Neural Cell Dissociation and Plating onto MEAs

This protocol assumes use of one multielectrode array (MEA) or other culture dish. Multiply supplies and materials as necessary. Instructions regarding electrode array may be disregarded for other culture dishes. All activity performed under sterile conditions, except where otherwise noted. Potter group uses laminar-flow hood to maintain sterility. Items brought into hood are first sprayed or wiped with 70% ethanol.

Before Beginning
Gather all supplies and materials. Ensure plating medium is mixed and equilibrated. Thaw PEI, papain, and DNAse in water bath (37 degrees C). Thaw laminin at room temperature; thawing in water bath will cause laminin to gel.

Prepare Dishes
Multielectrode array (MEA), or 35mm dia. glass-bottomed petri dish Petri dish, 100 x 15mm 1000µL pipetor & tips, or transfer pipet 100µL or 200µL pipetor & 100µL tips 20µL pipetor & tips Vacuum tube & 200µL pipet tips (optional, recommended) Polyethylene imine (PEI) in borate buffer, 100µL, pH 8.5 Sterile water, 1-2mL Laminin aliquot

Always take extreme care to avoid directly touching electrode array. Electrodes and leads are fragile and easily broken. When pipeting substances onto or off of array, safest technique is to rest pipet tip on finger of non-pipeting hand, and use body of pipetor as lever to control tip. Coat dish with PEI Place MEA in 100mm petri dish. Pipet 100µL PEI onto electrodes in center of MEA. Cover petri dish to prevent evaporation, and let PEI sit at least 30 minutes before removing. If plating medium is not yet made, mix now and place in incubator where cultures will be kept. Allow at least one hour for equilibration. See medium-preparation protocol for details. Remove PEI

Pipet l-2mL sterile water into the dish. (If plating more than a few dishes, it is convenient to use a serological pipet to dispense the water.) Using pipetor or vacuum tube, completely aspirate water from dish. Hovering tip over array should provide enough suction to vacuum up water without touching electrodes. Repeat process three more times (total of four rinse/aspirate cycles). Let dish dry, uncovered, for about 30 min. While dish dries, begin tissue dissociation and trituration. Continue with laminin coating when convenient. Coat dish with laminin Use 10-15µL of laminin. Pipet out the laminin until it forms a bulb at the edge of the pipet tip, then slowly, gently, touch bulb to center of array. Ensure laminin covers all electrodes. If not, add more laminin. Cover petri dish to prevent laminin evaporating, and place in the incubator. Let sit at least 20 minutes.
Neurons and glia do not naturally stick to the silicon nitrate substrate of the MEA. If not fixed in place, they have a tendency to (slowly) migrate around the dish. The characteristics of an action potential change depending on where it is measured, so if the cells are not stationary, it is impossible to say whether a change observed via the electrodes is due to network plasticity, or is simply an artifact of physical movement. It is therefor necessary to keep the neurons in constant position relative to the electrodes. Laminin is a naturally-occurring extracellular matrix protein used to glue the neurons in place relative to each other. Laminin will not easily stick to the substrate either, though, so the silicon nitrate is treated with polyethylene imine (PEI) to make it more hydrophilic, allowing the two substances to bind. Keeping the laminin in place helps keep the neurons in place relative to the electrodes, thereby increasing the validity of electrical measurements. Dishes are left in the humidified (65% RH) incubator to keep laminin from drying too quickly.

Prepare Cell Suspension
Dissected embryonic (E19) rat cortex Warm water bath, heated to 37 C 40µm cell strainer Pasteur pipet & squeeze bulb Four 15mL centrifuge tubes, labeled Unfiltered Cells, Filtered Cells, Balance, and Supernatant Papain solution, 2mL aliquot DNAse aliquot 5% bovine serum albumin (BSA) in phosphate-buffered saline (PBS), ~0.5mL Neurobasal plating medium

