Closing the system: production of viral antigen-presenting dendritic cells eliciting specific CD8+ T cell activation in fluorinated ethylene propylene cell culture bags.

BACKGROUND: A major obstacle to anti-viral and -tumor cell vaccination and T cell immunotherapy is the ability to produce dendritic cells (DCs) in a suitable clinical setting. It is imperative to develop closed cell culture systems to accelerate the translation of promising DC-based cell therapy products to the clinic. The objective of this study was to investigate whether viral antigen-loaded monocyte-derived DCs (Mo-DCs) capable of eliciting specific T cell activation can be manufactured in fluorinated ethylene propylene (FEP) bags. METHODS: Mo-DCs were generated through a protocol applying cytokine cocktails combined with lipopolysaccharide or with a CMV viral peptide antigen in conventional tissue culture polystyrene (TCPS) or FEP culture vessels. Research-scale (< 10 mL) FEP bags were implemented to increase R&D throughput. DC surface marker profiles, cytokine production, and ability to activate antigen-specific cytotoxic T cells were characterized. RESULTS: Monocyte differentiation into Mo-DCs led to the loss of CD14 expression with concomitant upregulation of CD80, CD83 and CD86. Significantly increased levels of IL-10 and IL-12 were observed after maturation on day 9. Antigen-pulsed Mo-DCs activated antigen-responsive CD8+ cytotoxic T cells. No significant differences in surface marker expression or tetramer-specific T cell activating potency of Mo-DCs were observed between TCPS and FEP culture vessels. CONCLUSIONS: Our findings demonstrate that viral antigen-loaded Mo-DCs produced in downscaled FEP bags can elicit specific T cell responses. In view of the dire clinical need for closed system DC manufacturing, FEP bags represent an attractive option to accelerate the translation of promising emerging DC-based immunotherapies.

No evidence of improved efficacy of covered stents over uncovered stents in percutaneous palliation of malignant hilar biliary obstruction: results of a prospective randomized trial.

OBJECTIVE: To investigate whether covered stents show a higher efficacy than uncovered stents in percutaneous treatment of malignant hilar biliary obstruction. METHODS: Patients with obstructive jaundice caused by an unresectable hilar malignancy were included after failed endoscopic intervention in a prospective randomized trial comparing expanded polytetrafluoroethylene and fluorinated ethylene propylene (ePTFE-FEP)-covered nitinol stents with uncovered nitinol stents. Exclusion criteria were as follows: primary tumors existing more than 3 months, a biliodigestive anastomosis, previous stenting, and a Karnofsky score of less than 50. Safety, clinical success, and adjuvant chemotherapy were compared as well as occlusion rate, patency, and survival. RESULTS: A total of 120 patients were included. One patient was post hoc excluded. Fourteen patients who died within 7 days and one patient without patency data were excluded from patency analysis. Serious adverse events (p = 0.4), 30-day mortality (p = 0.5), and clinical success (p = 0.8) were equivalent for both stent groups. Twenty-one out of 61 (34%) patients in the covered and 24/58 (41%) in the uncovered stent groups received adjuvant chemotherapy (p = 0.5). Occlusion rate was 54% (27/50) in the covered stent group and 57% (31/54) in the uncovered stent group (p = 0.8). Median patency was 229 days (95% CI 113-345) for covered stents and 130 days (95% CI 75-185) for uncovered stents (p = 0.1). Median survival in patients with covered stents was 79 days (95% CI 52-106) and with uncovered stents 92 days (95% CI 60-124) (p = 0.3). CONCLUSION: In malignant hilar biliary obstruction, there is no evidence that ePTFE-FEP-covered stents are superior to uncovered stents in terms of safety, clinical success, adjuvant chemotherapy, patency, or survival. KEY POINTS: • Percutaneous palliation of hilar biliary obstruction is feasible with both uncovered and covered stents. • Clinical success in terms of bilirubin decrease and adjuvant chemotherapy is achievable with both stents. • Thirty-day mortality is considerable when stenting is also offered to patients with a low performance status.

