[Survival of dopaminergic neurons that were hibernated in vitro for seven days]. / Supervivencia in vitro de neuronas dopaminérgicas hibernadas durante siete días.

INTRODUCTION: The use of fresh foetal tissue in neurotransplants entails considerable problems of logistics that limit its clinical applicability, something that can be resolved by the development of optimal tissue storage procedures that do not affect in vivo viability and survival of dopamine. AIMS. To determine whether 7 days' hibernation affects the survival of mesencephalic tissue in vitro, and to compare it to fresh tissue. MATERIALS AND METHODS: The midbrains of rats were hibernated for 1, 3, 5 and 7 days at 4 degrees C. A cellular suspension was prepared for culture throughout a 7-day period. The number of TH+ cells present in the fresh and hibernated cultures was determined. RESULTS: The morphology of the hibernated and cultured dopaminergic neurons was very similar to that of the fresh cells. Comparing the viability of the hibernated and fresh cells did not reveal any significant differences. No significant differences between the numbers of TH+ neurons were observed at any of the hibernation times. The lowest rate of TH+ cell survival was reached at seven days' hibernation. Significant differences (p < 0.05) were found between the number of TH+ neurons for fresh and hibernated tissue. CONCLUSIONS: Hibernation at 4 degrees C for up to five days guarantees the survival of TH+ cells in vitro, but it is affected by longer times. This procedure could be considered useful for preserving human tissue in clinical transplant applications. These results refer to in vitro conditions; therefore, studies must be conducted to investigate the survival and functionality of hibernated and transplanted neurons in animal models to enable us to evaluate its applicability in neurorestorative therapy.

[Marrow stromal cells cultured in N2-supplemented medium: implications on the generation of neural cells]. / Cultivo de las células estromales de la médula ósea en un medio suplementado con los nutrientes N2: implicaciones sobre la generación de células neurales.

INTRODUCTION: Most of the culture system for in vitro maintenance and neural differentiation of marrow stromal cells (MSCs) use synthetic media supplemented with 10 or 20% fetal bovine serum (FBS). Serum, however, is comprised of unknown quantities of undefined substances which could interfere the effect of exogenous substances on neural differentiation of MSCs. AIM. Here we describe survival of MSCs cultured in culture conditions where serum was reduced at 0.5 and 1% using Bottenstein and Sato's N2 formula (1979) and poly-L-lysine (PLL)-coated substrate. MATERIALS AND METHODS: Stromal cells isolated from rat femurs were cultivated in Dulbecco's modified Eagle medium at 10, 1, 0.5% FBS or in serum free medium containing N2 formula. In serum free medium or at low serum concentration culture surface was coated with PLL. Cell survival was determined by MTT method or by counting viable cells. RESULTS: Survival of MSCs cultured in N2 supplement was reduced at about 40% of that observed in 10% FBS containing medium. Under these conditions cell morphology was also affected. When N2 containing medium was supplemented with FBS at 0.5 or 1% a significant increase of survival with respect to that observed in N2-supplemented cultures was observed. Cells seeded on PLL-coated surface increased their survival by contrast with their homologous cultures seeded on uncoated surface. CONCLUSIONS: The culture system which combines N2 formula with FBS 1% and PLL-coated surface is useful for the maintenance of MSCs. These conditions offer advantages for the study of differentiation of these cells because they reduce the confounding influence of serum. The possible implication of this culture system for the study of neural differentiation by these cells is discussed.

Supervivencia in vitro de neuronas dopaminèrgicas hibernadas durante siete dias / s. t

El uso de tejido fetal fresco en el neurotrasplante presenta considerables dificultades logìsticas que limitan su aplicabilidad clìnica . Este aspecto podrìa solucionarse con el desarrollo de procedimientos optimos de almacenamiento del tejido que no afecten a la viabilidad y la supervivencia dopaminèrgica in vivo. Es objetivo del trabajao determinar si la hibernaciòn durante siete dìas influye sobre la supervivencia del tejido mesencefàlico in vitro y comparar el tejido hibernado con el fresco...(AU)

Supervivencia in vitro de neuronas dopaminérgicas hibernadas durante siete días / Survival of dopaminergic neurons that were hibernated in vitro for seven days

