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1.
The present study was undertaken to define the conditions for optimal cryopreservation of hepatocytes. Two different freezing procedures were analyzed: a slow freezing rate (SFR) (-2 degrees C/min down to -30 degrees C and then quick freezing to -196 degrees C) and a fast freezing rate (FFR) (direct freezing of tubes to -196 degrees C: -39 degrees C/min). Cells were frozen in fetal bovine serum containing 10% Dimethyl sulfoxide (DMSO). After rapid thawing at 37 degrees C, followed by dilution and removal of the cryoprotectant, cells were plated and several parameters were followed as criteria for optimal cryopreservation of cells. The FFR cells showed no apparent ultrastructural damage after 24 h of culture. Plating efficiency and spreading were similar as controls. Gluconeogenesis from pyruvate and fructose, tyrosine amino transferase induction by glucagon and dexamethasone, urea production, and plasma protein synthesis of FFR cells were similar to those found in control cultures. The FFR procedure, in comparison to the SFR method, seemed to render the best preserved hepatocytes. 相似文献
2.
Colauto Nelson Barros da Eira Augusto Ferreira Linde Giani Andrea 《World journal of microbiology & biotechnology》2011,27(12):3015-3018
The preservation of Agaricus blazei is generally done by mycelial subculturing, but this technique may cause genetic degenerations. Despite this, there is not
an efficient protocol established to preserve this fungus and cryopreservation could be an alternative. This study aimed to
evaluate two freezing protocols for cryopreservation at −80°C of A. blazei strains. Five fungus strains grown on rice grains with husk and were transferred to glycerol (10%) in cryovials. Next, the
cryovials were submitted to two freezing temperature protocols: (1) cryopreservation starting at 25°C, then at 8°C for 30 min
and kept at −80°C; (2) cryopreservation starting at 25°C, then 8°C for 30 min, −196°C for 15 min and kept at −80°C. After
1 year of cryopreservation, the cryovials were thawed in a water bath at 30°C for 15 min and transferred to malt extract agar
medium. It was concluded that the one-year cryopreservation process of A. blazei, grown on rice grains and cryopreserved at −80°C in glycerol 10%, is viable. The slow freezing, from 8 to −80°C, is effective
whereas the fast freezing, from 8 to −196°C and then to −80°C, is ineffective. The different genetic characteristics among
the strains of this fungus do not interfere in the cryopreservation process. 相似文献
3.
Josef Elster Peter Degma Ľubomír Kováčik Lucia Valentová Katarína Šramková Antonio Batista Pereira 《Biologia》2008,63(6):843-851
The freezing and desiccation tolerance of 12 Klebsormidium strains, isolated from various habitats (aeroterrestrial, terrestrial, and hydro-terrestrial) from distinct geographical
regions (Antarctic — South Shetlands, King George Island, Arctic — Ellesmere Island, Svalbard, Central Europe — Slovakia)
were studied. Each strain was exposed to several freezing (−4°C, −40°C, −196°C) and desiccation (+4°C and + 20°C) regimes,
simulating both natural and semi-natural freeze-thaw and desiccation cycles. The level of resistance (or the survival capacity)
was evaluated by chlorophyll a content, viability, and chlorophyll fluorescence evaluations. No statistical differences (Kruskal-Wallis tests) between strains
originating from different regions were observed. All strains tested were highly resistant to both freezing and desiccation
injuries. Freezing down to −196°C was the most harmful regime for all studied strains. Freezing at −4°C did not influence
the survival of studied strains. Further, freezing down to −40°C (at a speed of 4°C/min) was not fatal for most of the strains.
RDA analysis showed that certain Antarctic and Arctic strains did not survive desiccation at +4°C; however, freezing at −40°C,
as well as desiccation at +20°C was not fatal to them. On the other hand, other strains from the Antarctic, the Arctic, and
Central Europe (Slovakia) survived desiccation at temperatures of +4°C, and freezing down to −40°C. It appears that species
of Klebsormidium which occupy an environment where both seasonal and diurnal variations of water availability prevail, are well adapted to
freezing and desiccation injuries. Freezing and desiccation tolerance is not species-specific nor is the resilience only found
in polar strains as it is also a feature of temperate strains.
