of the neurons to the glass we need to first coat the surface normally you use a compound called poly l-lysine for this but we had an issue with the order right before filming so didn't have any on hand our friend David Ishii suggested using a compound called pei or polyethylene i mean which we normally use as a transfection agent to help get DNA into cells and so we happen to have some we found a paper that said it could be used as a substitute so we went with that to do the coating all we did was pipette NUMBER_TOKEN microliters of pei solution into the array and let it sit for NUMBER_TOKEN minutes before carefully removing the liquid to actually get the culturing started we pre warmed the culture media in a water bath until it was at about NUMBER_TOKEN degrees at that point we got the neurons out of the liquid nitrogen that they're store when not in use these were then placed in the water bath and when both things were warm we could see the array the neurons were first diluted with one milliliter of Culture Media and separately NUMBER_TOKEN microliters of Culture Media was added to each array at this point we noticed that one of the arrays was leaking so we had to discard it leaving us with only one array left to say that this was nerve-wracking was an understatement to the final array we added NUMBER_TOKEN microliters of the neuron solution so about a hundred and fifty thousand neurons then before we could close up the array and put them in the incubator to grow we had a quick look under the microscope at this point we realized that we'd messed up a bit and had seeded way too many neurons the pressure of all the things failing in such fast succession combined with us assuming that a bunch of the neurons would probably die made us overshoot oh well learning experience but otherwise things look good the electrodes looked fine and the neurons while very dense looked healthy and after that we just closed up the array and put it into the incubator to grow neurons are notorious for being delicate cells to grow and one of the issues you can have is the neurotransmitters that they secrete build up and eventually become toxic so the next day we added NUMBER_TOKEN microliters more of fresh Culture Media on the second day we left them alone and on the third day we changed the media entirely for NUMBER_TOKEN micro liters of fresh media at this point we also checked the cells again and sure enough they seemed to have adhered to the dish which is normally a really good sign we'd seeded the rest of the neurons we had left into a normal culture flask without the PEI pretreatment and after the same three day incubation they hadn't attached and all looked kind of dead so the array cells actually looked better which was really encouraging after about a week and two media changes we decided it was time to check and see if we could get any signal from the neurons to do this I'd search around online and found a company called backyard brains that makes a kit specifically for measuring signals from neurons though normally it's intended for a neuron in a cockroach leg I put together the kit and then it was time to test the array but before we got going I took a quick look at the cells to see how they looked and immediately noticed a huge problem some of the electrodes had begun to degrade and peel off the glass at this point there was only a few electrodes remaining intact so we marked them and when with the experiment anyway because the
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