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Introduction: For women diagnosed with leukemia, transplantation of cryopreserved ovarian tissue after disease remission is not advisable due to the risk of reintroduction of malignant cells. To restore fertility in these patients, a biodegradable artificial ovary that offers an environment allowing isolated follicles and stromal cells (SCs) to survive and grow needs to be developed. The aim of this study is therefore to test the survival and proliferation of isolated SCs encapsulated in an alginate-matrigel matrix. Materials and Methods: Four NMRI mice were ovariectomized and their ovaries were mechanically and enzymatically disrupted in order to isolate ovarian SCs. Groups of 50,000 SCs were embedded in an alginate-matrigel matrix for further fixation (fresh controls), one week of in vitro culture (IVC) or grafting. SC proliferation (Ki67), apoptosis (TUNEL), scaffold degradation, vessel formation (CD34) and inflammation (CD45) were analyzed. Results: SCs appeared round and none of them were found to be pyknotic in the alginate-matrigel beads from any of the three groups (fresh, IVC or grafting). After IVC or grafting, the beads degraded, losing their original round form. Infiltrating blood capillaries were observed in the grafted beads. CD34-positive cells were found around and inside the matrix. No fibrous capsules were seen in the grafts and 22% of CD45-positive cells were identified around the matrix. Proportions of 2.3%, 18.3% and 15.5% of cells were Ki67-positive in fresh, IVC and grafted beads respectively. The results from fresh control were statistically lower than the other two groups. The percentage of TUNEL-positive cells was 0%, 1.5% and 6.9% in fresh, IVC and grafted groups respectively. The results were statistically different between groups. Discussion: According to Ki67 and TUNEL immunostaining, SCs successfully survived and proliferated inside this matrix, which began to degrade, but did not elicit any major inflammatory reaction. This is the first assay to be conducted on the construction of a transplantable artificial ovary, representing the first step in a strategy that may be termed ‘ovarian tissue engineering’, which aims to restore endocrine activity and fertility to cancer patients. Our results showed that a biodegradable alginate-based matrix is a promising choice to encapsulate and graft isolated SCs. The next step will be incorporating isolated preantral follicles and ensuring their survival and interaction with SCs. Longer-term grafting will also be required to analyze the degradation kinetics of the matrix and its influence on follicle development.
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Vanacker, J., Dolmans, M.-M., des Rieux, A., Jaeger, J., Luyckx, V., Van Langendonckt, A., Donnez, J., & Andrade Amorim, C. (2012). Transplantation of an alginate-matrigel matrix containing isolated ovarian stromal cells: first step in developing an artificial ovary. 3rd International Conference “Strategies in Tissue Engineering”, Würzburg - Germany. https://hdl.handle.net/2078.5/204012