DIY-bioprinting

[Raptor Dan]

Hey all,

I found out about DIYbio from an article in the economist, and it
intersects nicely with some research I've been doing. Specifically, I
was working on a low-cost, simple way to manipulate cellular behavior
and did a small project based on hacking a $100 inkjet printer to
print different chemical solutions over fields of e. coli. The whole
process can be adapted for everything from printing chemicals on top
of cells to printing cells (mammalian or bacteria) onto other things.

An example of its use is found here:
http://www.plosone.org/article/info:doi%2F10.1371%2Fjournal.pone.0007086
and the supplemental information explains how to quickly set-up a
printer to do bioprinting. It's my own work and I have some shame
putting in a plug for it, but the system was meant to enable low-
budget bioprinting for all sorts of things and this seems like the
right type of community for that. I've been reading over the recent
postings here and I'm really impressed and I'd be interested to know
what people think of potential applications for this type of approach.

Best,

D

[Ben Gadoua]

   My one problem with that setup is that you can't autoclave it, it would have to be cold gassed to be serilized. You might be able to autoclave just the parts the cells contact... but that gets iffy. If you want to use it for mammalian cell culture you're going to need to find a way to sterilize it.

Ben

[Gene Hacker]

That's pretty cool. Though you might consider moving the print media
under the inkjet using some sort of linear slider mechanism. This
would allow printing on things thicker than CDs. This might entail
messing with the electronics though.

As far as sterilization is concerned you might do what NASA does to
"zero-g proof" their printers. In zero-g the salts in people's breath
can accumulate on PCBs and short connections, in order to prevent
this, all printers and electronics that go into space have their PCBs
coated in a protective non-conductive coating. Doing something similar
might allow one to apply sterilizing chemicals liberally without
worrying about damaging the electronics.

[Simon Quellen Field]

How long would you have to bath the device in UV light to sterilize it?

[Emmette Hutchison]

You could just UV all of the components very carefully.

Emmette

[JonathanCline]

On Sep 21, 11:56 pm, Raptor Dan <d.snrn...@gmail.com> wrote:

>
> An example of its use is found here: http://www.plosone.org/article/info:doi%2F10.1371%2Fjournal.pone.0007086
> and the supplemental information explains how to quickly set-up a
> printer to do bioprinting.

Can you comment on practical issues in using the system (outside that
mentioned in the formal publication), esp. if it is used long term or
across experiments. I have heard others who have tried inkjets
complain of clogging or leaking (for HP hacks), excessive clean cycles
(for EPSON hacks), etc. I have an EPSON cd-inkjet printer for this
type of project although I have not yet hacked the cartridges. I
assume you had to buy a reset chip? I wonder if that might also
workaround the trick mentioned in your Supp Info: "In the case of the
Epson R280, priming must be performed immediately prior to actually
printing, because this is the window where the printer drivers expect
that the system is fully primed and ready to print. Modifying the inks
during this phase essentially tricks the system." Presumably a good
reset chip (especially if it could be custom programmed) could bypass
this.


Any ideas how this step: "milled 800-micron deep wells in CDROM" can
be done DIY?


From reading several papers related to "conductive ink printing with
inkjets", some have measured the dot dispersal pattern and found it
best to modify the printer driver to optimize this. However these
effects don't seem to come into play unless measuring near nano
scales. Your Supp Info mentions this as well. Others have claimed
"modifications to open source driver for custom dot placement" however
I haven't found the original software or modified software mentioned
in the papers.

What hardware integration have you considered for the working system?
i.e. brainstorms about fitting it within a larger protocol flow,
especially an automated (robotics, microfluidics, etc) series?
Several other papers have used CDROMs to good advantage in slightly-
modified CD reader/writers for measurement. Connecting a couple of
these simple-teardown-and-modified systems together with an automated
flow might scale to a barebones "personal bio-analyzer" (idealized
brainstorm). Then, the "hidden" costs of each protocol (mostly,
human time and human error) can start to decrease.

> It's my own work and I have some shame
> putting in a plug for it,

Please plug your own work.

By the way, great paper, as the details of construction & measurement
are clear. (A great weakness in many bio papers. Though I see you're
an engineer & it's coming through.)

Can you upload your photoshop files?


## Jonathan Cline
## jcl...@ieee.org
## Mobile: +1-805-617-0223
########################

[Jake]

Sounds amazing! I remember a lot of potential being discussed when
the tek first came out.

