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., Cotman, C., et al. Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices. J. Vis. Exp. (9), e410, doi:10.3791/410 (2007).
Preparing Microfluidic Devices For Neuron Cell Culture
1) Clean Corning No.1 24 mm X 40 mm glass slides
Note: We have special metal trays that we load with the slides. The slides are separated individually and placed in slots in this metal rack. We can then place these metal loading trays in a glass dish that we fill with water, place in the water bath sonicator, and sonicate for 30 minutes. Subsequent washes are carried out in this dish.
2) Preparing the PDMS devices
Note: It is important to keep the devices face up. Initially, when we cut the PDMS away from the silicon wafer, the side in contact with the wafer is the clean side: it contains the microfluidic channels. We try to be as careful as we can as to not damage the device, and keep the clean side face up until we are ready for plasma bonding.
The device is now plasma bonded to the glass slide.
3) Coating The Devices With Poly L-Lysine (PLL)
=> 150 µl of PLL for the large wells and 30 µl PLL for the smaller wells. It does not have to be exact as long as the wells are full.
Note: If you look at a device you will see 4 wells: each two are connected. You want to put the PLL in one well so that it will flow through the device into the next well.
Note: You do not want to introduce air bubbles to the channels or chamber. Just vacuum out the excess PLL in the wells.
Figure 1 is a diagram of a microfluidic device. Notice how the Blue (Soma side) is connected and the Red (Axonal side) are connected. When we say put the PLL, or water or media, on one well on each side of the device, what we mean, for example, is: Place 150 ul of autoclaved dH2O in one Blue Well then place 150 µl of autoclaved dH20 into one Red Well. The water (or PLL, or Media, or Cells) will flow through the device to the other corresponding well.
Note: Please note that, from now on, when I say "place media, cells, water or PLL in the TOP wells", I am referring to the diagram above where the Soma would be on the left and the Axons will grow to the right.
Note: Due to the possibility that borate from the PLL can absorb into the PDMS, some in the Jeon Lab recommend incubating the devices overnight with autoclaved dH2O after a couple of initial quick rinses. This will ensure that all free borate will leach out of the PDMS prior to the loading of cells. If this is not done, there may be a release of borate into the media over time, which could lead to cytotoxicity.
Note: The devices can be incubated overnight and plated with cells the next day, or incubated a minimum of 3 hours with NMB + factors before plating the cells.
Preparing The Neurons For Loading Into The Devices
We use 18 day old fetal rat cortex that we either buy from a company called Brain Bits or that is prepared here on campus for us. We prefer to get the tissue fresh.
Plating The Devices
Supplemental On Using The Devices Without Plasma Bonding
Without plasma treating, Christina Tu, a technician, who does it successfully every week without plasma, says to just load the cells right away.
Remember to remove the media from both wells on the soma side (axon side won't matter if you leave the media in). It's the fluid flow that allows the cells to enter the main channel. If you have media in the wells, you won't get fluid flow.
Here we demonstrated how to use the neuron microfluidic device without needing a plasma cleaner. We refer to this as non-plasma bonding.
The authors have nothing to disclose.
|PDMS||Reagent||Dow Corning||Sylgard 184 with curing agent Please consult Dow Corning to find a vendor near you|
|Cornig No. 1 Cover Glass||Cover Glass||Corning||Corning No. X2935 244||Available through Fisher Scientific, Fish Catalog number 12-531D|
|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.|
|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.