CryoNet #31971 - #31973

CryoNet - Tue 15 Sep 2009

    #31972: Re: CAS freezing [Jeff Davis]
    #31973: Re: CAS freezing system, reply to information request [Jeff Davis]

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Message #31972
Date: Mon, 14 Sep 2009 14:50:20 -0700
Subject: Re: CAS freezing
From: Jeff Davis <[email protected]>

I've done some further Googling, and have found support for Brian's analysis.

This document:

Vitrification and glass transition of water: insights from spin probe ESR
http://arxiv.org/ftp/cond-mat/papers/0409/0409440.pdf

refers to a "no man's land" for vitrified water between 150 and 233
deg K, consistent with Brian's comments.

I feel the need to remind everyone that even if at times I can shovel
the jargon, that I'm not expert enough to speak authoritatively.  So
caveat emptor.

So, the "flash" transformation of supercooled water to an amorphous
(vitrified) state does not appear possible.  Brian's alternative
hypothesis of ice composed of smaller ice grains(crystals) is a better
hypothesis -- because it's possible and plausible.

Best, jeff davis

On Mon, Sep 14, 2009 at 2:00 AM, Brian Wowk <[email protected]> wrote:

Message #31970
Date: Sun, 13 Sep 2009 22:15:01 -0700
Subject: CAS freezing

      It occurs to me that solidifying pure water into an amorphous
solid (vitrifying it) at the temperatures and pressures used by CAS is
physically impossible.  The reason is as follows.  We already know the
physical properties of amorphous (vitrified) water because it is
possible to vitrify pure water in the laboratory by cooling small
droplets very fast (about a million degrees Celsius per minute).  In
particular, we know the glass transition temperature of vitrified
water.  It is approximately -130 degC.  When a vitrified solid is
warmed above its glass transition temperature, it becomes liquid.
Therefore any vitrified water sample is going to turn to a liquid at a
temperature warmer than about -130 degC.  What happens when vitrified
water turns to liquid water at -130 degC?  It freezes into ice
practically instantly.

      Therefore, if we assume that ice formation is prevented by
electromagnetic energy in a CAS system at -20 degC, water will only be
able to exist in two states when the power is turned off.  It can only
exist as supercooled liquid, or exist as ice.  It cannot become an
amorphous solid because the temperature is a 100 degrees higher than
the known temperature at which glassy water turns to liquid water (the
glass transition temperature).

      I read the CAS patents a couple of years ago.  My understanding
is that they claim water does freeze when the power is turned off, but
into smaller, less damaging crystals.  This is possible, and it might
help food preservation.  But it's not going to keep the water level
the same as when the water was a liquid.  Ice is still ice.  Put a
sample of CAS solidified water into liquid water at 0 degC, and I
guarantee you it will float.

---Brian Wowk

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Message #31973
Date: Mon, 14 Sep 2009 15:35:16 -0700
Subject: Re: CAS freezing system, reply to information request
From: Jeff Davis <[email protected]>

I received a reply to my request to the ABI folks in Japan.  Here it is:

Dear Mr. Jeff Davis

Thank you for your inquiry about CAS freezing system of September 10.
We are pleased to answer your request for information about research
into organ preservation using the CAS process.
The attached materials and following URLs should answer many of your
questions.

http://www.teethbank.jp/english.html  (Hiroshima Uni.)

We appreciate your interest in our company and its researches.

Yours very truly,

***************************************
ABI Co.,LTD.
President:  Norio Owada
238-1 Jyutaifu Nagareyama-City,
Chiba-Prefecture, 270-0133 JAPAN
e-mail: [email protected]
Phone: +81-4-7137-9131  fax: 04-7156-8662


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

Included were two attachments.  Here is one:

Cryopreservation of the entire ovary of cynomolgus monkeys in a magnetic field
environment without cryoprotectant

e-Poster: 493
Congress: ESHRE 2008
Type: Scientific poster

Topic: Female fertility: basic research

Authors: K. Kyono1, M. Hatori2, F. Sultana2, C. Nishinaka1, T. Kyoya1,
H. Uto1, S. Kanto1, M.

Kuchiki1, Y. Nakajo1, K. Fujii3, N. Owada3, T. Sankai2; 1Sendai/JP,
2Tsukuba/JP, 3Abiko/JP

MeSH:
Cryopreservation [E01.450.865.366.156]

Keywords: Autotransplantation, Magnetic field environment, Cynomolgus
monkey, Vascular


www.eshre.com

1. Purpose

In autotransplantation of ovarian cortical fragments, a large
proportion of follicles is lost, andovarian functions remain for a
short period of time. Theoretically, the best way is the
autotransplantation of an entire intact ovary with vascular
anastomosis. However, there has been no successful report of freezing
of an entire ovary yet. Here, we would like to describe the successful
freezing technique of the entire ovary without using any
cryoprotectant and seeding

procedure.

