BACKGROUND: The aim of our study was to assess the impact of different thawing protocols on morphological changes arising in cryopreserved human saphenous vein grafts. METHODS: The study was performed in 12 saphenous vein grafts harvested in brain death donors. Storage in the vapor phase of liquid nitrogen for 3 or 5 years followed. Two thawing protocols were tested: slow thawing in a refrigerator at temperature +4°C for 2 hr and rapid thawing-in a water bath at +37°C. Grafts were processed for scanning electron microscopy. Comparisons of continuous parameters under study between experimental groups were performed using the t-test (age, cold ischemia time, exposure to cryoprotectant, time of storage, total thawing time, mean thawing rate, morphology scoring of thawed HSVG) and the median test (HSVG length). Categorical parameters (sex and blood group) were formally tested using the chi-square test. RESULTS: All samples were evaluated according to morphological changes and scored in terms of morphologically intact endothelium, confluent endothelium with structural inhomogeneity, disruption of the intercellular contacts, separation of the endothelial cells, complete loss of the endothelium, and damage of the subendothelial layers. There is no statistically significant difference between the sample sets at the significance level of 0.05. There was no association with donors' age, sex, and time of storage. CONCLUSIONS: Human cryopreserved saphenous vein grafts in our experimental work showed no difference in terms of structural deterioration of the endothelial surface and basal membrane depending on different thawing protocols used.

• MeSH
• časové faktory MeSH
• dospělí MeSH
• endoteliální buňky účinky léků   transplantace   ultrastruktura   MeSH
• kryoprezervace * MeSH
• kryoprotektivní látky farmakologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• odběr tkání a orgánů MeSH
• přežití tkáně MeSH
• vena saphena účinky léků   transplantace   ultrastruktura   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

BACKGROUND: The aim of our experimental work was to assess morphological changes of arterial wall that arise during different thawing protocols of a cryopreserved human aortic root allograft (CHARA) arterial wall. METHODS: The experiment was performed on CHARAs. Two thawing protocols were tested: 1, CHARAs were thawed at a room temperature at +23°C; 2, CHARAs were placed directly into a water bath at +37°C. MICROSCOPIC SAMPLES PREPARATION: After fixation, all samples were washed in distilled water for 5 min, and dehydrated in a graded ethanol series (70, 85, 95, and 100%) for 5 min at each level. The tissue samples were then immersed in 100% hexamethyldisilazane for 10 minutes and air dried in an exhaust hood at room temperature. Processed samples were mounted on stainless steel stubs, coated with gold. RESULTS: Thawing protocol 1: All 6 (100%) samples showed loss of the endothelium and damage to the subendothelial layers with randomly dispersed circular defects and micro-fractures without smooth muscle cells contractions in the tunica media. Thawing protocol 2: All 6 (100%) samples showed loss of endothelium from the luminal surface, longitudinal corrugations in the direction of blood flow caused by smooth muscle cells contractions in the tunica media with frequent fractures in the subendothelial layer. CONCLUSION: All the samples thawed at the room temperature showed smaller structural damage to the CHARA arterial wall with no smooth muscle cell contraction in tunica media when compared to the samples thawed in a water bath.

• MeSH
• alografty * patologie   MeSH
• aorta patologie   transplantace   MeSH
• aortální chlopeň patologie   transplantace   MeSH
• chirurgická náhrada chlopně škodlivé účinky   metody   MeSH
• dárci tkání MeSH
• dospělí MeSH
• homologní transplantace škodlivé účinky   metody   MeSH
• kryoprezervace metody   MeSH
• lidé středního věku MeSH
• lidé MeSH
• srdeční chlopně umělé škodlivé účinky   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

