X-Message-Number: 32767
Date: Sat, 14 Aug 2010 00:38:04 -0700 (PDT)
From:
Subject: a proposal to stimulate cryonics research
Up till now funding for fully reversible organ cryopreservation (of which
cryonics is an offshoot) has been limited. Cryoprotectant toxicity has been the
primary roadblock to achieving this. However there are much less toxic
alternatives to commonly used cryoprotectants. Unfortunately there is virtually
no possibility for commercial applications using these superior cryoprotective
agents because they are too expensive. I submit that nothing succeeds better
than success in stimulating further funding. By pulling out all the stops,
expense be damned, and actually using these less toxic but pricey cryoprotective
agents such as ectoin, DESO, and KF7G; fully reversible liquid nitrogen
cryopreservation of entire animals is IMHO quite possible right now. Even
cheating a bit, and using a naturally freeze tolerant animal, would not much
reduce the impact of such a success. Seeing on national television even a frog
or snake surviving storage in liquid nitrogen would massively stimulate further
funding. With such an initial success then money would "suddenly" become
available to research ways and means to reduce the toxicity of affordible
cryoprotectants so that fully reversible organ cryopreservation (and even human
suspended animation) becomes a commercial reality in the near, as opposed to the
distant future. There is one Canadian billionaire signed up for cryonics, but
who is otherwise not active in funding research, and there are as yet no
American billionaires that are interested. All this could change once the
concept has been proven to work. Even Bill Gates might take notice.
Int J Artif Organs. 2010 Jun;33(6):370-80.
Alternatives to dimethylsulfoxide for serum-free cryopreservation of human
mesenchymal stem cells.
Grein TA, Freimark D, Weber C, Hudel K, Wallrapp C, Czermak P. University of
Applied Sciences Giessen-Friedberg, Institute of Biopharmaceutical Technology,
Giessen - Germany.
Abstract
Human mesenchymal stem cells (hMSCs) have some favorable characteristics
like high plasticity, multilineage differentiation potential, and comparably
easy handling in vitro, making them of interest for many clinical and
therapeutic approaches including cell therapy. For routine applications,
these cells have to be stored over a certain period of time without loss of
cell vitality and function. An easy way to preserve cells is to store them
at temperatures between -80 degrees C and -196 degrees C (liquid nitrogen).
To prevent cells from the damage caused by the cryopreservation process and
to achieve high cell recovery and vitality, cryoprotectants are used.
Typically dimethylsulfoxide, often in combination with serum, is used as a
cryoprotectant. However, for clinical approaches, the use of
dimethylsulfoxide and serum in patients is problematic for several reasons.
Therefore, the cryopreservation of human mesenchymal stem cells for cell
therapeutic applications without dimethylsulfoxide and serum demands
investigation. In this work, non-toxic alternatives to dimethylsulfoxide
such as glycerol or the compatible solutes, proline and ectoin, were
analyzed in a serum-free cryomedium with respect to their cryoprotective
properties. Different concentrations of the cryoprotectants (1-10% (w/v)
ectoin or proline, respectively, or 5-20% (v/v) glycerol) and certain
incubation times (0-60 minutes) were investigated with regard to post-thaw
cell vitality and cell growth. Our results showed that, in general,
cryopreservation with ectoin led to high post-thaw cell survival of up to
72% whereas after cryopreservation with glycerol and proline, the hMSC cells
were completely dead (glycerol) or had only poor cell survival (proline,
22%). Moreover, the morphology of the hMSC cells changed to a large and flat
phenotype after cryopreservation with proline. These results indicate that
glycerol and proline are not suitable for cryopreservation of hMSC. In
contrast, ectoin has the potential to replace dimethylsulfoxide as a
cryoprotectant in a serum-free cryomedium.
PMID: 20669142
Cryobiology. 2004 Aug;49(1):1-9.
Glass-forming property of the system diethyl sulphoxide/water and its
cryoprotective action on Escherichia coli survival.
Markarian SA, Bonora S, Bagramyan KA, Arakelyan VB. Department of Chemistry,
Yerevan State University, 375049 Yerevan, Armenia.