2. Methods and Materials
The methods applied for freezing the ovaries
Transplantation of ovaries after freezing-thawing
The theoretical principle of freezing system in a magnetic field
environment is that the vibration of particles avoids formation of ice
crystals as well as breakdown of the cells. All five monkeys (a-e)
used for the experiment had normal menstrual cycles at the start of
the experiment. We removed both ovaries from the five female
cynomolgus monkeys on the first day of menstruation. The ovaries were
immediately frozen in a magnetic field environment and

cryopreserved for about 1 month in liquid nitrogen. Frozen-thawed
ovaries were transplanted back into the skeletal muscles of the femur
(two monkeys) and under the kidney capsules (three monkeys). After
autotransplantation, the number of their menstruation and
gonadotrophin levels were recorded in 274 days.

3. Results

The menstrual cycle of one monkey recovered 57 days after
transplantation of the ovaries (A).

However, it was 206 days before the third menstruation that came with
progesteron level and estradiol level going up.

When the ovaries of the other four monkeys were stimulated by
injection of FSH, all the four monkeys recovered the menstrual cycles
(B, C, D, and E). In Monkey B, estradial level went up about 70 days
after transplantation, and progesteron level went up aruond 80 days
after transplantation. However, progesteron level remained low after
that. In Monkey C, the first menstruation was on day 68, following
estradial and progesteron levels going up. Regular menstrual cycles
were observed after that. Monkey D recovered the menstrual cycle with
hormone production over four months after transplantation; however,
menstruations were confirmed only twice. The reason for this is
unknown.

In the fifth monkey (E), the menstrual cycle did not recover for about
180 days. However, genital bleeding occurred twice on day 185 and 197
without hormonal changes.

Monkey a

autotransplantation in muscle

Monkey b

autotransplantation under kidney menbrane

Monkey c

autotransplantation under kidney menbrane

Monkey d

autotransplantation under kidney membrane

Monkey e

autotrasplantation in muscle

After frozen-thawed ovarian autotransplantation, four of the five
monkeys recovered their ovarian functions with hormone production and
the menstrual cycle. As the table below shows, the first menstruations
of Monkey A, B, and C occurred from 57 to 68 days after the
transplantation, and it indicates that it was derived from antral
follicles that survived in freezing in a magnetic field environment.
The first menstruations of Monkey D and E occurred about four months,
and it indicates that it was derived from primordial follicles.

Summary of all five monkeys transplantation place

FSH injection the first menstruation after transplantation

the number of menstruation in 274 days after transplantation

a muscle no 57 days 3

b kidney yes 57 days 3

c kidney yes 68 days 7

d kidney yes 129 days 2

e muscle yes 185 days 2

4. Conclusion

The advantages of this method are that it does not need any
cryoprotectant and seeding procedure, and that it can freeze an entire
ovary. It is suggested that both antral follicles and primordial
follicles could survive in freezing in a magnetic field environment.
In the near future, we hope to attempt the autotransplantation of an
entire intact ovary with vascular anastomosis.

This method of preserving intact internal organs would provide extra
time for transportation of an organ to transplant anywhere in the
world and would make possible the creation of banks for frozen
internal organs.

5. Mediafiles

Monkey a

autotransplantation in muscle

Monkey b

autotransplantation under kidney menbrane

Monkey c

autotransplantation under kidney menbrane

Monkey d

autotransplantation under kidney membrane

Monkey e

autotrasplantation in muscle

The methods applied for freezing the ovaries

Transplantation of ovaries after freezing-thawing

*****************i fi '******************

Effect of magnetic field supplementation during the freezing process
for porcine ovarian

tissue cryopreservation

e-Poster: 361

Congress: ESHRE 2008

Type: Scientific poster

Topic: ART, laboratory: cryopreservation of gonads

Authors: Y. Mochimaru1, N. Kuji1, M. Yamada1, T. Hamatani1, Y.
Yoshimura1, T. Sankai2, K.

Kyono3, M. Mihara1, C. Suzukamo4, N. Kashiwazaki4; 1Tokyo/JP,
2Tsukuba/JP, 3Sendai/JP, 4

Sagamihara/JP

MeSH:

Organ Preservation [E05.760.833.660]


www.eshre.com

1. Purpose

Growing evidence has been accumulated to suggest that supplementation
with an alternating

magnetic field might affect the freezing process of biological
materials, cells or tissues. In the

food industry, it has been shown that materials like meat, fish and
fruit frozen under the

influence of a magnetic field show less damage than those subjected to
ordinary freezing

without magnetic field supplementation. Furthermore, it has been
reported that the tooth had

been successfully cryopreserved using a slow-freezing protocol with a
magnetic field (Kaku et

al, 2007).

