X-Message-Number: 31973
Date: Mon, 14 Sep 2009 15:35:16 -0700
Subject: Re: CAS freezing system, reply to information request
From: Jeff Davis <>
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:
Phone: +81-4-7137-9131 fax: 04-7156-8662
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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
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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
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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.
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Best Jeff Davis
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