Growing Human Neurons Connected to a Computer

a large pizza with extra cheese that's how Peter Watts describes the pilot of an aircraft in his book starfish a square vanilla box half a meter on a side twice as thick as your wrists it was a smart gel but the characters called it head cheese cultured brain cells on a slab billions of neurons growing and connected up to a computer hardly more than goo and electrodes you can't program it but you can teach it just like any human student it's the latest in automation and was being used to phase out humans in various professions including in this case as pilots but such a thing couldn't really be made right well actually it can allow me to introduce headcheese version 1 neurons are amazing little creatures that when grown together are capable of learning communicating and sending signals long distances using a mixture of electrical impulses sudden release of various neurotransmitters and probably a dozen other mechanisms we're only just beginning to understand neurons pack a lot of computing power into a very tiny space put a few together and you've got enough to control tiny microscopic organisms a few hundred thousand can control a small animal a few billion and you've got the makings of a sentient intelligence capable of speech abstract thought mathematics and complex decision-making all contained in something the size of a grapefruit now if you could grow the neurons outside of an organism connected directly to a computer you could in theory use them directly to do a sort of computation and make decisions this is my homemade electrode array filled with actual human neurons today I want to take you through what is probably the craziest and most difficult project I've ever attempted and required filming in two countries and two very different labs because there are so many parts to this project the first attempt we'll talk about today was simply to set a baseline how far could we get before things failed could we build the electrode array could we keep the neurons alive could we get any signal out of them that's what we set out to find with this experiment so know we won't be flying a plane with this today but this won't be the last time you hear about this experiment with everything we've learned from it and we'll talk about in a moment we've got a clear set of goals to work towards and like the spider silk it'll be one of the big projects we come back to from time to time with updates as we make progress the idea actually came from a group of researchers that managed to do this successfully using custom-made micro electrode arrays they grew thousands of neurons on a special chip and then connected it up to a computer by sending in signals to the chip and listening for the neurons response they were able to train these tiny petri dishes of rat neurons to fly a plane in a simulation so let's start from the bottom and work our way up a few months ago I built this cool-looking device it's called a DC sputtering magnetron and it allows you to coat anything in a thin layer of metal using plasma and vacuum and yes it's made out of a jar for the whole build I've linked to the previous videos below in those videos we explored how we can lay down perfectly flat metallic mirrors or make intricate patterns for most of the testing of the system I was using microscope slides as my base layer and coated them in a variety of metals from silver to copper or gold and nickel by first applying a vinyl mask that I prepared with a vinyl cutter I can shield parts of the glass from the metal plasma and prevent it getting coated in metal when it's all done encoded I just peel off the vinyl and I'm left with an intricate metallic pattern on the glass after the first of the test making patterns I started to wonder if it was possible to make something actually functional with this rather than just pretty shapes sure I could make a simple electrical circuit but I wanted something more fun and as I was thinking about it I remembered the story about the rat neurons flying a plane so since then I've been working to give this a try the design for the electrode array itself I whipped up in Inkscape and based it on the original papers design I knew I couldn't get the extremely fine resolution that they did with my current setup as the vinyl is just too coarse so I went with 16 electrodes that terminated in rounded pads in a grid pattern the pattern was cut with the vinyl cutter and then covered with green tape to hold everything in place while the pattern is transferred to the glass microscope slide then after trimming the design all the areas where the electrodes would be had the vinyl removed I repeated this with a bunch more microscope slides as I wanted to have as many of these as I could and it's a good thing that I did because only one array survived the whole process from plasma to cell culture one at a time there prepared slides were coated first with a layer of silver and then with a layer of gold I let the silver layer grow for as long as possible so that the extremely thin wires would be as conductive as possible to make taking Reed it's easier the gold topcoat makes everything more biocompatible so that it's safe to grow the neurons on the electrodes we'll talk more about this later but my material selection here as well as the lack of any pre treatments to the glass gave a bunch of problems down the line so future versions will use a different material and treatments for this step because this was the first test the thick electrodes and the fact that the entire electrode is exposed to the culture solution is fine but for future iterations another step will be to coat everything but the very tips of the electrodes in an insulator so the readings come from a more distinct area and then switching from a vinyl mask to photoresist would allow for more and thinner electrodes to be made with the slides coated in metal and the vinyl removed I was left with four arrays of acceptable quality and so these could be turned into petri dishes to do that I started with 15 milliliter Falcon tubes and used my lathe to cut off the very top this is now a threaded piece of autoclavable plastic which once stuck to the microscope slide will make perfect little petri dishes to attach the piece of plastic to the glass I picked up some fda-approved implant grade two-part epoxy from Adam adhesives this stuff is autoclave able and bio safe so you could use it for something implanted into someone and it would remain inert and safe before this glueing step I cleaned everything off camera and brought all the slides into the bio lab so I had a clean dust free workspace the epoxy is activated by popping the internal separator of the packet and then mixing thoroughly I used the long sterile swab to coat the piece of Falcon tube which was then carefully placed onto a patterned microscope slide once these cured the electrode arrays are basically done and ready for neurons the lids can be carefully screwed on to keep the liquid protected and opened easily to work with the cells the finished arrays were then packed up and shipped to Indiana and my good friend Gabriel Athena's lab if you don't know Gabe he's a biologist that's done some really cool projects over the years recently you saw another project I filmed at his lab where we turned a grape into a meat berry he's doing a lot of cool stuff which we'll talk about in future videos but I've linked to his pages below if you're interested his day to day work involves working with mammalian cells so his lab is set up to grow things like the monkey cells we used in the meat berry video or in our case human neurons speaking of which for the neurons we had a few different options from different supply airs online but I wanted healthy human neurons which ranged from holy oh my god my wallet is on fire too still fairly expensive but not as bad we went with mixed cortical neurons which were derived from induced pluripotent stem cells that's a lot of big bio words that probably don't mean anything to most of you so let's break that down mixed cortical neurons means that it's a mixture of two different types of neurons that are normally found in the cerebral cortex of the brain the two types here are glutamatergic and gaba arabic which refer to the neurotransmitter that they interact with in this case glutamine and gaba respectively they're in about a 20 to 80 ratio likely taken from a pure stock of each and literally just mixed in a tube before they're sent out the induced pluripotent stem cell thing refers to how the neurons were created they weren't harvested from a living organism or at the least they didn't