X-Message-Number: 32270
Date: Wed, 30 Dec 2009 00:05:02 -0800 (PST)
From:
Subject: flavonoids protect against freezing and freeze-drying damage
[Although the results are impressive, flavonoid additions are still surprisingly
rare. The reason is not cost. For example, the distilled water in
cryoprotectant solutions could be replaced with filtered green tea at a
negligible cost.]
[Genistein reduces cryopreservation induced DNA damage.]
Hum Reprod. 2009 Sep;24(9):2061-70. Epub 2009 Jun 12.
Cryopreservation-induced human sperm DNA damage is predominantly mediated by
oxidative stress rather than apoptosis.
Thomson LK, Fleming SD, Aitken RJ, De Iuliis GN, Zieschang JA, Clark AM.
Fertility First, PO Box 807, Hurstville, NSW 2220, Australia.
BACKGROUND: Whereas studies have revealed that the cryopreservation of human
semen increases sperm DNA fragmentation, the mechanisms involved in this
type of cryo-injury are largely unknown. Elucidation of these mechanisms may
provide insight into preventing such injury. METHODS: We obtained 60 semen
samples from 60 men and conducted experiments to determine the cause of
cryopreservation-induced DNA fragmentation using
8-oxo-7,8-dihydro-2'deoxyguanosine (8OHdG) as a biomarker of oxidative
stress, percentage caspase positive cells as an indicator of apoptosis, the
potential antioxidant genistein and the caspase inhibitor Z-VAD(OMe)-FMK.
RESULTS: Cryopreservation led to a significant increase in percentage DNA
fragmentation, percentage 8OHdG and percentage caspase positive cells (P <
0.001). Percentage DNA fragmentation was positively correlated with
percentage 8OHdG before (r = 0.756, P < 0.001) and after cryopreservation (r
= 0.528, P = 0.017). The addition of 50 and 100 microM genistein to the
cryoprotectant had a significant protective effect on sperm DNA (P < 0.001)
although the caspase inhibitor demonstrated no difference to the control.
CONCLUSIONS: Human sperm DNA fragmentation is associated with an increase in
oxidative stress during cryopreservation, rather than the activation of
caspases and apoptosis. The estrogenic compound genistein may be useful in
reducing this effect but larger trials are needed to confirm this.
PMID: 19525298
[Green tea flavonoid ECGC comes to the rescue here.]
Cell Transplant. 2009;18(5):513-9.
Long-term preservation of rat skin tissue by epigallocatechin-3-o-gallate.
Kim H, Kawazoe T, Matsumura K, Suzuki S, Hyon SH. Department of Medical
Simulation Engineering, Research Center for Nano Medical Engineering, Institute
for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan.
Skin grafts can be preserved by cryopreservation and refrigerated storage at
4 degrees C. Epigallocatechin-3-O-gallate (EGCG) enhances the viability of
stored skin grafts and also extends the storage time up to 7 weeks at 4
degrees C. EGCG, the major polyphenolic constituent present in green tea,
has potent antioxidant, antimicrobial, antiproliferative, and free radical
scavenging effects. This study examined the effects of EGCG on skin
cryopreservation. Skin sample biopsy specimens from GFP rats were previously
treated with/without EGCG then moved to -196 degrees C. Skin samples were
transplanted to nude mice after 2, 8, and 24 weeks of preservation. Glucose
consumption was measured after thawing to assess the metabolic activity. Two
weeks later the transplanted skin grafts were excised and histologically
analyzed. Histological examinations revealed the degeneration of the
epidermal and dermal layers in all groups. In the EGCG groups, the grafts
showed higher integrity in the epidermal layer and dermal matrix. The
present findings suggest the future clinical usefulness of EGCG for skin
preservation; however, the mechanism by which EGCG promotes skin
preservation still remains unclear.
PMID: 19775511
[ECGC enables cells to survive freeze-drying. Its benefit is much greater than
that of trehalose, and a trehalose/ECGC combination offers a synergistic effect.
Before washing, cellular viability with this combination is over 90% after both
freeze-thawing and freeze-drying.]
PLoS One. 2009;4(4):e5240. Epub 2009 Apr 21.
Freeze-drying of mononuclear cells derived from umbilical cord blood followed by
colony formation.
Natan D, Nagler A, Arav A. Core Dynamics Ltd, Ness-Ziona, Israel.
BACKGROUND: We recently showed that freeze-dried cells stored for 3 years at
room temperature can direct embryonic development following cloning.
However, viability, as evaluated by membrane integrity of the cells after
freeze-drying, was very low; and it was mainly the DNA integrity that was
preserved. In the present study, we improved the cells' viability and
functionality after freeze-drying. METHODOLOGY/PRINCIPAL FINDINGS: We
optimized the conditions of directional freezing, i.e. interface velocity
and cell concentration, and we added the antioxidant EGCG to the freezing
solution. The study was performed on mononuclear cells (MNCs) derived from
human umbilical cord blood. After freeze-drying, we tested the viability,
number of CD34(+)-presenting cells and ability of the rehydrated
hematopoietic stem cells to differentiate into different blood cells in
culture. The viability of the MNCs after freeze-drying and rehydration with
pure water was 88%-91%. The total number of CD34(+)-presenting cells and the
number of colonies did not change significantly when evaluated before
freezing, after freeze-thawing, and after freeze-drying (5.4 x 10(4)+/-4.7,
3.49 x 10(4)+/-6 and 6.31 x 10(4)+/-12.27 cells, respectively, and
31+/-25.15, 47+/-45.8 and 23.44+/-13.3 colonies, respectively). CONCLUSIONS:
This is the first report of nucleated cells which have been dried and then
rehydrated with double-distilled water remaining viable, and of
hematopoietic stem cells retaining their ability to differentiate into
different blood cells.
PMID: 19381290
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2667668/pdf/pone.0005240.pdf
[Kaempferol-7-O-glucoside reduces vitrification solution toxicity.]
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.
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
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