A multi-channel electroporation microchip for gene transfection in mammalian cells

We developed a multi-channel electroporation microchip made of polydimethylsiloxane (PDMS) and glass for gene transfer in mammalian cells. This chip produces multiple electric field gradients in a single microchip by varying the lengths of the microchannels from 2 to 4 cm. Electric fields of 0.65, 0.57, 0.49, 0.41, and 0.33 kV/cm were simultaneously produced in a single chip when the voltage of 1.3 kV was applied. We transferred enhanced green fluorescent protein genes (pEGFP) into HEK-293 and CHO cells, which were cultured within the microchannels. The feasibility of our device was demonstrated because it was able to produce five different transfection rates and survival rates at different electric fields produced in a single microchip. This system is expected to optimize the experimental conditions in gene transfection research more easily and faster than conventional electroporation methods.     

Fabrication of a dual substrate display to test roles of cell adhesion proteins in vesicle targeting to plasma membrane domains

While much is known of the molecular machinery involved in protein sorting during exocytosis, less is known about the spatial regulation of exocytosis at the plasma membrane (PM). This study outlines a novel method, dual substrate display, used to formally test the hypothesis that E-cadherin-mediated adhesion directs basolateral vesicle exocytosis to specific sites at the PM. We show that vesicles containing the basolateral marker protein VSV-G preferentially target to sites of adhesion to E-cadherin rather than collagen VI or a control peptide. These results support the hypothesis that E-cadherin adhesion initiates signaling at the PM resulting in targeted sites for exocytosis.     

Antifouling Potential of Lubricious,Micro-engineered,PDMS Elastomers against Zoospores of the Green Fouling Alga Ulva (Enteromorpha)

The settlement and release of Ulva spores from chemically modified, micro-engineered surface topographies have been investigated using poly(dimethyl siloxane) elastomers (PDMSe) with varying additions of non-network forming poly(dimethyl siloxane) based oils. The topographic features were based on 5?μm wide pillars or ridges separated by 5, 10, or 20?μm wide channels. Pattern depths were 5 or 1.5?μm. Swimming spores showed no marked difference in settlement on smooth surfaces covered with excess PDMS oils. However, incorporation of oils significantly reduced settlement density on many of the surfaces with topographic features, in particular, the 5?μm wide and deep channels. Previous results, confirmed here, demonstrate preferences by the spores to settle in channels and against pillars with spatial dimensions of 5?μm, 10?μm and 20?μm. The combination of lubricity and pillars significantly reduced the number of attached spores compared to the control, smooth, unmodified PDMSe surfaces when exposed to turbulent flow in a flow channel. The results are discussed in relation to the energy needs for spores to adhere to various surface features and the concepts of ultrahydrophobic surfaces. A factorial, multi-level experimental design was analyzed and a 2nd order polynomial model was regressed for statistically significant effects and interactions to determine the magnitude and direction of influence on the spore density measurements between factor levels.     

Investigation of ifosfamide nephrotoxicity induced in a liver–kidney co‐culture biochip

In this article, we present a liver–kidney co‐culture model in a micro fluidic biochip. The liver was modeled using HepG2/C3a and HepaRG cell lines and the kidney using MDCK cell lines. To demonstrate the synergic interaction between both organs, we investigated the effect of ifosfamide, an anticancerous drug. Ifosfamide is a prodrug which is metabolized by the liver to isophosforamide mustard, an active metabolite. This metabolism process also leads to the formation of chloroacetaldehyde, a nephrotoxic metabolite and acrolein a urotoxic one. In the biochips of MDCK cultures, we did not detect any nephrotoxic effects after 72 h of 50 µM ifosfamide exposure. However, in the liver–kidney biochips, the same 72 h exposure leads to a nephrotoxicity illustrated by a reduction of the number of MDCK cells (up to 30% in the HepaRG‐MDCK) when compared to untreated co‐cultures or treated MDCK monocultures. The reduction of the MDCK cell number was not related to a modification of the cell cycle repartition in ifosfamide treated cases when compared to controls. The ifosfamide biotransformation into 3‐dechloroethylifosfamide, an equimolar byproduct of the chloroacetaldehyde production, was detected by mass spectrometry at a rate of apparition of 0.3 ± 0.1 and 1.1 ± 0.3 pg/h/biochips in HepaRG monocultures and HepaRG‐MDCK co‐cultures respectively. Any metabolite was detected in HepG2/C3a cultures. Furthermore, the ifosfamide treatment in HepaRG‐MDCK co‐culture system triggered an increase in the intracellular calcium release in MDCK cells on contrary to the treatment on MDCK monocultures. As 3‐dechloroethylifosfamide is not toxic, we have tested the effect of equimolar choloroacetaldehyde concentration onto the MDCK cells. At this concentration, we found a quite similar calcium perturbation and MDCK nephrotoxicity via a reduction of 30% of final cell numbers such as in the ifosfamide HepaRG‐MDCK co‐culture experiments. Our results suggest that ifosfamide nephrotoxicity in a liver–kidney micro fluidic co‐culture model using HepaRG‐MDCK cells is induced by the metabolism of ifosfamide into chloroacetaldehyde whereas this pathway is not functional in HepG2/C3a‐MDCK model. This study demonstrates the interest in the development of systemic organ–organ interactions using micro fluidic biochips. It also illustrated their potential in future predictive toxicity model using in vitro models as alternative methods. Biotechnol. Bioeng. 2013; 110: 597–608. © 2012 Wiley Periodicals, Inc.     

