X-Message-Number: 30199
Date: Sat, 22 Dec 2007 20:14:56 -0800 (PST)
From: 
Subject: a new vitrification solution is developed

  [Osmotic toxicity eliminated glycerol from this test. A
glycerol/methanol combination was not tested. Best solution overall
consisted of 20% DMSO, 10% propylene glycol, and 10% methanol. A straight
substitution of ethylene glycol for the propylene glycol was not
tested. With nine cryoprotectants (only five mentioned in the
abstract) under test at four concentrations (0%, 10%, 20%, 30%), a
complete test of all these combinations yielding a 40% total
concentration would have to test several thousand separate
solutions . This illustrates how futile it would be to try exhaustive
testing, without regard to or paying much attention to the
different modes of cryoprotectant toxicity. The following is my own
admittedly amateur attempt at factoring in toxicity considerations to
eliminate solutions from consideration.
  Since osmotic damage was a significant factor in this test, there was no
point in testing any solution which did not have at least some methanol
in it, since methanol was the only cryoprotectant which could rapidly
penetrate cell membranes, and limit this form of damage when nonramped
solutions are abruptly added to medium. However methanol itself has a high
extrinsic toxicity, as well as little ability to vitrify, so there is
also little point in testing solutions with more than 10% methanol in
them. 21'st Century Medicine has found that vitrification solutions with
ethylene glycol are less toxic than those containing propylene glycol,
possibly because of a reduced intrinsic (denaturing) toxicity. Therefore
there was no point in even considering any solution containing propylene
glycol. This all points out how little thought has been given to the three
different types of toxicity, which I can osmotic, intrinsic, and
extrinsic. Indeed some would even deny there are three different types of
toxicity, which gives an idea of how little is really known about the
nature of cryoprotectant toxicity. Aside from some attempts by Fahy et al,
the theoretic basis of cryoprotectant toxicity has been virtually ignored
in medical journals.]

Theriogenology. 2007 Sep 15;68(5):702-8. Epub 2007 Jul 2.
Preliminary studies on the vitrification of red sea bream (Pagrus
major) embryos.
  Ding FH, Xiao ZZ, Li J. Institute of Oceanology, Chinese Academy of
Sciences, Qingdao, China.
  The objective was to identify an appropriate cryoprotectant and protocol
for vitrification of red sea bream (Pagrus major) embryos. The toxicity
of five single-agent cryoprotectants, dimethyl sulfoxide (DMSO),
propylene glycol (PG), ethylene glycol (EG), glycerol (GLY), and methyl
alcohol (MeOH), as well as nine cryoprotectant mixtures, were investigated
by comparing post-thaw hatching rates. Two vitrifying protocols, a straw
method and a solid surface vitrification method (copper floating over
liquid nitrogen), were evaluated on the basis of post-thaw embryo
morphology. Exposure to single-agent cryoprotectants (10% concentration
for 15 min) was not toxic to embryos, whereas for higher concentrations
(20 and 30%) and a longer duration of exposure (30 min), DMSO and PG were
better tolerated than the other cryoprotectants. Among nine
cryoprotectant mixtures, the combination of 20% DMSO+10% PG+10% MeOH had
the lowest toxicity after exposure for 10 min or 15 min. High percentages
of morphologically intact embryos, 50.6+/-16.7% (mean+/-S.D.) and
77.8+/-15.5%, were achieved by the straw vitrifying method (20.5%
DMSO+15.5% acetamide+10% PG, thawing at 43 degrees C and washing
in 0.5M sucrose solution for 5 min) and by the solid surface
vitrification method (40% GLY, thawing at 22 degrees C and washing in
0.5M sucrose solution for 5 min). After thawing, morphological changes in
the degenerated embryos included shrunken yolks and ruptured
chorions. Furthermore, thawed embryos that were morphologically intact did
not consistently survive incubation.
PMID: 17606292

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