X-Message-Number: 4797
Newsgroups: sci.cryonics
Date: Mon, 21 Aug 1995 17:18:56 +0200 (MET DST)
From: Eugene Leitl <>
Subject: Fahy (II)
Message-Id: <>

essential Fahy cot'd... do not fold, mutilate or bit-mutate

-- Eugene


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[5] Practical Aspects of Ice-Free Cryopreservation (G. M. Fahy,
T. Takahashi, H. T. Meryman), <unknown source> (<unknown year>).

ABSTRACT  Low temperatures are used by biologists primarily
because they slow or prevent unwanted physical and chemical
events. Unfortunately, the utility of low temperatures is
usually compromised by the inconvenient fact that cooling also
leads to the crystallization of water and thereby creates new
and unwanted physical and chemical event which may injure the
system the biologist wishes to preserve. Although the penalties
imposed by freezing are in many cases acceptable, ice formation
renders biological preservation generally imperfect and
sometimes inconvenient.

Here we will consider an alternative to this situation. It is
now possible, after after suitable pretreatment, to prevent ice
formation in biological systems during cooling to liquid
nitrogen temperature in such a way that when these systems are
warmed rapidly, little or no injury is observed. Instead of
forming ice, these systems become glassy rather than
crystalline, and event known as vitrification.

CONCLUSION Vitrification has become a successful method for
cryopreserving human monocytes and erythrocytes, murine embryos,
and guinea pig smooth muscle and is likely to be useful for
other cell types and tissues relevant to blood banking. It can
be a simple, quick, inexpensive and reliable process when
carried out properly, but it must be executed carefully. Only
the future will decide the limits of applicability of this
relavely new approach to cryopreservation.

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[6] Simplified Calculation on Cell Water Content during Freezing
and Thawing in Nonideal Solutions of Cryoprotective Agents and
its Possible Application to the Study of "Solution Effects"
Injury, (Gregory M. Fahy) CRYOBIOLOGY 18, 473-482 (1981).

ABSTRACT In 1964 Mazur (10) introduced a second-order
differential equation, hereinafter referred to as the Mazur
equation, which permitted the calculation of cell water content
as a function of temperature, cooling rate, and cellular
properties (i.e., volume, surface area, solute content,
hydraulic conductivity). However, this formidable equation can
only be solved using an involved approximation method (the
Runge-Kutta method) with iteration 0.001 K. To the average
cryobiologist, the difficulty of solving the Mazur equation is
sufficient to preclude its use; certainly this was the
experience of the present author. A simplified method for
calculating the extent of cellular dehydration during freezing
at different rates was therefore sought. In addition, it was
desired to improve upon the Mazur equation by incorporating the
nonideal behaviour of glycerol and dimethyl sulfoxide (Me_2SO)
described elsewhere (2). Both of these goals have been achieved.
The derivation of the desired simplified equation for nonideal
cells is presented here. In addition, posible application of
the new equation to the study of "solutions effect" injury is
discussed.

CONCLUSION A simplified equation has been derived which reduces
the time and complexity of calculating subzero cell water
content during freezing and thawing as compared to caculation by
means of the Mazur equation. The simplified equation also allows
inclusion of the effects of nonideality of glycerol and
dimethyl sulfoxide aqueous electrolyte solutions. Furthermore, a
very simple, iterative method of solving the simplified equation
has been shown to give results which are equivalent to those
obtained using the far more difficult and involved Runge-Kutta
technique. It is hoped that these simplifications will make
calculation of cell water content accessible to more
cryobiologists. In addition, possible applications of such
calculations to mechanistic issues in the area of "solution
effeects" injure are discussed.

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[7] Microwave Hypothermia III; An Approach for Rapidly Rewarming
Cryogenically Preserved Organs, (Paul S. Ruggera, Gregory M.
Fahy), Proc. Ann. Int. Conf. of the IEEE Engineering in Medicine
& Biology Soc., 10: 884-885, (1988).

