Animal slaughter -- keeping it alive in a petri

[Nathan McCorkle]

I've been volunteering on a farm that aims for low-input (chems or
work) permaculture. The western Oregon climate is similar to that of
Japan and New Zealand, so lot's of rain and green plants all year
round! The butter is still yellow come winter!

Next week I'm watching/helping with a pig slaughter, so I was thinking
I might try spinning some blood and collecting the plasma and trying
to explant a chunk of heart. I don't have trypsin, though I do have
bromelain, and amazon.com has some digestive tablets that contain
trypsin. Though I don't have micron filters to filter the tablet
solution.

Anyway, I've done this with chicken embryo heart, can't remember what
media I used, but it was probably Eagle or DMEM.

Any ideas for making this work, am I missing something? Will plasma
sustain the cells for a day or so? I think my chicken heart cells
didn't start beating until after a day or two in culture previously.

--
-Nathan

[Patrik D'haeseleer]

Hi Nathan!

Just did some experiments in decellularization two weeks ago. We were going to do a whole pig heart, but the heart we'd gotten was missing to much of the vasculature to do the perfusion.

We did get to try some health food store digestive enzymes on heart slices - both bromelain and papain worked pretty darn well. We didn't try to grow any cells on them afterwards, of course...

Here's some pics - still need to add a bunch more:

http://www.flickr.com/groups/2303889@N24/pool

Patrik

[Nathan McCorkle]

Patrik, there are some great pics in there!

Do you happen to know if there's a 'before' pic in this arrangement?
http://www.flickr.com/photos/yeahsnos/10006833654/sizes/o/in/pool-2303889@N24/

What were you going for with the proteases? Wouldn't that destroy the
ECM, the scaffold?

I found some PBS (phosphate buffered saline) on amazon.com for
~$5/liter (when bought in 10L quantities)... but I think I'll just
make up some sterile isotonic salt solution with NaCl and KCl from the
food store (for rinsing the heart chunk of blood). Not sure how long
the cells would survive just in saline, though I haven't looked into
easy/DIY growth media. I figure I could spin down some blood and
collect the plasma, but I think I'd still need to filter that to get
'serum'.

Maybe it will be enough to keep the cells happy for a day or two... I
seem to remember my chicken explant and trypsinized culture started
beating within a day or two.

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--
-Nathan

[Nathan McCorkle]

I found this (1) paper on measuring action potentials of a pig heart.

They basically just mixed a 50:50 blood and a salt sultion called
Tyrode's Solution.
en.wikipedia.org/wiki/Tyrode's_solution

A few other papers also used similar balanced salt solutions, along
with 10% serum and maybe some antibiotics/antimycotics.

I got some MgCl2 _CaCO3 tablets and monobasic sodium phosphate saline
solution (has about 1/4 as much dibasic in it too) from the pharmacy.
I hope it'll approximate the Tyrode's solution well enough, I'll just
use food store NaCl and KCl and some dextrose I have too.

Then I plan on spinning down some blood at high-speed and mixing it
50:50 with the salt solution, then filtering through a 0.4 or 0.2
micron filter. Rinse the heart chunk a few times with sterile saline,
add to the serum+saline, and hope for the best.


1
http://xa.yimg.com/kq/groups/10355996/932940578/name/ST_TQ_during_ischemia.pdf

2
The Early Detection Research Network (EDRN)
Standard Operating Procedure (SOP) For Collection of Serum
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2655764/bin/NIHMS94453-supplement-1.pdf

--
-Nathan

[Eugen Leitl]

On Fri, Oct 11, 2013 at 01:18:34PM -0700, Patrik D'haeseleer wrote:
> Hi Nathan!
>
> Just did some experiments in decellularization two weeks ago. We were going
> to do a whole pig heart, but the heart we'd gotten was missing to much of
> the vasculature to do the perfusion.

Hey, I missed that one the first time, for some reason.

What is the procedure by which you obtained your heart? Is it legal
to obtain animal organs from slaugherhouse for research purposes
without a permit in your jurisdiction? How long are the ischemic times
in your experience, and how badly are the organs mangled to the
point where they're useless?

[Nathan McCorkle]

On Sat, Oct 19, 2013 at 7:48 AM, Eugen Leitl <eu...@leitl.org> wrote:
> On Fri, Oct 11, 2013 at 01:18:34PM -0700, Patrik D'haeseleer wrote:
>> Hi Nathan!
>>
>> Just did some experiments in decellularization two weeks ago. We were going
>> to do a whole pig heart, but the heart we'd gotten was missing to much of
>> the vasculature to do the perfusion.
>
> Hey, I missed that one the first time, for some reason.
>
> What is the procedure by which you obtained your heart? Is it legal
> to obtain animal organs from slaugherhouse for research purposes
> without a permit in your jurisdiction?