Presented at the International Symposium Biology and Taxonomy of Green Algae V, Smolenice, June 26–29, 2007, Slovakia.
This paper is dedicated to the memory of the late Dr. Bohuslav Fott (1908–1976), Professor of Botany at the Charles University
in Prague, to mark the centenary of his birth. 相似文献
4.
Meana A Martinez R Cañal P Arriaga MJ Román FS Llames S Orós C Moreno A Fernandez C 《Cell and tissue banking》2006,7(3):203-206
In order to transport and cryopreserve human tissues, it is essential to have an easy-to-use recipient where tissues can be
kept in sterile conditions. Here we show the results obtained by using Macopharma’s tissue freezing bags, an aluminium-polyethylene
multilayer bag, in our tissue bank of the Centro Comunitario de Sangre y Tejidos de Asturias. Five hundred and twenty-seven
cancellous bone homografts were obtained from hospitals located 120 km around our Bank. The homografts were submitted to bacteriological
controls and sent to our bank in these bags. They were stored at −70 °C and sent in dry ice to about 50 hospitals, where the
tissue was bacteriologically controlled and grafted. Furthermore, the behaviour of these bags at −140 °C (vapour nitrogen)
or −196 °C (liquid nitrogen) was tested. Our results indicate that Macopharma aluminium-polyethylene bags are suitable for
the transporting and cryopreserving of cancellous bone homografts. These bags could also be used for keeping tissues in nitrogen
containers. 相似文献
5.
K. Škrlep M. Bergant G. M. De Winter B. Bohanec J. Žel R. Verpoorte F. Van Iren M. Camloh 《Biologia Plantarum》2008,52(2):329-333
Different lines of cell suspension cultures of Taxus × media Rehd. and Taxus floridana Nutt. were cryopreserved with a two-step freezing method using a simple and inexpensive freezing container instead of a programmable
freezer. Four to seven days old suspension cell cultures were precultured in growth medium supplemented with 0.5 M mannitol
for 2 d. The medium was then replaced with cryoprotectant solution (1 M sucrose, 0.5 M glycerol and 0.5 M dimethylsulfoxide)
and the cells incubated on ice for 1 h. Before being plunged into liquid nitrogen, cells were frozen with a cooling rate of
approximately −1 °C per min to −80 °C. The highest post-thaw cell viability was 90 %. The recovery was line dependent. The
cryopreservation procedure did not alter the nuclear DNA content of the cell lines. The results indicate that cryopreservation
of Taxus cell suspension cultures using inexpensive freezing container is possible. 相似文献
6.
Catherine S. Ford Nicky B. Jones Johannes van Staden 《In vitro cellular & developmental biology. Plant》2000,36(5):366-369
Summary The protocol currently used to cryopreserve Pinus patula embryogenic tissue was investigated in an attempt to improve and optimize the recovery of tissue. This investigation describes
two aspects that influence tissue recovery after cryopreservation: (i) the effects of precooling tissue prior to immersion
into liquid nitrogen; and (ii) whether the choice of supports onto which the recovered tissue is suspended improved the recovery
rate. Results indicated that precooling tissue to −70°C prior to immersion into liquid nitrogen was superior to precooling
to −30°C or direct immersion in liquid nitrogen (−196°C). Tissue recovery improved when polyester grids were used as supports,
and was slowest on filter paper supports. 相似文献
7.
The first successful freezing of early embryos to −196°C in 1972 required that they be cooled slowly at ∼1°C/min to about
−70°C. Subsequent observations and physical/chemical analyses indicate that embryos cooled at that rate dehydrate sufficiently
to maintain the chemical potential of their intracellular water close to that of the water in the partly frozen extracellular
solution. Consequently, such slow freezing is referred to as equilibrium freezing. In 1972 and since, a number of investigators
have studied the responses of embryos to departures from equilibrium freezing. When disequilibrium is achieved by the use
of higher constant cooling rates to −70°C, the result is usually intracellular ice formation and embryo death. That result
is quantitatively in accord with the predictions of the physical/chemical analysis of the kinetics of water loss as a function
of cooling rate. However, other procedures involving rapid nonequilibrium cooling do not result in high mortality. One common
element in these other nonequilibrium procedures is that, before the temperature has dropped to a level that permits intracellular
ice formation, the embryo water content is reduced to the point at which the subsequent rapid nonequilibrium cooling results
in either the formation of small innocuous intracellular ice crystals or the conversion of the intracellular solution into
a glass. In both cases, high survival requires that subsequent warming be rapid, to prevent recrystallization or devitrification.
