Decellularizing tissue
  • chironexchironex March 2016
    So here's a fun one. Decellularization is the process by which you remove all (most) of the cellular material from a piece of tissue leaving only ECM (or cell wall if you're using plant tissue) behind. Normally this is done to animal tissues and is one of the ways they are exploring to make replacement hearts/organs for humans. Once you've got the decellularized tissue you can grow any new tissue on the scaffold. A researcher here in Ottawa spun the concept a bit and is decellularizing apples and growing human tissues on them. I've wanted to try this for a long time and fruit seemed like a great place to start so I gave it a try. I'm currently running a variety of fruit and veg and once that's done i'll move onto animal tissues. The thing that worked first was a couple slices of strawberry. I've also go kiwi, mushroom, a full strawberry, broccoli and apple slices going through the process and should be done in the next day or two. The process is super simple for fruit. Just slice, freeze for 20-40 minutes in a normal freezer and then soak in 0.5% SDS for a couple days. I change the sds every 5-6 hours to help draw as much out as possible. With animal tissue you gotta soak it in some other stuff first and it changes based on what sort of tissue. Also you should freeze it to -80 rather than -20. 

  • I heard a story awhile back about someone growing a finger back using ECM. It might be interesting to look at putting a thin layer on magnets.

    Is SDS cheap?
  • chironexchironex March 2016
    As glims so elegantly put it, SDS is science talk for soap. Stuff is dirt cheap, easy to get on amazon, especially if you live in the states. 
  • CassoxCassox March 2016
    I've wanted to do this for a long time. Dude, your awesome.
  • chironexchironex March 2016
    Thanks :) went to the grocery store today and they actually had some liver. Gonna do skin and liver for the next couple days. ought to be cool

  • glimsglims March 2016
    i ended up doing a horse heart about a month ago. remember to adjust your protocol for your materials accordingly. horse was way more difficult than pig, due to fat content and muscle density. you'll note that the strawberry seeds totally rejected the process. it is tho, really easy to do.

    i have some modified protocols for animal tissues that are in an errant lab notebook that is in austin (meaning no one responds to anything till sxsw is done).  The original process is found here : http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2945869/
    beyond that, there are a few modified versions online.

    notes:
    peracetic acid is just h2o2 and vinegar (check your molar amounts and adjust for concentrations to get a proper 1 to 1 ratio), sds is soap, trypsin is a protease and meat tenderizer will work in a pinch.
  • chironexchironex March 2016
    Ya as im experimenting with it im finding that different tissues are responding very differently. The strawberries were easy but the more woody/dense stuff is much slower. The mushrooms I tried released lots of cellular material but still remain mostly brown. Definitely going to need to modify the procedure for them. Excited to start messing with animal tissues. Was looking at a liver protocol. They used ammonia and triton x100 but I presume sds should work just fine in place of triton. May give the ammonia a try though. They used .1% ammonia and 1% triton. I was thinking of doing .1% ammonia like they did and switch in the .5% sds i've been using. If it doesn't work I'll increase the sds concentration or treat it with meat tenderizer first. Will take some experimentation but honestly I'm ok with that. 
  • chironexchironex March 2016
    So the liver upon further inspection is totally devoid of arteries/veins. Least any big enough to make use of. So for the first go I'll just soak the thing. I'm soaking it in weak meat tenderizer solution then gonna do a wash and switch to sds/ammonia. No clue if it'll work properly but worth a shot. But found a few leads on bigger/more complete organs to make this go better. Will keep updating. 
  • chironexchironex March 2016
    New pump arrived today. The liver is already looking incredibly pale compared to what it started as. I figure it'll be a nice white by tomorrow. It's also begun to float. Rather than the silvery stuff the comes out of fungi/plants the liver releases a red substance. Not really surprising but interesting. 

    My setup LINK
    Red stuff LINK
    Liver at the start LINK
    Current liver LINK1 LINK2(it floats!)

    Also the other fruit and stuff are basically done. Giving them one more day to be sure since they have a bit of color left. Also the mushrooms don't lose their color but do lose the cellular material. They go more transparent but not the nice white the strawberries do, so not much to look at. The kiwis are almost totally clear which is cool. Can almost see right through them. The skin retained all of its color though.  

