culturing on MEA chips

We have recently tried culturing rat cortical neurons on glass MEA chips (200µm/30µm int.ref). Prior to using these chips we successfully cultured rat cortical neurons on gass coverslips for electophysiolical recording. In brief we clean a used MEA chip by soaking overnight in a trypsin solution. The next day the chips are washed with dH2O. The day before the preparation we soak the chips in 100% EtOH for ~ 30mins (to sterilize). We then remove the chips and leave in the tissue culture hood until the EtOH has evaporated. We apply approx 1ml of Poly-D-lysine soln (0.01mg/ml) and leave for a minimum of 2 hrs. The polylysine is then washed off with sterile dH2O and left to air dry. We have been plating our cell suspension at various densities. Our problems are that the cells are clumping together instead of adhereing individually to the MEA chip. Hence we are not getting any networking. Also debris red blood cells etc is congregating right in the center of the chip where the electrodes are. We also appear to be getting a significant amount of cell death. My feeling is that the substrate is not working properly
If anyone has any tips on how to get good cultures we would be very grateful since without good cultures we are unable to test the system.

Dear Sally,

I asked users of the MEA system for advise. Here's their feedback:

Daniel Wagenaar, Caltech, USA:
Clumping is most likely the result of using poly-lysine. We get that
too. Cell death might well be a secondary result of clumping. I'd
recommend instead using poly-ethylene-imine (PEI). Put 0.05%
(weight/volume) PEI dissolved in a borate buffer (described below) on
MEA and leave for 45 min to 1 hour. Remove, and wash 4x with ddH2O.
(Dried PEI is toxic.) Leave to dry. Next, put a drop of laminin (20ug/ml in plating medium) on MEA and leave that for 30 minutes. Suck it off, don't wash, and plate cells over the location of the drop. Alternatively, laminin can be mixed in with the cells.

Good luck, Daniel.

PEI is Sigma P3143 Polyethyleneimine, 50% (w/v) aq.

Borate buffer:
3.10g boric acid (Fisher, A73-500)
4.75g borax (Sigma, B0127)
1L double distilled water
Adjust pH to 8.4

(Getting the boric acid & borax to dissolve takes a lot of effort.)

Laminin is Sigma L2020, 1mg/ml.


Steve Potter, Georgia Tech, USA:
Well the first step is for her to try to replicate our methods, published in
J. Neuroscience Methods 2001
Available at http://neuro.gatech.edu/groups/potter/publications.html
PEI was crucial for me to prevent that clumping problem.
Another useful paper includes methods for removing debris:
Potter, S. M., Pine, J. and Fraser, S. E. (1996) 'Neural transplant staining with DiI and vital imaging by 2-photon laser-scanning microscopy." Scanning Microscopy Supplement 10: 189-199. (Invited) Full HTML version . (Also on our Pubs page)


Shimon Marom, Technion, Haifa, Israel:
Ask her to contact my technician Ella Lyakhov at
lela@tx.technion.ac.il or Amir Minerbi at minerbi@tx.technion.ac.il to get a complete protocol. Shimon

I hope the above will help to solve your problem.
Also, thanks to Daniel, Steve and Shimon for their fast response and help.

Best regards,

Karl-Heinz

Thanks for all the advice. We are planing on using PEI and are hopefull that our problems will be resolved. Thanks to everyone for replying.
Sally

Dear Sally, I received another reply from Dr. Ger JA Ramakers, Amsterdam,:

This is my anwer to Sally Rumley's problem. Clumping is a clear sign of insufficient adhesion to the substratum. First of all, it may be better to perform heat sterilization, as hydrophobic components from the ethanol solution may become deposited onto the MEA's. Anything that makes the MEA's (or any glass surface) less hydrophilic will result in less adhesion of the substrate and consequently, the cells. I advize her to increase the poly L/D-lysine concentration to 40 ug/ml, and perform an overnight incubation. The MEA's should not run dry during the coating phase: it will be more difficult to remove excess poly-lysine, which will adversely affect the cells. Finally, she should wash the MEA's shortly (twice) before putting in the culture. The longer the substratum is drying, the less adhesive it will be. If these tips do not work satisfactorily, she should contact me directly.
Kind regards,
Ger Ramakers

Best regards,

Karl-Heinz

Dear Sally,

sometime brand new MEAs may present adhesion and spreading problems on the cell cultures. However in our protocol we use a double layer of promoting adhesion factor at higher concentration than yours.
1. Poly-D/L-Lysine solution 5mg in 50ml of sterile water (Sigma P9155 or P6407)
2. Laminin solution 1mg in 20ml of sterile water (Sigma L2020).

First put 80ul of laminin solution and incubate several hours. After replace laminin solution with 100ul of Poly-Lisine solution and incubate overnight. The day after, wash MEA with sterile water twice or three times and dry MEA surface under laminar flow before plating the cells.

I hope that my suggestion could help you. good luck!

Brunella

NBT-group, Dept. of Biophysical and Electronic Engineering - DIBE
University of Genova, Genova (Italy)