Poly(dimethylsiloxane) elastomers with tethered peptide ligands for cell adhesion studies

Poly(dimethylsiloxane) (PDMS) is the choice of material for a wide range of biological and non-biological applications because of its chemical inertness, non-toxicity, ease of handling and commercial availability. However, PDMS exhibits uncontrolled protein adsorption and cell adhesion and it has proved difficult to functionalize to present bioactive ligands. We present a facile strategy for functional surface modification of PDMS using commercial reagents to engineer polymer brushes of oligo(ethylene glycol) methacrylate that prevent cell adhesion and can be functionalized to display bioadhesive ligands. The polymer brushes resist biofouling and prevent cell adhesion and bioadhesive peptides can be tethered either uniformly or constrained to micropatterned domains using standard peptide chemistry approaches. This approach is relevant to various biomedical and biotechnological applications.     

Quantitative measurement and control of oxygen levels in microfluidic poly(dimethylsiloxane) bioreactors during cell culture

Microfluidic bioreactors fabricated from highly gas-permeable poly(dimethylsiloxane) (PDMS) materials have been observed, somewhat unexpectedly, to give rise to heterogeneous long term responses along the length of a perfused mammalian cell culture channel, reminiscent of physiologic tissue zonation that arises at least in part due to oxygen gradients. To develop a more quantitative understanding and enable better control of the physical-chemical mechanisms underlying cell biological events in such PDMS reactors, dissolved oxygen concentrations in the channel system were quantified in real time using fluorescence intensity and lifetime imaging of an oxygen sensitive dye, ruthenium tris(2,2’-dipyridyl) dichloride hexahydrate (RTDP). The data indicate that despite oxygen diffusion through PDMS, uptake of oxygen by cells inside the perfused PDMS microchannels induces an axial oxygen concentration gradient, with lower levels recorded in downstream regions. The oxygen concentration gradient generated by a balance of cellular uptake, convective transport by media flow, and permeation through PDMS in our devices ranged from 0.0003 (mg/l)/mm to 0.7 (mg/l)/mm. The existence of such steep gradients induced by cellular uptake can have important biological consequences. Results are consistent with our mathematical model and give insight into the conditions under which flux of oxygen through PDMS into the microchannels will or will not contribute significantly to oxygen delivery to cells and also provide a design tool to manipulate and control oxygen for cell culture and device engineering. The combination of computerized microfluidics, in situ oxygen sensing, and mathematical models opens new windows for microphysiologic studies utilizing oxygen gradients and low oxygen tensions. Electronic Supplementary Material is available in the online version of this article at     

Poly(dimethylsiloxane) (PDMS) and Silicon Hybrid Biochip for Bacterial Culture

In this study, a novel PDMS/silicon hybrid microfluidic biochip was fabricated and tested for the long-term batch culture of bacterial cells. The PDMS (poly(dimethylsiloxane)) cover with 3-dimensional micro-channels for flow was fabricated using Teflon tubing and hole-punch techniques, without photolithographic methods. The PDMS/silicon hybrid biochip was prepared by bonding of PDMS cover and a silicon chip that had electrodes and micro-fluidic channels defined. The absorption of liquid into PDMS cover was characterized and conditions to prevent drying of nutrient media within the micro-chamber were shown. The absorption of liquid from micro-chambers into the PDMS cover was reduced up to 2.5 times by changing the mixing ratio of PDMS and curing agent from 10 : 1 to 2.5 : 1. In addition, pre-saturation of the PDMS cover with media prior to the incubation resulted in the preservation of liquid in the micro-chambers for up to 22 hours. Optimization of the mixing ratio and pre-saturation of the PDMS cover reduced the drying time 10 times when compared to the unsaturated PDMS cover composed of 10 : 1 ratio of PDMS and curing agent. Listeria innocua and a strain of Escherichia coli, expressing green fluorescent protein (GFP), were successfully cultured in batch mode within the PDMS/silicon hybrid biochip.     

