Human amniotic membrane as an alternative source of stem cells for regenerative medicine.

The human amniotic membrane (HAM) is a highly abundant and readily available tissue. This amniotic tissue has considerable advantageous characteristics to be considered as an attractive material in the field of regenerative medicine. It has low immunogenicity, anti-inflammatory properties and their cells can be isolated without the sacrifice of human embryos. Since it is discarded post-partum it may be useful for regenerative medicine and cell therapy. Amniotic membranes have already been used extensively as biologic dressings in ophthalmic, abdominal and plastic surgery. HAM contains two cell types, from different embryological origins, which display some characteristic properties of stem cells. Human amnion epithelial cells (hAECs) are derived from the embryonic ectoderm, while human amnion mesenchymal stromal cells (hAMSCs) are derived from the embryonic mesoderm. Both populations have similar immunophenotype and multipotential for in vitro differentiation into the major mesodermal lineages, however they differ in cell yield. Therefore, HAM has been proposed as a good candidate to be used in cell therapy or regenerative medicine to treat damaged or diseased tissues.

Viability and histologic structure of porcine valves after cryopreservation.

BACKGROUND: Increased awareness of the limitations of current cardiac valve substitutes has generated a renewed interest in the use of allograft valves. The effects of currently used preservation techniques on the viability of the valve leaflets and the longevity of the implantation however remain controversial. The objective of this study is to analyze the influence of ischemic time, sterilization methods with or without fungicides, and storage procedures on the viability of the valve leaflets and on the histologic structure of the arterial wall, valve leaflet, and myocardium. METHODS: The tissue sources were hearts from 40 pigs with 1 hour of warm ischemic time. The aortic and pulmonary valves were dissected after 2 or 24 hours of cold ischemic time. They were stored in antibiotic solution for 20 hours at 4 degrees C with or without an antifungal agent. The samples were cryopreserved using a programmed temperature decrease method. After 1 week of storage in a liquid nitrogen tank, either in a gas or a liquid phase, the cardiac valves were slowly thawed and examined. RESULTS: Pulmonary valves showed greater viability than aortic valves. Decreased cellular viability was observed independent of cold ischemic time, treatment with amphotericin B, or the storage method used. Treatment with or without amphotericin B had no influence on cellular viability. Conversely it was observed that there was greater cellular viability among those valves stored in a liquid phase. As far as the histologic structure of the valve is concerned we did not observe any influence either in the treatment with amphotericin B or the storage method used although it was observed that reduction of the cold ischemic time minimized histologic injury. CONCLUSIONS: Optimization of preservation methods may decrease the negative effects of cryopreservation on cell viability and histologic structure of the valve.

Cryopreservation effect on proliferative and chondrogenic potential of human chondrocytes isolated from superficial and deep cartilage.

OBJECTIVES: To compare the proliferative and chondrogenic potential of fresh and frozen chondrocytes isolated from superficial and deep articular cartilage biopsies. MATERIALS AND METHODOLOGY: The study included 12 samples of fresh and frozen healthy human knee articular cartilage. Cell proliferation was tested at 3, 6 and 9 days. Studies of mRNA quantification, protein expression and immunofluorescence for proliferation and chondrogenic markers were performed. RESULTS: Stimulation of fresh and frozen chondrocytes from both superficial and deep cartilage with fetal bovine serum produced an increase in the proliferative capacity compared to the non-stimulated control group. In the stimulated fresh cells group, the proliferative capacity of cells from the deep biopsy was greater than that from cells from the superficial biopsy (0.046 vs 0.028, respectively, p<0.05). There was also a significant difference between the proliferative capacity of superficial zone fresh (0.028) and frozen (0.051) chondrocytes (p<0.05). CCND1 mRNA and protein expression levels, and immunopositivity for Ki67 revealed a higher proliferative capacity for fresh articular chondrocytes from deep cartilage. Regarding the chondrogenic potential, stimulated fresh cells showed higher SOX9 and Col II expression in chondrocytes from deep than from superficial zone (p<0.05, T student test). CONCLUSIONS: The highest rate of cell proliferation and chondrogenic potential of fresh chondrocytes was found in cells obtained from deep cartilage biopsies, whereas there were no statistically significant differences in proliferative and chondrogenic capacity between biopsy origins with frozen chondrocytes. These results indicate that both origin and cryopreservation affect the proliferative and chondrogenic potential of chondrocytes.

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.