ABSTRACT Wire-length resonant radio frequence (RF) coils [1],
which are based on equating a wavelength of RF with the wire
length used in winding a coil, were first developed at CDRH for
potential use in hypothermia treatment for cancer therapy.
Design modifications, coupled with a series of experiments [2],
established their potential use for rapidly and uniformly
rewarming canines following hypothermic cardiac surgery. This
paper details a new design which incorporates a single-end
coaxial input and RF shielding, allowing it to be used outside
of screen room environment. Data from early experiments to
investigate this system's usefulness in rewarming cryogenically
preserved donor organs are presented.

INTRODUCTION Vitrification has been under development by the
American Reed Cross as a means for organ cryopreservation. By
infusing a rabbit kidney with a cryoprotective fluid and cooling
it under extreme pressure, vitrification, i.e. solidification
without ice crystal formation has been demonstrated [3].
However, ice crystal formation during warming from the vitreous
state can only be avoided by using rapid and uniform heating.
The indications are that the conventional heating techniques do
not produce heating which is sufficiently rapid, uniform,
efficient, and readily applicable inside a high-pressure vessel.
Given the demonstrated deep, uniform heating capability of
wire-length resonant coils, and the desire to investigate
potential use of the longer wavelengths 10-100 MHz region of the
spectrum in this application, two coils were constructed and
tested at CDRH. Details of the construction and results of
testing thus far are presented here.

CONCLUSION The results of the heating peak's shift is of little
consequence in this application. Prior to vitrification, the
organ can be positioned in the container to align it with the
area of maximum heating. The available heating volume, however,
is of concern. The 12.5-cm coil's volume is too small for a
rabbit kidney, the first organ which will be investigated. The
16.5-cm coil's volume is adequate. However, as can be seen from
Figs. 2 and 3, the longer coild requires 33 percent more power
to attain a given temperature in given volume. The target
heating rate for vitrified organs is 1000 deg C/min. The data
indicated that this would require 4.2 kW for the coil of Fig,
and 5.6 kW for the coil of figure 3. It is interesting to note
that for this design the power increase required to achieve peak
temperatures may well be related to the ration of the coils'
lengths (i.e. 16.5/12.5 = 1.32). If this holds, it indicates
that for a coil length approaching the limit of the pressure
vessel's length, which will maximize the available heating
volume, RF power in the region of 10 kW will be required.

These results also suggest that the exposure system appears to
work well and, therefore, may be of use to other researchers who
ues this frequency range. It offers a high, uniform field
intensity with virtually no external emission and with
relatively low power requirements.

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[8] The Relevance of Cryoprotectant "Toxicity" to Cryobiology
(Gregory M. Fahy), CRYOBIOLOGY 23, 1-13 (1986)

ABSTRACT Cryoprotective agents are essential for the
cryopreservation of almost all biological systems. These
additives, however, do not usually permit 100 percent survival
after freezing and thawing, though from a theoretical point of
view they should be able to fully suppress all known types of
freezing injury. In view of the known biological and
physicochemical effects of cryoprotectants, it is suggested that
the toxicity of these agents is a key limiting factor in
cryobiology. Not only does this toxicity prevent the use of
fully protective levels of additive, but it may also be
manifested in the form of cryoinjury over and beyond the
cryoinjury due to classical causes. Evidence for this extra
injury ("cryoprotectant-associated freezing injury") is
reviewed. It is suggested that better supression of toxicity is
possible and will lead to advances in cryopreservation.

CONCLUSION Nevertheless, I submit that the detrimental effects
of cryoprotectants are almost as relevant to cryobiology as are
their cryoprotective effects, and that our understanding of
cryobiology will remain incomplete until we have finished
examining both sides of the cryoprotectant coin. We very much
neeed the services of talented biochemists and biophysicists who
can work out the mechanisms and the sites of injury so that we
might be permitted to protect cells againstt the "side effects"
of cryoprotectants. Of particular interest would be more studies
which explore those effects of cryoprotectants which have been
shown to be irreversible. We have substantial clues with regards
to both where and how to look for answers. We also have evidence
that deeper insight can lead to practical benefits (3, 13).

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.. to be continued

-- Eugene


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