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 JoVE General

Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device

1, 1, 2, 3, 3, 1

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)

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    Summary

    In this video we demonstrate the preparation of E18 Cortical Rat Neurons.

    Date Published: 10/01/2007, Issue 8; doi: 10.3791/305

    Cite this Article

    Harris, J., Lee, H., Tu, C. T., Cribbs, D., Cotman, C., Jeon, N. L. Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device. J. Vis. Exp. (8), e305, doi:10.3791/305 (2007).

    Abstract

    In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously dissected and prepared. The E18 cortex is, upon dissection, immediately dissociated into individual neurons. It is possible to store E18 cortex in Hibernate E buffer containing B27 at 4°C for up to a week before the dissociation is performed. However, there will be a drop in cell viability Typically we obtain our E18 Cortex fresh. It is transported to the lab in ice cold Calcium free Magnesium free dissection buffer (CMFM). Upon arrival, trypsin is added to the cortex to a final concentration of 0.125%. The cortex is then incubated at 37°C for 8 minutes. DMEM containing 10% FBS is added to the cortex to stop the reaction. The cortex is then centrifuged at 2500 rpm for 2 minutes. The supernatant is removed and 2 ml of Neural Basal Media (NBM) containing 2% B27 (vol/vol) and 0.25% Glutamax (vol/vol) is added to the cortex which is then re-suspended by pipetting up and down. Next, the cortex is triturated with previously fire polished glass pipettes, each with a successive smaller opening. After triturating, the cortex is once again centrifuged at 2500 rpm for 2 minutes. The supernatant is then removed and the cortex pellet re-suspended with 2 ml of NBM containing B27 and Glutamax. The cell suspension is then passed through a 40 um nylon cell strainer. Next the cells are counted. The neurons are now ready for loading into the neuron microfluidic device.

    Protocol

    Preparing E18 Fetal Rat Cortical Neurons for Compartmentalization

    Before starting out, it is important to warm all necessary media and reagents to 37°C.

    It is also important to sterilize everything that is used for preparing the cells (e.g., rubber bulbs, tube racks, media bottles, etc.), and that which is placed in the hood, by wiping down with 70% ethanol.

    1. Place two pieces of E18 Fetal Rat Cortex (one brain, previously dissected) in a 15 ml tube containing 1 ml ice-cold Calcium-free Magnesium-free dissection buffer.
    2. Add 1 ml of 0.25% Trypsin-EDTA to the cortex in dissection buffer, bringing the final volume to 2 ml and final trypsin concentration to 0.125%.
    3. Place the 15 ml tube in a 37°C water bath for 8 minutes.
    4. During this time, fire polish 3 glass Pasteur pipets, forming successively smaller openings, in a biosafety cabinet to help maintain sterility.
    5. After the 8 minute incubation of the cortex, add 10 ml of DMEM containing 10% FBS to the cortex to help stop the trypsin reaction.
    6. Centrifuge the 15 ml tube containing the cortex and DMEM/10% FBS at 2500 rpm for 2 minutes.
    7. In the biosafety cabinet, remove the supernatant from the cortex using a glass Pasteur pipet with vacuum suction attached. Be careful not to disturb or dislodge the pellet.
    8. Add 1 ml of NMB to the cortex pellet and gently pipet up and down. It is very important to avoid creating air bubbles while pipeting up and down, as the air bubbles can damage the cells by oxidation.
    9. Use the fire-polished Pasteur pipet with the widest opening to triturate the cortex by attaching a sterile rubber bulb to the end and pipeting up and down 5 times, again being careful not to introduce air bubbles. Continue this process with the other 2 glass pipets, each with a decreased opening size.
    10. After trituration, centrifuge down the cells again at 2500 rpm for 2 minutes.
    11. After centrifugation, once again remove the supernatant and resuspend the cell pellet in 2 ml of NBM.
    12. Filter the resuspended cell solution through a 45 um cell strainer.
    13. Stain the cells with Trypan Blue, counted, and loaded into the devices. We typically load 20 ul of cells per device.

    Note: the final concentration of cells is typically between 2.5 million cells/ml and 8 million cells/ml

    Loading the cells

    After the cells have been counted, it is time to load the cells in the device:

    1. Bring the previously prepared devices containing NBM into the biosafety cabinet to maintain sterility.
    2. Remove excess media from the wells by vacuum suction. However, be careful to avoid removing all of the media -- there must be some media in the main channel.
    3. Apply 20 ul of cells to the top left hand reservoir of the device (see diagram if necessary). The cells flow into the device and attach to the PLL treated surface.
    4. After loading, place the devices containing cells back in an incubator for 10 minutes to allow the cells to attach.
    5. After 10 minutes, fill the reservoirs with media and place devices back in the incubator.