Retinal pigment epithelium cell culture on thin biodegradable poly(DL-lactic-co-glycolic acid) films

Thin films of 50 : 50 and 75 : 25 poly(DL-lactic-co-glycolic acid) (PLGA) were manufactured with a controlled thickness of less than 10 μm. The effect of PLGA copolymer ratio on in vitro cell attachment, proliferation, morphology, and tight junction formation was evaluated using a human D407 retinal pigment epithelium (RPE) cell line. Almost complete cell attachment was achieved on both PLGA films after 8 h of cell seeding, which was comparable to that on tissue culture polystyrene (TCPS) controls. The initial cell seeding density affected attachment, and the optimal value for 50: 50 PLGA was 25 000 cells cm^-2. After 7 days of in vitro culture, cell density on 50 : 50 and 75 : 25 PLGA films increased 45 and 40 folds, respectively, and a 34-fold increase was observed on TCPS. The RPE cells cultured on PLGA films at confluence had a characteristic cobblestone morphology. Confluent RPE cells also developed normal tight junctions in vitro which were concentrated mainly at the apical surfaces of cell-cell junctions. These results demonstrated that thin biodegradable PLGA films can provide suitable substrates for human RPE cell culture, and may serve as temporary carriers for subretinal implantation of organized sheets of RPE.     

Retinal pigment epithelium cell culture on surface modified poly(hydroxybutyrate-co-hydroxyvalerate) thin films

There is currently no effective treatment for the retinal disorders caused by retinal pigment epithelium (RPE) degeneration. Transplantation of allografts is the main strategy towards correction of this malady. Tissue engineering could offer hope and involve the use of biodegradable polymeric templates to replace diseased or lost RPE. In this study PHBV8 film was chosen as a temporary substrate for growing retinal pigment epithelium cells as an organized monolayer before their subretinal transplantation. The surface of the PHBV8 film was rendered hydrophilic by oxygen plasma treatment to increase the reattachment of D407 cells on the film surface. Power and duration was changed, from 50 W, 10 min to 100 W, 20 min during plasma treatment. The effect of these two parameters on surface hydrophilicity, morphology, topography, surface composition of PHBV8 thin films was studied using AFM, SEM, and phase contrast microscopy. The effect of changes in surface characteristics on cell reattachment, spreading and cell growth rate was investigated. It was found that as the treatment level was increased the surface hydrophilicity increased and roughness was decreased probably due to ablation. The PHBV8 film treated with 100 W 10 min was found to be the most suitable for 24 h reattachment of D407 cells. The cells were also grown to confluency as an organized monolayer suggesting PHBV8 film as a potential temporary substrate for subretinal transplantation to replace diseased or damaged retinal pigment epithelium.     

Thermal Stable and Flexible Bio-Based Polybenzoxazine with Epoxy-Functionalized Poly(dimethylsiloxane) Hybrids

In this study, a monofunctionalized bio-based benzoxazine monomer (VAnBZ-CN) is synthesized by condensation of vanillin, formaldehyde, and aniline, and a bifunctional epoxy-functionalized poly(dimethylsiloxane) (PDMS-epoxy) is also prepared through hydrosilylation reactions with allyl glycidyl ether. By blending the inorganic PDMS-epoxy with the VAnBZ-CN benzoxazine monomer, an organic/inorganic hybrid material is formed, which exhibited enhanced thermal stability after thermal curing polymerization. The improved thermal stability observed in the hybrid material can be attributed to a combination of the formation of the triazine structure by VAnBZ-CN and the presence of the inorganic part of PDMS-epoxy on the surface. For example, the PDMS-epoxy/VAnBZ-CN = 1/1 hybrid showed the thermal decomposition temperature (T_d10) of 309 °C, the glass transition temperature (T_g) of 165 °C, and the char yield of 54 wt.% after the thermal curing polymerization process conducted at 240 °C based on thermogravimetric (TGA) and dynamic mechanical analyses (DMA). Furthermore, the char yield of the hybrid material with PDMS-epoxy/VAnBZ-CN = 1/3 is higher than that both of pure VAnBZ-CN and PDMS-epoxy after the thermal curing polymerization process. This result indicates that the addition of PDMS-epoxy resin can improve the formation of a char residue and then enhance the resistance to thermal decomposition for their overall thermal stabilities.     

Surface passivation of a microfluidic device to glial cell adhesion: a comparison of hydrophobic and hydrophilic SAM coatings.