Dissociate tissue Remove storage medium from tube containing tissue, and replace with 2mL papain. (Moving tissue into papain is an option, but subjects the tissue to more abuse due to extra pipeting.) Add 50µL DNAse. Place tube in water bath for 10-20 minutes (all papains are different; stronger papains require less time). Swirl every 5-7 minutes to bring fresh enzyme to the tissue.
Enzymatic dissociation is a recommended, though not absolutely necessary, prelude to mechanical separation of the cells in

the tissue. Papain's intended function is to dissolve the extra-cellular matrix binding the cells together. Left long enough, however, it will begin digesting the cells themselves, so it is important that the cells recieve only the minimum exposure necessary. As some of the cells break open, strands of DNA will be released into the papain solution. DNA is sticky, and will tie the cells together if not removed. DNAse attacks DNA specifically, and will not hurt intact cells. ~30µL should be enough, but 50µL won't hurt. Since papain will also attack the DNAse, add it only after combining the papain and tissue.

Halt dissociation When tissue is sufficiently digested (should appear fuzzy), remove papain and replace with 1mL of serum-containing plating medium. If tissue is pulled into pipet with papain, there are loose DNA strands binding it together. Add another 20-30µL DNAse. Tissue should fall apart within moments.
Different batches of papain can have different initial strengths, and all batches lose potency over time. You can never be certain how quickly the papain will act on the tissue, so physical appearance is a better indicator than immersion time of when the papain should be removed. Serum-containing medium is emphasized because is provides extra protein to "distract" the papain, reducing further enzymatic damage to the cells.

Triturate tissue Add 1mL plating medium to tube. (Tube now contains 2mL of medium.) Gently draw cells into pipet tip and dispense, three times. Spend about one second in each direction, on each pass. Triturating too violently will kill most of the cells. Let any visible brain pieces settle to bottom of tube. The supernatant is now a suspension of single cells. Pipet 1mL of suspension into Unfiltered Cells tube, so cells will not be subjected to more turbulence in repeated trituration steps. Repeat cycle (add medium, triturate, let settle, move cells) 3-4 more times. Pipet last milliliter of plating medium into Unfiltered Cells tube (should contain ~4-6mL suspension). Set aside dissociation/trituration tube, but do not yet discard.
Having used enzymatic dissociation to "loosen" the cells, we now use turbulence to mechanically separate them. Too much turbulence will damage the already-traumatized cells beyond recovery. Too little will fail to separate them. Getting the right amount is largely a matter of practice, but is not terribly difficult. If the cells don't come apart right away, just try pipeting a little harder. There are multiple opportunities during the cell suspension-preparation process to misplace cells. Keep all containers until you have confirmed suspension density in the cell-count step.

Filter large debris Place 40µm cell strainer in 35mm petri dish, and wet strainer with 1mL plating medium. Cap and invert suspension to resuspend cells. Gently pipet suspension into cell strainer. Hold pipet tip ~1cm above strainer, and dispense slowly enough that droplets form, rather than a stream. Remove cell strainer from dish, and transfer filtered suspension into Filtered Cells tube. Set aside Unfiltered Cells tube, but do not yet discard.
Large debris (clumps of tissue, meninges, blood cells) must be removed from suspension. Pre-wetting the strainer keeps cells from sticking to the dry fibers. Pipeting slowly avoids subjecting the cells to further turbulence. Seeing droplets indicates that you are pipeting slowly enough.

Filter small debris Bring total volume of suspension to at least 4mL. Using Pasteur pipet, layer ~0.5mL (~1cm) of 5% BSA solution at bottom of tube. Fill Balance tube with a volume of water equal to volume of cell suspension. (Balance tube & water need not be sterile, as they will never touch cells.) Place tubes

opposite each other in centrifuge and spin at 200RCF (200 x g) for 6 minutes. On removal, a small pellet of cells should be visible at bottom of tube. Clean and dry hemocytometer and coverslip while cells are spinning.
Small debris (nuclei, organelles, bits of cell membrane) is removed by rate-zonal centrifugation. Large particles (intact cells) will settle at a faster rate than smaller particles (debris). Raising the volume of suspension gives the cells more opportunity to get ahead of debris on the race to the bottom of the tube. BSA provides an extra bit of viscosity to slow down small debris even more, and also provides a bit of cushioning for the cells passing through.

