Zhengqin Yin

A high-throughput dielectrophoresis-based cell electrofusion microfluidic device†

A high‐throughput cell electrofusion microfluidic chip has been designed, fabricated on a silicon‐on‐insulator wafer and tested for in vitro cell fusion under a low applied voltage. The developed chip consists of six individual straight microchannels with a 40‐μm thickness conductive highly doped Si layer as the microchannel wall. In each microchannel, there are 75 pairs of counter protruding microelectrodes, between which the cell electrofusion is performed. The entire highly doped Si layer is covered by a 2‐μm thickness aluminum film to maintain a consistent electric field between different protruding microelectrode pairs. A 150‐nm thickness SiO2 film is subsequently deposited on the top face of each protruding microelectrode for better biocompatibility. Owing to the short distance between two counter protruding microelectrodes, a high electric field can be generated for cell electrofusion with a low voltage imposed across the electrodes. Both mammalian cells and plant protoplasts were used to test the cell electrofusion. About 42–68% cells were aligned to form cell–cell pairs by the dielectrophoretic force. After cell alignment, cell pairs were fused to form hybrid cells under the control of cell electroporation and electrofusion signals. The averaged fusion efficiency in the paired cells is above 40% (the highest was about 60%), which is much higher than the traditional polyethylene glycol method (<5%) and traditional electrofusion methods (∼12%). An individual cell electrofusion process could be completed within 10 min, indicating a capability of high throughput.

Polyimide Membrane Based Cell-electrofusion Chip

Abstract A novel flexible cell-electrofusion chip was fabricated using flexible printed circuits board (FPCB) technique, by which microelectrode array was fabricated on the copper foil laminated on the surface of the polyimide membrane. On this chip, cell electrofusion could be carried out at low voltage (

Electric Field Simulation of High-throughput Cell Electrofusion Chip

Abstract The electric field profile within a cell-fusion chip is of great significance for cell manipulation and cell-fusion efficiency, which is a main factor considered in chip design. This profile is mainly decided by the channel geometry and microelectrode structure. In the cell-fusion chip, the microelectrode array which was composed of a large number of microelectrodes was used to obtain high cell-fusion efficiency. Its simulation was difficult because there were many electrodes, complex channel geometry and microelectrode structure on this chip. ANSYS software was used in this study to simulate the electric field profile (strength and gradient) in the cell fusion chip. Comparison between different designs, the layout of electrodes was optimized and an interdigital, pectinate, rectangular microelectrode arrays were selected as the main components of the cell-electrofusion chip. In the preliminary experiments on this chip prototype, many plant protoplasts could be fused simultaneously. The fusion efficiency (about 40%) was much larger than those in traditional chemical induced fusion (

Perineuronal nets increase inhibitory GABAergic currents during the critical period in rats

AIM To investigate inhibitory γ-aminobutyric acid (GABA) ergic postsynaptic currents (IPSCs) and postsynaptic currents (PSCs) in layer IV of the rat visual cortex during the critical period and when plasticity was extended through dissolution of the perineuronal nets (PNNs). METHODS We employed 24 normal Long-Evans rats to study GABAA-PSC characteristics of neurons within layer IV of the visual cortex during development. The animals were divided into six groups of four rats according to ages at recording: PW3 (P21-23d), PW4 (P28-30d), PW5 (P35-37d), PW6 (P42-44d), PW7 (P49-51d), and PW8 (56-58d). An additional 24 chondroitin sulfate proteoglycan (CSPG) degradation rats (also Long-Evans) were generated by making a pattern of injections of chondroitinase ABC (chABC) into the visual cortex 1 week prior to recording at PW3, PW4, PW5, PW6, PW7, and PW8. Immunohistochemistry was used to identify the effect of chABC injection on CSPGs. PSCs were detected with whole-cell patch recordings, and GABAA receptor-mediated IPSCs were pharmacologically isolated. RESULTS IPSC peak current showed a strong rise in the age-matched control group, peaked at PW5 and were maintained at a roughly constant value thereafter. Although there was a small increase in peak current for the chABC group with age, the peak currents continued to decrease with the delayed highest value at PW6, resulting in significantly different week-by-week comparison with normal development. IPSC decay time continued to increase until PW7 in the control group, while those in the chABC group were maintained at a stable level after an initial increase at PW4. Compared with normal rats, the decay times recorded in the chABC rats were always shorter, which differed significantly at each age. We did not observe any differences in IPSC properties between the age-matched control and penicillinase (P-ase) group. However, the change in IPSCs after chABC treatment was not reflected in the total PSCs or in basic membrane properties in layer IV of the rat visual cortex. CONCLUSION Our results demonstrate that rather than rapidly increasing during the critical period for neuronal plasticity, IPSCs in layer IV of rat visual cortex are maintained at an immature level when PNNs are removed by chABC. This suggests that GABA receptor maturation involves the conformation of the CSPGs in PNNs.

