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PubMed1995

Effect of cryopreservation on semen quality in patients with testicular cancer.

OBJECTIVES: Current techniques in cryopreservation of human semen substantially decrease sperm quality. In addition, the pregnancy rate using cryopreserved sperm obtained from testicular cancer patients is lower than when sperm from normal fertile men is used. However, it is still unclear whether cryopreserved sperm from these patients is inherently defective or if the sperm loses its motility after thawing. This study was undertaken to assess the effect of cryopreservation on the quality and motion characteristics of semen from patients with testicular cancer before definitive therapy compared with semen from normal volunteers. METHODS: We compared the sperm quality before and after cryopreservation in samples from 34 patients with testicular cancer and 30 normal volunteers who were referred for sperm banking over a 7-year period. The effects of cancer stage and histologic type on various semen parameters were also examined. A computer-assisted semen analysis was performed before and after cryopreservation on each specimen. The nitrogen vapor technique using Test yolk buffer with glycerol as a cryoprotective agent was used for cryopreservation. The motile sperm count and motion characteristics (motility, velocity, linearity, amplitude of the lateral head movement, motility index) were analyzed before and after cryopreservation and compared between the groups. RESULTS: Semen quality did not significantly differ among patients with Stage I, II, or III cancer. However, semen quality tended to be poorer at higher cancer stages. In general, semen quality was better among patients with pure seminomas than with pure embryonal tumors; quality was worst among patients with mixed germ cell tumors. However, 71.4% of patients with mixed tumors presented with Stage III disease, whereas all patients with seminomas presented with Stage I disease. Significant differences were also seen in prefreeze motility (median, 42% [interquartile range, 24 to 51] versus 60.5% [range, 49 to 73]; P = 0.0004) and motile sperm count (6.7 x 10(6)/mL [range, 3.4 to 14.4] versus 50.0 [range, 24.6 to 72.0]; P = 0.0001) in patients compared with controls, respectively. The motile sperm count and percent motility significantly decreased in both patients and controls after cryopreservation (P = 0.0001). However, the percentage decline in motile sperm count and motion characteristics after cryopreservation did not differ significantly between patients and controls (P > 0.01). CONCLUSIONS: We conclude that the effect of cryopreservation on sperm quality in patients with testicular cancer is identical to its effect on sperm from normal fertile men. Differences in values after preservation are explained by poor semen characteristics before freezing; semen quality declines with more extensive disease. Stage I patients also had poorer quality than control subjects. Thus, we recommend that routine sperm banking be encouraged among all patients with testicular cancer before the initiation of specific medical treatment. We also recommend that future efforts be focused on improving the technique of sperm banking.

Adolescent↗
PubMed2003

Heterotopic transplantation of cryopreserved tracheae in a rat model.

INTRODUCTION: The successful use of cryopreserved tracheal allografts in canine models suggests their use in humans. The grade of genetic difference, the mechanism of revascularisation and the method of cryopreservation are not clearly defined. The purpose of our study was to investigate the rejection of tracheal transplants in a standardised heterotopic rat model using different forms of cryopreservation. METHODS: Tracheae from Brown Norway rats were implanted into the omentum from Brown Norway rats or Lewis rats. We transplanted fresh isografts or allografts and pretreated isografts or allografts. Cryopreservation was performed in a medium containing 10% dimethyl sulphoxide at -80 degrees C for 28 days (I) or -196 degrees C for 84 days (II) or without medium at -80 degrees C for 28 days (III). The transplants were excised after 7 and 21 days, respectively. RESULTS: Histological examinations revealed normal structure and function of isografts after 21 days. In the cryopreserved isograft, the epithelium had disappeared and the tracheal lumen was partially obstructed by a non-compact fibrous tissue. In the fresh allografts, the epithelium was replaced by aggressive fibrous tissue, infiltrating the membranous part of the trachea and occluding the tracheal lumen. The cartilage was vital without any sign of rejection. In the cryopreserved allografts, the tracheal lumen was obstructed by dense fibrous tissue with an inflammatory reaction. The cartilage of cryopreserved allografts (II) and (III) had lost the nuclei corresponding to non-vital tissue. Only in the cryopreserved allografts (I) did we find nodular regeneration at the edges of the cartilaginous bow. CONCLUSIONS: The heterotopic transplantation model allows the study of the mechanisms leading to tracheal obstruction. Cryopreservation was found to have no clear advantage in reducing transplant immunogenicity. Cryopreservation leads to significant damage to the cartilage, the intensity of which is dependent on the mode of cryopreservation.