The physical/ chemical analysis developed for initially nondehydrated cells appears generally applicable to these other nonequilibrium
procedures as well. 相似文献
8.
Andrade MG Sá CN Marchionni AM dos Santos Calmon de Bittencourt TC Sadigursky M 《Cell and tissue banking》2008,9(4):279-287
Allograft bone has been widely used for reconstruction of different portions of the skeleton. The fragment of bone harvested
must be kept under low temperatures. The cryopreservation also contributes to decrease the antigenic potential of the tissue.
Although this technique is considered safe, there is little information about the morphological modifications that the medullary
and cortical portions of bone suffer after freezing. Hence, the aim of this study was to investigate the morphology of bone
tissue after freezing under different temperatures and periods. Twelve rabbits were used to analyze the effects of two temperatures,
−20°C and −70°C, during four periods of time: 30, 60, 90, 120 days. Tissues were analyzed by HE, picro-sirius stains and also
by Feulgen’s reaction, through qualitative and morphometric ways, considering the area occupied by cells and nuclei, medullary
and cortical portions, as well as by collagen expression at cortical. The differences among the treatments were analyzed by
Tukey′s test, at 5% significance level. Bone freezing increased cellular and nuclear areas at cancellous bone and diminished
nuclear area at the cortical bone. Cortical bone collagen suffered denaturation proportionally to temperature decrease and
to freezing duration. These alterations compromised the morphology of tissues after 90 or 120 days of freezing at the temperature
of −70°C. Cells necrosed during freezing, contributing to reduce bone antigenicity. 相似文献
9.
J. P. Costanzo A. L. Allenspach R. E. Lee Jr. 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1999,169(4-5):351-359
We investigated function and ultrastructure of sciatic nerves isolated from wood frogs (Rana sylvatica) endemic to the Northwest Territories, Canada, following freezing at −2.5 °C, −5.0 °C, or −7.5 °C. All frogs frozen at −2.5 °C,
and most frogs (71%) frozen at −5.0 °C, recovered within 14 h after thawing began; however, frogs did not survive exposure
to −7.5 °C. Sciatic nerves isolated from frogs frozen at −7.5 °C were refractory to electrical stimulation, whereas those
obtained from frogs surviving exposure to −2.5 °C or −5.0 °C generally exhibited normal characteristics of compound action
potentials. Frogs responded to freezing by mobilizing hepatic glycogen reserves to synthesize the cryoprotectant glucose,
which increased 20-fold in the liver and 40-fold in the blood. Ultrastructural analyses of nerves harvested from frogs in
each treatment group revealed that freezing at −2.5 °C or −5.0 °C had little or no effect on tissue and cellular organization,
but that (lethal) exposure to −7.5 °C resulted in marked shrinkage of the axon, degeneration of mitochondria within the axoplasm,
and extensive delamination of myelin sheaths of the surrounding Schwann cells.
Accepted: 28 April 1999 相似文献
10.
The autecology and ecophysiology of two selected periphytic species of Xanthophyceae (Tribonema fonticolum and T. monochloron) were studied from seasonal pools of the inundation area, in the upper part of the Lužnice River (Třeboňsko Biosphere Reserve,
Czech Republic) during winter–spring flood. Our studies have shown that these species differ in their ecological requirements
(their temperature and light optima; inorganic carbon sources for photosynthesis; and also their ability to survive freezing
and desiccation injuries). In our experiments, the optimal growth temperatures for both strains were higher than the temperatures
of the water they were collected and isolated from. Tribonema monochloron has the rate of photosynthesis several times higher than T. fonticolum. In addition, the optimal growth temperatures were about 3–4°C lower for Tribonema monochloron than for T. fonticolum. From our results, we concluded that both strains of Tribonema prefer low intensities of irradiance. Both Tribonema strains were determined as CO2 users, but we revealed the ability of T. fonticolum to use HCO3− in small amounts. In both Tribonema strains, 100% of the cells survived freezing down to −4°C. The cells’ viability after freezing at −40, −100 and −196°C was
much higher for T. monochloron (about 40%) than for T. fonticolum (about 4%). With respect to desiccation damages, at temperatures of +4 and +20°C, T. monochloron (the species better adapted to low temperatures) did not survive. In contrast, about 80% cells of T. fonticolum survived desiccation at both temperatures.