I'd be interested in if it could perform maneuvers similar to a colony
picker, but in reverse form. Say you want to lay out a pattern of
mostly isolated bacteria or spores in order to examine or pick them
more easily. Would the heat from the print head spraying process
destroy your culture? Also what types of solvents or carrier systems
are used? Are they expensive? Is there a cheap bio-compatible
delivery solvent? I realize standard ink had a good bit of water in
it, but will straight dH2O work?


-Jake

[Nathan McCorkle]

On Tue, Sep 22, 2009 at 10:09 AM, Gene Hacker <cam...@gmail.com> wrote:
>
> That's pretty cool. Though you might consider moving the print media
> under the inkjet using some sort of linear slider mechanism. This
> would allow printing on things thicker than CDs. This might entail
> messing with the electronics though.
>
> As far as sterilization is concerned you might do what NASA does to
> "zero-g proof" their printers. In zero-g the salts in people's breath
> can accumulate on PCBs and short connections, in order to prevent
> this, all printers and electronics that go into space have their PCBs
> coated in a protective non-conductive coating.

In a lab I work in now, we make buoys for sensing stuff in rivers, and
we coat the PCBs with a silicone spray to avoid water or salt
failure.... NASA could have something different, but it also seems
like a pretty solid method. I'll get the name of the spray tonight or
tomorrow.

> Doing something similar
> might allow one to apply sterilizing chemicals liberally without
> worrying about damaging the electronics.

I guess it depends how well the silicone spray cures... would not be
hard to find out!

--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics

[William Heath]

I have heard and seen an inkjet printer altered to print heart cells in such a way that it created a 3d object, they said they were using some special "adhesive" to cause the cells to bind together that was bio compatible.  At the end of the printing the heart actually began to beat!  I would love to reproduce this.  Any ideas how I can do that? 

[Nathan McCorkle]

On Tue, Sep 22, 2009 at 11:24 AM, JonathanCline <jnc...@gmail.com> wrote:
>
> On Sep 21, 11:56 pm, Raptor Dan <d.snrn...@gmail.com> wrote:
>
>>
>> An example of its use is found here: http://www.plosone.org/article/info:doi%2F10.1371%2Fjournal.pone.0007086
>> and the supplemental information explains how to quickly set-up a
>> printer to do bioprinting.
>
> Can you comment on practical issues in using the system (outside that
> mentioned in the formal publication), esp. if it is used long term or
> across experiments.  I have heard others who have tried inkjets
> complain of clogging or leaking (for HP hacks), excessive clean cycles
> (for EPSON hacks), etc.  I have an EPSON cd-inkjet printer for this
> type of project although I have not yet hacked the cartridges.  I
> assume you had to buy a reset chip?  I wonder if that might also
> workaround the trick mentioned in your Supp Info: "In the case of the
> Epson R280, priming must be performed immediately prior to actually
> printing, because this is the window where the printer drivers expect
> that the system is fully primed and ready to print. Modifying the inks
> during this phase essentially tricks the system."   Presumably a good
> reset chip (especially if it could be custom programmed) could bypass
> this.
>
>
> Any ideas how this step: "milled 800-micron deep wells in CDROM"  can
> be done DIY?
>

Apply a photoresist with a spincoater. Perform lithography, wash away
non-solidified material, etch exposed areas for a length of time (this
may or may not be previously characterized, so experimentation with
etch times will probably be needed), wash away photoresist.

depending on how wide the channels are will determine if you could do
this with a laser printer or not, but quite possibly. Somewhere in the
archive here, I believe the width of a toner molecule was discussed,
as well as the minimum line width too.

[Nathan McCorkle]

On Tue, Sep 22, 2009 at 1:39 PM, William Heath <wgh...@gmail.com> wrote:

> I have heard and seen an inkjet printer altered to print heart cells in such
> a way that it created a 3d object, they said they were using some special
> "adhesive" to cause the cells to bind together that was bio compatible.  At
> the end of the printing the heart actually began to beat!  I would love to
> reproduce this.  Any ideas how I can do that?

That it began beating is quite hard for me to believe. The heart can't
be made of homogeneous cell types, can it? I mean, muscle fibers vs
ventricle walls must be expressing different proteins... to me organs
are things that must start out as a stem cell (or group of them),
differentiate, and expand into the final shape

Do you have a citation for that "heart printing"?

[William Heath]

On Tue, Sep 22, 2009 at 10:43 AM, Nathan McCorkle <nmz...@gmail.com> wrote:

On Tue, Sep 22, 2009 at 1:39 PM, William Heath <wgh...@gmail.com> wrote:

> I have heard and seen an inkjet printer altered to print heart cells in such
> a way that it created a 3d object, they said they were using some special
> "adhesive" to cause the cells to bind together that was bio compatible.  At
> the end of the printing the heart actually began to beat!  I would love to
> reproduce this.  Any ideas how I can do that?