Resuspend cells Transfer supernatant to Supernatant tube. Remove as much medium as possible without disturbing pellet. Set aside Supernatant tube, but do not yet discard. Add 0.5mL (per half-brain) of plating medium to pellet. Resuspend cells by gentle trituration or by flicking bottom of tube. Cells are now ready for counting.

Count Cells
hemocytometer and coverslip Phase-contrast microscope Manual counter 20µL pipetor and tips

Clean and dry hemocytometer and coverslip thoroughly. Place coverslip on hemocytometer. Use gentle trituration or inversion to resuspend cells immediately before pipetting onto hemocytometer. Pipet 10µL of cell suspension into both triangular grooves on the hemocytometer. Under the microscope, count cells in central block of squares, or count several squares, depending on density of suspension. It is typical to see about 10 cells per box, or 250 in the 5x5 array. The dimensions of the array are 1mm x 1mm x 100 microns, or 0.1ul. Cells in array x 10 = cells/ul. 2500 cells/µL is ideal. If necessary, suspension can be diluted with plating medium, or concentrated by further centrifugation. If cell count is substantially lower than 2500/µL (<1000/µL), cells may have been lost in an earlier step. Check contents of used tubes under microscope to determine if recovery is possible. Cells viewed under microscope are no longer sterile. Discard them.
The hemocytometer and coverslip may have cells left on them from when they were last used. Ensure the accuracy of your count by thoroughly cleaning both. Be sure that cells are evenly distributed throughout the suspension before attempting count.

Cell Plating

MEA lid (Teflon ring w/ gas-permeable FEP membrane) Neurobasal plating medium, 1-2mL

Cell drop Just before plating, pipet away most of the laminin, leaving a wet spot. Pipet 10-20µL (20-50,000 cells) of concentrated suspension onto MEA, centered on the electrodes. Place FEP lid on MEA, and cover petri dish. Note to self: May want to re-write to indicate set volume of density-adjusted suspension.
Do not remove all laminin; it is there to hold cells in place.

Attachment Leave culture in incubator ~30 minutes while cells attach to laminin. Observe sealed culture under microscope. If cells are not touching every electrode, add more concentrated suspension and let settle as before.
Move the dish to incubator carefully; sudden movements may cause cell suspension droplet to roll off of laminin. During attachment period, cells are slowly falling through suspension medium and laminin to the MEA substrate. Once they are settled, confirm plating density. Cultures with too few neurons will have difficulty forming a network, and will provide fewer useful data.

Flooding Gentley pipet 1mL of plating medium into dish (2mL for 35mm glass-bottomed dishes). Aim for edge of MEA well, so as not to dislodge cells. Replace MEA and petri dish lids, and return culture to incubator.
This medium is meant to feed the culture until it is established enough to switch to regular feeding medium. Think of it as baby food used until the cultures are old enough to be "weaned."

Luer-tip syringe w/ 0.2µm filter Jimbo's feeding medium, 1-2mL

24-36 hours after flooding, replace Neurobasal plating medium with Jimbo's feeding medium. Draw feeding medium into syringe, then attach filter. Pipet old medium from dish, and replace with filtered medium from syringe, always being careful not to dislodge cells. Photograph culture under phase-contrast microscope, and note condition. Replace half the feeding medium twice a week. Replacing all the medium once a week is an option, but not recommended.
All media should be prepared and kept in a sterile environment. The inside of a medium jar starts out sterile, and, if only

opened under the hood, should remain sterile. That said, our culture media are kept in the same humidified incubator that houses the cultures, and incubators are natural breeding grounds for mold, bacteria, and other contaminants. It is not unheard of for cultures to become infected, possibly due to contaminants transmitted from the outside to the inside of the jar. Filtering the medium immediately before use is a highly-recommended precautionary measure.

Banker G, Goslin K. 1998. Culturing Nerve Cells, 2nd Edition. Cambridge, Mass.: MIT Press Potter, S.M., DeMarse, T. B. (2001) "A new approach to neural cell culture for long-term studies." J. Neurosci. Methods 110: 17-24. Brewer GJ, Torricelli JR, Evege EK, Price PJ (1993) Optimized survival of hippocampal-neurons in B27-supplemented neurobasal(tm), a new serum-free medium combination. Journal Of Neuroscience Research 35:567-576.

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