    Discussion

    Cell densities can be varied depending on the application. However, seeding primary neurons at too low of a density typically leads to cell death. Depending on the incubator and the level of humidity, media may need to be changed in the devices every 2 to 3 days. When changing media, it is important after removing the media from the reservoirs, to never remove media from the main channels. Further, it is good practice to place fresh media in the top wells and allow some fresh media to flow through the device and into the main channels before filling up all the reservoirs.

    Disclosures

    Materials

    Name Type Company Catalog Number Comments
    NBM medium Invitrogen 21103-049 Neural Basal Media is obtained by Invitrogen under their Gibco line of cell culture reagents.
    E18 rat embryos Animal Obtained from pregnant Sprague Dawley Rats. The pregnant rat is sacrificed and the E18 fetal rat pups dissected to obtain the E18 fetal rat cortex.
    CMFM dissection buffer HBSS buffer ice-cold Calcium-free Magnesium-free dissection buffer HBSS based.
    15 ml Tube Tool BD Biosciences Falcon No. 352097 Available through Fisher Scientific catalog number 14-959-70C
    0.25% Trypsin-EDTA Reagent Invitrogen
    37˚C water bath
    Pasteur pipets Fisher Scientific glass
    DMEM medium Invitrogen
    FBS Reagent Invitrogen serum, used at 10% in DMEM
    centrifuge set at 2500 rpm
    Trypan Blue Invitrogen for counting live/dead cells
    BD Falcon Cell Strainer 40 um Tool BD Biosciences Falcon cat# 352340 Available through Fisher Scientific. Fisher catalog# 08-771-1
    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.

    References

    1. Park, J.W., Vahidi, B., Taylor, A.M., Rhee, S.W., Jeon, N.L. Microfluidic culture platform for neuroscience research. Nat Protoc. 2006;1(4):2128-36.

    2. Taylor, A.M., Blurton-Jones, M., Rhee, S.W., Cribbs, D.H., Cotman, C.W., Jeon, N.L. A microfluidic culture platform for CNS axonal injury, regeneration and transport. Nat Methods. 2005 Aug;2(8):599-605.

    Comments

    6 Comments

    This is quite impressive.  How do you avoid the neurons in the small channels sticking to the PDMS and peeling off when you remove the slabs before fixation?  Is there some sort of special treatment for the PDMS or technique used before removal so the axons are not disturbed?
    Reply

    Posted by: AnonymousJune 20, 2008, 2:05 PM

    Hello Allyson first, there are two ways to bond the device to glass, plasma bonding and by non-plasma bonding.  There are two other videos demonstrating this on JOVE that may interest you. If a device is plasma bonded to the glass it cannot be removed.  A non-plasma bonded device can be removed from the glass but if not done carefully axons in the microgrooves will get lifted off.  Some researchers have said that placing the device on ice for 5 minutes after fixing prior to removing the device helps keep the neurons on the glass. Most researchers including our lab just stain the neurons in the device and image them in the device.  For many applications this is sufficient enough.  If you wish to remove the device before staining then non-plasma bonding will have to be done and the device chilled briefly after fixing before removing.  Usually if one is going to remove the device for staining it is best to prepare a few samples in the likely hood that in some cases the neurons will be disturbed upon removing the device.
    Reply

    Posted by: Joseph H.June 20, 2008, 3:07 PM

    Just wanted to let everyone know a company has formed to make and sell the neuron device. Please contact xonamicrofluidics@gmail.com for more information.
    Reply

    Posted by: AnonymousJuly 4, 2008, 1:03 PM

    Hi, I was wondering- do the channels between the two compartments form a diffusion barrier? I would like to stimulate one of the culture chambers and not the other (using KCl)... Cheers
    Reply

    Posted by: AnonymousDecember 19, 2008, 5:03 AM

    Hello John Brown Sorry for the late response.  Work has been done with gradients in microfluidic devices but the horizonatal channels were much, much wider.  I am not sure if in the Neuron Device a gradient is created.  There might be one on a small channel that would probably be hard to measure.  I would suggest searching PubMed by Dr. Noo Li Jeon.  He has published several papers with devices used to make gradients.  Feel free to contact me at xonamicrofluidics@gmail.com if you have other questions. JŒ
    Reply

    Posted by: AnonymousFebruary 13, 2009, 12:55 PM

    Hello, I want to know the process of dissection of Fetal Rat Cortexone brain.Because I should the experiment,but nobody would teach me.Thank you.
    Reply

    Posted by: Jun L.July 6, 2012, 8:44 AM

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