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
Materials
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

Procedure
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

Materials
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

Procedure

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

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

Procedure

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."

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

Procedure
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.

References
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. http://www.neuro.gatech.edu/groups/potter/publications.html

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.