Handling editor: J. Padisak. 相似文献
11.
Conditions were evaluated for optimum cryopreservation of primary chicken embryo kidney (CEK) cells. The recovery of viable
CEK cells was best (50.8% viability) when the concentration of dimethyl sulfoxide (DMSO) in the freezing medium was 20% (v/v).
The viability of primary CEK cells was not influenced by the concentration of calf serum in the freezing medium, the duration
of storage at −70°C before storage in liquid nitrogen, cell concentration, or the method of addition or dilution of DMSO.
Thawed cells recovered and grew in complete growth medium similarly to cells freshly isolated from kidney, and influenza viruses
produced plaques in the monolayer. The cryopreservation procedures described here may facilitate maintenance of a standard
stock of primary CEK cells for laboratories where preparation of primary CEK cells is not an option. 相似文献
12.
Determination of the kinetics of permeation of dimethyl sulfoxide in isolated corneas 总被引:2,自引:0,他引:2
Corneal cryopreservation requires that endothelial cells remain viable and intercellular structure be preserved. High viability
levels for cryopreserved endothelial cells have been achieved, but preserving intercellular structure, especially endothelial
attachment to Descemet's membrane, has proved difficult. Cell detachment apparently is not caused by ice, suggesting osmotic
or chemical mechanisms. Knowledge of the permeation kinetics of cryoprotectants (CPAs) into endothelial cells and stroma is
essential for controlling osmotic and chemical activity and achieving adequate tissue permeation prior to cooling. Proton
nuclear magnetic resonance (NMR) spectroscopy was used to assess the permeation of dimethyl sulfoxide (DMSO) into isolated
rabbit corneas. Corneas with intact epithelia were exposed to isotonic medium or 2.0 mol/L DMSO for 60 min and subsequently
transferred to 2.0 or 4.0 mol/L DMSO, respectively, at 22, 0, or −10°C. DMSO concentration in the cornea was measured vs time.
The Kedem-Katchalsky model was fitted to the data. Hydraulic permeability (m3/N·s) is 7.1×10−13+216%-11% at 22°C, 8.2×10−13+235%−21% at 0°C, and 1.7×10−14+19% −16% at −10°C. The reflection coefficient is 1.0+2%−1% at 22°C and 0°C, and 0.9±5% at −10°C. Solute mobility (cm/s) is
5.9×10−6+6%–11% at 22°C, 3.1×10−6+12%−11% at 0°C, and 5.0×10−8 cm/s+59%−40% at −10°C. 相似文献
13.
Rhodamine-phalloidin staining of winter oilseed rape suspension cells revealed that the structure of actin cytoskeleton changes
with the phase of cell growth. In small, 4-day-old cells, entering the exponential phase of growth, a dense and uniformly
distributed cortical microfilament networks was seen. In six-day-old vacuolated cells, which reached the stationary phase
of growth, the actin cytoskeleton was composed of thicker microfilament cables in irregular arrangements. In cells acclimated
in cold for 7 days a dense, uniformly distributed and cortical microfilament network was still seen. The fine microfilament
network was sensitive to extracellular freezing since the structures underwent depolymerization at −3 °C (in the presence
of extracellular ice), both in non-acclimated and cold-acclimated cells. The thicker transvacuolar cables in cells of the
stationary growth phase resisted freezing to −7 °C. Acclimation of suspensions at 2 °C resulted in slowing down growth of
cells and in the increased freezing tolerance of cells as indicated by a decrease of LT50 from −11 °C to −17.5o or to −25 °C when determined 7 or 20 days after the beginning of the cold treatment, respectively. Freezing tolerance of
non-acclimated cells decreased from −11 °C to −8 °C during subculture, showing a transient increase to −17 °C on the day 6.