Cell adhesion in a microfluidic structure can lead to catastrophic flow problems due to the comparable size of the cell with the microfabricated device. Such issues are important in the growing research area involving the merging of biological materials and MEMS devices. We have examined the surface compatibility of uncoated and coated microfabricated glass and semiconductor surfaces under static solution (cell culture) and flow experiments (microfluidic device) using glial (astrocyte and glioblastoma) cells. Bare semiconductor and glass surfaces were most attractive to cell adhesion, promoting biofouling under both static and flow conditions. Passivation of the surfaces was performed with silane coupling agents octadecyltrimethoxysilane (OTMS) or N-(triethoxysilylpropyl)-O-polyethylene oxide urethane (TESP) on SiO2 surfaces via self-assembled monolayer (SAM) deposition. The hydrophilic TESP coating was effective at inhibiting biofouling of the microfluidic structure, allowing greater than several minutes of fluid flow. The hydrophobic OTMS coating, on the other hand, promoted cell adhesion leading to restricted flow within a few minutes. Interestingly, under cell culture conditions the TESP surface exhibited biocompatible properties for glial cell adhesion and proliferation, in contrast to the OTMS surface which resisted cell growth. These studies suggest that cell adhesion is dependent upon the time domain of the cell-surface interaction.     

Poly(N-isopropylacrylamide) copolymer films as vehicles for the sustained delivery of proteins to vascular endothelial cells

The aim of this study was to establish the capacity of thermoresponsive poly(N-isopropylacrylamide) copolymer films to deliver bioactive concentrations of vascular endothelial growth factor (VEGF165) to human aortic endothelial cells (HAEC) over an extended time period. Films were prepared using a 50:50 (w/w) mixture of non-crosslinkable and crosslinkable copolymers of the following monomer compositions (w/w): 85:15, N-isopropylacrylamide (NiPAAm):N-tert-butylacrylamide (NtBAAm); and 85:13:2 NiPAAm:NtBAAm:acrylamidobenzophenone (ABzPh, crosslinking agent), respectively. After crosslinking by UV irradiation, the ability of films to incorporate a fluorescently labeled carrier protein (FITC-labeled BSA, 1 mg loaded per film), at 4 degrees C, was first established. Incorporation into the matrix was confirmed by the observation that increasing film thickness from 5 to 10 microm increased release from collapsed films at 37 degrees C (1.76 +/- 0.15 and 10.98 +/- 3.38 microg/mL, respectively, at 24 h postloading) and that this difference was maintained at 5 days postloading (1.81 +/- 0.25 and 13.8 +/- 2.3 microg/mL, respectively). Incorporation was also confirmed by visualization using confocal microscopy. When 10-microm films were loaded with a BSA solution (1 mg/mL) containing VEGF165 (3 microg/mL), sustained release of VEGF165 was observed (10.75 +/- 3.11 ng at 24 h; a total of 31.32 +/- 8.50 ng over 7 days). Furthermore, eluted VEGF165 increased HAEC proliferation by 18.2% over control. The absence of cytotoxic species in medium released from the copolymer films was confirmed by the lack of effect of medium (incubated with copolymer films for 3 days) on HAEC viability. In conclusion this study has shown that NiPAAm:NtBAAm copolymers can be loaded with a therapeutic protein and can deliver bioactive concentrations to human vascular endothelial cells over an extended time period.     

Poly (ethylene glycol) hydrogel scaffolds with multiscale porosity for culture of human adipose-derived stem cells

Three-dimensional (3?D) hydrogel scaffolds are an attractive option for tissue regeneration applications because they allow for cell migration, fluid exchange, and can be synthesized to closely mimic the physical properties of the extracellular matrix environment. The material properties of hydrogels play a vital role in cellular migration and differentiation. In light of this, in-depth understanding of material properties is required before such scaffolds can be used to study their influence on cells. Herein, various blends and thicknesses of poly (ethylene glycol) dimethacrylate (PEGDMA) hydrogels were synthesized, flash frozen, and dried by lyophilization to create scaffolds with multiscale porosity. Environmental scanning electron microscopy (ESEM) images demonstrated that lyophilization induced microporous voids in the PEGDMA hydrogels while swelling studies show the hydrogels retain their innate swelling properties. Change in pore size was observed between drying methods, polymer blend, and thickness when imaged in the hydrated state. Human adipose-derived stem cells (hASCs) were seeded on lyophilized and non-lyophilized hydrogels to determine if the scaffolds would support cell attachment and proliferation of a clinically relevant cell type. Cell attachment and morphology of the hASCs were evaluated using fluorescence imaging. Qualitative observations in cell attachment and morphology of hASCs on the surface of the different hydrogel spatial configurations indicate these multiscale porosity hydrogels create a suitable scaffold for hASC culture. These findings offer another factor of tunability in creating biomimetic hydrogels for various tissue engineering applications including tissue repair, regeneration, wound healing, and controlled release of growth factors.     