No Advantage of Expanded Polytetrafluoroethylene and Fluorinated Ethylene Propylene-Covered Stents over Uncovered Nitinol Stents for Percutaneous Palliation of Malignant Infrahilar Biliary Obstruction: Results of a Single-Center Prospective Randomized Trial.

PURPOSE: To prove that covered stents are more efficacious than uncovered stents regarding patency, safety, enabling of chemotherapy, and survival in percutaneous palliation of malignant infrahilar biliary obstruction. MATERIALS AND METHODS: After failed endoscopic treatment, 154 patients with obstructive jaundice caused by unresectable infrahilar malignancy were randomly allocated to receive an expanded polytetrafluoroethylene and fluorinated ethylene propylene-covered or an uncovered nitinol stent. Occlusion rate, patency, and survival were assessed. Safety and clinical success in terms of chemotherapy were compared. RESULTS: Three patients were excluded post hoc. Fifteen patients died within 7 d and were excluded from patency analysis. Occlusion rates were 32% (21 of 66) for covered and 29% (20 of 70) for uncovered stents (P = .7). Estimated median patency durations were 308 d (95% confidence interval [CI], 178-438 d) for covered and 442 d (95% CI, 172-712 d) for uncovered stents (P = .1). Serious adverse events (P = 1.0) and 30-day mortality (P = .5) were equivalent between groups. At hospital discharge, median bilirubin reduction of 8 mg/dL was found in both groups (P < .001). In the covered stent group, 35 patients (48%) received palliative chemotherapy, vs 29 (37%) in the uncovered stent group (P = .2). Estimated median survival times were 96 days (95% CI, 68-124 d) with covered stents and 75 days (95% CI, 42-108 d) with uncovered stents (P = .6). CONCLUSIONS: In malignant infrahilar biliary obstruction not amenable to endoscopy, no improvement in patency or survival with percutaneously placed covered stents could be confirmed. Covered and uncovered stent types exhibit similar safety profiles and clinical success rates.

Wearable Device Oriented Flexible and Stretchable Energy Harvester Based on Embedded Liquid-Metal Electrodes and FEP Electret Film.

In this paper, a flexible and stretchable energy harvester based on liquid-metal and fluorinated ethylene propylene (FEP) electret films is proposed and implemented for the application of wearable devices. A gallium liquid-metal alloy with a melting point of 25.0 °C is used to form the stretchable electrode; therefore, the inducted energy harvester will have excellent flexibility and stretchability. The solid-state electrode is wrapped in a dragon-skin silicone rubber shell and then bonded with FEP electret film and conductive film to form a flexible and stretchable energy harvester. Then, the open-circuit voltage of the designed energy harvester is tested and analyzed. Finally, the fabricated energy harvester is mounted on the elbow of a human body to harvest the energy produced by the bending of the elbow. The experimental results show that the flexible and stretchable energy harvester can adapt well to elbow bending and convert elbow motion into electric energy to light the LED in a wearable watch.

Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope.

Light sheet microscopy techniques, such as selective plane illumination microscopy (SPIM), are ideally suited for time-lapse imaging of developmental processes lasting several hours to a few days. The success of this promising technology has mainly been limited by the lack of suitable techniques for mounting fragile samples. Embedding zebrafish embryos in agarose, which is common in conventional confocal microscopy, has resulted in severe growth defects and unreliable results. In this study, we systematically quantified the viability and mobility of zebrafish embryos mounted under more suitable conditions. We found that tubes made of fluorinated ethylene propylene (FEP) filled with low concentrations of agarose or methylcellulose provided an optimal balance between sufficient confinement of the living embryo in a physiological environment over 3 days and optical clarity suitable for fluorescence imaging. We also compared the effect of different concentrations of Tricaine on the development of zebrafish and provide guidelines for its optimal use depending on the application. Our results will make light sheet microscopy techniques applicable to more fields of developmental biology, in particular the multiview long-term imaging of zebrafish embryos and other small organisms. Furthermore, the refinement of sample preparation for in toto and in vivo imaging will promote other emerging optical imaging techniques, such as optical projection tomography (OPT).

An ultralow background substrate for protein microarray technology.