The vitrification of human embryos is more and more frequently being utilized as a method of assisted reproduction. For this technique, gentle treatment of the embryos after thawing is crucial. In this study, the balance of amino acids released to/consumed from the cultivation media surrounding the warmed embryos was observed in the context of a cultivation environment, which was with the atmospheric oxygen concentration ≈20% or with a regulated oxygen level-hysiological (5%). It is the first time that total amino acid turnover in human embryos after their freezing at post compaction stages has been evaluated. During this study, progressive embryos (developed to blastocyst stage) and stagnant embryos (without developmental progression) were analyzed. It was observed that the embryos cultivated in conditions of physiological oxygen levels (5% oxygen) showed a significantly lower consumption of amino acids from the cultivation media. Progressively developing embryos also had significantly lower total amino acid turnovers (consumption and production of amino acids) when cultured in conditions with physiological oxygen levels. Based on these results it seems that a cultivation environment with a reduced oxygen concentration decreases the risk of degenerative changes in the embryos after thawing. Therefore, the cultivation of thawed embryos in an environment with physiological oxygen levels may preclude embryonal stagnation, and can support the further development of human embryos after their thawing.

• Publikační typ
• časopisecké články MeSH

Introduction: The rate of thawing of cryopreserved human iliac arteries allografts (CHIAA) directly affects the severeness of structural changes that occur during this process. Method: The experiment was performed on ten CHIAA. The 10% dimethylsulphoxide in 6% hydroxyethyl starch solution was used as the cryoprotectant; all CHIAA were cooled at a controlled rate and stored in the vapor phase of liquid nitrogen (-194°C). Two thawing protocols were tested: (1) placing the CHIAA in a water bath at 37°C, and (2) the CHIAA were thawed in a controlled environment at 5°C. All samples underwent analysis under a scanning electron microscope. Testing of the mechanical properties of the CHIAA was evaluated on a custom-built single axis strain testing machine. Longitudinal and circumferential samples were prepared from each tested CHIAA. Results: Ultrastructural analysis revealed that all five CHIAA thawed during the thawing protocol 1 which showed significantly more damage to the subendothelial structures when compared to the samples thawed in protocol 2. Mechanical properties: Thawing protocol 1-longitudinal UTS 2, 53 ± 0, 47 MPa at relative strain 1, 27 ± 0, 12 and circumferential UTS 1, 94 ± 0, 27 MPa at relative strain 1, 33 ± 0, 09. Thawing protocol 2-longitudinal ultimate tensile strain (UTS) 2, 42 ± 0, 34 MPa at relative strain 1, 32 ± 0, 09 and circumferential UTS 1, 98 ± 0, 26 MPa at relative strain 1, 29 ± 0, 07. Comparing UTS showed no statistical difference between thawing methods. Conclusion: Despite the significant differences in structural changes of presented thawing protocols, the ultimate tensile strain showed no statistical difference between thawing methods.

• MeSH
• alografty účinky léků   fyziologie   MeSH
• arteria iliaca účinky léků   fyziologie   MeSH
• dimethylsulfoxid farmakologie   MeSH
• dospělí MeSH
• kryoprezervace metody   MeSH
• kryoprotektivní látky farmakologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Ciel štúdie: Získanie klinických skúseností s modifikovanou metódou vitrifikácie, ktorá bola použitá na zmrazovanie nadbytočných ľudských embryí po in vitro fertilizácii (IVF) a intracytoplazmatickej injekcii spermie (ICSI). Názov a sídlo pracoviska: Centrum asistovanej reprodukcie pri II. gynekologicko-pôrodníckej klinike LF UPJŠ a FN L. Pasteura, Košice. Metodika: Modifikovanou metódou vitrifikácie bolo zmrazených 215 ľudských embryí po IVF-ICSI, získaných v 42 cykloch. Embryá boli zmrazené po 48-hodinovej in vitro kultivácii. Modifikovaná metóda vitrifikácie spočívala v použití série stúpajúcich koncentrácii kryoprotektíva (etylénglykol a sacharóza) na konečnú koncentráciu 40% etylénglykol a 1 M sacharóza. Ako zmrazovací nosič bola použitá pipetovacia 100 μl „špička“. Embryá boli rozmrazené pri izbovej teplote a kryoprotektívum bolo vymyté v troch krokoch, v klesajúcich koncentráciach sacharózy (1 M, 0,5 M a čisté médium). Bola hodnotená morfológia embryí pred zmrazením a po rozmrazení. Po rozmrazení boli embryá kultivované in vitro 24 hodín a iba tie embryá, u ktorých sa rozdelila aspoň jedna blastoméra boli prenesené. Výsledky: Frekvencia prežívania embryí po rozmrazení bola 69,8 %, 48 % embryí sa po rozmrazení ďalej vyvíjalo. Priemerný počet prenesených vyvíjajúcich sa embryí bol 2,9±1,4. Frekvencia klinických gravidít na rozmrazený cyklus bola 19,0 %, frekvencia klinických gravidít na transfer bola 27,6 %. Porodilo 7 pacientiek a narodilo sa 8 detí. Záver: Na základe výsledkov bolo dokázané, že modifikovaná metóda vitrifikácie ľudských embryí je spoľahlivá a predstavuje jednoduchšiu, kratšiu a lacnejšiu alternatívu pomalého zmrazovania.