Animals↗
PubMed2001

Effect of cryopreservation period on rat tracheal allografts.

BACKGROUND: The effect of cryopreservation on tracheal allogenicity is still unclear. Therefore, in this study, we assessed the effect of cryopreservation period on tracheal allografts in 62 rats. METHODS: Each transplant consisted of a 3-ring segment of the trachea harvested from 8 Lewis rats, immersed in preservation solution, and cryopreserved and stored in a Bicell biofreezing vessel in a deep freezer at -80 degrees C. Six tracheal grafts without cryopreservation that underwent a heterotopically syngeneic transplantation into the omentum served as controls. Forty-eight tracheal segments were randomly assigned to 8 groups according to period of cryopreservation, which ranged from 0 to 12 months (0, 0.5 [2 weeks], 1, 2, 3, 6, 9 and 12 months). The cryopreserved grafts were then thawed and heterotopically implanted into the omentum of recipient Brown-Norway rats. After 28 days, the tracheal segments were then evaluated histologically. RESULTS: All isografts in Group 1 were intact, whereas the allografts undergoing a particularly shorter period of cryopreservation showed a more stenotic lumen. Prolonged periods of cryopreservation tended to show decreasing tendencies of viability of chondrocytes, mononuclear cell infiltration and sub-epithelial thickness, whereas all allografts showed a uniformly denuded epithelium, irrespective of the length of cryopreservation. CONCLUSIONS: A longer period of cryopreservation may help to maintain a better patency of tracheal allografts by preventing an allogeneic response. Reduced tracheal allogenicity may be associated with a decreased viability of chondrocytes by cryopreservation.

Animals↗
PubMed1997

The effect of cryopreservation on the lethal mutation rate in Drosophila melanogaster.

Although cryopreservation is routinely used for the storage of a range of biological organisms, few studies have been conducted to determine whether cryopreservation increases the frequency of mutation. A procedure for the cryopreservation of Drosophila melanogaster embryos has recently been developed. Cryopreservation of D. melanogaster is of special interest to geneticists and evolutionary biologists because it would make it possible to assay control and experimental populations simultaneously during long-term studies. Before cryopreserved embryos can be used for such studies, it is first necessary to show that cryopreservation is not mutagenic. We tested for mutagenic effects or cryopreservation in D. melanogaster embryos with an X-linked, recessive lethal assay. The mutation rates of cryopreserved and control flies were not significantly different. We can be 95% certain that cryopreservation does not increase mutation by a factor greater than 2.39. This is the first quantitative estimate of the mutagenic effect of cryopreservation on the germ line of a metazoan. The results are reassuring when considering the genetic impact of cryopreservation on mammalian gametes and embryos.

Animals↗
PubMed1999

Controlling transplant vasculopathy in cryopreserved vein grafts with polyethylene glycol and glutathione during transport.

BACKGROUND: the biological characteristics of cryopreserved allografts are poorly understood, although many factors are known to influence their outcome. This study examines the development of transplant vasculopathy in both fresh and cryopreserved vein allografts and specifically assesses the efficacy of a transport solution containing 10% polyethylene glycol and 10 microM glutathione (PEG/GSH). METHODS: jugular veins were harvested from control donor rabbits and transplanted as interposition carotid bypass grafts in 30 New Zealand White (NZW) rabbits. Ten received the fresh jugular veins (fresh). Ten animals received jugular veins which had been harvested, transported in a physiological solution, cryopreserved and stored in a standard fashion (cryopreserved). Ten animals received jugular veins which had been harvested, transported in the same solution with the addition of PEG/GSH, cryopreserved and stored in a standard fashion (PEG/GSH). Cryopreserved jugular veins were stored for 6 weeks before transplantation. All animals were sacrificed 28 days postoperatively. Vein grafts were perfusion-fixed and wall dimensions were determined by planimetry. RESULTS: all transplanted grafts were patent at harvest. The control cryopreserved vein grafts showed a 54% increase in mean intimal thickness (63+/-10 micron vs. 41+/-3 micron p<0.05) but no change in mean medial thickness (125+/-9 micron vs. 119+/-13 micron; p = N.S. ) compared to the fresh allograft. Transport of the grafts in PEG/GSH solution resulted in the abolition of the increase in intimal thickness (41+/-4 micron; p <0.01) associated with cryopreservation without a change in medial thickness (140+/-15 micron; p = N.S.) compared to the cryopreserved allograft. CONCLUSION: cryopreserved vein grafts develop significant intimal hyperplasia compared to freshly transplanted grafts. The use of PEG/GSH in the transport solution significantly reduces this transplant graft intimal hyperplasia to that which develops in fresh grafts and may lead to improvements in the clinical use of cryopreserved veins.

