X-Message-Number: 30337
Date: Sun, 20 Jan 2008 23:57:02 -0800 (PST)
From: 
Subject: The lowest toxicity cryoprotectant is... (part 1/2)

[It may be 1,3-propanediol. (also called trimethylene glycol)
1,3-propanediol has been tested just three times as a cryopreservative, in
2006 (PMID: 17256068), 1989 (2708506), and 1980 (7431324). The reason it
has  been largely over-looked in the field of cryopreservation appears to be
because it is a poor glass former, while the most effective cryoprotectants
tend to good glass formers. However good glass formers have a strong
tendancy to exhibit increased toxicity in vitrification solutions (see
Fahy et al in 14969679). As a consequence vitrification solutions tend to
contain small amounts of good glass formers, supplemented by larger
amounts of relatively poor glass formers to eliminate ice formation, while
incurring a reduced toxicity penalty. An extract from table 1 of
(10788314) implies that 1,3-propanediol is the lowest toxicity
cryoprotectant. Fahy found that substituting ethylene glycol for
1,2-propanediol reduced vitrification toxicity. If Fahy is correct then
further substituting 1,3-propanediol for ethylene glycol should further
lower toxicity.

Critical Cooling Rates to Avoid Any Crystalization
45% solute         C/minute)
L2,3-butanediol     <2.5
1,3-butanediol       7
1,2-propanediol     10
1,2,3- butanetriol  11
dimethylsulfoxide   14
dimethylformamide   23
diethylformamide    48
1,4-butanediol      71
ethylene glycol    260
glycerol           380
1,3-propanediol   1100

There exists independent data confirming that 1,3-propanediol is the lowest
toxicity cryoprotectant. In (7431324) the effect of various concentrations
of cryoprotectants on rabbit spermatozoa motility at 20 C for 30 minutes was
documented in table 1 below. At the highest dosage tested 1,3-propanediol
showed the highest motility, indicating that it was the least toxic.

Cryoprotectant    0.31M 0.93M 1.55M 2.17M 3.10M
1,3-propanediol   50    52    54    54    52
Dimethylsulfoxide 62    63    61    60    38
Lactamide         61    62    57    51    35
Acetamide         60    61    54    45    30
Ethylene glycol   48    50    50    39    27
Glycerol          47    23     7     3     2
Dimethylformamide 63    55    33    18     0
Propionamide      44    46    19     6     0
Malomide          61    33     7     5     0
Formamide         53    39    16     4     0
Butyramide        30    17     0     0     0

The effect on motility after freezing and thawing was also measured, as
shown below. Here 2.0M solutions were always significantly more toxic than
1.0 M solutions, except those for 1,3-propanediol. However 1,3-propanediol
still did not have the best results even at the most concentrated 2.0M
solution. This highlights the difference between most effective, and least
toxic. If one were developing a vitrification solution based solely on the
results shown here, one would base it on the most effective cryoprotectant,
which was 1.0M lactamide. The rest of the vitrification solution would
then consist of 3.1M plus of 1,3-propanediol.

Cryoprotectant    0.125M 0.25M 0.5M 0.75M 1.0M 1.25M 1.5M 2.0M
1,3-propanediol   19     11    11   13    17   23    16   15
Dimethylsulfoxide 25     31    31   38    44   36    28   17
Lactamide          5      5    29   42    53    -    47   31
Acetamide          9     12    24   37    40   33    25    2
Ethylene glycol   12     17    24   22    22   20    22   15
Glycerol          12     10     8    4     2   <1    <1   <1
Dimethylformamide 14     27    32   30    19    -     -    -
Propionamide       6      4     6   22    12    2     0    0
Malomide          12      5    <1    -     -    -     -    -
Formamide          4      6     6    3    <1   <1    <1   <1
Butyramide         2      0     0    -     -    -     -    -
Without(control)  <1

Below are some relevant abstracts.]

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