Introducción. El uso de tejido fetal fresco en el neurotrasplante presenta considerables dificultades logísticas que limitan su aplicabilidad clínica. Este aspecto podría solucionarse con el desarrollo de procedimientos óptimos de almacenamiento del tejido que no afecten a la viabilidad y la supervivencia dopaminérgica in vivo. Objetivo. Determinar si la hibernación durante siete días influye sobre la supervivencia del tejido mesencefálico in vitro, y comparar el tejido hibernado con el fresco. Material y métodos. El mesencéfalo de rata se hibernó 1, 3, 5 y 7 días a 4 °C. Se preparó la suspensión celular para cultivar durante siete días. Se determinó el número de células TH+ presentes en los cultivos frescos e hibernados. Resultados. La morfología de las neuronas dopaminérgicas hibernadas y cultivadas fue muy similar a la de las células frescas. La comparación de la viabilidad de las células hibernadas con la de las frescas mostró diferencias no significativas. No hay diferencias significativas entre el número de neuronas TH+ observadas en todos los tiempos de hibernación. La supervivencia de células TH+ más baja se alcanzó a los siete días de hibernación. Existen diferencias significativas (p < 0,05) entre el número de neuronas TH+ del tejido fresco y el hibernado. Conclusiones. La hibernación a 4 °C hasta cinco días garantiza la supervivencia in vitro de las células TH+; tiempos mayores, la afecta. Este procedimiento podría considerarse útil para la conservación del tejido humano aplicable en el trasplante clínico. Estos resultados se refieren a condiciones in vitro; por tanto, se requiere estudiar la sobrevivencia y funcionalidad de las neuronas hibernadas y trasplantadas en modelos animales para evaluar su aplicación en la terapia neurorrestaurativa

Introduction. The use of fresh foetal tissue in neurotransplants entails considerable problems of logistics that limit its clinical applicability, something that can be resolved by the development of optimal tissue storage procedures that do not affect in vivo viability and survival of dopamine. Aims. To determine whether 7 days’ hibernation affects the survival of mesencephalic tissue in vitro, and to compare it to fresh tissue. Materials and methods. The midbrains of rats were hibernated for 1, 3, 5 and 7 days at 4 °C. A cellular suspension was prepared for culture throughout a 7-day period. The number of TH+ cells present in the fresh and hibernated cultures was determined. Results. The morphology of the hibernated and cultured dopaminergic neurons was very similar to that of the fresh cells. Comparing the viability of the hibernated and fresh cells did not reveal any significant differences. No significant differences between the numbers of TH+ neurons were observed at any of the hibernation times. The lowest rate of TH+ cell survival was reached at seven days’ hibernation. Significant differences (p < 0.05) were found between the number of TH+ neurons for fresh and hibernated tissue. Conclusions. Hibernation at 4 °C for up to five days guarantees the survival of TH+ cells in vitro, but it is affected by longer times. This procedure could be considered useful for preserving human tissue in clinical transplant applications. These results refer to in vitro conditions; therefore, studies must be conducted to investigate the survival and functionality of hibernated and transplanted neurons in animal models to enable us to evaluate its applicability in neurorestorative therapy

Cultivo de las celulas estromales de la mèdula òsea en un medio suplementado con los nutrientes N2: implicaciones sobre la generaciòn de cèlulas neurales / s. t

Most of the culture system for in vitro maintenance and neural differentiation of marrow stromal cells (MSCs) use synthetic media supplemented with 10 or 20 percent fetal bovine serum (FBS). Serum, however, is comprised of unknown quantities of undefined substances which could interfere the effect of exogenous substances on neural differentiation of MSCs, AIM. Here we describe survival of MSCs cultured in culture conditions where serum was reduced at 0,5 and 1 percent using Bottenstein and Sato's N2 formula (1979) and poly-L-lysine (PLL)-coated substrate...(AU)

Cultivo de las células estromales de la médula ósea en un medio suplementado con los nutrientes N2: implicaciones sobre la generación de células neurales / Marrow stromal cells cultured in n2-supplemented medium: implications on the generation of neural cells