Replica-moulded polydimethylsiloxane culture vessel lids attenuate osmotic drift in long-term cell cultures

An imbalance in medium osmolarity is a determinant that affects cell culture longevity. Even in humidified incubators, evaporation of water leads to a gradual increase in osmolarity over time. We present a simple replica-moulding strategy for producing self-sealing lids adaptable to standard, small-size cell-culture vessels. They are made of polydimethylsiloxane (PDMS), a flexible, transparent and biocompatible material, which is gas-permeable but largely impermeable to water. Keeping cell cultures in a humidified 5% CO₂ incubator at 37°C, medium osmolarity increased by +6.86 mosmol/kg/day in standard 35 mm Petri dishes, while PDMS lids attenuated its rise by a factor of four to changes of +1.72 mosmol/kg/day. Depending on the lid membrane thickness, pH drifts at ambient CO₂ levels were attenuated by a factor of 4 to 9. Comparative evaporation studies at temperatures below 60°C yielded a 10-fold reduced water vapour flux of 1.75 g/day/dm² through PDMS lids as compared with 18.69 g/day/dm² with conventional Petri dishes. Using such PDMS lids, about 2/3 of the cell cultures grew longer than 30 days in vitro. Among these, the average survival time was 69 days with the longest survival being 284 days under otherwise conventional cell culture conditions. Electronic Supplementary Material  Supplementary material is available for this article at and is accessible for authorized users. Supplementary material pertaining to this article is available on the Journal of Biosciences Website at     

Development of Whispering Gallery Mode Polymeric Micro-optical Electric Field Sensors

Optical modes of dielectric micro-cavities have received significant attention in recent years for their potential in a broad range of applications. The optical modes are frequently referred to as "whispering gallery modes" (WGM) or "morphology dependent resonances" (MDR) and exhibit high optical quality factors. Some proposed applications of micro-cavity optical resonators are in spectroscopy¹, micro-cavity laser technology², optical communications^3-6 as well as sensor technology. The WGM-based sensor applications include those in biology⁷, trace gas detection⁸, and impurity detection in liquids⁹. Mechanical sensors based on microsphere resonators have also been proposed, including those for force^10,11, pressure¹², acceleration¹³ and wall shear stress¹⁴. In the present, we demonstrate a WGM-based electric field sensor, which builds on our previous studies^15,16. A candidate application of this sensor is in the detection of neuronal action potential.The electric field sensor is based on polymeric multi-layered dielectric microspheres. The external electric field induces surface and body forces on the spheres (electrostriction effect) leading to elastic deformation. This change in the morphology of the spheres, leads to shifts in the WGM. The electric field-induced WGM shifts are interrogated by exciting the optical modes of the spheres by laser light. Light from a distributed feedback (DFB) laser (nominal wavelength of ~ 1.3 μm) is side-coupled into the microspheres using a tapered section of a single mode optical fiber. The base material of the spheres is polydimethylsiloxane (PDMS). Three microsphere geometries are used: (1) PDMS sphere with a 60:1 volumetric ratio of base-to-curing agent mixture, (2) multi layer sphere with 60:1 PDMS core, in order to increase the dielectric constant of the sphere, a middle layer of 60:1 PDMS that is mixed with varying amounts (2% to 10% by volume) of barium titanate and an outer layer of 60:1 PDMS and (3) solid silica sphere coated with a thin layer of uncured PDMS base. In each type of sensor, laser light from the tapered fiber is coupled into the outermost layer that provides high optical quality factor WGM (Q ~ 10⁶). The microspheres are poled for several hours at electric fields of ~ 1 MV/m to increase their sensitivity to electric field.     

Feasibility of on-chip detection of endotoxin by LAL test

The LAL (Limulus amebocyte lysate) test for the detection and quantification of endotoxin is based on the gelation reaction between endotoxin and LAL from a blood extract ofLimulus polyphemus. The test is labor intensive, requiring dedicated personnel, a relatively long reaction time (approximately 1 h), relatively large volumes of samples and reagents and the detection of the end-point is rather subjective. To solve these problems, a miniaturized LOC (labon-a-chip) prototype, 62 mm (L)×18 mm (W), was fabricated using PDMS (polydimethylsiloxane) bonded to glass. Using this prototype, in which 2 mm (W)×44.3 mm (L)×100 μm(D) microfluidic channel was constructed, turbidometric and chromogenic assay detection methods were compared, and the chromogenic method was found the most suitable for a small volume assay. In this assay, the kinetic-point method was more accurate than the end-point method. The PDMS chip chickness was found to be minimized to around 2 mm to allow sufficient light transmittance, which necessitated the use of a glass slide bonding for chip rigidity. Due to this miniaturization, the test time was reduced from 1 h to less than 10 min, and the sample volume could be reduced from 100 toca. 4.4 μL. In summation, this study suggested that the LOC using the LAL test principle could be an alternative as a semi-automated and reliable method for the detection of endotoxin.