Poly(N-isopropylacrylamide) co-polymer films as potential vehicles for delivery of an antimitotic agent to vascular smooth muscle cells.

INTRODUCTION: Local delivery of antimitotic agents is a potential therapeutic strategy for protection of injured coronary vasculature against intimal hyperplasia and restenosis. This study sought to establish the principle that thermoresponsive poly(N-isopropylacrylamide) co-polymer films can be used to deliver, in a controlled manner, an antimitotic agent to vascular smooth muscle cells (VSMC). METHODS: A series of co-polymer films was prepared, using varying ratios (w/w) of N-isopropylacrylamide (NiPAAm) monomer to N-tert-butylacrylamide (NtBAAm) and loaded with the antimitotic agent colchicine (100 nmol/film) at room temperature. RESULTS: The extent of colchicine release at 37 degrees C was inversely proportional to the amount of NtBAAm in co-polymer films: release after 48 h from 85:15, 65:35 and 50:50 (NiPAAm:NtBAAm) films was 26, 17 and 0.5 nmol, respectively. In cytotoxicity studies, when medium incubated with co-polymers for 24 h (in the absence of colchicine) was further incubated with target bovine aortic smooth muscle cells (BASMC), no loss of cell viability occurred. Colchicine released from all three co-polymer films significantly inhibited proliferation and random migration of BASMC: 100 nM colchicine (released from 65:35 NiPAAm:NtBAAm) reduced cell proliferation to 25.7+/-1.7% of levels seen in the absence of colchicine (control) and random cell migration to 37.7+/-5.7% of control (mean+/-S.E.M., n = 3, P < .01 and P < .05, respectively). The magnitudes of these effects were comparable to those seen in separate experiments with native colchicine and were observed in samples of released colchicine which had been stored at -20 degrees C for up to 6 months. CONCLUSIONS: This study has shown that the release of the antimitotic agent colchicine, from NiPAAm/NtBAAm co-polymer films can be manipulated by changes in co-polymer composition. Furthermore, such drug released at 37 degrees C retains comparable bioactivity to that of native colchicine.     

Poly(dimethylsiloxane)-poly(ethylene oxide)-heparin block copolymers. II: Surface characterization and in vitro assessments

Amphiphilic block copolymers containing poly(dimethylsiloxane), poly(ethylene oxide), as well as heparin-coated glass beads and tubes were evaluated for the amounts and activities of surface-immobilized heparin. Because the amphiphilic copolymer system is thermodynamically predicted to demonstrate low-energy phase enrichment on the surfaces of air-cast films, studies were also undertaken to understand the in vitro results. Solvent-cast copolymer films have a heterogeneous microphase-separated structure according to transmission electron micrographs. Wilhelmy plate contact angle analysis indicates significant surface restructuring occurs upon hydration. Attenuated total reflectance infrared spectroscopy studies of the desiccated and hydrated films at two different sampling depths show compositional heterogeneity as a function of depth, as well as near surface restructuring allowing surface enrichment of the high-energy segments following contact with water. Significant concentrations of heparin are detected on the surface of these coatings by toluidine blue assays. In addition, a portion of the surface-bound heparin maintains its original bioactivity as determined by recalcification times, thrombin times, and Factor Xa assays. These substrates were also tested for platelet adhesion and activation reactions in vitro using polymer-coated beads in rabbit platelet-rich plasma. Heparinized polymers promoted low levels of platelet adhesion and serotonin release. Surface concentrations of heparin from bioactivity assays were then correlated with platelet adhesion and the extent of platelet release to assess the efficacy of this heparin-immobilized copolymer as a blood-compatible material or coating.     

