X-Message-Number: 26129
Date: Tue, 3 May 2005 20:50:41 -0700 (PDT)
From: Doug Skrecky <>
Subject: cryoprotectant/methanol combinations

[This abstract looks at various combinations of propylene glycol (low
chemical toxicity, high osmotoxicity), with methanol (high chemical
toxicity, low osmotic toxicity).]

Theriogenology. 2005 Mar 1;63(4):1207-19.
Cryopreservation of flounder (Paralichthys olivaceus) embryos by vitrification.
  Conventional cryopreservation of complex teleost embryos has been
unsuccessful, possibly because their large size (1-7 mm diameter),
multi-compartmental structure and low water permeability lead to
intracellular ice formation and chilling injury. To overcome these
obstacles, we have developed a vitrification procedure for
cryopreservation of flounder (Paralichthys olivaceus) embryos. In initial
toxicity tests, propylene glycol (PG) and methanol (MeOH) were less toxic
to embryos than dimethylformamide (DMF) or dimethyl sulfoxide (Me2SO),
whereas ethylene glycol (EG) and glycerol (Gly) were toxic to all tested
embryos. Embryos between four-somite and tail bud stages were more
tolerant to vitrifying solutions than embryos in other developmental
stages. Four vitrifying solutions (FVS1-FVS4) were prepared by combining
a basic saline solution (BS2) and cryoprotectants PG and MeOH in
different proportions (FVS1: 67, 20 and 13%; FVS2: 60, 24 and 16%; FVS3:
55, 27 and 18%; FVS4: 50, 30 and 20% of BS2, PG and MeOH, respectively).
Their impact on flounder embryos was then compared. FVS1 produced the
highest survival rate; whereas deformation rate was highest for FVS4.
Five-step equilibration of embryos in FVS2 resulted in higher survival
rates than equilibration in 4, 3, 2 or 1 steps. Flounder embryos varying
from the 14-somite to the pre-hatching stage were cryopreserved in the
four vitrifying solutions in liquid nitrogen for 1-7 h. From eight
experiments, 20 viable thawed embryos were recovered from 292
cryopreserved embryos. Fourteen larvae with normal morphology hatched
successfully from the 20 surviving frozen-thawed embryos from five
experiments. Embryos at the tail bud stage exhibited greater tolerance to
vitrification than embryos at other stages. These results establish that
cryopreservation of flounder embryos by vitrification is possible. The
technology has many potential applications in teleost germplasm resource
conservation.

[Although the abstract below does not mention this, the best results were
with 20% glycerol, and 5% methanol. My hypothesis with regard to this
fact, relates to the relatively low chemical toxicity, but very high
osmotic toxicity of glycerol. Since methanol rapidly penetrates cellular
membranes, it probably acted to reduce glycerol induced osmotic
toxicity.]

Theriogenology. 2005 Feb;63(3):763-73
Toxicity and protective efficiency of cryoprotectants to flounder
(Paralichthys olivaceus) embryos.
  With the purpose of finding an ideal cryoprotectant or combination of
cryoprotectants in a suitable concentration for flounder (Paralichthys
olivaceus) embryo cryopreservation, we tested the toxicities, at culture
temperature (16 degrees C), of five most commonly used
cryoprotectants-dimethyl sulfoxide (Me2SO), glycerol, methanol (MeOH),
1,2-propylene glycol (PG) and ethylene glycol (EG). In addition,
cryoprotective efficiency to flounder embryos of individual and combined
cryoprotectants were tested at -15 degrees C for 60 min. Five different
concentrations of each of the five cryoprotectants and 20 different
combinations of these cryoprotectants were tested for their protective
efficiency. The results showed that the toxicity to flounder embryos of
the five cryoprotectants are in the following sequence: PG < MeOH < Me2SO
< glycerol < EG (P < 0.05); whereas the protective efficiency of each
cryoprotectant, at -15 degrees C for a period of 60 min, are in the
following sequence: PG > Me2SO approximately MeOH approximately glycerol
> EG (greater symbols mean P < 0.05, and approximate symbols mean P >
0.05). Methanol combined with any one of the other cryoprotectants
gave the best protection, while ethylene glycol combined with any one of
the other cryoprotectants gave the poorest protection at -15 degrees C.
Toxicity effect was concentration dependent with the lowest concentration
being the least toxic for all five cryoprotectants at 16 degrees C. For
PG, MeOH and glycerol, 20% solutions gave the best protection at -15
degrees C; whereas a 15% solution of Me2SO, and a 10% solution of EG,
gave the best protection at -15 degrees C.

