Isolation and characterization of the human ovarian cell population for transplantation into an artificial ovary

To support survival and growth of follicles, the transplantable artificial ovary should mimic the original organ, offering a physical (3D matrix) and biological support (cells). In order to replicate the ovarian cell populations, the aim of this study is to assess the proportions of stromal and endothelial cells in the ovarian cortex. To this end, ovarian biopsies were obtained from six women (mean age: 49 years). The epithelial layer and medulla were carefully removed. The cortex was finely minced and enzymatically digested and the isolated cells were fixed. For cell characterization, immunostaining for CD31 (for endothelial cells) and inhibin-α (for granulosa cells) was performed. Positive cells in each staining were counted and the proportion of the different cell populations was estimated from the total number of isolated cells. Since there is no specific marker for ovarian stromal cells, we estimated the proportion of these cells by performing a vimentin immunostaining and subtracting the proportions of CD31- and inhibin-α-positive cells. Immunostaining showed that 84% of isolated cells were vimentin-positive. From this pool, 3% were endothelial cells and 1% granulosa cells. Consequently, the population of ovarian stromal cells was 80%. In conclusion, our findings show that stromal cells represent the larger population of cells in the human ovarian cortex. While this ensures follicle survival and development in a normal ovary, we believe that the low proportion of endothelial cells could have a negative impact on the angiogenesis in the artificial ovary after the first days of transplantation.

Isolation and characterization of the human ovarian cell population for transplantation into an artificial ovary

To support survival and growth of follicles, the transplantable artificial ovary should mimic the original organ, offering a physical (3D matrix) and biological support (cells). In order to replicate the ovarian cell populations, the aim of this study is to assess the proportions of stromal and endothelial cells in the ovarian cortex. To this end, ovarian biopsies were obtained from six women (mean age: 49 years). The epithelial layer and medulla were carefully removed. The cortex was finely minced and enzymatically digested and the isolated cells were fixed. For cell characterization, immunostaining for CD31 (for endothelial cells) and inhibin-α (for granulosa cells) was performed. Positive cells in each staining were counted and the proportion of the different cell populations was estimated from the total number of isolated cells. Since there is no specific marker for ovarian stromal cells, we estimated the proportion of these cells by performing a vimentin immunostaining and subtracting the proportions of CD31- and inhibin-α-positive cells. Immunostaining showed that 84% of isolated cells were vimentin-positive. From this pool, 3% were endothelial cells and 1% granulosa cells. Consequently, the population of ovarian stromal cells was 80%. In conclusion, our findings show that stromal cells represent the larger population of cells in the human ovarian cortex. While this ensures follicle survival and development in a normal ovary, we believe that the low proportion of endothelial cells could have a negative impact on the angiogenesis in the artificial ovary after the first days of transplantation.(AU)

Isolamento e caracterização das células mesenquimais derivadas da membrana amniótica dos gatos domésticos / Isolation and characterization of Mesenchymal cells from cat amniotic membrane

As células tronco mesenquimais derivadas do âmnio (AMSCs) são células multipotentes com alto potencial para se diferenciar em múltiplas linhagens. Podem ser isoladas sem recurso a procedimentos invasivos e usadas sem levantar quaisquer implicações éticas. O presente estudo visa isolar e caracterizar as células mesenquimais progenitoras da membrana amniótica de gatos domésticos para futura aplicação em terapia celular. As células foram isoladas de quatro membranas fetais, coletadas durante as campanhas rotineiras de castração em gatas no último terço de gestação, após anestesia geral. A porção dorsal do âmnio foi separada mecanicamente, lavada com PBS e submetida à digestão com colagenase. As células coletadas foram propagadas em cultivo (DMEN-F12/-MEM) e criopreservadas em várias passagens enquanto se efetuava a avaliação da cinética de crescimento e das características morfológicas. Em cultivo, as AMSCs demonstraram aderência à placa e uma morfologia similar a dos fibroblastos.

The amnion derived mesenchymal stem cells (AMSCs) are multipotent cells with a high ability to differentiate into multiple lineages. They can be obtained by non-invasive methods and therefore are exempt from the normal ethical problems involving stem cell use. The aim of this study was to isolate and characterize the progenitor mesenchymal cells from the cat amniotic membrane for future application in cell therapy. The cells were isolated from four fetal membranes collected after a routine ovarian hysterectomy process from cats in their third gestational trimester, under general anesthesia. The dorsal portion of amnion was mechanically separated, washed with PBS and subjected to collagenase digestion. The isolated cells were propagated in culture media (DMEMF12 or -MEM) and frozen in various passages while the growing kinetics and cell morphology were analyzed. In culture medium, AMSCs were adherent to the plastic culture dish and had a morphology similar to fibroblasts.

Isolamento e caracterização das células mesenquimais derivadas da membrana amniótica dos gatos domésticos / Isolation and characterization of Mesenchymal cells from cat amniotic membrane

As células tronco mesenquimais derivadas do âmnio (AMSCs) são células multipotentes com alto potencial para se diferenciar em múltiplas linhagens. Podem ser isoladas sem recurso a procedimentos invasivos e usadas sem levantar quaisquer implicações éticas. O presente estudo visa isolar e caracterizar as células mesenquimais progenitoras da membrana amniótica de gatos domésticos para futura aplicação em terapia celular. As células foram isoladas de quatro membranas fetais, coletadas durante as campanhas rotineiras de castração em gatas no último terço de gestação, após anestesia geral. A porção dorsal do âmnio foi separada mecanicamente, lavada com PBS e submetida à digestão com colagenase. As células coletadas foram propagadas em cultivo (DMEN-F12/-MEM) e criopreservadas em várias passagens enquanto se efetuava a avaliação da cinética de crescimento e das características morfológicas. Em cultivo, as AMSCs demonstraram aderência à placa e uma morfologia similar a dos fibroblastos. (AU)

The amnion derived mesenchymal stem cells (AMSCs) are multipotent cells with a high ability to differentiate into multiple lineages. They can be obtained by non-invasive methods and therefore are exempt from the normal ethical problems involving stem cell use. The aim of this study was to isolate and characterize the progenitor mesenchymal cells from the cat amniotic membrane for future application in cell therapy. The cells were isolated from four fetal membranes collected after a routine ovarian hysterectomy process from cats in their third gestational trimester, under general anesthesia. The dorsal portion of amnion was mechanically separated, washed with PBS and subjected to collagenase digestion. The isolated cells were propagated in culture media (DMEMF12 or -MEM) and frozen in various passages while the growing kinetics and cell morphology were analyzed. In culture medium, AMSCs were adherent to the plastic culture dish and had a morphology similar to fibroblasts. (AU)