Explorative 3-D culture of early secondary follicles in a time lapse system for up to 36 days gives valuable, but limited insights in follicular development.

BACKGROUND

Cryopreservation of ovarian cortical tissue is an important option for female fertility preservation. This is particularly valuable for cancer patients who need to be treated urgently with chemotherapy, leaving no time for hormonal stimulation. The transfer of malignant cells in certain cancers remains as a potential risk after freezing, thawing and transplantation of ovarian tissue while isolation and in vitro growth (IVG) of follicles could be a safe alternate approach of female fertility protection.

METHODS

Ovarian cortex tissue was frozen, thawed and cultured for 8 days prior to isolating and embedding of early secondary follicles in a 3D matrix, suitable for time lapse monitoring for up to 36 days. Continuous growth of a theca-like cell layer and extrafollicular protrusions were visually evaluated with a permanent monitoring system facilitating real-time follicular development without deviations in the culture conditions. Occurrence of theca cell growth was visually characterized by extrafollicular formation of cells, beyond the outer follicle boundaries. To validate the results observed by time-lapse monitoring, live cell imaging was conducted and determined with immunofluorescence staining.

RESULTS

Individual follicles significantly increased in size over time. Time-lapse video monitoring revealed extending and retracting of filopodia-like structures in the outer follicular region adjacent to the 3D environment. Theca-like cells and actin components of filopodia-like structures were identified based on immunofluorescence staining.

CONCLUSIONS

Time lapse monitoring of 3-D cultured follicles is a promising explorative approach to obtain valuable visual insights regarding the many facets of follicular growth and to optimize follicular culture conditions towards a clinical application. As the study is limited by a lack of mechanistic insights into theca cell differentiation and filopodia function, additional studies are necessary to validate the preliminary results of this approach.