[This abstract illustrates the low osmotic toxicity of methanol relative
to glycerol.]

Theriogenology. 2004 Sep 15;62(6):1153-9.
Methanol as a cryoprotectant for equine embryos.
  Equine embryos (n=43) were recovered nonsurgically 7-8 days after
ovulation and randomly assigned to be cryopreserved in one of two
cryoprotectants: 48% (15M) methanol (n=22) or 10% (136 M) glycerol
(n=21). Embryos (300-1000 microm) were measured at five intervals after
exposure to glycerol (0, 2, 5, 10 and 15 min) or methanol (0, 15, 35, 75
and 10 min) to determine changes (%) in diameter over time (+/-S.D.).
Embryos were loaded into 0.25-ml plastic straws, sealed, placed in a
programmable cell freezer and cooled from room temperature (22 degrees C)
to -6 degrees C. Straws were then seeded, held at -6 degrees C for 10 min
and then cooled to -33 degrees C before being plunged into liquid
nitrogen. Two or three embryos within a treatment group were thawed and
assigned to be either cultured for 12 h prior to transfer or immediately
nonsurgically transferred to a single mare. Embryo diameter decreased in
all embryos upon initial exposure to cryoprotectant. Embryos in methanol
shrank and recovered slightly to 76+/-8 % of their original diameter;
however, embryos in glycerol continued to shrink, reaching 57+/-6 % of
their original diameter prior to cryopreservation. Survival
rates of embryos through Day 16 of pregnancy were 38 and 23%,
respectively (P>0.05) for embryos cryopreserved in the presence of
glycerol or methanol. There was no difference in pregnancy rates of mares
receiving embryos that were cultured prior to transfer or not cultured
(P>0.05). Preliminary experiments indicated that 48% methanol was not
toxic to fresh equine embryos but methanol provided no advantage over
glycerol as a cryoprotectant for equine blastocysts.

[Osmotic toxicity is a major limitation with most vitrification solutions.]

Hum Reprod. 2004 May;19(5):1148-54. Epub 2004 Apr 7.
The effect of osmotic stress on the metaphase II spindle of human
oocytes, and the relevance to cryopreservation.
  BACKGROUND: Knowing osmotic tolerance limits is important in the design
of optimal cryopreservation procedures for cells. METHODS: Mature human
oocytes were exposed to anisosmotic sucrose solutions at concentrations
of 35, 75, 150, 600, 1200, or 2400 (+/-5) milliosmolal (mOsm) at 37
degrees C. A control treatment at 290 mOsm was also utilized. Oocytes were
randomly allocated to each experimental treatment. After the treatment,
the oocytes were cultured for 1 h, then fixed in cold methanol.
Immunocytochemical staining and fluorescence microscopy were used to
assess the morphology of the metaphase II (MII) spindle. Logistic
regression was used to determine if media osmolality had a significant
effect on spindle structure. RESULTS: Osmolality was a significant
predictor of spindle morphology. Hyposmotic effects at 35, 75, and 150
mOsm resulted in 100, 67, and 56% of oocytes having abnormal spindles,
respectively. Hyperosmotic effects at 600, 1200, and 2400 mOsm resulted
in 44, 44, and 100% of the spindles with abnormal structure,
respectively. CONCLUSIONS: Anisosmotic conditions lead to disruption of
the MII spindle in human oocytes. Applying this fundamental knowledge to
human oocyte cryopreservation should result in increased numbers of cells
maintaining viability.

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