X-Message-Number: 7678
Date: Tue, 11 Feb 1997 23:50:56 +0100 (MET)
From: Eugene Leitl <>
Subject: FYI: (looong) Cryobiology now online (at last!)

It's pretty bare, but it's a start...


Things which can be found there, e.g.:

The Effect of Cooling Rate and Temperature on the Toxicity of Ethylene
Glycol in the Rabbit Internal Carotid Artery Monica Wusteman, Serena Boylan,
David E. Pegg

The smooth muscle and vascular endothelium of small elastic arteries (the
rabbit common carotid artery) are injured by exposure to 40% ethylene glycol
(EG) at 4=B0C, and additional damage occurs when the arteries are cooled
without freezing to -20=B0C. This paper reports attempts to reduce this injury
by altering the cooling rate and temperature of exposure to the
cryoprotectant. Very slow cooling (0.1=B0C/min) removed all residual smooth
muscle and endothelial function when assessed in vitro after rewarming and
removal of the cryoprotectant. Very rapid cooling to -20=B0C also increased the
injury, both to the endothelium and to the smooth muscle.  Reducing the

temperature of exposure to 40% EG from +4=B0C to -20=B0C had no beneficial 
on the smooth muscle but enabled the vascular endothelium to retain some
functional activity.  These data suggest that the mechanism responsible may
be related to the physical properties of ethylene glycol rather than to a
biochemical interaction with metabolic processes, and that it is a mechanism
which is highly specific for the cell types involved. It also underlines the
difficulties involved in the successful cryopreservation of complex tissues
and organs.  Cryobiology, v 33, n 4, August 1996, p423-429 (ID CY960042)
Copyright 1996 Academic Press, Inc.


An Experimental Study of the Mechanical Response of Frozen Biological Tissues
at Cryogenic Temperatures YOED RABIN, Paul S. Steif, Michael J. Taylor,
Thomas B. Julian, Norman Wolmark

An experimental study of the mechanical response of frozen soft biological
tissues to applied compressive stresses is presented. This study is related
to the mechanical stresses that develop due to the contraction of frozen
tissues in cryopreservation as well as in cryosurgical procedures. The main
concept in this study is that the stresses associated with the constrained
contraction of the frozen tissue, i.e., due to temperature variations within
the frozen tissue, can be simulated by an external mechanical load which is
applied to the frozen tissue while the tissue is maintained at a uniform
temperature. A new apparatus for measuring compressive stresses and strains
of frozen biological tissues in cryogenic temperature range is presented. A
new technique for processing the fresh biological tissue into a cylindrical
frozen sample for mechanical testing is introduced.  Results of compression
tests on rabbit liver, kidney, and brain are presented and are compared with
available data from the literature on sea ice and single ice crystals. An
unusual response of frozen biological tissues to compressive stress was
observed: after the initial, roughly linear elastic portion there was a
series of sudden stress drops at constant strain, each followed by a linear
increase of stress with strain to the next drop. This phenomenon, which is
attributed to the accumulation of microcracks, broadly resembles plastic
deformation, and thus provides some support for simple mechanical models
invoked in theoretical studies.  Cryobiology, v 33, n 4, August 1996,
p472-482 (ID CY960048) Copyright 1996 Academic Press, Inc.