Results indicate that the arrangement of actin microfilaments and their sensitivity to freezing-induced depolymerization depends
on the phase of cell growth rather than on cell acclimation status. Possible mechanisms involved in the freezing-induced depolymerization
of actin microfilaments are discussed.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
14.
Cryopreservation of sperm is an extremely important issue in the field of male infertility as freezing can have detrimental
effects on a variety of sperm functions, some of them not accessible to the traditional semen quality analysis. In this study,
chromatin structure variations in human spermatozoa in semen were studied with the sperm chromatin structure assay (SCSA),
both before and after cryopreservation. Samples were divided into two aliquots: the first was analysed without further treatment,
while the second was stored in liquid nitrogen at −196 °C using standard cryopreservation techniques. The fresh and thawed
aliquots were also assessed by light and fluorescence microscopy (after Acridine Orange staining, AO), and computer-assisted
semen analysis (CASA) of motility. Overall sperm quality was found to deteriorate after cryopreservation. When thawed spermatozoa
were subjected to an extra swim-up round, a general improvement in nuclear maturity was seen in post-rise spermatozoa. 相似文献
15.
Transgenic suspension cells of Oryza sativa L. cv. Dongjin utilized as a host for producing recombinant human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig)
were preserved in liquid nitrogen (−196 °C) after slow prefreezing in a deep freezer (−70 °C). The development of an optimal
procedure for long-term storage was investigated by the addition of various concentrations of cryoprotectant mixture and osmoticum
in preculture media before cooling. A pre-deep-freezing time of 120 min was the most effective for maintaining cell viability.
Compared with mannitol, sorbitol, trehalose, and NaCl under the same osmotic conditions, 0.5 M sucrose was found to be the
best osmoticum for preculture media. The cryoprotectant comprising sucrose, glycerol, and dimethylsulfoxide (DMSO) was applied
to the precultured cells, and a combination of 1 M sucrose, 1 M glycerol, and 1 M DMSO provided the best result. The viability
with this optimized condition was 88% after cryocell-banking for 1 day. The expression of hCTLA4Ig in recovered callus from
cryopreservation was also kept stable, and the production level was similar to that observed in noncryopreserved cultures. 相似文献
16.
Coutts M Hinton S Zheng J Scharp DW 《In vitro cellular & developmental biology. Animal》2007,43(1):2-6
Pancreatic acinar cells rapidly lose their characteristic features when cultured in vitro. No successful cryopreservation
methods have been reported. To solve the problem of storing pancreatic acinar material, we found that it could be preserved
at nonfreezing, cold temperatures: above the freezing point of cell culture medium (−0.6°C) or at typical refrigeration temperatures
(6.0–8.0°C) for up to 7 d. Under the conditions we defined, we determined that there was no significant dedifferentiation
and no significant decrease in cell health. Good viability and enzyme content were realized after storage, as determined by
growth in culture, histological evaluation, and enzyme content by ELISA (lipase and amylase). 相似文献
17.
Settled zoospores of the green macroalga Enteromorpha intestinalis were subjected to several different freezing and storing
treatments at both cryogenic and non-cryogenic temperatures after which their viability was assessed using a spore germination
bioassay. Three different cooling rates were tested: slow cooling at –1°C min−1 and –0.5°C min−1 to end temperatures in the range –20°C to –40°C, and a two-step procedure whereby the spores were frozen to –30°C at a rate
of –1°C min−1 prior to immersion in liquid nitrogen at –196°C. Spore viability was also investigated using the cryoprotectants glycerol
and dimethyl suphoxide (DMSO), a reduced saline medium and various storage times. In the majority of experiments, the use
of a cryoprotectant during the freezing process significantly increased the viability of the spores, with DMSO affording slightly
greater protection than glycerol. All treatments produced high viabilities (ranging from 75.3–100.0%) after 5-min storage
at the different end temperatures. However, progressively longer storage up to 7 days generally resulted in a marked reduction
in viability. This was with the exception of spores frozen in a reduced saline medium; a medium of 75% seawater and either
5 or 10% DMSO greatly increased spore viability, with values of > 40% recorded for spores stored at –20°C for up to 5 weeks.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
18.