Introducción. La mayoría de los sistemas de cultivo para el mantenimiento in vitro y la diferenciación neural de las células estromales de la médula ósea (CEM) utilizan medios sintéticos suplementados con suero fetal bovino (SFB) al 10 o al 20%. Sin embargo, el suero se compone de cantidades desconocidas de sustancias no definidas que podrían interferir el efecto de las sustancias exógenas sobre la diferenciación neural de estas células. Objetivo. En este trabajo describimos la supervivencia de las CEM en condiciones de cultivo donde se redujo la concentración del SFB al 0,5 y al 1% y se utilizó la fórmula N2 de Bottenstein y Sato (1979) y un sustrato tratado con poli-L-lisina (PLL). Materiales y métodos. Se cultivaron células estromales aisladas de los fémures de rata en el medio de Eagle modificado por Dulbecco, con concentraciones de SFB del 10, el 1 y el 0,5% o en medio libre de suero, que contenían la fórmula N2. En los cultivos crecidos en medios libres de suero o con baja concentración de éste, la superficie de cultivo se trató con PLL. La supervivencia celular se midió por el método del MTT o por recuento de las células vivas. Resultados. La supervivencia de las CEM cultivadas en la fórmula N2 disminuyó hasta aproximadamente el 40% de la observada en el medio con SFB al 10%, y se afectó la morfología celular. Un aumento significativo de la supervivencia con respecto al cultivo en N2 se produjo cuando, además de este nutriente, se añadió SFB al 0,5 y al 1%. En los cultivos sembrados sobre superficies tratadas con PLL la supervivencia celular aumentó en comparación con los sembrados sobre superficies no tratadas. Conclusiones. Este sistema de cultivo que combina la fórmula N2 con SFB al 1% y emplea un sustrato tratado con PLL, es adecuado para el mantenimiento de las CEM. Estas condiciones son ventajosas para estudiar la diferenciación neural de estas células, ya que reducen la interferencia del suero. Se discute la posible implicación de este sistema de cultivo para los estudios de diferenciación neural en estas células

Introduction. Most of the culture system for in vitro maintenance and neural differentiation of marrow stromal cells (MSCs) use synthetic media supplemented with 10 or 20% fetal bovine serum (FBS). Serum, however, is comprised of unknown quantities of undefined substances which could interfere the effect of exogenous substances on neural differentiation of MSCs. Aim. Here we describe survival of MSCs cultured in culture conditions where serum was reduced at 0.5 and 1% using Bottenstein and Sato’s N2 formula (1979) and poly-L-lysine (PLL)-coated substrate. Materials and methods. Stromal cells isolated from rat femurs were cultivated in Dulbecco’s modified Eagle medium at 10, 1, 0.5% FBS or in serum free medium containing N2 formula. In serum free medium or at low serum concentration culture surface was coated with PLL. Cell survival was determined by MTT method or by counting viable cells. Results. Survival of MSCs cultured in N2 supplement was reduced at about 40% of that observed in 10% FBS containing medium. Under these conditions cell morphology was also affected. When N2 containing medium was supplemented with FBS at 0.5 or 1% a significant increase of survival with respect to that observed in N2-supplemented cultures was observed. Cells seeded on PLL-coated surface increased their survival by contrast with their homologous cultures seeded on uncoated surface. Conclusions. The culture system which combines N2 formula with FBS 1% and PLL-coated surface is useful for the maintenance of MSCs. These conditions offer advantages for the study of differentiation of these cells because they reduce the confounding influence of serum. The possible implication of this culture system for the study of neural differentiation by these cells is discussed

[Stromal cell transplant in the 6-OHDA lesion model]. / Trasplante de células estromales en el modelo de lesión por 6-OHDA.