This study was designed to examine the suitability of immature horse oocytes
for vitrification.  Immature oocytes derived from slaughtered horse ovaries
were transferred to a vitrification solution (EFS; 40% ethylene glycol, 18%
Ficoll, and 0.3 M sucrose in modified phosphate-buffered saline) directly
(Groups 1 and 4) or were first exposed to 20% ethylene glycol solution for 10
min (Groups 2 and 5) or 20 min (Groups 3 and 6). Oocytes were handled at 20=B0C
(Groups 1, 2, and 3) or 30=B0C (Groups 4, 5, and 6). After vitrification and
warming, their viability was assessed by maturation culture for 32 h. The
percentages of oocytes reaching the metaphase II stage after the in vitro
maturation in Groups 2, 3, 5, and 6 (16.0, 16.7, 10.0, and 8.2%,
respectively) were higher than those in Groups 1 and 4 (2.2 and 1.9%,
respectively). In untreated control oocytes, 55.6% completed meiosis in
vitro. Transmission electron microscopy was used to compare the fine
structure of vitrified oocytes (treated as Group 2) with those of untreated
control oocytes and EFS-exposed, nonvitrified oocytes (n =3D 10 each). The
viability of EFS-exposed oocytes, assessed by in vitro maturation, was 27.7%.
Vitrification induced some ultrastructural changes, such as the swelling of
mitochondria together with reduced matrix density, the destruction of
communication between oocytes and their surrounding cumulus cells, and the
presence of vacuoles located in the periphery of the ooplasm. However, these
changes were not always observed. Exposure of the oocytes to EFS solution
induced similar ultrastructural changes in mitochondria and cell-cell
communication but to a lesser extent. However, the exposure to EFS induced
vacuoles in the periphery of the ooplasm to the same extent as did the
vitrification.  Thus, immature horse oocytes can be cryopreserved by
vitrification with EFS solution. Reduced viability of EFS-exposed and/or
vitrified horse oocytes may relate to morphological changes such as
destruction of the intercellular communications between cumulus cells and
oocytes.  Cryobiology, v 33, n 3, June 1996, p300-310 (ID CY960030) Copyright
1996 Academic Press, Inc.



Successful cryopreservation of tissues will ultimately require a more
detailed understanding of how the in situ environment modifies cell responses
during cooling and warming.  Low-temperature responses of porcine
split-thickness skin and isolated basal keratinocytes were compared after
various cooling protocols and in the presence and absence of cryoprotectants.
Recovery was assessed by measuring oxygen consumption kinetics in skin and
tetrazolium reduction in isolated cells. Freeze substitution was used to
visualize ice nucleation and growth in skin. The results indicated that the
time required for diffusion of water in split-thickness skin delayed osmotic
responses in the basal keratinocytes and resulted in increased intracellular
and intercellular ice formation. Rapid cooling (-200=B0C/min) in the presence
of cryoprotectants resulted in a reduction in the number of cells containing
ice and the size of the intercellular ice crystals and an increase in tissue
recovery. These observations support other reports which suggest that
cell-to-cell and cell-to-substrate interactions are sensitive sites for
cryoinjury. A practical recommendation from this study is that high recovery
of split-thickness skin may be achieved with protocols using high cooling
rates.  Cryobiology, v 33, n 3, June 1996, p376-389 (ID CY960038) Copyright
1996 Academic Press, Inc.



The effect of sugars or reduced saccharides trehalose, sucrose, sorbitol, or
mannitol on the glass-forming tendency during cooling and the stability of
the wholly amorphous state during warming has been studied with
2,3-butanediol, 1,2-propanediol, or 1,3-butanediol in three different carrier
solutions. The 2,3-butanediol contained 96.7% (w/w) racemic mixture of the
levo and dextro isomers and 3.1% (w/w) of the meso isomer (called
2,3-butanediol 97% dl). The carrier solutions were water, a
phosphate-buffered saline, and two organ preservation solutions (Euro-Collins
and Saint Thomas). The latter two were chosen because they are often used for
kidney and heart preservation, respectively. The concentrations of
2,3-butanediol, 1,2-propanediol, and 1,3-butanediol varied respectively from
25 to 34, 30 to 35, and 30% (w/w).  The concentrations of saccharides were 4
or 5% (w/w). In the absence of saccharides, for a given 2,3-butanediol
concentration, the glass-forming tendency increased in the following order:
water, Saint Thomas, the phosphate buffer, Euro-Collins. Addition of 4 or 5%
(w/w) saccharide resulted in a large increase in the glass-forming ability of
the solution during cooling and increased the stability of the glass during
warming; but replacement of 4 or 5% diol by an equivalent weight (percentage)
of a saccharide decreased, though to a lesser extent, these properties.
Cryobiology, v 33, n 3, June 1996, p363-375 (ID CY960037) Copyright 1996
Academic Press, Inc.