Evaluación de la motilidad espermática usando dos crioprotectores / Evaluation of sperm motility using two cryoprotectants

La infertilidad, es un problema que afecta a una gran cantidad de parejas. Una de sus causas es la disminución de la calidad seminal debido, por ejemplo, a tratamientos gonadotóxicos. La criopreservación seminal es la técnica que permite conservar y almacenar espermatozoides sin que pierdan su capacidad fecundante; siendo esta una herramienta fundamental en reproducción asistida. El objetivo de este trabajo ha sido optimizar la técnica de criopreservación. Para ello se llevó a cabo un estudio, sobre muestras de pacientes en estudio por problemas de fertilidad, en el que se compararon dos medios de criopreservación (SpermCryo™All-round y CryoSperm™) y la aplicación o no de un baño en nitrógeno líquido a las muestras (previo a su almacenamiento); así como el efecto del tiempo que transcurre desde la eyaculación hasta el procesado sobre la calidad de la muestra. Las posibles variaciones fueron estudiadas con un analizador automático, mediante la realización de test pre- y post-congelación para comprobarla movilidad espermática

Infertility is a problem that affects a lot of couples. One of its causes is a decreased semen quality due to, for example, gonadotoxic treatments. The cryopreservation of human semen is the technique that allows sperm preserving and storing without losing their fertilizing capacity; being a fundamental tool in assisted reproduction. The aim of this study was to optimize the cryopreservation technique. To this end, a study carried out on samples of patients under study by fertility problems, in which two cryoprotectant media (SpermCryo™ All-round and CryoSperm™) and the execution or non-execution of an immersion of the samples in liquid nitrogen (before storage) were compared; and the effect of the time between ejaculation and the processing on the quality of the sample. Variations were studied with an automatic analyzer by performing pre- and post-thaw sperm motility tests. The results show no difference between the two cryoprotectants media, but seems to have a tendency to obtain better postthaw mobility with either depending on sample characteristics. Moreover, the liquid nitrogen bath had no apparent effects on post-thaw results. However, we must highlight the importance of time in the processing of semen samples once liquefied, to avoid decreased sperm quality. To improve post-thaw outcomes the key lies in the necessity to adjust the freezing protocol to the sample characteristics and a correct implementation of the protocol cryopreservation (selection and addition of cryoprotectant media...); favoring the management of infertility and the success of assisted reproduction techniques

Programa de preservación de la fertilidad en pacientes oncológicos / Fertility preservation program in cancer patientes

La infertilidad, es un problema que a día de hoy afecta a una gran cantidad de parejas. Una de las posibles causas es la disminución a nivel mundial de la calidad seminal debido a factores como pueden ser la alimentación, el consumo de alcohol y tabaco, altos niveles de estrés o factores ambientales. Además, el aumento de la incidencia de enfermedades tumorales en pacientes menores de 35 años, pone de manifiesto la necesidad de preservar la fertilidad de estos pacientes mediante técnicas de criopreservación. Es importante, que ésta se realice antes de que reciban ningún tipo de tratamiento (quimioterapia y radioterapia). En este trabajo, se llevará a cabo un estudio comparativo entre las muestras de varones que acuden al Hospital Materno Infantil por problemas de fertilidad y aquellos pacientes oncológicos que antes de iniciar el tratamiento realizan una criopreservación de la muestra seminal analizando las posibles variaciones de estas últimas muestras, mediante un test de post-congelación para comprobar la viabilidad de las muestras criopreservadas. Los parámetros que se van a analizar son: edad del paciente, volumen de la muestra, concentración espermática, porcentaje de espermatozoides móviles e inmóviles, móviles progresivos y espermatozoides tipo 'a'

The infertility, is a problem affecting a large number of couples today. One of the possible causes is decreasing around the world of the seminal ability due to factors such as food, alcohol and tobacco consumption, high levels of stress or environmental factors. In addition, the increase in the incidence of tumour diseases in patients younger than 35 years, highlights the need to preserve the fertility of these patients through cryopreservation techniques. It is important that this be done before they receive any kind of treatment (chemotherapy and radiotherapy). In this work, we will take place a comparative study between the samples of male couples who come to the mother and child Hospital for fertility problems and cancer patients before treatment carried out a Cryopreservation of spermatozoa by analysing possible variations of these latest samples, using a test of post-congelacion to check the viability of samples criopreserved. The parameters will be analyzed are: the patient's age, volume of sample, sperm concentration, percentage of progressive mobile and stationary, mobile sperm and sperm type 'a'