Microfluidic pool structure for cell docking and rapid mixing

A microfluidic pool structure for cell docking and rapid mixing is described. The pool structure is defined as a microchamber on one structural layer of a bilayer chip and connects with two or more individual microchannels on the other structural layer. In contrast to the turbulent flow in a macroscale pool, laminar streams enter and exit this microfluidic pool structure with definite and controllable direction that may be influenced by the location and geometry of the pool. A simple microfluidic model was used to validate this hypothesis. In this model, a microscale pool structure was made on the lower layer of a chip and connected with three parallel microchannels in the upper layer. Simulation and experimental results indicated that the flow profile within the pool structure was determined by its geometry and location. This could be used as a flow control method and it was simpler than designs based on microvalve, hydraulic pressure, or electrokinetic force, and has some important applications. For example, controllable streams within this structure were used to immobilize biological cells along the microchannel walls. When different solution streams flowed through the pool, rapid diffusion of analytes occurred for short diffusion distance between vertical flow laminas. Furthermore, desired dilution (mixing) ratio could be obtained by controlling the geometry of the microfluidic pool.

Optogenetics: a novel optical manipulation tool for medical investigation.

Optogenetics is a new and rapidly evolving gene and neuroengineering technology that allows optical control of specific populations of neurons without affecting other neurons in the brain at high temporal and spatial resolution. By heterologous expression of the light-sensitive membrane proteins, cell type-specific depolarization or hyperpolarization can be optically induced on a millisecond time scale. Optogenetics has the higher selectivity and specificity compared to traditional electrophysiological techniques and pharmaceutical methods. It has been a novel promising tool for medical research. Because of easy handling, high temporal and spatial precision, optogenetics has been applied to many aspects of nervous system research, such as tactual neural circuit, visual neural circuit, auditory neural circuit and olfactory neural circuit, as well as research of some neurological diseases. The review highlights the recent advances of optogenetics in medical study.

Optical control after transfection of channelrhodopsin-2 recombinant adenovirus in visual cortical cells

Channelrhodopsin-2 ectopically expressed in the retina can recover the response to blue light in genetically blind mice and rats, but is unable to restore visual function due to optic nerve or optic tract lesions. Long Evans rats at postnatal day 1 were used for primary culture of visual cortical cells, and 24 hours later, cells were transfected with recombinant adenovirus carrying channelrhodopsin-2 and green fluorescent protein genes. After 2–4 days of transfection, green fluorescence was visible in the cultured cells. Cells were stimulated with blue light (470 nm), and light-induced action potentials were recorded in patch-clamp experiments. Our findings indicate that channelrhodopsin-2-recombinant adenovirus transfection of primary cultured visual cortical cells can control the production of action potentials via blue light stimulation.

Study of the inhibitory effect of fatty acids on the interaction between DNA and polymerase β

The binding of human DNA polymerase β (pol β) to DNA template-primer duplex and single-stranded DNA in the absence or presence of pol β inhibitors has been studied using a surface plasmon resonance biosensor. Two fatty acids, linoleic acid and nervonic acid, were used as potent pol β inhibitors. In the interaction between pol β and DNA, pol β could bind to ssDNA in a single binding mode, but bound to DNA template-primer duplexes in a parallel mode. Both pol β inhibitors prevented the binding of pol β to the single strand overhang and changed the binding from parallel to single mode. The affinities of pol β to the template-primer duplex region in the presence of nervonic acid or linoleic acid were decreased by 20 and 5 times, respectively. The significant inhibitory effect of nervonic acid on the pol β-duplex interaction was due to both a 2-fold decrease in the association rate and a 9-fold increase in the dissociation rate. In the presence of linoleic acid, no significant change of association rate was observed, and the decrease in binding affinity of pol β to DNA was mainly due to 7-fold increase in the dissociation rate.