Eugen, not sure if Patrik's experience differs, but from what I've
found online the Animal Welfare Act (anti animal abuse laws, including
research) only cover how an animal 'feels', so presumably none of it's
laws cover after the feeling center (brain) has been gouged (shot,
cored with a pneumatic bolt, etc). I emailed some law firm who deals
with animal rights, I'll let you know if they respond.

I think in my case at least, since the animal was raised for food, I
would simply be playing with a food product... so I don't think animal
research applies at all. Obtaining it is me going to the farm on a
slaughter day when the customer hasn't requested to buy the heart.

[Cathal Garvey (Phone)]

I'm surprised by the query. In Ireland, a visit to a butcher will get you high quality hearts no problem; they're eaten by some as a delicacy.

--
Sent from my Android device with K-9 Mail. Please excuse my brevity.

[Eugen Leitl]

On Sat, Oct 19, 2013 at 09:20:08PM +0100, Cathal Garvey (Phone) wrote:

> I'm surprised by the query. In Ireland, a visit to a butcher will get you high quality hearts no problem; they're eaten by some as a delicacy.

A sane person would indeed assume that there would be
no difference between using slaughterhouse organs for
food or research. In Germany, there is a difference.
You need to get a permit -- probably pro forma, but
I'm not even sure about what the requirements are
until we ask.

So much for sanity.

[Nathan McCorkle]

Is there a difference if you go directly to the farmer? Here in the
U.S. you can sell live animals, and slaughter them for your own
consumption without any govt relations. If you want to sell that meat
in 'cuts' by the pound, you need a govt approved slaughter house to do
the job.

[Eugen Leitl]

On Sun, Oct 20, 2013 at 10:57:11AM -0700, Nathan McCorkle wrote:

> Is there a difference if you go directly to the farmer? Here in the
> U.S. you can sell live animals, and slaughter them for your own
> consumption without any govt relations. If you want to sell that meat

German farmers cannot legally slaughter their animals, they need a (EU-regulation-compliant,
I think kosher/halal butchers are exempt, which results in cruel and unusual
punishment versus a relatively low-stress and quick electrostun/exsanguination kill)
butcher to do it.

Despite all the recent improvements, there are a lot of double standards and hypocrisy
prevalent in treatment of food versus experimental animals, especially in the
companion animal category.

[Patrik D'haeseleer]

I got the original hearts via Cameron, one of our BioCurious members whose sister has a farm where they raise pigs. Unfortunately, the heart was butchered to be used as food, which means they cut off the major arteries, and put a slit through the chambers to let the blood drain. Sigh...

I'm getting five (5!) fresh pig hearts tomorrow, from a meat wholesaler in Berkeley that was recommended bythe good folks at the California Academy of Sciences. Apparently, the trick is to ask for a "bio-heart", meaning a heart that is intended for scientific experiments. The tradeoff is that they are not certified as food-grade - presumably because USDA regulations specify that you have to cut off the inedible bits.

Patrik

[Stephen Van Sickle]

On Mon, Oct 21, 2013 at 10:44 PM, Patrik D'haeseleer <pat...@gmail.com> wrote:

 

I'm getting five (5!) fresh pig hearts tomorrow, from a meat wholesaler in Berkeley that was recommended bythe good folks at the California Academy of Sciences. Apparently, the trick is to ask for a "bio-heart", meaning a heart that is intended for scientific experiments. The tradeoff is that they are not certified as food-grade - presumably because USDA regulations specify that you have to cut off the inedible bits.

Let us know how it goes.  Do you have any idea how old the hearts are when the wholesaler gets them?

steve vs 

[Patrik D'haeseleer]

On Tuesday, October 22, 2013 6:19:12 PM UTC-7, sjv wrote:

Let us know how it goes.  Do you have any idea how old the hearts are when the wholesaler gets them?

No idea whatsoever - I haven't asked, since it really doesn't matter for what I'm doing. I assume they've been slaughtered the same or the previous day, but you'd probably want to know an answer in hours, not days.

Also, since I'm dealing with a wholesaler, I'm not a terribly high priority for them. Apparently they screwed up and didn't even put in my order, so now I'm getting the hearts only on Thursday. Well - one more week to get the ghost heart done by Halloween!

Patrik