Objective: To obtain clinical experience with a modified method of vitrification used to freeze supernumerary human embryos following in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Setting: Centre of Assisted Reproduction, 2nd gynaecological-obstetrical clinic of Faculty hospital of L. Pasteur and Medical Faculty of the University of P. J. Šafárik, Košice. Methods: A modified method of vitrification was used to freeze 215 human embryos after IVF-ICSI, obtained in 42 cycles. The embryos were frozen at 48 h after in vitro culturing. The modified method of vitrification consisted in the use of a series of solutions with increasing cryoprotectant concentrations (ethylene glycol and sucrose) up to the final concentration of 40% ethylene glycol and 1 M sucrose. A 100 μl pipetting „tip” was used as a freezing container. The embroys were thawed at room temperature and the cryoprotectant was washed out three consecutive steps with sucrose solutions of decreasing concentrations (1 M, 0.5 M, and the pure medium). The morphology of embryos was evaluated before and after thawing. After thawing, the embryos were cultured in vitro for 24 hours and only the embryos with at least one cleaved blastomere were used for transfer. Results: The percentage of embryos which survived thawing and were capable of further development was 69.8% and 48%, respectively. The mean number of transferred and developing embryos was 2.9±1.4. The percentage of clinical pregnancies per thawing cycle and per transfer was 19.0% and 27.6%, respectively. Seven patients delivered 8 children. Conclusion: The results showed that our modification of vitrification procedure of human embryos is reliable and represents a simpler, shorter and cheaper alternative to slow-rate freezing.

• MeSH
• asistovaná reprodukce MeSH
• dospělí MeSH
• fertilizace in vitro metody   MeSH
• finanční podpora výzkumu jako téma MeSH
• infertilita terapie   MeSH
• kryoprezervace metody   MeSH
• lidé MeSH
• reprodukční lékařství MeSH
• viabilita buněk MeSH
• výzkum embrya MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• ženské pohlaví MeSH

BACKGROUND: The aim of our experimental work was to assess the impact and morphological changes that arise during different thawing protocols on human aortic valve (AV) leaflets resected from cryopreserved aortic root allografts (CARAs). OBJECTIVES: Two thawing protocols were tested: 1. CARAs were thawed at a room temperature (23°C); 2. CARAs were placed directly into a water bath at a temperature of 37°C. After all the samples were thawed, non-coronary AV leaflets were sampled from each specimen and fixed in a 4% formaldehyde solution before they were sent for morphological analysis. MATERIAL AND METHODS: All the samples were washed in distilled water for 5 min and dehydrated in a graded ethanol series (70%, 85%, 95%, and 100%) for 5 min at each level. The tissue samples were then immersed in 100% hexamethyldisilazane (HMDS) for 10 min, and then air-dried in an exhaust hood at room temperature. Processed samples were mounted on stainless steel stubs and coated with gold. Histological analysis was performed with the use of an electron microscope on a scanning mode operating at 25 kV - BS 301. RESULTS: Thawing protocol 1 (room temperature at 23°C): 6 (100%) samples showed loss of the endothelial covering of the basal membrane with no damage to the basal lamina. Thawing protocol 2 (water bath at 37°C): 5 (83%) samples showed loss of the endothelial covering of the basal membrane with no damage to the basal lamina. One (17%) sample showed loss of the endothelial covering the basal membrane with significant damage to the basal membrane. CONCLUSIONS: Based on our experimental work, we can clearly conclude that cryopreserved AV leaflet allografts show identical structural changes at different rates of thawing.

