordably and the CTF extraction technique does not require expensive equipment. Thus, we believe that the developed method can provide an easy-to-use and affordable platform for CTF characterization in clinical settings and laboratories.     

Ultrathin crosslinked membranes at the interface between oil and water

The tensio-active properties of different types of diesters can be used to synthesize two-dimensional model networks at the interface between oil and water. We have systematically studied rubber-elastic, glass-like and transient membranes, which are stabilized and crosslinked by physical or chemical contacts. The kinetics of surface gelation and the mechanical properties of the crosslinked membranes were investigated by measuring two-dimensional rheological parameters, such as the shear modulus or the surface viscosity. The experimental data of these investigations are in fairly good agreement with the theoretical predictions of percolation theories. Further informations on the molecular structure of the crosslinked membranes can be obtained from electron spin resonance (ESR) measurements. Using spin labels of significantly different sizes, which are diffusing from the aqueous environment into the oil phase, it is possible to determine the average mesh size of the interfacial network structure. The systematic study of these ultrathin membranes offers new insights into aspects of current research, and opens interesting new technical applications.     

RGD peptide-conjugated poly(dimethylsiloxane) promotes adhesion, proliferation, and collagen secretion of human fibroblasts

A novel technique for conjugating Arg-Gly-Asp (RGD) peptides to poly(dimethylsiloxane) (PDMS) surfaces as well as its application to cell culture is presented in this paper. This technique performs RGD conjugation to PDMS through photochemical immobilization of functional NHS groups to PDMS surface followed with linking RGD peptide to the surface via coupling reaction with NHS. A bifunctional photolinker, N-sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-SANPAH), was used to conjugate RGD peptide to the surface. Compared to existing methods for peptide conjugation to PDMS, this technique is convenient, efficient, and free of organic contamination to PDMS surfaces. It can also be used to conjugate other peptides or proteins to most polymeric materials. In addition, cell culture studies showed that the RGD-conjugated PDMS surfaces promoted the adhesion, proliferation, and collagen production of human skin fibroblasts (HSFs). Finally, the RGD-conjugated PDMS surfaces are resistant to autoclaving and UV irradiation, which enables them to be repeatedly used in cell culture studies.     

Synthesis of macroporous poly(dimethylsiloxane) scaffolds for tissue engineering applications

Macroporous, biostable scaffolds with controlled porous architecture were prepared from poly(dimethylsiloxane) (PDMS) using sodium chloride particles and a solvent casting and particulate leaching technique. The effect of particulate size range and overall porosity on the resulting structure was evaluated. Results found 90% v/v scaffolds and particulate ranges above 100?μm to have the most optimal open framework and porosity. Resulting hydrophobic PDMS scaffolds were coated with fibronectin and evaluated as a platform for adherent cell culture using human mesenchymal stem cells. Biocompatibility of PDMS scaffolds was also evaluated in a rodent model, where implants were found to be highly biocompatible and biostable, with positive extracellular matrix deposition throughout the scaffold. These results demonstrate the suitability of macroporous PDMS scaffolds for tissue engineering applications where strong integration with the host is desired.     

Suspension arrays of hydrogel microparticles prepared by photopatterning for multiplexed protein-based bioassays

Suspension arrays for protein-based assays have been developed using shape-coded poly(ethylene glycol) (PEG) hydrogel microparticles to overcome the problems with current systems which use color-coded rigid microparticles as protein supports. Various shapes of hydrogel microparticles were fabricated by a two-step process consisting of photopatterning and flushing using a poly(dimethylsiloxane) (PDMS) channel as a molding insert. Hydrogel microparticles with lateral dimensions ranging from 50 to 300 μm were fabricated using different molecular weights of PEG (700, 3,400, and 8,000 Da), by which the water content and swelling behavior of the hydrogel microparticles could be controlled. Protein-entrapped hydrogel microparticles were prepared in a suspension array format, and PEG hydrogel could encapsulate proteins without deactivation for a week due to its high water content and soft nature. The sequential bienzymatic reaction of hydrogel-entrapped glucose oxidase (GOX) and peroxidase (POD) was successfully investigated using fluorescence detection, demonstrating one possible application of suspension arrays. Furthermore, a mixture of two different shapes of hydrogel microparticles containing GOX/POD and alkaline phosphatase (AP), respectively, was prepared and the shape-coded suspension array was used for simultaneous characterization of two different enzyme-catalyzed reactions.     

