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William R. Ledoux Ii
09-11-1996, 05:04 AM
Last month I posted the following question:

I am interested in the effects of freezing on biological tissues.
Specifically, does long term freezing (6 to 18 months) have any
detrimental effects on the mechanical properties of soft tissue? I am
concerned that the tissue may suffer from 'freezer burn'. However, if
this problem is avoided by proper handling and wrapping procedures, is it
possible to assume that any damage to the tissue will occur during the
initial freezing process and the length of time the specimens remain
frozen does not matter?

A summary of the responses:
several relevant references are given below
the colder you freeze the better
the faster you thaw the better
freezing is accepted as a necessary and unavoidable part of experiments.

The full text is included below.

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I don't know about 6-18 months but we have done some studies on the effects
of freezing on the compressive creep properties of the porcine
intervertebral disc and we have seen significant differences in the
time-dependent properties after three weeks of freezing. The
time-independent properties, however, were not affected. We looked at the
data using a 3-parameter viscoelastic model (see 42nd ORS p677) and using a
3-parameter fluid transport model (data to be presented at the ASME meeting
in Atlanta in November).

Elisa Bass
bass@euler.me.berkeley.edu

************

Concerning your enquiry about the effects of freezing on the mechanical
properties of the soft tissue, we did a study of the human spine specimens
some of which had been frozen upto 230 days. The referance is given below:

Panjabi MM, Krag MH, Summers D, Videman T. Biomechanical Time-Tolerance of
Human Spine Specimens. J Orthop Rsch 3:292-300, 1985.

Manohar M. Panjabi
panjabi@biomed.med.yale.edu

************

There is a huge literature on freezing tissues listed in "biocryogenics".
The major problem concerning the cellular component is the rate of cooling
(rate of freezing) and the rate of rewarming where osmotic effects can
rupture cell membranes. If you are intersted in extracellular things like
collagen and elastin then the packaging becomes the main concern since
the mechanical properties of these proteins depend on the hydration level.

Freezer burn will definitely change mechanical properties of collagen and
elastin but can be prevented by hermetically sealing in a dry or inert gas
atmosphere.

Once you have optimized the cooling rate and storage conditions you should
be able to keep tissues in stable condition for a very long time.

Marvin Sherebrin
email: sherebrin@uwovax.uwo.ca

************

I had a professor in biomechanics at Michigan State
University that was involved in tissue research who
might be able to shed some light on your quiry. His
name is Roger C. Haut PhD. You can find him by
contacting the engineering department or the
biomechanics department in the College of
Osteopathic Medicine at MSU.


J. Tim Zipple PT, MS

************

off the top of my head, i know that RC Haut has looked into this issue. i
will look through my notes and if i can find additional info, i will let
you know.

Hugh Magen
hemagen@ucdavis.edu

************

I'm in the latter stages of completing my Master's on this topic.
Cryobiologists, who are quite concerned with cell survival and not so
much with the retention of mechanical properties, suggest that tissue can
be immobilized indefinitely (theoretically) if frozen in liquid nitrogen
(-196C) or at any temperature below the vitrification point (-130C). If
you are freezing in an ultracool biological freezer (-76C to -86C), the>
tissue will probably be stable, but there is a slight uncertainty. If
you are freezing in a regular freezer (-20C), cross your fingers.>
In terms of retaining mechanical properties (which I am looking t), I
have not yet completely analysed my data, but intuition would sa, the
colder the better. Also, be aware that most of the damage occurs in the
freezing and thawing stages, especially the latter. The rate that you
warm your tissue may be critical. If you are starting at a very low
temperature, you will want to thaw quickly. However, there is a bit of a
grey area in deciding what rate to thaw at from arelatively high sub-zero
temperature.

In addition, consider how much of a change in properties is acceptable.
0.5%? 5%? 50%? Don't plan on only getting a 0.5% change at any temp.
Probably any temp. will give less than a 50% change. Once again, it is
grey around 5-15% difference from fresh.

Anyway, good luck. Hope this has been helpful.

P.S. Proper handling techniques should avoid freezer burn at low
temperatures, and I think that freezing is so quick (for small specimens
like tendons or ligaments) in liquid nitrogen that wrapping procedures
are not even required. (Wrapping is mainly to prevent dehydration of the
tissue.)

Jeff Cassin
cassin@conn.me.queensu.ca

************

To: wrledoux@eniac.seas.upenn.edu
Subject: Re: effect of longterm freezing on tissue

Hi Bil,

I too would be interested in hearing about any posting you might get. I
think that some important variables would be the temperature which you
store the specimens, the humidity, and another thing would be the number
of freeze/thaw cycles. Our lab hasn't done much in the way of testing
the effects of freezing. Most people I know accept freezing as a necessary
part of the experiment, unavoidable, and generally don't seem to much
worried by the process.

Douglas Chang
dchang@sdcc3.ucsd.edu