Cold tolerance and dehydration in Enchytraeidae from Svalbard 总被引:4,自引:1,他引:3
L. Sømme T. Birkemoe 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1997,167(4):264-269
When cooled in contact with moisture, eight species of arctic Enchytraeidae from Svalbard were killed by freezing within
minutes or hours at −3 and −5 °C; an exception was Enchytraeus kincaidi which survived for up to 2 days. When the temperature approached 0 °C the enchytraeids apparently tried to escape from the
moist soil. The supercooling capacity of the enchytraeids was relatively low, with mean supercooling points of −5 to −8 °C.
In contrast, specimens of several species were extracted from soil cores that had been frozen in their intact state at −15 °C
for up to 71 days. Compared to freezing in a moist environment, higher survival rates were obtained during cooling at freezing
temperatures in dry soil. Survival was recorded in species kept at −3 °C for up to 35 days, and in some species kept at −6 °C
for up to 17 days. Slow warming greatly increased survival rates at −6 °C . The results strongly suggest that arctic enchytraeids
avoid freezing by dehydration at subzero temperatures. In agreement with this, weight losses of up to ca. 42% of fresh weight
were recorded in Mesenchytraeus spp. and of up to 55% in Enchytraeus kincaidi at water vapour pressures above ice at −3 to −6 °C. All specimens survived dehydration under these conditions.
Accepted: 12 December 1997 相似文献
19.
The ability to survive freezing and thawing is a key factor for the existence of life forms in large parts of the world. However,
little is known about the freezing tolerance of mycorrhizal fungi and their role in the freezing tolerance of mycorrhizas.
Threshold temperatures for the survival of these fungi have not been assessed experimentally. We grew isolates of Suillus luteus, Suillus variegatus, Laccaria laccata, and Hebeloma sp. in liquid culture at room temperature. Subsequently, we exposed samples to a series of temperatures between +5°C and
−48°C. Relative electrolyte leakage (REL) and re-growth measurements were used to assess the damage. The REL test indicated
that the lethal temperature for 50% of samples (LT50) was between −8.3°C and −13.5°C. However, in the re-growth experiment, all isolates resumed growth after exposure to −8°C
and higher temperatures. As many as 64% of L. laccata samples but only 11% in S. variegatus survived −48°C. There was no growth of Hebeloma and S. luteus after exposure to −48°C, but part of their samples survived −30°C. The fungi tolerated lower temperatures than was expected
on the basis of earlier studies on fine roots of ectomycorrhizal trees. The most likely freezing tolerance mechanism here
is tolerance to apoplastic freezing and the concomitant intracellular dehydration with consequent concentrating of cryoprotectant
substances in cells. Studying the properties of fungi in isolation promotes the understanding of the role of the different
partners of the mycorrhizal symbiosis in the freezing tolerance. 相似文献
20.
The adaptive fitness of a freeze-tolerant insect may be mediated by both endogenous and exogenous interactions. The aim of
the study presented here was to characterize the freeze tolerance of alpine Tiger moth caterpillars (Metacrias huttoni) and highlight two poorly explored indices of the potential attrition of fitness: (1) downstream development and reproduction;
(2) parasitism. Caterpillars survived temperatures as low as −16°C and demonstrated >90% 72-h survival after exposures to
−10°C. Two-week acclimations at 5, 10, and 20°C had no effect on body water content, haemolymph osmolality or survival of
equilibrium freezing, but there was a significant elevation of the temperature of crystallization (T
c) in those caterpillars acclimated to 5°C. Cell viability of fat body tissue was resilient to freezing (−10 to −16°C), but
midgut and tracheal cells showed significant degradation. Pupation and eclosion were unaffected by freezing at −5 or −10°C.
Likewise, there were no significant differences in egg production or the proportion of eggs that hatched between control and
frozen insects. By contrast, the ability of tachinid larvae to survive freezing within their hosts means that parasitism plays
an important role in regulating population size. Mean parasitism of caterpillars by tachinids was 33.3 ± 7.2%. Pupation and
imago emergence of tachinids after host ‘endo-nucleation’ was >75%. Eclosed adult tachinids showed a non-significant increase
in the incidence of wing abnormalities in relation to low temperature exposure. 相似文献