start as neurons that would be what's called primary cortical neurons which refer to them being freshly harvested from something rather than lab-created to make them scientists start with one of a variety of cell types from some organism lots have been used but honestly it doesn't really matter that much let's say skin cells from a person for example or as we normally call them human fibroblasts to turn fibroblasts into stem cells you first get them growing in a culture flask like we've shown in previous videos then you transfer them which means you take a piece of DNA and put it into the cells that special piece of DNA is readily available and something you buy premade it's a little circle of DNA that once it's inside the cell kicks on four different proteins these set off a massive symphony of biochemical pathways in the cell that makes it revert back to a stem cell-like form what is a stem cell well normally these are sort of your base level cells they can make essentially infinite copies of themselves and can differentiate to become any other cell type in theory once you've got stem cells you can use different molecules to trigger the differentiation process and if you deliver the right triggers at the right time you end up with a cell type you want in this case the two neuron types we just talked about these cells are a terminal cell lines so they shouldn't divide anymore so however many neurons you have in the tube is the number you have to work with for our experiment we purchased the smallest available quantity which was one million and came in this tiny tube to allow the neurons to grow we need special growth media for them this will contain all the nutrients and other chemicals that they need to survive we went with neuro basal media and a b27 supplement from thermo Fisher the supplement should contain a variety of different growth factors and hormones like nerve growth factor to keep the neurons happy we also added glutamine as the protocol for the media preparation called for it and finally we added antibiotics to make sure that the neurons were protected from any bacterial contamination I tell you more about what's in the media but honestly it's such a witch's brew of stuff that I really don't know what a lot of it is or does and just followed the recipe on the site I bought it from before we can put any neurons into the electrode arrays we first need to add an air filter to them to allow the cells to breathe and then sterilize them so they're perfectly clean and won't contaminate the neurons the filters I made out of these luer lock syringe filters that I normally use to sterilize media the pores are small enough to prevent anything from getting in but will let air through these are epoxy din 2 pre-drilled holes in the lids to sterilize the arrays we used an autoclave but this is where we hit a big snag we put the arrays into an empty pipette tip box to protect them while they were in the machine but the heat warped the box and most of the arrays got stuck in the bottom on top of that the epoxy just failed without even sticking on two of them so by the time we got things unstuck we were down to two remaining arrays and had no way to make more with the remaining arrays it was time to finally put some neurons in them and to try and get them growing to improve the adhesion 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 200 microliters of pei solution into the array and let it sit for 20 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 37 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 250 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 150 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 250 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 250 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 metal is so delicate I used conductive tape to act as a bridge wire between the array and the detector but no matter which arrangement I tried on the electrodes no signal came out of the array I also checked with an oscilloscope and didn't see any activity the last test was just to measure the voltage and there was a slight voltage between some of the electrodes but what that means I don't really know so in the end this first test was a flop but considering the goal is making head cheese and neither of us have worked with neurons before I think we did pretty well and we did learn an enormous amount along the way which was really the point of all this I knew going in this was going to be a ridiculously difficult project so with this baseline set we know what to do for a second attempt we'll be working towards that second attempt over the next few months and we'll revisit this project hopefully a couple times a year until we get something that works before we do that though various bits of a hardware or need to be upgraded and we'll need to do more research so to wrap this video up let's go over all the things we'll be changing for the next time and learned from this test first the electrodes obviously need to be improved instead of silver as a base layer will be using titanium which we demonstrated we could do in a recent video it's a stickier metal and is often used in industry as a base coat for exactly this application and reason then we'll still do gold on top to make sure it remains conductive before any coating is applied though next time we plan on pre treating the slides by first soaking them in hot piranha solution this is a nasty combination of sulfuric acid and hydrogen peroxide which will destroy any surface contamination and make sure the glass is more hydrophilic and hopefully will make the metal stick better the epoxy will probably need to be changed out or we'll need to sterilize with something other than an autoclave but I like the Falcon tube thing and will probably stick with that for now I think the epoxy might just be a bit too brittle and doesn't stick to glass well but perhaps the piranha solution pretreatment will help it stick better for the next attempt we're gonna do a lot more destructive testing to make sure that whatever we go with won't fail spectacularly like this batch did for the neurons themselves we'll be sure to seed less of them and also experiment with some different media formulations to see if there's something else that lets them grow better we'll also get some special stains to visualize the neurons better as they're so tiny it's hard to see their morphology from recent reading it looks like if the neurons are too they don't actually form connections properly so next time with far fewer cells we should see some nice axon and dendrite outgrowth as we expect with neuron culture which should mean actually getting some signal from it clearly and finally for the measurement we may build our own detector or amplifier or maybe we might just get the nicer model from backyard brains but if we make those changes hopefully it'll end with at least getting some signal out of the neurons from there we can go and start working on interacting with the neurons and improving the electrode array and maybe someday get to the point where we can start training them to do things like fly a plane in a sim before we can though there's a lot of work ahead of us to improve the coatings means a total rebuild of the magnetron and more mammalian work means I'll need to heavily stress test everything before flying out to Indiana again so be sure to subscribe is it'll be a little while till we come back to this and I've got projects planned that will help act as sort of a bridge to set up the infrastructure to give this another go before I close out I need to say a massive thank you to my amazing patrons channel members and supporters on Kofi this project was pretty expensive so to fully get it working will probably take a lot more time and money when I first started making videos I never imagined that there would be so many amazing people out there that want to support my work and it could let me afford trying something so crazy so thanks a million and if you'd like to help keep the flow of science videos coming then consider kicking a buck or two my way if you like this video be sure to hit that like button and of course subscribe and I've also linked to some of my other more Frankenstein Emad science projects below if all this mammalian cell culture stuff is new to you then I'd highly suggest checking out the meat Barre video as I went into a lot more detail about handling these sorts of cells than I could in this video and of course be sure to head over to my other social media platforms to see these projects long before they end up in videos that's all for now and I'll see you next time

