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)
Harris, J., Lee, H., Vahidi, B., Tu, C., Cribbs, D., Jeon, N. L., et al. Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons. J. Vis. Exp. (7), e261, doi:10.3791/261 (2007).
In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80°C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.
Part 1: Preparing the microfluidic neuron device
Note: If a vacuum dessicator is not available, the master can be left at room temperature for several hours. The air bubbles will eventually dissipate on their own. If a hot plate is not available, the PDMS will cure at room temperature after 24 hours.
Note: It is also important to make sure that the master is level during the curing process.
Part 2: Cutting out the neuron microfluidic device
Part 3: Plasma bonding the devices to glass cover slips.
In this video we have demonstrated how to make and prepare a PDMS microfluidic device that we use to culture neurons in. The device allows us to obtain a pure axonal fraction separated by microgrooves from the compartment containing a mix of cell bodies, dendrites, and axons. These devices allow the researcher to study the effects of various treatments as well as provide a platform to carry out physical injury on the axons and observe what effects these treatments have on the neurons. The device also provides a level of order to culturing neurons that helps facilitate transport studies. In addition, the microgrooves, which separate the cell culture compartment from the axon compartment, allows for fluidic isolation between the two compartments making it possible to treat one side of the device without effecting the other side of the device directly with the treatment. This property allows the researcher to study effects such as signal transduction and transport that result from the treatment.
|PDMS||Reagent||Dow Corning||Sylgard 184 with curing agentPlease consult Dow Corning to find a vendor near you|
|Plasam Cleaner||Tool||Harrick Scientific Products, Inc.||http://www.harrickplasma.com/|
|Neural Basal Media||Reagent||Invitrogen||21103-049||Neural Basal Media is obtained by Invitrogen under their Gibco line of cell culture reagents.|
|B27||Reagent||Invitrogen||17504-044||B27 is a proprietary supplement available through Invitrogen under their Gibco line of cell culture reagents.|
|Glutamax||Reagent||Invitrogen||35050-061||Glutamax is available through Invitrogen under their Gibco line of celll culture reagents.|
|Cornig No. 1 Cover Glass||cover glass||Corning||Corning No. X2935 244||Available through Fisher Scientific, Fish Catalog number 12-531D|
|60 mm Petri Dish||Tool||Fisher Scientific||08-757-13A||60 mm polystyrene sterile petri dishes are used to house the device bound to glass.|
|BD Falcon 50 ml Tube||Tool||BD Biosciences||Falcon No. 352098||Available through Fisher Scientific catalog number 14-959-49A|
|BD Falcon 15 ml tube||Tool||BD Biosciences||Falcon No. 352097||Available through Fisher Scientific catalog number 14-959-70C|
1. Park JW, Vahidi B, Taylor AM, Rhee SW, Jeon NL. Microfluidic culture platform for neuroscience research. Nat Protoc. 2006;1(4):2128-36.
2. Taylor AM, Blurton-Jones M, Rhee SW, Cribbs DH, Cotman CW, Jeon NL. A microfluidic culture platform for CNS axonal injury, regeneration and transport. Nat Methods. 2005 Aug;2(8):599-605.