    Broccolli LINK
    Kiwi LINK
  • ElectricFeelElectricFeel March 2016
    But what do they taste like?
  • ZerbulaZerbula March 2016
    And will it blend? :o

    But really, nice work. :3
  • chironexchironex March 2016
    Finally done the fruit and veg and made a video going through the whole process LINK


    The liver is being a pain and taking a lot longer than I'd hoped. I ended up moving it into a sonication bath and have been treating it for 15-20 minutes at a time then allowing it to cool back down to room temp. Lots more cellular debris is coming out now and it's getting much more transparent. I'll post pictures when I have something exciting to show. Once it's closer to done i'll do a last wash in peracitic acid to remove fats and such then into di before preservation.
  • glimsglims March 2016
    Are you using the alternating high saline and then di washes? it seems to really help, yknow, osmatically destroying the cells. A lot of the steps seem arbitrary, but once you break it all down, they are all there for a reason. Especially that peracetic one. I was concerned nothing was working right till i got to that point.
  • chironexchironex March 2016
    The ultrasound really seems to be helping. Doing a high saline wash right now then di. It went really white after a minute or two in the saline so it seems to be helping. I'll keep you posted.
  • ChrisBotChrisBot March 2016
    I know this is very childish, but I would love to see you grow some muscle tissue in the berries, and some plant tissue in the liver. I am sure you could associate it with some artistic meaning...something along the lines of "Everything you can imagine is real"

    Meat berries, and veggie liver!
  • chironexchironex March 2016
    Childish? LOL i have been thinking the same thing. This whole thing is weird and fun and exciting and messes with your perception of things. Was thinking decellularizing a steak and growing some plant tissue on it. Would make for the most confusing veg burger of all time.
  • chironexchironex March 2016
    Ok so after high saline/di and another sds wash, I moved on to peracitic acid. WOW did it help. Here's where it's currently at LINK

    I've got it in di and am gonna see how it looks in a little while. May need to do one more wash cycle with sds or saline or something to finish it off but it looks so much better. 
  • JohnDoeJohnDoe March 2016
    Another childish thing to ask.... Have you thought about trying to grill the barries after you grow the tissue in them? Or even just grill the cell walls? This is making me hungry....
  • chironexchironex March 2016
    Ok so here's a video going through the whole decell process. As I mention in the video this whole process has been far from ideal and is more a practice run than anything, but Im pretty happy with how it turned out.

  • chironexchironex May 2016
    So the heart I ordered arrived today...... LINK1 LINK2

    Thing only cost me 5 bucks XD halfway through the process and it's already looking way lighter. Will keep updating as I make progress. The picks are from the beginning of the process. My friend who was filming the whole ordeal almost passed out. It was amazing.
  • chironexchironex May 2016
    Process is totally done!!!!! I'm now the proud owner of a decellularized heart. Check this out: LINK
  • Sweet! When do you start making cyber-hearts?
  • Awesome.
  • So I'm curious what the purpose of decellularizing a heart is or other tissue?

    And after seeing your video of ghost fruit it reminds me very much of a tomato at 90ft below the surface in the ocean. Still cool though.
  • chironexchironex May 2016
    so there are a few reasons to decell tissue. The most obvious is that you could re-seed the scaffold with your own cells and grow yourself replacement parts. The other is that the scaffolds themselves retain some of the biomarkers that tell cells "this cell type goes here". So if you decell skin, and freeze dry it, you could use the resulting powder to make topic stuff for healing. I'm working this concept into my transdermal paint for example. 
  • I see. My question then would be how do you preserve said organ while removing the cells in order put yours in its place then use the organ/tissue?