We herein report an ultralow background substrate for protein microarrays. Conventional protein microarray substrates often suffer from non-specific protein adsorption and inhomogeneous spot morphology. Consequently, surface treatment and a suitable printing solution are required to improve the microarray performance. In the current work, we improved the situation by developing a new microarray substrate based on a fluorinated ethylene propylene (FEP) membrane. A polydopamine microspot array was fabricated on the FEP membrane, with proteins conjugated to the FEP surface through polydopamine. Uniform microspots were obtained on FEP without the application of a special printing solution. The modified FEP membrane demonstrated ultralow background signal and was applied in protein and peptide microarray analysis.

Batch versus flow photochemistry: a revealing comparison of yield and productivity.

The use of flow photochemistry and its apparent superiority over batch has been reported by a number of groups in recent years. To rigorously determine whether flow does indeed have an advantage over batch, a broad range of synthetic photochemical transformations were optimized in both reactor modes and their yields and productivities compared. Surprisingly, yields were essentially identical in all comparative cases. Even more revealing was the observation that the productivity of flow reactors varied very little to that of their batch counterparts when the key reaction parameters were matched. Those with a single layer of fluorinated ethylene propylene (FEP) had an average productivity 20% lower than that of batch, whereas three-layer reactors were 20% more productive. Finally, the utility of flow chemistry was demonstrated in the scale-up of the ring-opening reaction of a potentially explosive [1.1.1] propellane with butane-2,3-dione.

Photovoltaic and thermal properties of electrolytes based on electrospun poly(vinylidene fluoride-hexafluoro propylene)/poly(methyl methacrylate) nanofibers for dye-sensitized solar cells.

We prepared electrospun polymer nanofibers by electrospnning method and investigated about their applications to dye-sensitized solar cells (DSSCs). Electrospun polymer nanofibers applied to the polymer matrix in electrolyte for DSSCs. To improve the stiffness of polymer nanofiber, poly(vinylidene fluoride-hexafluoro propylene)/Poly(methyl methacrylate) (PVDF-HFP/PMMA) blend nanofibers were prepared and examined. In the electrospun PVDF-HFP/PMMA (1:1) blend nanofibers, the best results of VOC, JSC, FF, and efficiency of the DSSC devices showed 0.71 V, 12.8 mA/cm2, 0.61, and 5.56% under AM 1.5 illumination.

Percutaneous use of ePTFE/FEP-covered metallic stent for palliation of malignant biliary obstruction.

INTRODUCTION: Placement of self-expanding metallic stents has been a standard palliative treatment for patients with inoperable biliary malignancy, aiming at improving quality of life via a minimally invasive procedure. This study aimed to evaluate the efficacy of percutaneous placement of expanded polytetrafluoroethylene/fluorinated ethylene propylene-covered metallic stents for palliation of inoperable biliary malignancy. MATERIAL AND METHODS: Between February 2012 and April 2013, 11 patients with inoperable malignant biliary obstruction were treated with stent implantation. Serum bilirubin and liver enzyme levels were measured before/immediately after stent placement and at one, three, and six months. Patient survival and stent patency were calculated using Kaplan-Meier analysis. RESULTS: No migration occurred. Patients showed clinical improvement immediately and one month after stent placement, with a significant reduction in mean serum bilirubin level (p < 0.05). At six months, bilirubin levels were <1.5 mg/dL in all cases. One patient developed acute liver failure four days after stent implantation, leading to death. Survival rates were 90% at three months and 70% at six months. Primary patency rates were 100%, 100%, and 90% at one, three, and six months, respectively. CONCLUSION: Percutaneous placement of expanded polytetrafluoroethylene/fluorinated ethylene propylene-covered metallic stents offered an effective palliative therapy for patients with inoperable biliary malignancy.

Extracorporeal bypass model of blood circulation for the study of microvascular hemodynamics.