• MeSH
• alografty MeSH
• aortální chlopeň transplantace   MeSH
• homologní transplantace metody   MeSH
• kryoprezervace metody   MeSH
• lidé MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

OBJECTIVES AND DESIGN: At the present time there are two waiting list for patients with vascular prosthetic infection indicated for arterial transplantation in the Czech Republic. The inclusion of each patient for cold-stored or cryopreserved arterial transplantation is the preference of indicating surgeon. In this experimental work we studied the immunogenicity of rat aortal allografts treated by our new clinical cryopreservation/slow thawing protocol. MATERIAL AND METHODS: Brown-Norway (BN) (N = 6, 203-217 g) or Lewis (LEW) (N = 6, 248-254 g) abdominal aortal grafts treated in accordance with our new clinical cryopreservation/slow thawing protocol were orthotopically transplanted to Lewis recipients (N = 12, 191-245 g). Aortal wall histology and infiltration by recipient immune cells, as well as donor specific anti MHC class I and II antibodies in recipient serum were studied in both isografts and allografts on day 30 postransplant. Core data of cryopreserved allografts were compared to our previous data of cold-stored aortal allografts treated in accordance with our clinical cold-storage protocol. RESULTS: Cryopreserved allografts showed regular morphology of aortal wall with clear differentiation of all three basic anatomical layers on day 30 postransplant. Intimal layer showed no hyperplasia, luminal surface was covered by endothelial cells. No statistical difference was observed in tunica media thickness between isografts and allografts. The medial layer showed no necrosis, shrinkage or immunoglobuline G deposition in any experimental group. The adventitial infiltration by immune cells was significantly higher (P<0.05) in allografts. Cryopreserved allografts showed significant lower activation of both cell- and antibody mediated immunity compared to historical data of cold-stored allografts. CONCLUSION: Aortal wall histology of rat allografts treated by our new standardized clinical cryopreservation/slow thawing protocol was comparable to that of the cryopreserved isografts on day 30 posttranspant. The immunogenicity of cryopreserved aortal allografts was significantly lower compared to that of cold-stored aortal allografts.

• MeSH
• alografty fyziologie   MeSH
• aorta transplantace   MeSH
• arterie transplantace   MeSH
• homologní transplantace metody   MeSH
• kryoprezervace metody   normy   MeSH
• krysa rodu rattus MeSH
• modely u zvířat MeSH
• potkani inbrední BN MeSH
• potkani inbrední LEW MeSH
• rejekce štěpu imunologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH

The authors present their contribution to the improvement of methods suitable for the detection of the freezing and thawing damage of cells of cryopreserved venous grafts used for lower limb revascularization procedures. They studied the post-thaw viability of cells of the wall of cryopreserved venous grafts (CVG) immediately after thawing and after 24 and 48 h culture at +37 °C in two groups of six CVG selected randomly for slow thawing in the refrigerator and rapid thawing in a water bath at +37 °C. The grafts were collected from multi-organ and tissue brain-dead donors, cryopreserved, and stored in a liquid nitrogen vapor phase for five years. The viability was assessed from tissue slices obtained by perpendicular and longitudinal cuts of the thawed graft samples using in situ staining with fluorescence vital dyes. The mean and median immediate post-thaw viability values above 70% were found in using both thawing protocols and both types of cutting. The statistically significant decline in viability after the 48-h culture was observed only when using the slow thawing protocol and perpendicular cutting. The possible explanation might be the "solution effect damage" during slow thawing, which caused a gentle reduction in the graft cellularity. The possible influence of this phenomenon on the immunogenicity of CVG should be the subject of further investigations.