Although the precise mechanism by which a magnetic field exerts its
beneficial effect in tissue

cryopreservation has not been elucidated yet, it may correlate with
the maintenance of

super-cooling state of the solution (since alternating magnetic field
vibrates the H2O molecules,

formation of the ice crystals are inhibited). Consequently, the size
of the ice crystals formed

during freezing under a magnetic field was smaller than that found
during ordinary freezing in

the absence of a magnetic field.

In this study, we investigated the effect of magnetic field
supplementation for porcine ovarian

tissue cryopreservation, since methods for improved efficacy of
ovarian tissue cryopreservation

are still being sought.

2. Methods and Materials

1. Porcine ovaries, retrieved from a slaughterhouse, were kept at 4
degree Celsius before being

frozen. The process of freezing the ovaries is shown in Figure 1.

After cryopreservation for 1 week in liquid nitrogen, the tissues were
directly immersed in a

water bath at 37 degrees Celsius. The formardehyde-fixed tissues were
examined under a light

microscope after HE staining, to determine the maintenance of
morphological normality in both

the follicles and other structures of the interstitial tissues.
Normality of follicular structures was

scored microscopically, according to parameters shown in Figure 2.

2. To investigate the difference in the efficacy of the freezing
process used with or without

magnetic field supplementation, the freezing temperature (at which
latent heat generation was

observed) during the slow-freezing process was investigated for both
distilled water and the

cryoprotective solution (1.5M DMSO in PBS) utilized for the ovarian
tissue freezing.

3. Results

1. it was found that more primordial follicles remained
morphologically intact after thawing with

the use of the freezing protocol with magnetic field supplementation
than with the use of the

protocol without magnetic field supplementation (grade 1 follicle ;
17% vs. 3%, p<0.001, Figure

3and Figure 4).

Furthermore, defects in the interstitial tissues, which are thought to
occur as a result of large ice

crystal formation, were less evident in the ovaries frozen in the
presence of a magnetic field (

Figure 5).

2. The mean freezing temperatures of distilled water with and without
the application of a

magnetic field were -4.5 and -4.6 degree Celsius, and those of the
cryoprotective solution (1.5M

DMSO in PBS) with and without the application of a magnetic field were
-11.5 and -11.4 degree

Celsius, respectively (Table 1).

Thus, there were no differences in the freezing temperature of either
of these solutions used for

freezing of the porcine ovary, either in the presence or absence of
magnetic field

supplementation.

4. Conclusion

Magnetic field supplementation seems to be advantageous for
cryopreservation of porcine

ovarian tissue. However, the cryoprotective mechanisms in the presence
of magnetic field

supplementation might not be correlated with the stabilizing effect of
the super-cooling state of

the cryoprotective solution.

5. References

1) Kaku M, Kamata H, Kawata T, Tai M, et al. Cryopreservation of PDL
cells by use of program

freezer with magnetic field for teeth banking. Dentistry in Japan.
2007 Mar; 43: 82-86

2) Paynter SJ, Cooper A, Fuller BJ, Shaw RW. Cryopreservation of
bovine ovarian tissue:

structural normality of follicles after thawing and culture in vitro.
Cryobiology. 1999 Jun; 38(4):

301-9

                 ************************************************************
The second attachment is a JPEG file, a scan of the first page of an
article entitled:

"MRI, Magnetic Resonance Influenced, organ freezing method under magnetic field"

Here's a link:

http://translate.google.ca/translate?hl=en&sl=ja&u=http://blog.goo.ne.jp/wakate-a/e/33fdd5a1318097afbc63b445a844524d&ei=T8KuStWODpDusQPv3NzDCw&sa=X&oi=translate&resnum=1&ct=result&prev=/search%3Fq%3D%2522MRI,%2BMagnetic%2BResonance%2BInfluenced%2Borgan%2Bfreezing%2Bmethod%2Bunder%2Bmagnetic%2Bfield%2522%26hl%3Den

Here's the abstract:

A scientific approach towards organ preservation was initiated by
Alexis Carrel, who was awarded the Nobel Prize in 1904. Subsequently,
cryopreservation of organs gained momentum in the 1950s. However,
organ cryopreservation has long been considered impossible, because
the cells in the organs suffer from substantial damage due to
ice-crystal formation and generation of osmotic-pressure gradients in
the tissue. Meanwhile, in gynecology, freezing techniques for sperms,
ova, fertilized eggs, etc., have been developed to ensure retention of
fertility. Cells and tissue fragments can be frozen with a high
probability of success. Some studies on the clinical applications of
these approaches in humans have been published. In this study, we
tried to cryopreserve organs, and not the cells or tissues, by using a
freezing technology that employed a variable magnetic field. This
freezing technology was principally developed by a Japanese company
engaged in the development of food-freezing technologies. The
combination of different techniques, ie, the integration of this
freezing technology with our technique, Super-Microsurgery, is
expected to expedite basic experimental research on cryopreservation
of small organs, particularly reproductive organs, which was hitherto
considered impossible.

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

Best Jeff Davis

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