Many studies have been performed to better understand the hemodynamics in microvessels, such as arterioles and venules. However, due to the heterogeneous features of size, shape, blood-flow velocity, and pulsatility of microvessels, conducting a systematic study on these factors has been almost impossible. Although in vitro studies have been performed for this purpose, the usefulness of in vitro data is limited by the fact that the rheological properties of blood are changed as blood is exposed to in vitro environments. The purpose of the present study is to investigate the feasibility of a rat extracorporeal bypass model that combines in vivo and in vitro models. An arteriovenous shunt loop with a sub-bypass loop of fluorinated ethylene propylene (FEP) microtube was constructed between the jugular vein and femoral artery of a rat. Three pinch valves were installed in the main loop. Microscopic images of the blood flow in the FEP tube were sequentially captured with a high-speed camera, and the whole velocity field information was obtained using a micro-particle image velocimetry technique. Experimental results reveal that the velocity fields of the blood flow inside the microtube are well measured because the FEP tube is transparent and has nearly the same refractive index as water. The flow velocity and the pulsatility index of the blood flow in the microtube can be controlled by adjusting the three pinch valves installed upstream, midstream, and downstream of the bypass loop. This hybrid model that combines in vivo and in vitro models can be useful in studying microvascular hemodynamics.

Fast construction of polymer monolithic columns inside fluorinated ethylene propylene (FEP) tubes for separation of proteins by reversed-phase liquid chromatography.

This paper describes the preparation of polymer monolithic columns in the confines of fluorinated ethylene propylene (FEP) tubes. These tubes are cheap, chemically stable, and widely used in flow analysis laboratories. UV-initiated grafting with 5 wt% benzophenone in methanol for 1 h activated the internal surface walls, thus enabling the further covalent binding of ethylene glycol dimethacrylate (EDMA) from a 15 wt% solution in methanol, also via photografting. Both steps used 254 nm radiation under a potency of 120 mJ cm2. ATR-FTIR measurements revealed the presence of carbonyl, alkyl and vinyl groups in the functionalized FEP. The density of vinyl groups was high enough to firmly attach a poly(lauryl methacrylate-co-ethylene glycol dimethacrylate) monolith in 120 × 1.57 mm i.d. tubes, prepared via photopolymerization. The total preparation lasts less than 2-h. The columns were permeable, (1.58 ± 0.06) × 10-13 m2, providing reproducible chromatographic parameters of retention times, retention factor, selectivity, and resolution. The monoliths were stable at flow rates of 500 µL min-1, collapsing only at flow rates >700 µL min-1, a condition that increased the backpressure over 1000 psi (experiments at the room temperature). The separation of proteins by reversed-phase liquid chromatography demonstrated the efficiency of the columns. Determination of egg white proteins (ovalbumin and lysozyme) and myoglobin in spiked urine proved the applicability to the analysis of real samples.

Influence of photo-initiators in the preparation of methacrylate monoliths into poly(ethylene-co-tetrafluoroethylene) tubing for microbore HPLC.

In this study, poly(butyl methacrylate-co-ethyleneglycol dimethacrylate) polymeric monoliths were in situ developed within 0.75 mm i.d. poly(ethylene-co-tetrafluoroethylene) (ETFE) tubing by UV polymerization via three different free-radical initiators (α,α'-azobisisobutyronitrile (AIBN), 2,2-dimethoxy-2-phenylacetophenone (DMPA) and 2-methyl-4'-(methylthio)-2-morpholinopropiophenone (MTMPP). The influence of the nature of each photo-initiator and irradiation time on the morphological features of the polymer was investigated by scanning electron microscopy, and the chromatographic properties of the resulting microbore columns were evaluated using alkyl benzenes as test substances. The beds photo-initiated with MTMPP gave the best performance (minimum plate heights of 38 µm for alkyl benzenes) and exhibited a satisfactory reproducibility in the chromatographic parameters (RSD < 11%). These monolithic columns were also successfully applied to the separation of phenylurea herbicides, proteins and a tryptic digest of ß-casein.

Room-temperature intermediate layer bonding for microfluidic devices.