Animals↗
PubMed2000

High conservation of both phase I and II drug-metabolizing activities in cryopreserved rat liver slices.

1. Xenobiotic-metabolizing enzymes, including both cytochrome P450 and phase II-conjugating systems, have been characterized in rat liver slices cryopreserved in 12 or 18% dimethylsulphoxide (DMSO). 2. Several cytochrome P450 isoforms in rat liver slices metabolized testosterone to a variety of hydroxylated products. The rates of formation of these same products were well maintained during cryopreservation of the slices in both 12 or 18% DMSO. 3. After cryopreservation of rat liver slices in 18% DMSO, the rates of metabolism of ropivacaine to 3-hydroxyropivacaine, 4-hydroxyropivacaine and PPX (all catalysed by different cytochrome P450 isoforms) were approximately 94, 79 and 82% respectively of the corresponding rates observed with fresh slices. 4. The rates of conjugation of 7-hydroxycoumarin and 1-naphthol by rat liver slices were significantly decreased after cryopreservation in 12% DMSO, but they were maintained when the concentration of this cryopreservant was increased to 18% 5. After cryopreservation in 12% DMSO, the mitochondrial reduction of the tetrazolium salt MTT by rat liver slices was significantly lowered. In contrast, slices cryopreserved in 18% DMSO demonstrated no significant decrease in their capacity to reduce MTT. 6. Thus, in agreement with previous studies, it was found that cytochrome P450-dependent activities are retained after cryopreservation of liver slices. Although phase II-conjugating enzyme activities are more sensitive to cryopreservation, it was shown that increasing the concentration of DMSO present during cryopreservation could circumvent the problem. This modification improves the usefulness of cryopreserved rat liver slices as a tool in drug metabolism studies.

Amides↗
PubMed1997

Cryopreservation of rat pancreatic islets: effect of ethylene glycol on islet function and cellular composition.

BACKGROUND: Inasmuch as cryopreservation can facilitate clinical islet transplantation by providing a means of storing supplemental islets in order to augment marginally adequate grafts, protocols are needed to allow for a minimal loss in viable beta cells. By replacing the cryoprotectant dimethyl sulfoxide (DMSO) with ethylene glycol (EG), a more simplified cryopreservation protocol was developed, which resulted in improved survival and function of rat pancreatic islets. METHODS: Nonfrozen islets, islets cryopreserved in DMSO, and EG-cryopreserved islets were compared for percent recovery, cellular composition, in vitro viability, and metabolic function after transplantation. RESULTS: After cryopreservation in DMSO or EG, islet yield was similar to that of nonfrozen controls; however, islets cryopreserved in DMSO exhibited lower cellular DNA, insulin, and glucagon content, as well as an impaired insulin secretory capacity in vitro than the nonfrozen controls. When compared with controls, islets cryopreserved in DMSO contained a higher proportion of beta cells but a lower number of glucagon-positive cells, whereas cryopreservation with EG resulted in similar DNA/hormone contents, in vitro viability, and cellular composition. Transplantation of islet grafts composed of comparable numbers of beta cells (2.1-2.3 million) corrected diabetes in 100% (6/6; nonfrozen controls), 92% (10/11; DMSO), and 100% (14/14; EG) of the recipients; however, those who received DMSO-treated islets took longer to achieve euglycemia and remained glucose-intolerant. CONCLUSIONS: These results demonstrate that EG allows for the successful cryopreservation of rat islet beta and a cells with the same yield and quality as nonfrozen islets. The observation that alpha-cell survival was better after cryopreservation with EG may explain the improved functional viability of these grafts. Further studies are needed to assess whether this protocol provides any advantage for cryopreserving large numbers of human islets.

Animals↗
PubMed2003

Apoptotic cell death and function of cryopreserved porcine hepatocytes in a bioartificial liver.

