Research

PDMS microfluidic device for long-term axonal electrophysiology

Electrophysiology on thin and intricate axonal branches  poses experimental challenges. To reduce experimental complexity, we coupled microelectrode arrays (MEAs) to  microchannel devices for the long-term in vitro tracking of axonal morphology and activity with high spatiotemporal resolution. Our model allowed the long-term multisite recording from pure axonal branches in a microscopy-compatible environment. Electrophysiological data over 95 days in vitro (DIV) showed an age-dependent increase of axonal conduction velocity, which was positively correlated with, but independent of evolving burst activity over time. Conduction velocity remained constant at chemically increased network activity levels. In contrast, low frequency (1 Hz, 180 repetitions) electrical stimulation evoked amplitude-dependent direct (5-35 ms peri-stimulus) and polysynaptic (35-1,000 ms peri-stimulus) activity with temporarily (<35 ms) elevated propagation velocities along the axonal branches. The experimental paradigm may lead to new insights into stimulation-induced axonal plasticity.


Habibey R, Latifi S, Mousavi H, Pesce M, Arab-Tehrany E, Blau A. A multielectrode array microchannel platform reveals both transient and slow changes in axonal conduction velocity. Sci Rep. 2017 Aug 17;7(1):8558. doi: 10.1038/s41598-017-09033-3.

Rouhollah Habibey

                             


Natural lecithin nanoliposomes promotes neural networks development

Phospholipids in the brain cell membranes contain different polyunsaturated fatty acids (PUFAs), which are critical to nervous system function and structure. In particular, brain function critically depends on the uptake of the so-called “essential” fatty acids such as omega-3 (n-3) and omega-6 (n-6) PUFAs that cannot be readily synthesized by the human body. Natural lecithin rich in various PUFAs were transformed it into nanoliposomes, which increased neurite outgrowth, network complexity and neural activity of cortical rat neurons in vitro. We also observed an upregulation of synapsin I (SYN1), which supports the positive role of lecithin in synaptic development and maturation. Enhancd neuronal development offeres devising new lecithin delivery strategies for therapeutic applications.



Latifi S, Tamayol A, Habibey R, Sabzevari R, Kahn C, Geny D, Eftekharpour E, Annabi N, Blau A, Linder M, Arab-Tehrany E. Natural lecithin promotes neural network complexity and activity. Sci Rep. 2016;6:25777. doi: 10.1038/srep25777. 


Comparison of gelling agents for long-term 3D neural cell culture & electrophysiology 

In classic monolayer cell culture, the world is flat. We assessed a selection of natural gelling agents of non-animal origin (ι- and κ-carrageenan, gellan gum, guar gum, locust bean gum, sodium alginate, tragacanth and xanthan gum) in serum-free medium at 1–4% (w/v) concentration for their suitability as a natural 3D culture environment for brain-derived cells. Their biophysical properties (viscosity, texture, transparency, gelling propensity) resemble those of the extracellular matrix (ECM). They not only protected neurons in cell culture from shear forces and medium evaporation, but also stabilized cellular microenvironment. Among the selected gels, guar gum and locust bean gum with intercalated laminin allowed for cortical cell embedment. Neurons plated on and migrating into gellan gum survived and differentiated even without the addition of laminin. Guar gum supported the functional survival of 3D cortical culture over a period of 79 days in a proof-of-concept long-term microelectrode array (MEA) electrophysiology study.



Wilk N, Habibey R, Golabchi A, Latifi S, Ingebrandt S, Blau A (2016). Selective comparison of gelling agents as neural cell culture matrices for long-term microelectrode array electrophysiology. Oilseeds & Fats, Crops and Lipids, 23 (1).


Combined PDMS microdevice and laser microdissection for selective axonal injury

A miniaturized and thin PDMS microchannel device was combined with a MEA chip and a laser microdissection (LMD) setup to selectively dissect axonal projections and investigate the electrophysiological and morphological response in distinct network compartments over 45 days in vitro (45 DIV).  Locally extended areas along the microchannel, so-called working stations, forced axonal bundles to branch out and thereby allowed for their repeatable and controllable local, partial or complete dissection. Microscopy images confirmed progressive antrograde degeneration of distal axonal segments over four weeks after surgery.

Pulsed sub nanosecond laser (355 nm PNV-001525-040) is the light source of the LMD arrangement setup.  MEA–PDMS assembly was mounted on motorized stage of the microscope.





Habibey R, Golabchi A, Latifi S, Difato F, Blau A. A microchannel device tailored to laser axotomy and long-term microelectrode array electrophysiology of functional regeneration. Lab on a Chip, 2015, DOI: 10.1039/C5LC01027F

Patterned 3D cortical networks on MEA substrate

Due to the limited number of electrodes (59-255) on MEAs, most of the network activity cannot be captured because the majority of the cells are placed far from the extracellular recording electrodes. To overcome this limitation, PDMS devices with 80-120 µm diameter wells, were designed to seed cortical neurons directly on top of the electrodes to increase the number of units from which network activity can be recorded. In addition, neurons in one of the well modules could extend their axons through microchannels to connect to the other modules. The connectivity pattern could be defined by shape, number and size of interconnecting microchannels. Furthermore, we coated the walls of each well with cell adhesion mediators and produced 50 -100 µm thick cell assemblies, both containing neurons and glia cells, without any supporting scaffold. Such 3D structures resembling in vivo neuronal network structures and showed rich electrophysiological properties compared to 2D cultures with the same overall number of neurons.

Crossing microchannels and multiple axonal compartments

PDMS microchannel tiles on MEAs for neural network compartmentalization A) A schematic cross-section of a microchannel and the reservoirs shows how microchannels selectively let axons grow on top of an electrode row while preventing cell bodies to enter. B) PDMS microstructure including four big reservoirs interconnected by an 8 × 8 matrix of channels. C) Cells seeded in a somal compartment (left) had grown their axons after 9 DIV through the entire length of a microchannel into the empty axonal compartments. D) Magnified view of the axons inside the channels between electrodes 15-14 and 25-24. E) Magnified view of axons entering the axonal compartment. Black arrows indicate at an electrode and green arrows point at axons. 





Habibey R, Golabchi A, Blau A. (2015). Microchannel scaffolds for neural signal acquisition and analysis. Springer Series on Computational Neuroscience. 13:47-64.

An incubator-independent perfusion platform for automation of the cell culture tasks

A simple benchtop cell culture perfusion system adapted to commercial microelectrode arrays (MEAs) recording equipment and standard microscope stages for simultaneous and uninterrupted extracellular electrophysiology and time-lapse imaging at ambient CO2 levels. The concept relies on a transparent, replica-molded, gas-permeable polydimethylsiloxane (PDMS) perfusion cap, gravity- or syringe-driven perfusion, and pre-conditioning of pH-buffered serum-free cell culture medium to ambient CO2 levels at physiological temperature. Network formation imaged right after cell plating. Extracellular recordings revealed details on sudden as well as gradual changes of spontaneous activity in maturing neural networks with large intra-day fluctuations.  Time-lapse imaging unveiled a rather static macroscopic network architecture while local morphology underwent spatial oscillations on the timescale of minutes.

 


Saalfrank D, Konduri A, Latifi Sh, Habibey R, Golabchi A, Martiniuc A, Knoll A, Ingebrandt S, and Blau A. (2015). Incubator-independent cell-culture perfusion platform for continuous long-term microelectrode array electrophysiology and time-lapse imaging. R. Soc. open sci. 2 (6), 150031.

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