Targeted cell adhesion on selectively micropatterned polymer arrays on a poly(dimethylsiloxane) surface

Herein, we introduce the fabrication of polymer micropattern arrays on a chemically inert poly(dimethylsiloxane) (PDMS) surface and employ them for the selective adhesion of cells. To fabricate the micropattern arrays, a mercapto-ester—based photocurable adhesive was coated onto a mercaptosilane—coated PDMS surface and photopolymerized using a photomask to obtain patterned arrays at the microscale level. Robust polymer patterns, 380 μm in diameter, were successfully fabricated onto a PDMS surface, and cells were selectively targeted toward the patterned regions. Next, the performance of the cell adhesion was observed by anchoring cell adhesive linker, an RGD oligopeptide, on the surface of the mercapto-ester—based adhesive-cured layer. The successful anchoring of the RGD linker was confirmed through various surface characterizations such as water contact angle measurement, XPS analysis, FT-IR analysis, and AFM measurement. The micropatterning of a photocurable adhesive onto a PDMS surface can provide high structural rigidity, a highly–adhesive surface, and a physical pathway for selective cell adhesion, while the incorporated polymer micropattern arrays inside a PDMS microfluidic device can serve as a microfluidic platform for disease diagnoses and high-throughput drug screening.     

Hepatocyte spheroid culture on a polydimethylsiloxane chip having microcavities

A two-dimensional microarray technique of spherical multicellular aggregates (spheroids) using a microfabricated polydimethylsiloxane (PDMS) chip and the expression of liver-specific functions of primary rat hepatocytes on the chip were investigated. The PDMS chip, which was fabricated by a photolithography-based technique, consisted of approximately 2500 cylindrical microcavities (approximately 1100 cavities/cm²) in a triangular arrangement of 330 μm pitch on a PDMS plate (20 × 20 mm); each cavity measured 300 μm in diameter and 100 μm in depth. Most hepatocytes on the PDMS chip gradually gathered and subsequently formed a single spheroid in each cavity until 3 days of culture. A part of the spheroid was attached to the bottom or wall surface of the microcavity, and the spheroid configuration was maintained for at least 14 days of culture. Albumin secretion, ammonia removal and ethoxyresorufin O-dealkylase (EROD) activity, which is a cytochrome P-450-dependent reaction, of hepatocytes on the PDMS chip were higher than those of a monolayer dish or a flat PDMS dish without microcavities, and were maintained for at least 10 days of culture. The spheroid microarray technique appears to be promising in the development of cell chips and microbioreactors.     

PDMS‐Modified Polyurethane Films with Low Water Contact Angle Hysteresis

Summary: Polyurethane (PU) films with low water contact angle hysteresis (CAH) were prepared by employing less than 0.2 wt.‐% of mono‐ or bihydroxyl‐functionalized polydimethylsiloxane (PDMS) of 60–70 DMS repeat units. A Si content of as low as 0.03 wt.‐% was sufficient to lead to a CAH of 20°. The curing temperature demonstrated strong effects on the wetting behavior of the PDMS‐modified PU films: a relatively low curing temperature (40 °C) resulted in stronger surface segregation of PDMS than higher curing temperatures, giving rise to high water receding CAs and thus low CAH. It was found from angle‐resolved X‐ray photoelectron spectroscopy analysis that the surface silicon content was greater when cured at a lower temperature. The surface of PDMS‐modified PU films was very smooth from AFM observations, but the Si content had strong effects on the surface phase contrast. The chain length of 60–70 DMS repeating units for both mono‐ and bifunctional PDMS appeared to be long enough to prevent the surface reorganization during the CA measurements.

Schematic, simplified illustration of PDMS chains at the surface of PU films modified by PDMS₆₀? OH and PDMS₇₀? (OH)₂.     

Selective Removal of Dilute Benzene from Water by Various Cross‐Linked Poly(dimethylsiloxane) Membranes Containing tert‐Butylcalix[4]arene

Summary: This paper focuses on the addition of tert‐butylcalix[4]arene (CA) to cross‐linked poly(dimethylsiloxane) (PDMS) membranes derived from poly(dimethylsiloxane) di(methyl methacrylate) macromonomer (PDMSDMMA) and various divinyl cross‐linker compounds, on the pervaporation characteristics for the removal of dilute benzene from an aqueous solution. When an aqueous solution of 0.05 wt.‐% benzene was permeated through the cross‐linked PDMSDMMA membranes containing CA they showed high benzene/water selectivity and permeability. Both the benzene/water selectivity and permeability of the membranes were enhanced by increasing both the divinyl compound cross‐linker content and the amount of CA, and were significantly influenced by the type of divinyl compound. Addition of CA to PDMSDMMA membranes cross‐linked with divinylperfluorohexane (DVF) showed the best pervaporation performance. The normalized permeation rate, benzene/water separation factor, and pervaporation separation index of these membranes were 1.86 × 10⁻⁵ kg · m/(m² · h), 5 027, and 9 350, respectively. The pervaporation characteristics of the cross‐linked PDMSDMMA membranes containing CA were studied with respect to their chemical and physical structure. Furthermore, the mechanism of the selective permeation of benzene over water through these membranes was investigated.