X-Message-Number: 4793
Date: Fri, 18 Aug 1995 18:14:24 +0200 (MET DST)
From: Eugen Leitl <>
Subject: basic Fahy papers abstracts

Cryonics/cryobiology Fahy et al. paper abstract/conclusion
digest (Thanks, C.P.!) - basic bibliography crossection in ASCII
form. Excuse numerous typos.

-- Eugene

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[1] Cryoprotectant Toxicity and Cryoprotectant Toxicity
Reduction: In Search of Molecular Mechanisms. (Gregory M. Fahy,
Terence H. Lilley, Helen Linsdell, Mary St. John Douglas, Harold
T. Meryman) CRYOBIOLOGY 27, 247-8 (1990).

ABSTRACT Cryoprotectant toxicity is a fundamental obstacle to
the full potential of artificial cryopreservation, yet it
remains in general a poorly understood phenomenon.
Unfortunately, most relevant biochemical studies to date have
not met the basic criteria for demonstrating mechanisms for
toxicity. A model biochemical study of cryoprotectant toxicity
was that of Baxter and Lathe, which demonstrated that alteration
of a specific enzyme (fructose diphosphatase, or FDPase) was the
cause of impaired glycolysis after treatment with and removal of
dimethyl sulfoxide (D). FDPase alteration by D was reported  to
be preventable by the simultaneous presence of amides. This
protection could be due to a "counteracting solute" effect
similiar to that employed by nature, but we find no meaningful
correlation between the general protein stabilizing or
destabilizing tendency of the cryoprotectant medium and its
toxicity. Baxter and Lathe postulated that the effect of D
arises from hydrogen bonding between D and the epsilon amino
groups of surface lysine residues on FDPase, and it was found
that molecule which resembled this group could block the
alteration induced by D, presumably by competing with lysine
residues for assotiation with D. However, we find that the
interaction between D and and lysine in the presence of water is
actually thermodynamically repulsive, and that the presence of
formamide does not affect the interaction beteen D and lysine,
implying no useful complex formation between formamide and D. We
were also unable to demontrate that the locking compounds
consistently reduce toxicity when added to D rather than
substituting for D, contrary to predictions based on complex
formation between blocking compounds and D. In summary, it seem
that present concepts of cryoprotectant toxicity are in need of
serious revision.

CONCLUSION Despite the critical relevance of cryoprotectant
toxicity to cryobiology, the mechanisms of toxicity pertinent to
the different cryoprotectants remain elusive. The biochemical
tools needing for determining mechanisms of toxicity as well as
many suggestive leads are available, but as of 1989, fully 18
years after the first comprehensive study by Baxter and Lathe,
almost nothing has been done. We hope that this review will
encourage others to develop answers to the many open questions
presented by the observations considered here and by the overall
enigma of cryoprotectant toxicity.

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[2] Vitrification as an Approach to Cryopreservation (G. M.
Fahy, D. R. MacFarlane, C. A. Angell, H. T. Meryman) CRYOBIOLOGY
21, 407-4 (1984)

ABSTRACT Recent developments have opened the possibility that
the problems of freezing and thawing organs might eventually be
overcome by an alternative approach to organ cryopreservation,
namely, vitrification. Here we will review some of the
principles of vitrification, describe the current state of the
art, consider how a practical vitrification scheme might work,
and conclude by noting how the principles of vitrification
relate to and illuminate the principles and practices of
freezing.

CONCLUSION In conclusion, although many formidable problems
remain to be solved or even addressed, vitrification is an
intriguing possibility for indefinite preservation of complex
biological systems in general. It has the advantage of
presenting problems that are well-defined and limited in number.
It also seems to us to be closer to fruition than is organ
cryopreservation by freezing. But we would also like to
emphasize that the pursuit of vitrification may lead to improved
freezing techniques as well. For example, the problems of
cryoprotectant toxicity we must face with this approach may also
be at the heart of an explanation for "solution effects" injury
in many systems. In fact, the use of cryoprotectant toxicity
neutralization has already improved the freeze-thaw recovery of
kidney tissue (17). The problem of introducing high
concentrations of cryoprotectant must be faced in any event in
organ freezing procedures in order to prevent mechanical damage
from ice (57), so the problems of cryoprotectant toxicity are
immediate and practical ones for both procedures. Additional
connections between vitrification and freezing are described in
Fig. 17. The T_h/T_g intersection point (Fig. 17A) should be
relevant for defining the temperature from which a slowly frozen
cell or a cell cooled by a step procedure can best survive a
plunge into liquid nitrogen, subject, of course, to several
secondary consideration. Using this guide, it has in fact
recently been possible to document the first substantial
recovery of kidney tissue frozen to liquid nitrogen temperature
(17). It also appears that the principles of vitrification may,
as indicated here, provide a deeper understanding of the optimal
cooling rate, which, as suggested here (Fig. 17B), may be that
cooling rate which comes closest to bringing the cell's
temperature to the cell's T_g at an intracellular concentration
just high enough to avoid homogenous nucleation.

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[3] Vitrification as an Approach to Organ Cryopreservation:
Past, Present, and Future. (G. M. Fahy), Cryopreservation and
Low Temperature Biology in Blood Transfusion (C. Th. Smit
Sibinga, P. C. Das, H. T. Meryman, Eds.) Kluwer Academic
Publishers, Boston 1990: pp. 255-8.

ABSTRACT The concept of preserving organs in a viable condition
outside of the human body dates at least from 1812, when
LeGallois first proposed that after death of the body the human
head (and therefore the individual) could be kept alive by
removing it and supporting it by normothermic artificial machine
perfusion [1]. History, however, has gone instead in the
direction of hypothermic organ preservation, which seems to be a
better option for both biological and economic reasons. The
subject of this paper still lies in the future, and that is
"cryothermic" preservation, i.e., preservation of organs at
temperatures below -100 deg C. The primary advantage of this
approach, should it prove to be possible, is that preservation
times should become indefinite at such temperatures, thereby
opening up many new and significant opportunities in
transplantation medicine. This paper reviews the many steps
towards the goal of organ cryopreservation by vitrification
which have been taken since the beginning of this field in 1980,
and also provides a very brief sketch of the rather ironic
history of this area of research.

CONCLUSION Although vitrification and successful reimplantation
of an animal kidney has yet to be achieved, success with single
cells or small multicellular specimens has already been
demonstrated and reports of success with increasingly complex
tissue can be anticipated in the near future. There is a large
variety of potentially transplantable tissues what awaits only
improved techniques for preventing graft rejection to become
clinically useful. Some of the most promising advances in
transplantation immunology involve the induction of
donor-specific tolerance through the transfusion of
appropriately modified leukocytes from the donor. Vitrified
cadaver allografts of the future may well be banked in
conjunction with cryopreserved blood cells from the donor. The
necessity for immunological preparation of the recipient over an
interval of two or more weeks will mandate cryopreservation of
the allografts. Although conventional cryoprotectant freezing
may be an acceptable method for some tissues where a degree of
injury can be tolerated, the inevitable mechanical damage from
ice formation will place a restrictive limit on the use of
freezing, particularly of highly vasularized tissues in which
the vascular endothelium is particularly vulnerable to freezing
injury. Vitrification avoids he problems assotiated with ice
formation, cell dehydration and the control of freezing rates
and thus should have many applications. Much of the challenge
assotiated with vitrification now concerns the toxicity with of
vitrifiable perfusion solutions, but this is a relatively new
topic, as yet poorly understood and with much opportunity for
developments of practical importance. No persuasive evidence has
as yet been presented to contradict thhe expectation that the
cryopreservation of tissues and organs is indeed possible and
that we are on the right track.

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[4] Physical Problems with the Vitrification of Large Biological
Systems (Gregory M. Fahy, Joseph Saur, Robert J. Williams),
CRYOBIOLOGY 27, 429-510 (1990).

ABSTRACT Vitrification is an attractive potential pathway to the
successful cryopreservation of mature mammalian organs, but
modern cryobiological research on vitrification to date has been
devoted mostly to experiments with solutions and with biological
systems ranging in diameter from about 6 through about 100 um.
The present paper focuses on concerns which are particularly
relevant to large biological systems, i.e. those systems ranging
in size from approximately 10 ml to approximately 1.5 liters.
New qualitative data are provided on the effect of sample size
on the probability of nucleation and the ultimate size of the
resulting ice crystals as well as on the probability of fracture
at or below T_g. Nucleation, crystal growth, and fracture depend
on cooling velocity and the magnitude of thermal gradients in
the sample, which in turn depend on the sample size, geometry
and cooling technique (environmental thermal history and thermal
uniformity). Quantitative data on thermal gradients, cooling
rates, and fracture temperatures are provided as a function of
sample size. The main conclusions are as follows. First, cooling
rate (from about 0.2 to about 2.5 deg C/min) has a profound
influence on the temperature-dependant processes of nucleation
and crystal growth in 47-50% (w/w) solutions of propylene
glycol. Second, fracturing depends strongly on cooling rate and
thermal uniformity and can be postponed to about 25 deg C below
T_g for a 482-ml sample if cooling is slow and uniform. Third,
the presence of a carrier solution reduces the concentration of
cryoprotectant needed for vitrification (C_v). Hoever, the C_v
of samples larger than about 10 ml is significantly higher than
the C_v of smaller samples whether a carrier solution is present
or not.

CONCLUSION In this paper we have provided preliminary results on
the problems of vitrifying large specimens. The primary problems
considered here are fracturing and crystallization during
cooling. It seems likely that fracturing can be prevented by
careful sample handling even for samples as large as a human
kidney. Crystallization is a more serious problem, and much more
must be learned about the magnitude of this problem in different
circumstances. It is likely that crystallization can be reduced
by the removal of heterogenous nucleating agents and the use of
specific crystal-growth inhibitors, but it is not clear whether
these growth inhibitors can lower C_v back to values which have
acceptable toxicity or whether they can themselves induce damage
(1, 9). However, rabbit kidneys are small enough to be cooled at
rates compatible with vitrification at concentrations studied in
the past, so the study of cryopreservation of this organ by
vitrification can continue as the more formidable problems of
vitrifying much larger organs are addressed.

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.. to be continued...

-- Eugene

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