Channel: The Thought Emporium

Views: 2,154,965

Rating: 4.932003 out of 5

Keywords: neuron, ai, human neurons, cell culture, biology, science, tissue culture, sterile, aseptic, electrode, plasma, metal coating, plasma coating, growth media, thermofischer, gibco, electrode array, mea, multi electrode array, bci, brain computer interface, brain, brain cell, cortical neuron, glutamine, gaba, design, tutorial, educational, diy

Id: V2YDApNRK3g

Length: 17min 18sec (1038 seconds)

Published: Tue Dec 03 2019

Reddit Comments

[u/pucakee]

When you don’t have brain cells so you grow your own.

[u/txmail]

I fixed the leg on my table so it is no longer is wobbly. This is cool too though.

[u/lake_chutes]

I saw a video like this a while ago. Some dude grew arrays of neurons on Petri dishes with input and output stages for signals. He eventually trained them like a neural network and hooked them up to a computer so that they could play a jet simulator game... pretty well.

It’s probably one of the coolest things I’ve ever seen I need to find it again

[u/Eyehavequestions]

Didn’t watch video yet.

Does the guy poke it with a tiny stick to see if it’s alive?

[u/G3TR34L]

I did this in my lab today!

[u/spigot66]

What are your thoughts? I don't know let me ask my neuron

[u/asoap]

As someone that knows very little about this stuff. I am super impressed.