We have previously shown that cryopreservation leads to increased apoptotic death of porcine hepatocytes intended for use in a bioartificial liver (BAL). This study was designed to determine if a broad-spectrum caspase inhibitor, IDN-1965, reduced apoptosis and increased function of cryopreserved porcine hepatocytes in static culture or in a BAL. Porcine hepatocytes were studied immediately after isolation and after 2 weeks of cryopreservation in liquid nitrogen using medium supplemented with 25 micromol/L IDN-1965 or vehicle. Both apoptotic and necrotic cells were observed in cultures of fresh and cryopreserved hepatocytes, but the percentage of apoptotic cells increased after cryopreservation. Cryopreservation in IDN-1965 improved hepatocyte viability and reduced apoptotic cell death determined by TUNEL assay. Cryopreservation of hepatocytes in IDN-1965 was also associated with reduced caspase 3-like activity, decreased release of cytochrome c from mitochondria, and a slower decline in mitochondrial membrane potential after thawing. These markers of apoptosis were lowest after cryopreservation when IDN-1965 was added to both the culture and cryopreservation medium. Functional markers of hepatocyte activity (albumin production, diazepam metabolism, urea production) were also increased after cryopreservation and culture of hepatocytes in medium supplemented with 25 micromol/L IDN-1965. Cryopreservation of porcine hepatocytes in the presence of caspase inhibitor IDN-1965 was associated with reduced apoptosis and improved function of porcine hepatocytes in both static culture and a perfused BAL. These data demonstrate that inhibition of apoptosis also preserves cell function.

Albumins↗
PubMed2005

The effects of cryopreservation on human hepatocytes obtained from different sources of liver tissue.

Successful cryopreservation of human hepatocytes is important to establish hepatocyte banks for clinical use or in vitro research. The availability of donor tissue from unused liver segments/lobes and non-heart-beating donors (NHBD) has provided newer sources of hepatocytes. The quality of hepatocytes at the time of cryopreservation is important as cells isolated from liver tissue of borderline quality may not withstand the stresses associated with cryopreservation and subsequent thawing. Human hepatocytes were cryopreserved after isolation from mainly donor tissues (n = 40). In vitro assessment of the viability and function of the fresh and thawed cryopreserved hepatocytes was performed. Viability, attachment efficiency, enzyme activity, and albumin production of hepatocytes were all significantly decreased, and LDH leakage significantly increased, on thawing after cryopreservation. The viability of cryopreserved hepatocytes isolated from tissue rejected for orthotopic liver transplantation (36 +/- 15%) was significantly lower than those isolated from tissue where part was used for liver transplantation (47 +/- 14%, p = 0.002), but there were no significant differences in functional parameters. The viability of cryopreserved hepatocytes isolated from NHBD tissue (29 +/- 9%, p = 0.001) and from steatotic donor tissue (35 +/- 11%, p = 0.019) was significantly lower than those isolated from normal donor tissue (49 +/- 14%). There was no difference in functional parameters, except for albumin production of hepatocytes from NHBD tissue (2.9 +/- 1.0 microg/h/mg protein) being significantly lower than those from normal donor tissue (4.8 +/- 2.8 microg/h/mg protein, p = 0.03). The viability and attachment efficiency of cryopreserved hepatocytes isolated from liver tissue from resections for tumors was significantly higher, and the LDH leakage significantly lower, than those isolated from all donor tissue. Hepatocytes isolated from NHBD and steatotic tissue were more vulnerable to the effects of cryopreservation. Further research is required to improve hepatocyte isolation and cryopreservation protocols for different types of liver tissue.

Adolescent↗
PubMed2000

Increased post-thaw viability and phase I and II biotransformation activity in cryopreserved rat liver slices after improvement of a fast-freezing method.

An existing cryopreservation method for liver slices applies 12% dimethylsulfoxide and rapid freezing. We found that cells in rat liver slices cryopreserved in this manner deteriorated rapidly upon culturing. To improve this cryopreservation method, we varied the dimethylsulfoxide concentration (0, 12, 18, and 30%), the cryopreservation medium (Williams medium E, fetal calf serum, and University of Wisconsin medium), slice thickness, and the storage period at 4 degrees C during slice preparation before cryopreservation. After thawing, slices were cultured for 4 h at 37 degrees C before their viability was evaluated by their potassium content and the number of intact cells determined histomorphologically. The biotransformation capacity of liver slices cryopreserved by the improved method was assessed by testosterone oxidation, hydroxycoumarin sulfation, and glucuronidation. Best results were obtained with 18% dimethylsulfoxide in Williams medium E: the potassium content of cryopreserved slices was higher than 65%, and the number of intact cells was higher than 60% of that in fresh slices; with 12% dimethylsulfoxide, potassium content was less than 40%, and the number of intact cells was less than 30%. Results did not differ between the three cryopreservation media. Viability of thin slices (8-10 cell layers) was better maintained than that of thicker slices (>14 cell layers). Storage at 4 degrees C of slices before cryopreservation decreased viability after cryopreservation. Both oxidative and conjugation activities were better than 60% of fresh values. Although results varied, slices cryopreserved with this improved method and cultured for 4 h retained viability between 50 and 80%, and biotransformation activity between 60 and 90% of fresh slices.

Animals↗
PubMed2001

Cryopreservation of suckling pig hepatocytes.

To determine the best and simplest method for cryopreservation of pig hepatocytes, we compared immediate cryopreservation with cryopreservation after short-term culture. Suckling pig hepatocytes were isolated by a modified 2-step in situ collagenase perfusion method, suspended in serum-free medium, and preserved for 10 da by two cryopreservation methods. Serial measurements were made of cell viability, LDH release, synthesis of protein, urea and glucose, glucose-6-phosphatase (G-6-Pase) activity, and diazepam transformation after thawing. These measurements were performed on both groups of cultured hepatocytes, and on freshly isolated hepatocytes, which served as a control. High viability (>95%)of thawed hepatocytes was obtained and maintained in both cryopreservation groups. There were no significant differences in cell viability, protein synthesis, glucose synthesis, G-6-Pase activity, or diazepam transformation between the two cryopreservation groups. In the immediate cryopreservation group, urea synthesis was less than in the group with cryopreservation after short-term culture. Protein synthesis, glucose synthesis, and diazepam transformation were lower in both cryopreserved groups than in the controls. The results showed that a protocol of immediate cryopreservation of hepatocytes in RPMI-1640 medium containing 10% DMSO, hormones, growth factors, and 10% newborn bovine serum, together with rate-controlled freezing and rapid thawing, provides indices of cell viability and function during subsequent serum-free culture that are comparable to hepatocytes cryopreserved after short-term culture, except for lower urea production. This simple procedure can be used in studies of bioartificial liver and hepatocyte transplantation.

Animals↗
PubMed2003

Optimal conditions for heart cell cryopreservation for transplantation.

Cultured myocyte transplantation into an infarcted myocardium has been shown to improve contractile function. Cryopreservation of cultured muscle cells or heart tissue will be important for the technology to be practical. This study, using fetal cardiomyocytes, evaluated the optimal conditions for muscle cell cryopreservation. Study 1: Fetal rat cardiomyocytes were isolated and cultured. The freshly isolated and passage 1, 2, 3 and 4 cells were cryopreserved in a solution containing 70% IMDM, 20% FBS and 10% DMSO and stored in -196 degrees C for 1, 2, 4, 8, 12 and 24 weeks. The cells were thawed and cultured. Cell number and contractility were evaluated at 0, 2, 4, 6, 8 and 10 days of culture. Study 2: Rat myocardium was cryopreserved in sizes of 0.2, 2 and 6 mm3 for 1 week. The tissue was thawed and cells were isolated. Cell growth and contractility were evaluated. (1) Cardiomyocytes grew and contracted after cryopreservation. Storage time did not affect cell survival rate, beating cell numbers and beating rates. Increasing cell passage prior to cryopreservation decreased the percentage of beating cells. (2) Cells isolated from cryopreserved tissue grew in vitro and contracted normally. Cell yield decreased with increased cryopreserved tissue size. Fetal rat cardiomyocytes survived and functioned after in vitro cryopreservation. Viable cells can be isolated from cryopreserved myocardium and cultured. Cryopreservation of small pieces of myocardium is preferred for maximal cell yields.

Animals↗
PubMedDate not supplied

Functional changes in canine saphenous veins after cryopreservation.

This study was designed to determine the functional characteristics of the endothelium, smooth muscle and nerve terminals of cryopreserved veins. Freshly harvested and cryopreserved canine saphenous veins were cut into rings. In some rings, the endothelium was removed. Cryopreserved veins were stored at -196 degrees C for at least 3 weeks prior to use. All rings contracted in a concentration-dependent manner to depolarization with KCl and to alpha-adrenergic stimulation; the maximal tensions were significantly less in cryopreserved than in freshly harvested veins. Calcium ionophore A23187 caused greater relaxations in rings with than without endothelium in freshly harvested and cryopreserved veins. These relaxations were reduced significantly by methylene blue and NG-monomethyl-L-arginine (L-NMMA) only in fresh veins. Cocaine-sensitive uptake of H3-norepinephrine was reduced following cryopreservation. Immediately after cryopreservation, the production of prostacyclin was elevated. The calcium ionophore A23187 stimulated production of prostacyclin only in freshly harvested veins. Tissue content of endothelin did not change following cryopreservation. These results suggest that cryopreservation of canine saphenous veins alters nerve terminals and decreases the ability of the smooth muscle to contract. The endothelium releases an endothelium-derived relaxing factor and prostanoids following cryopreservation but the ability to synthesize nitric oxide is probably reduced. These changes following cryopreservation may affect patency of the veins when used as arterial grafts.

Animals↗
PubMed1998

Cytoskeleton and polyploidy after maturation and fertilization of cryopreserved germinal vesicle-stage mouse oocytes.

PURPOSE: Our purpose was to assess the effect of cryopreservation on cytoskeleton of germinal vesicle (GV) mouse oocytes and determine whether irreversible spindle damage and related digyny associated with cryopreservation of metaphase II (MII) oocytes can be avoided. METHODS: The GV oocytes were cryopreserved using a slow-cooling (0.5 degree C/min) and slow-thawing (8 degrees C/min) protocol in 1.5 M dimethylsulfoxide supplemented with 0.2 M sucrose and analyzed before and during fertilization by multiple-label fluorescence and differential interference contrast microscopy techniques. RESULTS: When examined after in vitro maturation, the vast majority (> 95%) of cryopreserved and control oocytes displayed normal microfilament and microtubule organization. With respect to barrel-shaped spindle and normal chromosome alignment, no significant differences were observed between cryopreservation (78 and 86%, respectively) and control (85 and 95%, respectively) groups. In fertilization experiments, spindle rotation, formation of the second polar body, and pronuclear migration were displayed by similar percentages of cryopreserved (96, 94, and 37%, respectively) and control (98, 97, and 45%, respectively) oocytes, indicating normal functionality of the cytoskeleton during this period. However, pronuclear formation was significantly inhibited by cryopreservation (81%) compared with controls (100%). Regarding digyny and polyspermy, no significant increase was observed after cryopreservation (3 and 10%, respectively) compared with controls (3 and 6%, respectively). CONCLUSIONS: Cryopreservation of mouse oocytes at the GV stage is particularly advantageous to circumvent the spindle damage and increased digyny noted after cryopreservation of MII oocytes.

Actin Cytoskeleton↗
PubMed2005

The thermodynamic principles of isochoric cryopreservation.

The goal of this study is to introduce the fundamental thermodynamic principles of isochoric (constant volume) cryopreservation for low temperature preservation of biological materials. Traditionally, cryopreservation is performed in an isobaric process (constant pressure) at 1 atm, because this is our natural environment and it is most convenient experimentally. More than half a century of studies on cryopreservation shows that the major mechanism of damage during isobaric cryopreservation is the increase in intracellular ionic concentration during freezing, which presumably causes chemical damage to the components of cells. Cryoprotectants as well as hyperbaric pressures have been developed as methods to reduce the extent of chemical damage during freezing. The theoretical studies in this paper show that in isochoric cryopreservation, the increase in solution concentration during freezing is lower at each temperature by almost an order of magnitude from that in isobaric cryopreservation. This suggests that isochoric cryopreservation could be a preferential alternative to isobaric cryopreservation. The technology for isochoric cryopreservation is very simple; freezing in a constant volume chamber. Using a simple isochoric cryopreservation device, we confirm the theoretical thermodynamic predictions.

Cryopreservation↗
PubMed2006

Smooth muscle cell injury after cryopreservation of human thoracic aortas.

The cryopreservation protocol we use for arterial reconstructive surgery has been studied to evaluate smooth muscle cell (SMC) structural integrity and viability before implantation. Samples of human thoracic aortas (HTA) were harvested from five multi-organ donors. Sampling included unfrozen and cryopreserved specimens. Cryopreservation was performed using RPMI with human albumin and 10% Me(2)SO in a controlled-rate freezing apparatus. Thawing was accomplished by submerging bags in a water bath (39 degrees C) followed by washings in cooled saline. In situ cell preservation as investigated by light and transmission electron microscopy showed that SMCs from cryopreserved HTA had nuclear and cytoplasmic changes. A TUNEL assay, performed to detect DNA fragmentation in situ, showed increased SMC nuclear positivity in cryopreserved HTA when compared to unfrozen samples. 7-AAD flow cytometry assay of cells derived from cryopreserved HTA showed that an average of 49+/-16% cells were unlabeled after cryopreservation. Organ cultures aimed to study cell ability to recover cryopreservation damage showed a decreasing number of SMCs from day 4 to day 15 in cryopreserved HTA. In conclusion, the cryopreservation protocol applied in this study induces irreversible damage of a significant fraction of arterial SMCs.

Aorta, Thoracic↗
PubMed2005

Role of apoptotic signaling pathway in metabolic disturbances occurring in liver tissue after cryopreservation: Study on rat precision-cut liver slices.

Precision-cut liver slices in culture (PCLS) appears as a useful and widely used model for metabolic studies; the interest to develop an adequate cryopreservation procedure, which would allow maintaining cell integrity upon incubation, is needed to extend its use for human tissues. We have previously shown that cryopreservation of rat PCLS leads to caspase-3 activation and early alterations of their K+ content upon incubation. In this study, we tested the hypothesis that counteracting intracellular K+ loss and/or interfering with cell death signaling pathways could improve the viability of cryopreserved PCLS. PCLS were prepared from male Wistar rat liver and cryopreserved by rapid freezing before incubation. The addition of a caspase inhibitor-Z-DEVD-FMK (2.5 microM)-in the culture medium did not improve viability of cryopreserved PCLS. Incubation of cryopreserved PCLS in a K+ rich medium (135 mM) increased K+ content and avoided caspase-3 activation, but did not improve cell viability. Caspase-3 inhibition, a decrease in cell lysis, and improvement of glycogen content were observed in cryopreserved PCLS after addition of LiCl (100 mM) in the incubation medium. These results indicate that, even if caspase-3 activation is linked to the K+ loss in cryopreserved PCLS, its inhibition does not allow restoring the metabolic capacities. LiCl, acting on a target upstream of caspase-3 inhibition, improves cell viability and allows glycogen accumulation when added in culture medium of cryopreserved PCLS; and could thus be considered as an interesting adjuvant in the culture of cryopreserved PCLS.

Animals↗
PubMed2005

Effect of cryopreservation on mitochondrial DNA of zebrafish (Danio rerio) blastomere cells.

Cryopreservation has been extensively used in human reproductive medicine, aquaculture and conservation programmes for endangered species. However, despite the growing successes of cryopreservation, post-thaw recovery of reproductive and embryonic cells very often remains poor. Many studies have been devoted to the mechanisms of cryodamage. It is known that cryopreservation causes extensive damage to membranes; reduce the metabolic activity of cells; and disturbs the mitochondrial bioenergetical processes of cells. But few investigations on the genetic stability of cells during cryopreservation have been performed, and the role of any genetic impact cryopreservation needs to be determined. Some indirect data in the literature suggests that progress in this field might come from investigating freezing damage to mitochondrial DNA (mtDNA), nuclear DNA and other genome-related structures. In this study, zebrafish (Danio rerio) blastomeres were treated in three different ways: control suspension of blastomere cells in phosphate buffered saline; equilibration of blastomeres with 2M dimethyl sulfoxide (Me2SO) for 1h at room temperature and cryopreservation using Me2SO as a cryoprotectant. Mitochondrial DNA was analysed in fresh cells and after the different treatments. Two different loci of mtDNA were amplified with the help of PCR and sequenced. The sequences were analysed and nuclear base substitutions were counted for both control and treated samples. The results showed that cryopreservation significantly increased the frequency of mutations (0.78+/-0.27% in comparison to 0.16+/-0.25% of control), whilst 2M Me2SO treatment did not bring a significant increase in frequency of mutations (0.24+/-0.28%). The distributions of the mutation locations were analysed. More investigations are needed to determine whether optimisation of cryopreservation protocol is possible to reduce these adverse effects; whether such mutations interfere with overall function of the cells; whether similar changes also occur in the nuclear DNA and whether such mutations happen in other species. Meanwhile, it is important to be cautious in making judgements of the effect of cryopreservation technique in assisted reproduction. This is the first report on the effect of cryopreservation on mtDNA.

Animals↗