浓度与压力梯度可调的三维细胞培养微流控芯片的研制

目的制作化学浓度梯度与压力梯度可调的细胞三维培养微流控芯片,构建可模拟在体细胞生长所处动态微环境的体外模型。方法利用光刻成型技术、模塑法以及等离子键合工艺,制作3通道结构的微流控细胞培养芯片。通过微注射泵控制微通道内溶液流动生成浓度梯度,利用液面高度差生成压力梯度,并通过骨架染色比较二维培养与三维培养下的细胞形态。结果获得了化学浓度梯度与压力梯度可调的微流控细胞培养芯片。在2μL.min-1的流速下,中间通道的浓度梯度3 h后可达到相对稳定。100 Pa的压力差在中间通道生成的视在压力梯度为0.11 Pa/μm,从而驱动三维支架内间隙渗流的生成。并在微流控芯片内实现脐静脉内皮细胞稳定的三维培养。结论该芯片结构简单,制作方便,能灵活调控细胞生长所处的微环境,可进一步用于研究不同的微环境参数对细胞行为的影响。     

Perineurial and glial cells in the tickBoophilus microplus (Acarina: Ixodidae): freeze-fracture and tracer studies

Summary In the cattle tickBoophilus microplus, the cells of the perineurium are characterized by accumulations of glycogen which increase dramatically after feeding. Gap junctions couple both these perineurial cells which ensheath the C.N.S. and the underlying glial cells. No tight junctions have been found between perineurial cells and there is in consequence no blood-brain barrier. Using ionic lanthanum as a tracer the extensive gap junctions are shown to have no occluding effect and lanthanum penetrates through the perineurium and glial layers to the level of the axonal surfaces. By colloidal lanthanum impregnation and freeze-fracture studies, the gap junctions appear to be typical of arthropods in that their particles show a characteristic diameter (13 nm in freeze-fracture), are distributed relatively loosely within the junctional plaques and fracture onto the E face of the junctional membranes. Semi-ordered particle arrays are found on E face membranes of adjacent axons and glia which may represent axoglial junctions.On leave from C.S.I.R.O., Division of Entomology, Australia.     

Initiated chemical vapor deposition of thermoresponsive poly(N-vinylcaprolactam) thin films for cell sheet engineering

Poly(N-vinylcaprolactam) (PNVCL) is a thermoresponsive polymer known to be nontoxic, water soluble and biocompatible. Here, PNVCL homopolymer was successfully synthesized for the first time by use of a one-step vapor-phase process, termed initiated chemical vapor deposition (iCVD). Fourier transform infrared spectroscopy results showed that radical polymerization took place from N-vinylcaprolactam monomers without damaging the functional caprolactam ring. A sharp lower critical solution temperature transition was observed at 31 °C from the iCVD poly(N-vinylcaprolactam) (PNVCL) film. The thermoresponsive PNVCL surface exhibited a hydrophilic/hydrophobic alteration with external temperature change, which enabled the thermally modulated attachment and detachment of cells. The conformal coverage of PNVCL film on various substrates with complex topography, including fabrics and nanopatterns, was successfully demonstrated, which can further be utilized to fabricate cell sheets with aligned cell morphology. The advantage of this system is that cells cultured on such thermoresponsive surfaces could be recovered as an intact cell sheet by simply lowering the temperature, eliminating the need for conventional enzymatic treatments.     

Parametric control of collision rates and capture rates in geometrically enhanced differential immunocapture (GEDI) microfluidic devices for rare cell capture

The enrichment and isolation of rare cells from complex samples, such as circulating tumor cells (CTCs) from whole blood, is an important engineering problem with widespread clinical applications. One approach uses a microfluidic obstacle array with an antibody surface functionalization to both guide cells into contact with the capture surface and to facilitate adhesion; geometrically enhanced differential immunocapture is a design strategy in which the array is designed to promote target cell–obstacle contact and minimize other interactions (Gleghorn et al. 2010; Kirby et al. 2012). We present a simulation that uses capture experiments in a simple Hele-Shaw geometry (Santana et al. 2012) to inform a target-cell-specific capture model that can predict capture probability in immunocapture microdevices of any arbitrary complex geometry. We show that capture performance is strongly dependent on the array geometry, and that it is possible to select an obstacle array geometry that maximizes capture efficiency (by creating combinations of frequent target cell–obstacle collisions and shear stress low enough to support capture), while simultaneously enhancing purity by minimizing non-specific adhesion of both smaller contaminant cells (with infrequent cell–obstacle collisions) and larger contaminant cells (by focusing those collisions into regions of high shear stress).     