Crystallization of Ice in Aqueous Solutions of Glycerol and Dimethyl
Sulfoxide.  1. A Comparison of Mechanisms J. M. HEY, D. R. MACFARLANE

The crystallization of ice from aqueous solutions of glycerol and dimethyl
sulfoxide (Me2SO) has been studied using differential scanning calorimetry.
In particular, the ice crystallization behavior of glycerol and Me2SO
solutions containing approximately the same mole percent solute concentration
(i.e., approximately 16 mol%) has been compared. These solutions (45 w/w%
Me2SO (15.9 mol%) and 50 w/w% glycerol (16.4 mol%)) were shown to exhibit
markedly different ice crystallization properties. For example, the peak
homogeneous nucleation temperature of the Me2SO solution was observed to be
3=B0C above Tg, whereas the peak homogeneous nucleation temperature of the
glycerol solution was shown to be 20=B0C above Tg.  Further, the 50 w/w%
glycerol solution was shown to devitrify at temperatures close to those of
the peak nucleation rate, whereas the Me2SO solution was found to devitrify
at temperatures much higher than the peak nucleation temperature. This, along
with evidence from emulsion-based calorimetry experiments, indicates that the
nucleation leading to devitrification in 45 w/w% Me2SO solutions is largely
heterogeneous in nature.  Cryobiology, v 33, n 2, April 1996, p205-216 (ID
CY960021) Copyright 1996 Academic Press, Inc.


Analysis of Thermal Stresses around a Cryosurgical Probe YOED RABIN, PAUL SSTEIF

Large thermal stresses easily exceeding the tissue yield strength may develop
in the frozen region around a cryosurgical probe. A new integrodifferential
solution for the heat transfer problem of biological tissues freezing around
a cryosurgical probe is presented in this article. This solution is suitable
for cases of high Stephan numbers and for a temperature-dependent forcing
function at the cryoprobe. A new solution for the thermal stresses around a
cryosurgical probe is also presented, based on an elastic-perfectly plastic
model. It is proposed that thermal stresses beyond the elastic limit of the
frozen region may sharply increase the mechanical damage to the cell
membranes due to plastic deformation. It was found that plastic deformation
always starts at the cryoprobe surface; however, plastic deformation may also
be formed near the freezing front at high cooling rates and large cryoprobes.
It is demonstrated that under some conditions plastic deformation may occur
in the entire frozen region. A parametric study to identify the best cooling
protocol for maximal plastic deformation is presented.  Cryobiology, v 33, n
2, April 1996, p276-290 (ID CY960028) Copyright 1996 Academic Press, Inc.


A New Cryosurgical Device for Controlled Freezing YOED RABIN, AVRAHAM SHITZER

A new cryosurgical device utilizing liquid nitrogen, which is a modification
of an existing commercial system, was developed. In the new
computer-controlled cryodevice the temperature of the cryoprobe is controlled
by means of an electrical heating element.  The desired temperature-forcing
function is calculated to ensure a specified constant cooling rate at the
freezing front. The new device facilitates real-time data processing, and, in
particular, simulation of the heat transfer processes. A series of tests was
performed to study the characteristics of the cryodevice and to validate the
underlying assumptions. These tests were performed using organic tissue,
i.e., potatoes, as an in vivo simulating medium of biological tissue. The
differences between experimental data and computed results were found to be
within =B10.5=B0C, which falls within the uncertainty range of the experimental
temperature measurements. A typical control error of the new device is within

=B10.3=B0C, prior to the formation of the freezing front, and =B10.6=B0C 
which is of the same order of magnitude as the uncertainty range of the
temperature measurements. The new device is capable of producing maximal
cooling rates of 50=B0C/min down to temperatures of -165=B0C and a maximal
heating rate of 300=B0C/min. The maximal cooling power of the cryoprobe, due to
LN2 boiling, is 80 W; the maximal electrical heating power of the cryoprobe
is 160 W. Precooling of the device requires about 30 min, and it can be
operated continuously for about 3 h. Initial results of experimental in vivo
cryosurgery performed on rabbit hindlimbs, including histological
observations and thermal analysis, are presented in the second part of this
study.  Cryobiology, v 33, n 1, February 1996, p82-92 (ID CY960009) Copyright
1996 Academic Press, Inc.

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