• MeSH
• alografty diagnostické zobrazování   účinky léků   MeSH
• apoptóza účinky léků   MeSH
• dárci tkání MeSH
• dimethylsulfoxid farmakologie   MeSH
• fluorescenční barviva * MeSH
• konfokální mikroskopie metody   MeSH
• kryoprezervace metody   MeSH
• kryoprotektivní látky farmakologie   MeSH
• lidé MeSH
• optické zobrazování metody   MeSH
• transplantace cév metody   MeSH
• vena femoralis diagnostické zobrazování   účinky léků   MeSH
• vena saphena diagnostické zobrazování   účinky léků   MeSH
• viabilita buněk účinky léků   MeSH
• zmrazování * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The resistance of Yersinia enterocolitica O:9, Escherichia coli O157:H7 and natural microflora against lactic acid (LA), ascorbic acid (AA), and freezing-thawing in noninoculated and inoculated fresh sausages was studied. Samples were stored at –18 °C for 28 days and thawed in microwave (MW), at room temperature (RT), in refrigerator (R) and under flowing of tap water (F) on days 7, 14, 21 and 28. Plate Count Agar (PCA), Sorbitol Mac Conkey agar (SMC) and Mac Conkey agar (MC) were used for microbial counts. A maximal reduction of 1.57 log in mesophilic aerobes and no significant changes in total and fecal coliform levels with respect to the initial counts in natural microflora were observed along storage. In inoculated fresh sausages, reductions of 1.37 log on PCA and 2.17 log on SMC were obtained in E. coli O157:H7 populations as compared to the control groups on day 0. Similarly, reductions of 1.69 log on PCA and 2.79 log on MC as compared to the initial level were observed in counts of Y. enterocolitica inoculated samples. Salmonella Anatum, P. aeruginosa, Y. enterocolitica B1A O:7,8-8-8,19 and E. coli non O157:H7 strains were recovered from the natural microflora by enrichment techniques. Thawing in refrigerator was more frequently related to the best reductions of total mesophilic aerobe, E. coli O157:H7 and Y. enterocolitica O:9 counts than the other thawing methods. Reductions of microbial populations observed in LA treated samples were similar to those observed in AA treated samples. Although the acidic and freezing treatments might reduce the microbial levels in natural microflora of fresh sausages, they appeared to be ineffective in the total elimination of high inocula of pathogens like E. coli O157:H7 and Y. enterocolitica O:9.

• MeSH
• Escherichia coli patogenita   MeSH
• kyseliny analýza   MeSH
• manipulace s potravinami MeSH
• potravinářská mikrobiologie MeSH
• techniky in vitro MeSH
• Yersinia enterocolitica patogenita   MeSH
• zmrazování MeSH

BACKGROUND: The aim of the study was to evaluate the in vitro quality of cryopreserved red blood cells obtained from different sources with or without leucodepletion and stored at 4±2 °C in AS-3 for up to 21 days. MATERIALS AND METHODS: Red blood cells were collected by four methods: double erythrocytapheresis, whole blood collection with buffy coat removal, double erythrocytapheresis with in-line leucofiltration, or whole blood collection with in-line leucofiltration. All four types of red blood cells were frozen in 40% glycerol after collection and stored at a temperature below -65 °C for at least 30 days, thawed, deglycerolised and subsequently reconstituted in AS-3. The in vitro haematological and biochemical properties of the thawed red blood cells were tested on days 0, 7, 14, and 21 after deglycerolisation and reconstitution. RESULTS: Overall, 72 units were processed. Leucodepletion of cryopreserved red blood cells units reduced haemolysis, lowered ammonia concentration, preserved pH and osmolality and led to sustained higher concentrations of ATP. In contrast, the source of red blood cells (apheresis or whole blood) did not affect their quality. DISCUSSION: The quality of all investigated red blood cells units was the same as or even better than that of erythrocytes obtained from double erythrocytapheresis with a 24-hour survival of at least 86% after up to 3 weeks of storage in AS-3.

• MeSH
• adenosintrifosfát krev   MeSH
• amoniak krev   MeSH
• cytaferéza metody   MeSH
• erytrocyty * účinky léků   MeSH
• glycerol farmakologie   MeSH
• hemoglobiny analýza   MeSH
• hemolýza MeSH
• koncentrace vodíkových iontů MeSH
• konzervace krve * MeSH
• kryoprezervace * MeSH
• kryoprotektivní látky farmakologie   MeSH
• leukoredukce * MeSH
• lidé MeSH
• osmolární koncentrace MeSH
• techniky in vitro MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH