Apoptosis in fresh and cryopreserved cardiac valves of pig samples.

To analyse the influence of cold ischemic time (CIT) (2-24 h) and of cryopreservation (liquid phase) on the viability of the valvular fibroblasts and in the presence of apoptosis. Cardiac valves from 10 pigs were evaluated by anatomo-pathological study of the wall, muscle and leaflet. At the same time, the presence of cellular death due to apoptosis was investigated in two ways; directly on tissue by Apodetec system and by two-colour flow cytometry assay analyzing a suspension of fibroblast from valve leaflets using Anexina V and propidium iodure (PI). We established three groups of samples to compare different experimental conditions: 2 h of ischemia (group 1), 24 h of ischemia (group 2), and a programme of cryopreservation (-1 degrees C/min) after 2 h of ischemia, followed by storage in liquid nitrogen during a week and thawing was performed (group 3). The analysis of viabilities showed slight differences between all three groups. The results indicated CIT of 24 h undergoing more structural affectation than CIT of 2 h. Flow cytometry analysis did not show important differences between groups; however cryopreserved samples (group 3) slightly less viability and a higher percentage of death by apoptosis than group 1 and 2 using flow cytometry. Apoptosis was confirmed on tissue from all valves but mainly in samples of group 2 and group 3. In summary, the viability of the valves in the case of ischemic times of 2 h, 24 h or after cryopreservation/thawing differs slightly. The death of the cells is mainly mediated by necrosis and not by apoptosis.

Functional assessment of cryopreserved human femoral arteries for pharmaceutical research.

An established method for cryopreservation that might preserve the vascular and endothelial responses of human femoral arteries (HFAs) to be transplanted as allografts was studied. HFAs were harvested from multiorgan donors and stored at 4 degrees C in saline solution before cryostorage. Thirty HFA rings were isolated and randomly assigned to one control group of unfrozen HFAs (eight rings) and one group of cryopreserved HFAs (22 rings). Cryopreservation was performed in RPMI solution containing dimethylsulfoxide (DMSO) and the rate of cooling was -1 degrees C/min until -40 degrees C and faster rates until -150 degrees C was reached. The contractile and relaxant responses of unfrozen and frozen/thawed arteries were assessed in organ bath by measurement of isometric force generated by the HFAs. After thawing, the maximal contractile responses to the contracting agonist tested (noradrenaline) were in the range of 43% of the responses in unfrozen HFAs. The endothelium-independent responses to sodium nitroprusside were not altered whereas the endothelium-dependent relaxant responses to acetylcholine were weakly altered. The cryopreservation method used provided a limited preservation of contractility of HFAs, a good preservation of the endothelium-independent relaxant responses, and a good preservation of endothelium-dependent relaxation. It is possible that further refinements of the cryopreservation protocol, such as a slower rate of cooling and a more controlled stepwise addition of DMSO, might allow better post-thaw functional recovery.

Functional assessment of cryopreserved pig aortas for pharmaceutical research.

Several in vitro studies have demonstrated diminished post-thaw functional activity. Therefore, the aim of this study was to investigate the consequences of thawing and storage method used on the post-thaw functional activity of cryopreserved pig aortas with the aim of adjusting the freezing and thawing protocol so that the vascular segments are preserved in the best possible state, maintaining structure and functionality so that they can later be transplanted with success. In vitro responses of frozen, thawed pig aortas were used to investigate the functional activity after thawing at 15 degrees C and 100 degrees C/min and after storage in gas or liquid phase of liquid nitrogen. Cryopreservation was performed in RPMI 1640 medium + 10% dimethylsulfoxide and the rate of cooling was -1 degrees C/min, until -150 degrees C was reached. After thawing the maximal contractile responses to all the contracting agonists tested (KCl, noradrenaline) were in the ranges of 13-27% compared with the responses in unfrozen pig aortas. Contractile responses were slightly better when thawing was performed at 15 degrees C/min compared with 100 degrees C/min. The endothelium independent relaxant responses to sodium nitroprusside were reduced ( P < 0.05). Cryostorage of pig arteries also resulted in a loss of the endothelium-dependent relaxant response to acetylcholine. The cryopreservation method used provided a limited preservation of pig aorta contractibility, a reduction of the endothelium independent relaxant responses, and no apparent preservation of the endothelium-dependent relaxation. It is possible that further refinements of the cryopreservation protocol might allow better post-thaw functional recovery of pig aortas.

Functional assessment of cryopreserved human aortas for pharmaceutical research.

We evaluated the impact of standard cryopreservation on functional properties of human aortic homografts. From seven human donors, the thoracic descending aorta was obtained. Effects of cryopreservation on contractibility and endothelium function were tested. After cryopreservation no endothelium-dependent or endothelium-independent relaxation was found and the contractibility was strongly affected. Arteries showed no function and loss of endothelial integrity after cryopreservation and thawing.

Functional assessment of human femoral arteries after cryopreservation.

An established method for the cryopreservation of human femoral arteries for subsequent transplantation as allografts has been studied with particular attention to preservation of smooth muscle and endothelium. Human femoral arteries (HFAs) were harvested from multi-organ donors. Two groups were established; a control group of unfrozen HFAs and a group of cryopreserved HFAs. Cryopreservation was performed using RPMI solution containing dimethyl sulfoxide and the rate of cooling was 1 degrees C/min to -40 degrees C and faster thereafter until -150 degrees C was reached. The contraction and relaxation responses of unfrozen and frozen/thawed arteries were assessed by measurement of the isometric force generated by the HFAs in an organ bath. After thawing (warming was at 15 degrees C/min) the maximal contractile response to noradrenaline was 43% of the response of unfrozen HFAs. The endothelium-independent response to sodium nitroprusside was not altered, whereas the endothelium-dependent relaxation response to acetylcholine was slightly altered. The cryopreservation method used provided limited preservation of the contractility of human femoral arteries, and good preservation of both endothelium-independent and endothelium-dependent relaxation responses.

Cambios histopatológicos en arterias humanas sometidas a procesos de isquemia fría y criopreservación / Histopathological changes in human arteries following cold ischemia and cryo preservation processes

Introducción. La creciente demanda de injertos vasculares ha provocado una búsqueda continua del método `ideal' para minimizar los daños vasculares durante el proceso de conservación. Objetivo. Estudiar la repercusión sobre la histología arterial del proceso de preservación del tejido en sus distintas fases. Pacientes y métodos. Hemos analizado 86 segmentos arteriales (ilíaca y femoral superficial) procedentes de 50 donantes. De cada segmento se obtuvieron tres muestras en distintas fases del proceso, y se establecieron otros tantos grupos de estudio: arterias frescas; postisquemia fría y poscriopreservación. El tiempo máximo de isquemia fria fue de 20 horas y las muestras se mantuvieron en solución antibiótica a 4 °C. La criopreservación se realizó en una solución con dimetil sufóxido (DMSO) con descenso térmico programado de 1 ºC / min con almacenamiento en fase gas (-150 °C a -190 °C). Se valoraron parámetros como la preservación del endotelio, la intensidad de cambios degenerativos (degeneración mixoide, apoptosis) en la pared arterial y la presencia de fracturas, comparando los resultados entre los distintos grupos. Se estudiaron igualmente las causas que llevaron al fracaso de algunos injertos. Resultados. El 81,4 por ciento de las arterias criopreservadas mostró una pérdida prácticamente total del endotelio (3,5 por ciento en los otros dos grupos) y más del 50 por ciento, importantes cambios degenerativos en su pared frente al 3,5 y 8,1 por ciento de los grupos 1 y 2. Conclusiones. El proceso de criopreservación provoca una importante pérdida endotelial y cambios degenerativos en la pared arterial. El tiempo de isquemia fría que se empleó en nuestro estudio no tiene repercusión en la estructura arterial (AU)

Efecto del almacenamiento en fase gaseosa sobre la viabilidad celular, la apoptosis y la actividad funcional en aortas de cerdo criopreservadas. Estudio preliminar / The effect of gas phase storage on cell viability, apoptosis and functional activity in cryopreserved aortas from pigs a preliminary study

Objetivos. Analizar las consecuencias del protocolo de congelación y del método de almacenamiento utilizado sobre la actividad funcional, la estructura anatomopatológica y el grado de apoptosis de las aortas abdominales de cerdo criopreservadas durante seis meses, tras la descongelación. Materiales y métodos. Se obtuvieron injertos arteriales de cerdos y cada aorta se dividió en dos fragmentos. Grupo 1: segmentos en fresco o bien fijados en formaldehído tras la toma de la muestra (grupo control). Grupo 2: segmentos fijados tras incubación antibiótica y criopreservación durante seis meses para un estudio anatomopatológico y de apoptosis, o bien utilizados directamente tras su descongelación para un estudio funcional. La incubación antibiótica se realizó en medio de un cultivo RPMI suplementado con antibióticos. Después de la incubación antibiótica, la criopreservación se llevó a cabo en medio RPMI con 10 por ciento de dimetilsulfóxido (DMSO). La tasa de enfriamiento fue de 1 °C/min, y el posterior almacenamiento se realizó en fase gaseosa. Resultados. No hay diferencia en cuanto al grado de apoptosis en los dos grupos, ni diferencias significativas en el grado de fragmentación de las elásticas (algo mayor en el grupo 2). Se observa un aumento tanto del grado de despegamiento como del desprendimiento endotelial en el grupo congelado con respecto al grupo control. Después de la descongelación, las máximas respuestas a los vasoconstrictores probados (KCl y noradrenalina) fueron del 13 y el 24 por ciento de las respuestas de las aortas que se obtuvieron en fresco. Las respuestas relajantes independientes del endotelio al nitroprusiato sódico estaban reducidas y se produjo una importante reducción de las respuestas de relajación dependientes del endotelio a la acetilcolina. Conclusiones. El método de criopreservación que se empleó disminuyó las respuestas de contracción y relajación a los seis meses y produjo cambios morfológicos importantes en cuanto a la conservación del endotelio y de las elásticas, pero no alteró el grado de apoptosis (AU)

Effect of cryopreservation on human articular chondrocyte viability, proliferation, and collagen expression.

Autotransplantation of human chondrocytes is an alternative therapeutic treatment for focal lesions of cartilage. During the process of isolation and culture of chondrocytes some problems that render the implantation of the cells unsuitable can occur. For security, some cells must be stored using cryopreservation. The objective of this study was to analyze the effect of cryopreservation on cellular viability, proliferation, and collagen expression of human chondrocytes. Human osteoarthritic cartilage (n = 23) was obtained and transferred to a sterile flask containing Dulbecco's modified Eagle's medium (DMEM) and antibiotics. Chondrocytes were isolated, cultured for 3-4 weeks, and frozen in DMEM containing 10% human serum and 10% dimethyl sulfoxide by use of three different protocols. A cellular fraction was frozen directly to -80 degrees C (Protocol I). Another fraction was directly frozen to -80 degrees C and 24 h later introduced into liquid nitrogen (Protocol II). The last aliquot was frozen with controlled freezing using a freezing rate of -1 degrees C/min to a temperature of -40 degrees C, 2 degrees C/min to -60 degrees C, and 5 degrees C/min to -150 degrees C (Protocol III). Cells were cryopreserved for 2 weeks. Cells from each cryopreservation method were then cultured for 7 days and cellular proliferation was evaluated by the counting of the total cells in each flask. Cryopreservation had a negative effect on chondrocyte survival and proliferation. The survival after cryopreservation with the three protocols was 70-75%. There was no significative difference between the methods used to cryopreserve (P = 0.4117). However, there was a significant difference among the donors (P = 0.0111). Cellular proliferation of chondrocytes was reduced by cryopreservation (P = 0.024). The rate of proliferation of different groups was control samples 6.56, Protocol I 4.66, Protocol II 4.69, and Protocol III 5.58. Statistical analysis showed that the programmed protocol was the best method to preserve cellular functions. Chondrocytes were able to express collagen type II 1 week after cryopreservation. Cryopreservation modifies the survival and proliferation of chondrocytes. Of all protocols used to cryopreserve, the programmed protocol seems to be the best technique. Cryopreservation does not alter the collagen type II expression.