Abstract
In this work the thermal properties of diethyl sulphoxide (Et2SO), as well
as its cryoprotective ability are studied and related to other well-known
cryoprotectant substances, like dimethyl sulphoxide (Me2SO). We have
investigated the thermal properties of Et2SO/water systems using
Differential Scanning Calorimetry at a very low heating/cooling rate (2
degrees C/min). Liquid/solid or glassy/crystalline transitions have been
observed only for the solutions with content of Et2SO ranging from 5 up to
40% w/w and/or greater than 85%. In the 45-75% w/w Et2SO range we have found
a noticeable glass-forming tendency and a great stability of the amorphous
state to the reheating. In samples with Et2SO content ranging from 80 to
85%, we observed a great stability of the glass forming by cooling, but a
lesser stability to the subsequent reheating. The glass-forming tendency of
these solutions is discussed in terms of existing competitive interactions
between molecules of Et2SO, on the one hand, and Et2SO and water molecules,
on the other hand. The results are well explainable on the basis of the
model structure of water/Et2SO solutions, deduced by Raman and infrared
studies [J. Mol. Struct. 665 (2003) 285-292]. The cryoprotective ability of
Et2SO on Escherichia coli survival has been also investigated, and a
comparison among Et2SO and other widely used cryoprotectants, like Me2SO and
glycerol has been done. Survival of E. coli, determined after
freezing-thawing process, was maximal at 45% w/w Et2SO (more than 85%
viability). It should be noted that at the same concentration the survival
is only about 35% in the presence of Me2SO and not more than 15% in the
presence of glycerol. These features are well consisted with the
glass-forming properties of Et2SO.
PMID: 15265712
Cryobiology. 2008 Dec;57(3):242-5. Epub 2008 Sep 15.
Improved cryopreservation by diluted vitrification solution with
supercooling-facilitating flavonol glycoside.
Kami D, Kasuga J, Arakawa K, Fujikawa S. National Agricultural Research Center
for Hokkaido Region, Sapporo 062-8555, Japan.
Abstract
The effect of kaempferol-7-O-glucoside (KF7G), one of the
supercooling-facilitating flavonol glycosides which was originally found in
deep supercooling xylem parenchyma cells of the katsura tree and was found
to exhibit the highest level of supercooling-facilitating activity among
reported substances, was examined for successful cryopreservation by
vitrification procedures, with the aim of determining the possibility of
using diluted vitrification solution (VS) to reduce cryoprotectant toxicity
and also to inhibit nucleation at practical cooling and rewarming by the
effect of supplemental KF7G. Examination was performed using shoot apices of
cranberry and plant vitrification solution 2 (PVS2) with dilution.
Vitrification procedures using the original concentration (100%) of PVS2
caused serious injury during treatment with PVS2 and resulted in no regrowth
after cooling and rewarming (cryopreservation). Dilution of the
concentration of PVS2 to 75% or 50% (with the same proportions of
constituents) significantly reduced injury by PVS2 treatment, but regrowth
was poor after cryopreservation. It is thought that dilution of PVS2 reduced
injury by cryoprotectant toxicity, but such dilution caused nucleation
during cooling and/or rewarming, resulting in poor survival. On the other
hand, addition of 0.5mg/ml (0.05% w/v) KF7G to the diluted PVS2 resulted in
significantly (p<0.05) higher regrowth rates after cryopreservation. It is
thought that addition of supercooling-facilitating KF7G induced
vitrification even in diluted PVS2 probably due to inhibition of ice
nucleation during cooling and rewarming and consequently resulted in higher
regrowth. The results of the present study indicate the possibility that
concentrations of routinely used VSs can be reduced by adding
supercooling-facilitating KF7G, by which more successful cryopreservation
might be achieved for a wide variety of biological materials.
PMID: 18824164
Am J Physiol. 1990 Mar;258(3 Pt 2):R559-68.
Life in a frozen state: adaptive strategies for natural freeze tolerance in
amphibians and reptiles.
Storey KB. Institute of Biochemistry, Carleton University, Ottawa, Ontario,
Canada.
Abstract
Winter survival for various species of amphibians and reptiles that
hibernate on land depends on freeze tolerance, the ability to survive for
long periods of time with up to 65% of total body water as extracellular
ice. Freeze tolerance has been described for four species of frogs, one
salamander, and hatchlings of the painted turtle. A very limited tolerance
also occurs in garter snakes. Studies of freeze tolerance in vertebrates
have primarily focused on the wood frog Rana sylvatica and have assessed the
regulation of cryoprotectant synthesis, cryoprotectant action in freezing
preservation of isolated cells and tissues, metabolism and energetics under
the ischemic conditions imposed by freezing, and the role of ice-nucleating
agents in blood. The adaptations that preserve life at subzero temperatures
for these animals illustrate the principles of vertebrate organ
cryopreservation and may have important applications in the development of
technology for the freezing preservation of transplantable human organs.
PMID: 2180324
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