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In JoVE (2)
- Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons
- Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices
Other Publications (5)
Articles by Behrad Vahidi in JoVE
JoVE General
Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons
1Department of Biomedical Engineering, University of California, Irvine (UCI), 2Stem Cell Research Center, University of California, Irvine (UCI), 3Institute for Brain Aging and Dementia, University of California, Irvine (UCI)
In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.
JoVE General
Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices
1Department of Biomedical Engineering, University of California, Irvine (UCI), 2Stem Cell Research Center, University of California, Irvine (UCI), 3Institute for Brain Aging and Dementia, University of California, Irvine (UCI)
In this video we demonstrate how to use the neuron microfluidic device without plasma bonding.
Other articles by Behrad Vahidi on PubMed
Microfluidic Culture Platform for Neuroscience Research
This protocol describes the fabrication and use of a microfluidic device to culture central nervous system (CNS) and peripheral nervous system neurons for neuroscience applications. This method uses replica-molded transparent polymer parts to create miniature multi-compartment cell culture platforms. The compartments are made of tiny channels with dimensions of tens to hundreds of micrometers that are large enough to culture a few thousand cells in well-controlled microenvironments. The compartments for axon and somata are separated by a physical partition that has a number of embedded micrometer-sized grooves. After 3-4 days in vitro (DIV), cells that are plated into the somal compartment have axons that extend across the barrier through the microgrooves. The culture platform is compatible with microscopy methods such as phase contrast, differential interference microscopy, fluorescence and confocal microscopy. Cells can be cultured for 2-3 weeks within the device, after which they can be fixed and stained for immunocytochemistry. Axonal and somal compartments can be maintained fluidically isolated from each other by using a small hydrostatic pressure difference; this feature can be used to localize soluble insults to one compartment for up to 20 h after each medium change. Fluidic isolation enables collection of pure axonal fraction and biochemical analysis by PCR. The microfluidic device provides a highly adaptable platform for neuroscience research and may find applications in modeling CNS injury and neurodegeneration. This protocol can be completed in 1-2 days.
Generation of Stable Concentration Gradients in 2D and 3D Environments Using a Microfluidic Ladder Chamber
We have developed a simple microfluidic device for generating stable concentration gradients in 2D and 3D environments. The device, termed the Ladder Chamber, uses a two-compartment diffusion system to generate steady state gradients across flow-free channels that connect the source and sink channels. To demonstrate the utility of the Ladder Chamber for cell migration, neutrophil chemotaxis was successfully observed in soluble chemoattractant (IL-8) gradient. The Ladder Chamber's simple design and experimental implementation make it an attractive approach for investigating cell migration and other biological experiments in well-defined gradients in 2D surfaces as well as in 3D gels.
Microfluidic-based Strip Assay for Testing the Effects of Various Surface-bound Inhibitors in Spinal Cord Injury
This paper describes a novel microfluidic-based assay for spinal cord injury (SCI) research. Conventional methods such as neurite outgrowth and strip assays cannot recapitulate the organized structure of the spinal cord and thus poorly simulate the injury microenvironment. In addition, it is difficult to obtain quantitative results to compare subtle differences on a chemical's effect on normal growth and regeneration. In SCI, the cell bodies are often located away from the immediate lesion, while the damaged and regenerating axons are exposed to the inhibitory milieu of the scar-tissue. We combined micropatterning and microfluidics to selectively place high purity CNS neurons on favorable substrate but allow only axons to interact with permissive (i.e. polylysine) and inhibitory substrates (i.e. aggrecan) presented in alternating strips. On patterned surfaces, axons were confined on permissive lanes and consistently avoided inhibitory strips. Since processes are expected to proceed in a pre-defined direction/geometry, even small deviations, indicative of the drug's effectiveness, can be readily detected. To demonstrate the potential utility of the method in drug screening for SCI, we used chondroitinase-ABC as a model drug to overcome the inhibitory effects of aggrecan. Enzymatic treatment promoted axons to cross onto the nerve-inhibitory strips and extend randomly across the pattern. Such effects can be easily observed and confidently quantitated to obtain objective comparison. This approach is amenable for high throughput screening and may be used to study the effects of pharmaceuticals that suppress inhibitors of neuronal growth/regeneration.
Examination of Axonal Injury and Regeneration in Micropatterned Neuronal Culture Using Pulsed Laser Microbeam Dissection
We describe the integrated use of pulsed laser microbeam irradiation and microfluidic cell culture methods to examine the dynamics of axonal injury and regeneration in vitro. Microfabrication methods are used to place high purity dissociated central nervous system neurons in specific regions that allow the axons to interact with permissive and inhibitory substrates. Acute injury to neuron bundles is produced via the delivery of single 180 ps duration, lambda = 532 nm laser pulses. Laser pulse energies of 400 nJ and 800 nJ produce partial and complete transection of the axons, respectively, resulting in elliptical lesions 25 mum and 50 mum in size. The dynamics of the resulting degeneration and regrowth of proximal and distal axonal segments are examined for up to 8 h using time-lapse microscopy. We find the proximal and distal dieback distances from the site of laser microbeam irradiation to be roughly equal for both partial and complete transection of the axons. In addition, distinct growth cones emerge from the proximal neurite segments within 1-2 h post-injury, followed by a uniform front of regenerating axons that originate from the proximal segment and traverse the injury site within 8 h. We also examine the use of EGTA to chelate the extracellular calcium and potentially reduce the severity of the axonal degeneration following injury. While we find the addition of EGTA to reduce the severity of the initial dieback, it also hampers neurite repair and interferes with the formation of neuronal growth cones to traverse the injury site. This integrated use of laser microbeam dissection within a micropatterned cell culture system to produce precise zones of neuronal injury shows potential for high-throughput screening of agents to promote neuronal regeneration.
Integrated Microfluidics Platforms for Investigating Injury and Regeneration of CNS Axons
We describe the development of experimental platforms to quantify the regeneration of injured central nervous system (CNS) neurons by combining engineering technologies and primary neuronal cultures. Although the regeneration of CNS neurons is an important area of research, there are no currently available methods to screen for drugs. Conventional tissue culture based on Petri dish does not provide controlled microenvironment for the neurons and only provide qualitative information. In this review, we introduced the recent advances to generate in vitro model system that is capable of mimicking the niche of CNS injury and regeneration and also of testing candidate drugs. We reconstructed the microenvironment of the regeneration of CNS neurons after injury to provide as in vivo like model system where the soluble and surface bounded inhibitors for regeneration are presented in physiologically relevant manner using microfluidics and surface patterning methods. The ability to control factors and also to monitor them using live cell imaging allowed us to develop quantitative assays that can be used to compare various drug candidates and also to understand the basic mechanism behind nerve regeneration after injury.