INTRODUCTION: A good deal of evidence currently exists to show that transplanting foetal mesencephalic tissue can produce symptomatic benefits both in patients and in disease models. Nevertheless, the technical and ethical difficulties involved in obtaining enough suitable foetal cerebral tissue have been a serious obstacle to its application. Stromal cells derived from bone marrow, due to their potential capacity to generate different types of cells, could be an ideal source of material for cell restoration in neurodegenerative diseases. AIMS: Our aim was to evaluate the effect of transplanting stromal cells derived from bone marrow on the behaviour of 6-OHDA rats, when they are inserted into the striatum. MATERIAL AND METHODS: In this study we used rats with a lesion in the substantia nigra induced by 6-hydroxydopamine, divided into several experimental groups. Rotary activity induced by D-amphetamine (5 mg/kg, intraperitoneally) was evaluated before and throughout the three months following the transplant in all the experimental groups, except in the group of healthy controls. Hemiparkinsonian rats received a total of 350 000 foetal ventral mesencephalic cells and 8 x 10(4) stromal cells/microL, which were implanted in the striatum. RESULTS AND CONCLUSIONS: Animals with stromal cells transplanted in the body of the striatum significantly reduced the number of turns induced by amphetamine (p < 0.05); yet this reduction was not greater than that induced by foetal mesencephalic cell transplants. We were also unable to demonstrate any significant improvement in the motor skills of the forelimbs.

[In vitro viability and glutathione levels in mesencephalic neurons after seven days hibernation]. / Viabilidad in vitro y niveles de glutati n en neuronas mesencef licas hibernadas durante siete d as.

In embryonic mesencephalic transplant in patients with Parkinson s disease dopaminergic survival is low (5 10%), and for this reason the use of multiple donors has been considered. The difficulty of obtaining more tissue determines the need for a procedure that enables human nigral tissue to be stored for a time without affecting its physiological state in any significant way. This study was designed to determine whether hibernation of tissue fragments has any influence on viability, how the viability of the mesencephalic cells behaves after 7 days hibernation and the glutathione levels in the hibernated tissue (HT). The viability of the HT in pieces (82.37 2.12) was found to be higher than the value for the whole mesencephalon (70.29 3.43). Viability of the HT, seven days at 4 C, at different post dissociation times, did not differ significantly. Despite the significant differences found between hibernated and fresh tissue at t= 0, this procedure does not seem to affect the mesencephalic tissue in any significant way, as it conserved a 94% viability after hibernation. No evidence was found of increased glutathione content as an antioxidizing response to the damage that might be caused by hibernation. These results suggest that since hibernation does not have any significant effect on the state of the cells it could be considered a useful procedure for conserving tissue to be used in clinical transplants. Moreover, further research is needed on survival and functionality of hibernated cells after being transplanted into animal models in order to evaluate their potential for use in cell therapy.

[Effects of simultaneous transplant of foetal mesencephalic cells in the striatum and the subthalamic nucleus of hemiparkinsonian rats]. / Efectos del trasplante simultáneo de células mesencefálicas fetales en el estriado y el núcleo subtalámico de ratas hemiparkinsonianas.

INTRODUCTION: The main strategy followed in neural transplants as a method of treatment for Parkinson s disease, both experimental and clinical, has been to introduce foetal mesencephalic cells into the target area: the striatum. However, when the dopaminergic cells in the substantia nigra degenerate, not only is the dopaminergic innervation of the striatum affected but also other nuclei: globus pallidus, substantia nigra, substantia nigra pars reticulata and subthalamic nucleus. A series of data from pharmacological and physiological studies offer strong evidence that the dopamine released in these nuclei may play an important role in regulating the output nuclei of the basal ganglia. AIM: To evaluate the effect of transplanting foetal mesencephalic cells on the behaviour of 6 OH DA rats when introduced into the striatum and the subthalamic nucleus. MATERIALS AND METHODS: 6 OH DA was used to induce lesions in the substantia nigra of rats, which were divided into several experimental groups. The rotating activity induced by D amphetamine (5 mg/kg, intraperitoneally) and apomorphine (0.05 mg/kg, subcutaneously) was evaluated before and three months after the transplant in all the experimental groups, except in the control group of healthy rats. The hemiparkinsonian rats received a total of 350,000 foetal ventral mesencephalic cells, which were implanted within small deposits in the striatum (8) and in the subthalamic nucleus (4). RESULTS AND CONCLUSIONS: Rotation induced by both drugs was significantly lower (p= 0.05) in animals that had had dopaminergic cells transplanted into the striatum body. No significant improvement in this behaviour was to be found when transplants were limited to just the subthalamus or, simultaneously, also to the striatum. A significant increase in rotating behaviour induced by apomorphine was observed in the group which received a transplant in just the subthalamus.