In this work a novel room-temperature bonding technique based on chemically activated Fluorinated Ethylene Propylene (FEP) sheet as an intermediate between chemically activated substrates is presented. Surfaces of silicon and glass substrates are chemically modified with APTES bearing amine terminal groups, while FEP sheet surfaces are treated to form carboxyl groups and subsequently activated by means of EDC-NHS chemistry. The activation procedures of silicon, glass and FEP sheet are characterized by contact angle measurements and XPS. Robust bonds are created at room-temperature by simply pressing two amine-terminated substrates together with activated FEP sheet in between. Average tensile strengths of 5.9 MPa and 5.2 MPa are achieved for silicon-silicon and glass-glass bonds, respectively, and the average fluidic pressure that can be operated is 10.2 bar. Moreover, it is demonstrated that FEP-bonded microfluidic chips can handle mild organic solvents at elevated pressures without leakage problems. This versatile room-temperature intermediate layer bonding technique has a high potential for bonding, packaging, and assembly of various (bio-) chemical microfluidic systems and MEMS devices.

On-line preconcentration and enantioseparation of thalidomide racemates by CEC with the hyphenation of octyl and norvancomycin monoliths.

A method was developed for simultaneous preconcentration and chiral separation of thalidomide enantiomers in human urine by CEC in combination with self-concentration and solvent gradient effects. A 4 cm long octyl (C8) monolithic column was hyphenated with a 15 cm long norvancomycin (NVC)-bonded monolithic column via a fluorinated ethylene-propylene interface. Sample solution was injected into the C8 monolithic column, the two thalidomide enantiomers were first preconcentrated on the C8 monolithic column, and then separated with a further concentration on the NVC-bonded monolithic column by CEC. Injection of 34.8 mm plug of sample solution gave 278- and 298-fold enhancement in sensitivity, and detection limits of 90 and 94 microg/L for the two thalidomide enantiomers. Peak areas of the two isomers were linear in a range of 0.5-50 mg/L. The precision for five replicate injections of 10 mg/L were 0.8-0.9 and 1.1-2.3% for the migration time and peak height, respectively. The developed method was applied to the determination of racemic thalidomide in spiked human urine samples.

Argon plasma-treated fluorinated ethylene propylene: Growth of primary dermal fibroblasts and mesenchymal stem cells.

Surface modification is an important step in making a synthetic polymer cytocompatible. We have previously reported improved cytocompatibility of immortalized human keratinocytes (HaCaT) with the otherwise bioinert fluorinated ethylene propylene (FEP) upon treatment with argon plasma discharge. In this article, we show that FEP modified with Ar plasma with the power of 3 and 8 W for 40 and 240 s served as a suitable material for cultivation of primary human dermal fibroblasts (HDF), which showed significantly improved proliferation and spreading comparable to standard tissue culture polystyrene. We also evaluated focal adhesions formed by HDF cells on modified FEP, which were far more numerous compared to pristine FEP. Moreover, we attempted spontaneous osteogenic differentiation of adipose-derived mesenchymal stem cells modified with human telomerase reverse transcriptase on Ar plasma-modified FEP. While the spontaneous osteogenic differentiation was unsuccessful, the cells were able to adhere and differentiated on tested matrices upon the administration of osteodifferentiation medium. These combined findings suggest that the treatment of FEP with Ar plasma comprises and efficient method to enable the adhesion and proliferation of various cell types on an otherwise largely bioinert material.

Teflon films for chemically-inert microfluidic valves and pumps.

We present a simple method for fabricating chemically-inert Teflon microfluidic valves and pumps in glass microfluidic devices. These structures are modeled after monolithic membrane valves and pumps that utilize a featureless polydimethylsiloxane (PDMS) membrane bonded between two etched glass wafers. The limited chemical compatibility of PDMS has necessitated research into alternative materials for microfluidic devices. Previous work has shown that spin-coated amorphous fluoropolymers and Teflon-fluoropolymer laminates can be fabricated and substituted for PDMS in monolithic membrane valves and pumps for space flight applications. However, the complex process for fabricating these spin-coated Teflon films and laminates may preclude their use in many research and manufacturing contexts. As an alternative, we show that commercially-available fluorinated ethylene-propylene (FEP) Teflon films can be used to fabricate chemically-inert monolithic membrane valves and pumps in glass microfluidic devices. The FEP Teflon valves and pumps presented here are simple to fabricate, function similarly to their PDMS counterparts, maintain their performance over extended use, and are resistant to virtually all chemicals. These structures should facilitate lab-on-a-chip research involving a vast array of chemistries that are incompatible with native PDMS microfluidic devices.

Mechanisms and Characterization of the Pulsed Electron-Induced Grafting of Styrene onto Poly(tetrafluoroethylene-co-hexafluoropropylene) to Prepare a Polymer Electrolyte Membrane.

During the pulsed-electron beam direct grafting of neat styrene onto poly(tetrafluoroethylene-co-hexafluoropropylene) (FEP) substrate, the radiolytically-produced styryl and carbon-centered FEP radicals undergo various desired and undesired competing reactions. In this study, a high-dose rate is used to impede the undesired free radical homopolymerization of styrene and ensure uniform covalent grafting through 125-µm FEP films. This outweighs the enhancement of the undesired crosslinking reactions of carbon-centered FEP radicals and the dimerization of the styryl radicals. The degree of uniform grafting through 125-µm FEP films increases from ≈8%, immediately after pulsed electron irradiation to 33% with the subsequent thermal treatment exceeding the glass transition temperature of FEP of 39°C. On the contrary, steady-state radiolysis using 60Co gamma radiolysis, shows that the undesired homopolymerization of the styrene has become the predominant reaction with a negligible degree of grafting. Time-resolved fast kinetic measurements on pulsed neat styrene show that the styryl radicals undergo fast decays via propagation homopolymerization and termination reactions at an observed reaction rate constant of 5 × 108 l · mol-1 · s-1. The proton conductivity of 25-µm film at 80°C is 0.29 ± 0.01 s cm-1 and 0.007 s cm-1 at relative humidity of 92% and 28%, respectively. The aims of this work are: 1. electrolyte membranes are prepared via grafting initiated by a pulsed electron beam; 2. postirradiation heat-treated membranes are uniformly grafted, ideal for industry; 3. High dose rate is the primary parameter to promote the desired reactions; 4. measurement of kinetics of undesired radiation-induced styrene homopolymerization; and 5. The conductivity of prepared membranes is on par or higher than industry standards.

Technical report: effects of PUVA treatment on the optical properties of blood/tissue storage bags during extracorporeal photochemotherapy.

Extracorporeal photochemotherapy (photopheresis, ECP) is a novel therapeutic method for patients who do not respond to immunosuppressive medications, and gaining interest in the treatment of Graft-vs-Host Disease. This paper is focused on the optical transmission properties of plastic bags which can be used in an independent (off-line) method of ECP, and reports the results of spectral measurements on various bags of different chemical compositions, with and without PUVA treatment. Regarding their higher and more uniform UVA transmission values, FEP based bags perform superior to the others. Considering its UVB absorption and UVA transmission properties, the EVA bag is a good choice, while Polyimide Kapton-FEP plastic film should not be considered for use in ECP. PUVA treatment of blood bags may affect their optical behaviour, and causes reduction of transmission of the material in UV range of the spectrum.

Novel Substrates for Microarrays.

In the microarray platform, the surface substrate is critical to the result quality in terms of signal consistency and detection sensitivity. Traditional substrates such as glass and nitrocellulose often entail complicated preparation processes such as the activation and functionalization of the reaction spots and surface blocking to prevent nonspecific molecule adsorption. In addition, coffee-ring morphology of the spots is a common issue in the traditional substrates. To address these issues, we introduced a novel substrate based on fluorinated ethylene propylene (FEP) membrane for microarrays.

Surface free energy effect on bacterial retention.

Bacterial infection is one of the most frequent and severe complications in the long-term effectiveness of medical implants and devices, greatly increasing treatment cost and inconvenience to the patient. Surface physical and chemical properties are known to influence the extent and form of bacterial infection, although the exact correlation with specific properties is difficult due to the complexity of the system. One approach in the attempt to reduce the bacterial colonisation is to modify the surface energy and chemistry, so as to influence the interactions between the surface and the bacteria that come into contact with it. Five types of coatings were investigated in this study, together with silicone, and polished and non-polished stainless steel 316L. Surfaces were tested for retention of Pseudomonas aeruginosa AK1 after 1h. A good correlation (>90%) was found between P. aeruginosa AK1 retention and total surface free energy, as well as its polar and dispersive components. The minimum level of P. aeruginosa AK1 retention was found for a range of total surface free energy in the range 20-27 mN/m.