That it began beating is quite hard for me to believe. The heart can't
be made of homogeneous cell types, can it? I mean, muscle fibers vs
ventricle walls must be expressing different proteins... to me organs
are things that must start out as a stem cell (or group of them),
differentiate, and expand into the final shape

Do you have a citation for that "heart printing"?

I have something better than a citation, an actual video of them doing it:

http://singularityhub.com/2009/06/08/growing-organs-in-the-lab/  (look at the video at the bottom, it says Daily motion on it)

This video actually shows a hewlett packard inkjet printer that is modified as follows:

1.  The three ink chambers of the printer ink cartridge are populated with cells

2.  A heart is printed one layer at a time (They simply print on a 2d plane, then lower the plane slightly for the next layer, until the 3d heart is created)

3.  He actually shows the heart beating after 4 hours of the bio printed heart

Help me to reproduce this, its amazing!!!!  Give me practical knowledge, what is the medium he is printing into, what is that solution?  Is he using chicken heart cells, rat heart cells, what?  What is he putting in the inkjet printer chambers exactly?  This is so killer to me! 

[Paul Anderson]

On 2009-09-22, at 1:43 PM, Nathan McCorkle wrote:

>
>>
>
> That it began beating is quite hard for me to believe. The heart can't
> be made of homogeneous cell types, can it?

I recall this as well. Mind, he said beating, not that it was pumping
anything. Heart cells have a sympathetic mechanism that can, in some
circumstances, cause them to beat even without a signal from the
nervous system.

---
Paul Anderson
wacky...@gmail.com
http://www.andersonloco.com

[William Heath]

On Tue, Sep 22, 2009 at 5:19 PM, Paul Anderson <wacky...@gmail.com> wrote:

On 2009-09-22, at 1:43 PM, Nathan McCorkle wrote:

>
>>
>
> That it began beating is quite hard for me to believe. The heart can't
> be made of homogeneous cell types, can it?

I recall this as well.  Mind, he said beating, not that it was pumping
anything.  Heart cells have a sympathetic mechanism that can, in some
circumstances, cause them to beat even without a signal from the
nervous system.

The video shows it beating, it is not debatable :>

[Raptor Dan]

Thanks for the comments, and I hope this system proves useful!

Sterility: It wasn't much a problem for bacteria. 70% EtOH wipe downs
between trials seemed to be sufficient. Mammalian cultures are a whole
different story, and this set-up can't easily print on mammalian cells
unless they are cultured in a hydrogel.

Printer Logistics:

I flushed all the print lines with sterile DI. You can also use
various alcohols, and I have seen reports that methanol is effective.
Hot water can also greatly help.

As for reset-chips, they are (a) illegal and (b) ineffective with
certain models, including the R280. Moreover, it's hardly worth
hacking the system if all you need to do is load it at a specific
time. However, if your system is compatible with a chip, it may be
more efficient...

Milling is convenient, but laser cutting would have been better. In a
DIY context, you can hand-cut out a window and glue transparency film
the bottom to make a reservoir. Alternately, this can be done
(carefully and slowly) on a drill press.

System integration would be fun, and definitely possible. It really
depends on what you want to do. I am planning to integrate an optical
system into it to image in real time, and I am also intending to mass
produce the CD cartridges with a laser-cutter, thereby allowing me to
print much large surface areas.

Files: I'll upload the printer templates once I dig them out of my
archives.

Printer-dynamics: Epson uses piezo print heads which have no
appreciable thermal effects, so they are 'safe' in that sense.
However, viable cells of all kinds have been printed with thermal jet
systems, so I wouldn't worry too much on that score. In terms of the
suspension media, I mention that I printed saturated sugar solutions.
The key is that the viscosity of the fluid must lie in the range of
the print head. A lot of the optimization that goes on with printer
design/hacking is to optimize ink settings based on expected
viscosity.

Organ-printing: I am also looking into this, but this is not something
you can do at home without incubators, expensive media, hydrogels,
microscopes, and an extremely sterile field (i.e. hood). You can,
however, print bacterial cells with no problem, although I admit that
is less exciting.


As a quick safety warning--DMSO is very dangerous and will penetrate
straight through gloves and skin. If you smell a garlicy smell when
you aren't near any DMSO, it may mean that it's gotten into your
blood. I had no problems and it won't eat you from the inside out or
anything, but it will carry whatever is mixed with it into your
tissues.