Poly(amidoamine) dendronized hollow fiber membranes: synthesis, characterization, and preliminary applications as drug delivery devices

Poly(amidoamine) (PAMAM) dendrons were prepared from hollow fiber membranes (HFM) consisting of bromomethylated poly(2,6-dimethyl-1,4-phenylene oxide) (BPPO) in a stepwise manner. The prepared HFM were characterized by Fourier transform infrared spectroscopy, elemental analysis, and scanning electron microscopy. The drug loading efficiency and release behavior of the PAMAM dendronized HFM were evaluated using sodium salicylate, sodium methotrexate, and Congo red as model drugs. The results suggest that PAMAM dendronized HFM can be effectively loaded with a variety of drugs and prolong the release of these drugs. The drug loading and release characteristics of the HFM depend on the generation of PAMAM dendrons grafted on the membranes. The prepared PAMAM dendronized BPPO HFM are promising scaffolds in drug delivery and tissue engineering.     

Analysis of murine hepatitis virus (JHM strain) tropism toward Lewis rat glial cells in vitro. Type I astrocytes and brain macrophages (microglia) as primary glial cell targets

The murine hepatitis virus, JHM strain, causes a relapsing subacute demyelinating encephalomyelitis in Lewis rats after intracranial infection. The disease process involves both virus persistence within glial cells and the induction of autoimmunological attack of myelin, however, the relative importance of these features involved in chronic relapsing demyelination remains to be determined. In this report, we analyze the tropism of JHM virus to various neural cell types present within primary Lewis rat central nervous system cultures. Infection of primary cultures with JHM virus revealed that type I astrocytes and brain macrophages are the initial target cells of infection and that the myelin-forming oligodendrocytes are comparatively resistant, becoming infected only rarely through virus mediated cell fusion with previously infected cells. In addition, infection of cultures after removal of oligodendrocytes by various means had no effect on the tropism of JHM virus for the cultures. Cytopathic effects of JHM virus proceed rapidly by cell fusion within the astrocyte-macrophage monolayer, leaving the oligodendrocyte population largely unaffected. Therefore, the highly selective infection of type I astrocytes and macrophages appears to form the basis of JHM virus neurotropism in Lewis rats. These results indicate that JHM virus infection of astrocytes and brain macrophages may be more important in inducing chronic relapsing demyelinating processes than direct infection of the myelin-forming oligodendrocytes. Other possible pathways leading to chronic demyelination in rats involving type I astrocytes and brain macrophages are discussed.     

A novel biomaterial: Poly(dimethylsiloxane)-polyamide multiblock copoly mer I. Synthesis and evaluation of blood compatibility

Aramid-silicone resins (PASs) consisting of aromatic polyamide (aramid) and poly(dimethylsiloxane) (PDMS) segments were synthesized by low temperature solution polycondensation. For the evaluation of blood compatibility in vitro, two kinds of experiments were carried out. One was the thromboxane B₂(TXB₂) release test from platelets attaching to PAS and Biomer®. The other was the observation of the platelet adhesion on the surfaces of PAS by scanning electron microscopy (SEM). The results indicated that PAS was bio-inert in vitro. The surface chemical composition of PAS films was investigated by means of electron probe micro analysis (EPMA), X-ray photoelectron spectroscopy (XPS), and dynamic contact angle measurements. The relationship between blood compatibility and surface composition of PAS is discussed.     

Poly(DL-lactic acid) film surface modification with heparin for improving hemocompatibility of blood-contacting bioresorbable devices

This work describes a simple method to immobilize heparin by covalent bonding to the surface of poly(lactic acid) film with the aim of showing improved hemocompatibility. Carboxyl groups present in heparin molecules were activated by reaction with N-hydroxy-succinimide and allowed to react with free amino groups created at the surface of poly(DL-lactic acid) films by controlled aminolysis. Contact angle measurements and XPS analysis confirmed the binding. Quantification was determined by radioactivity using heparin labeled with tritium. The surface exhibited anti factor Xa activity, thus confirming the presence of bounded heparin that kept some biological activity. Finally platelets adhesion showed less platelet adhesion on heparin modified films as well as preserved morphology.