    Would this be strictly for artificial so you have less chance for rejection?
  • chironexchironex May 2016
    that what the decellularization process does. that white is just connective tissue with all the cells removed. Seeding it with new cells is really straightforward from that point. The only change is that I'd use different regaents to do the process in order to better preserve the scaffold. So switch SDS with triton x100, peracitic with deoxycholic and bromolain with trypsin. Once all the cells are removed there's nothing to reject. You seed if with your own cells. It is literally you when all is said an done. 
  • Ok so by removing the cells it is preserving the organ. I was under the impression that the organ is actually dying during that whole process and is dead once finished. (I mean un useable in the human body).
  • chironexchironex May 2016
    well, it was pretty dead when I got it, what with it being outside of the thing it used to be attached to. This isn't like a heart transplant where you need to get it into the recipient asap. This is totally different. Once it's decellularized, there's nothing left other than collagen and other connnective tissue. When you seed it with your cells, they colonize the scaffold and eventually form a new heart. Then from there you get it beating again. Once it's fully formed and beating you can transplant it. It starts "dead" and you put life back into it in a sense. 
  • JohnDoeJohnDoe May 2016
    When you say "dead" I assume you mean that there is just nothing to animate it right?
  • Nothing to animate it, and nothing to animate in the first place.
  • Ok i think got it now haha. When it comes to biology i know almost nothing.
  • chironexchironex May 2016
    And many hours of editing later, the video documenting the whole process is finally done. LINK
  • Is there a specific brand or type of soap you would use?
  • That's damn cool. I've been reading about custom built meshes from organic materials for growing organs, but this could work out so much better. I don't know much about biology (first degree was geology) so potential stupid question: Would this process have better prospects for getting around rejection issues in organ transplants or are the things (proteins?) responsible for rejection still left over after decellularization?

    Love the bismuth geode (browsing your Instagram). I still can't get my geodes to come out how I want. I think it's an issue of them cooling too fast. My regular crystals come out nice and big because they form in a larger pan and cool more slowly, but some idiot built the AC vent to blow right on the stove, which makes controlling the temperature difficult. I need to invest in a hot plate.
  • chironexchironex May 2016
    you need pure sds or triton. The former you can usually get online very easily. 
  • chironexchironex May 2016
    @katzevonstich yup, that's why this technique is getting so much attention. You'd literally grow yourself a new organ. And it is you since you use your own cells to seed it so no risk of rejection. 

    I use a blow torch to heat my stuff up so it's pretty toasty when I pour it into the moulds. I think I'll make a video showing my process this weekend. I already have the footage anyway. 
  • So after you decellularized the organ, how do you recell it? Do you just inject it with stem cells? Or is it more complicated?
  • chironexchironex May 2016
    Here's a link to a paper going over the process LINK
  • chironexchironex November 2016
    Just finished writing an article about my time working on decellularization. 
    LINK
  • misslittymisslitty November 2016
    This is beautiful and a great read!
  • BenbeezyBenbeezy November 2016
    I was actually looking this stuff up and re found the forum hah I'm going to start working on printing the structure based off of CT scans of healthy organs. And see if I can get parts printed and re cell the structure with something like you said, cow cells.
  • CassoxCassox November 2016
    Yeah. Beautiful pics.
  • _Larry__Larry_ November 2016
    Can you seed it with cells from that particular organ? Or could you use your stem cells? I was thinking about the possibility of growing a new pancreas for diabetic patients. Since this isn't a "medical" study you can take many more liberties than the fda would allow.
  • ThomasEgiThomasEgi November 2016
    judged from number of people benefiting I'd vote for liver. Alcohol and obesity damaged way too many of those all around the globe. DIY organ kits would be quite fun to see. get that second heart and other timey whimey things.
    In all seriousness I'd be very much interested in how this progresses. Are stem cells really required or would a bunch of healthy adult cells populate too?
  • chironexchironex November 2016
    Adult cells will probably work. Though I think for some cell types it would be easier to start with stem cells, seed the scaffold and then transform them into the final cell type. Like neurons for example. Would be far more mobile when they aren't all intertwined or have an axon hanging off them. I think myocytes would be the best first thing to try
  • glimsglims November 2016
    Gotta go stem with this. If you just try normal human adult cells, they aren't going to grow the way you want. And if you use immortalized cells, then they aren't yours and they're weird.
    But you can't just scrape out some cells and keep them growing on a substrate. 
  • tbaleanntbaleann November 2016
    I was wondering if you could make a coating out of decellularized tissue then seed it with your cells for trans dermal implants that would be without the microscopic gap due to your skin bonding directly with coating.
  • chironexchironex November 2016
    I mean that's basically what we're hoping to do, but rather than worry about seeding it with cells, we're making the coating so that cells just grow into it naturally
  • ZwytechhackerZwytechhacker November 2016
    @cironex But what about the tissue how do yu get it to actually coat the implant?
  • TheGreyKnightTheGreyKnight November 2016
    If you want to get technical, they aren't coating. They're growing into extracellular matrix. 

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