Keep on brainstorming, and try out the system! The expensive
components are the X-Gal and DMSO. You don't even need a microscope
for large samples.

[J. S. John]

On Tue, Sep 22, 2009 at 8:26 PM, William Heath <wgh...@gmail.com> wrote:
> The video shows it beating, it is not debatable :>
>

Thinking about this, the cells may be only providing structural
support? For a heart to beat on its own, you would need the pace maker
cells [SA and AV nodes] and the nerves. I think the pumping may be due
to artificial means of moving fluid through the chambers. It's not too
clear from the video. Any ideas?

--
Folding@Home
http://folding.stanford.edu/
Team Maximum PC Magazine (Team ID: 11108)
Rank 3

[Ben Gadoua]

  John;


 Cardiomyocytes will start to spontaneously beat on their own. They usually beat in unison because of calcium uptake and regulation. The rhythm of the heart in the video is certainly not a sinus rhythm, but it is a beat. Many times you can see groups in culture beat in unison from primary cardiomyocytes or from immortalized cardiomyocytes such as HL-2 cells.

Ben

[Tito Jankowski]

Here's an example of a hacker doing bioprinting with plant tissue,
thanks to Yashas for showing me:
Allison Kudla: http://www.washington.edu/dxarts/profile_research.php?who=kudla&project=EdenBumber

This system uses a computer controlled four-axis positioning table to
"print" intricate bio-architectural constructions out of moss and
seeds. Suspended in a clear gel growth medium, the moss continues to
grow and the seeds sprout. The algorithmically-generated patterns
drawn by the system are based on the Eden growth model and leverage
mathematical representations of both urban growth and cellular growth,
thereby connecting the concept of city with the concept of the
organism. This project is working to make concrete the idea of dynamic
and fluid computer space altering the expression and formation of a
living and growing biological material, via its collaboration with an
engineering mechanism. -- from Allison's site

Tito

[William Heath]

Hi Tito and All,

Thanks soooo much for this link!  This answers some of my major questions on how to do the following:

1.  Load the ink wells with the bio paper (agar)
2.  Load the ink wells with cells

My other big questions which I continue to be puzzled about is how to print 3d structures.  Based on the video I posted about the heart printing they are printing into a well with some kind of fluid in it.  Is that fluid hydrogel?  There is also a platform that lowers for the next layer of cells.  I am confused how to do this part.  Any mechanical engineers who know how I might accomplish that goal on an inkjet printer?  I guess I would need to drill some kind of hole to put the well in and then somehow move the platform in x-y based on the paper feeding etc... and also lower the platform in z between that.  I am quite open to ideas on the best way to do that!

-Tim

P.S.

I was told the best way to design this would be with solid works but I am a mere mortal.

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[Jake]

> As for reset-chips, they are (a) illegal

Don't know where you are but in every country in the free world that I know of they are perfectly legal.

As far as sterilization... Why not just run it like a flow hood or sterile transfer room. You have a UV light and mostly leave it on all day except for when you're actually using it. You can get UV bulbs of just about any size, with many of them in the $10-15 range. I got one from 1000bulbs.com awhile back for about that price. It might be hard on the plastic or eventually damage any optical sensors in the system, but the actual printer is pretty cheap. I could get pallets of random used inkjets for basically nothing at surplus. In lots of places you actually have to pay to have them recycled, which a lot of people do when they find out it's almost cheaper to buy a new one rather than replace the cartridges.

-Jake
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[Nathan McCorkle]

On Tue, Dec 29, 2009 at 1:55 AM, Jake <DIYbio.li...@o-bio.org> wrote:

> As for reset-chips, they are (a) illegal

Don't know where you are but in every country in the free world that I know of they are perfectly legal.

As far as sterilization... Why not just run it like a flow hood or sterile transfer

This is something I don't always understand exactly why people complain about... you can electroporate an antibiotic resistance plasmid and culture them in drug media. You would need figure out how long until the cell kicks out the plasmid and try to adjust your drug concentration accordingly, but if you use a system that breaks down the drug instead of pumping it out, I think it would work well.

 

room.  You have a UV light and mostly leave it on all day except for when you're actually using it.  You can get UV bulbs of just about any size, with many of them in the $10-15 range.  I got one from 1000bulbs.com awhile back for about that price.  It might be hard on the plastic or eventually damage any optical sensors in the system, but the actual printer is pretty cheap.  I could get pallets of random used inkjets for basically nothing at surplus.  In lots of places you actually have to pay to have them recycled, which a lot of people do when they find out it's almost cheaper to buy a new one rather than replace the cartridges.


-Jake
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