Multipotent mesenchymal stromal stem cells derived from waste human ovarian follicular fluid: bioengineering applications in vitro

Modern tissue engineering strategies combine living cells and scaffold materials to develop biological substitutes that can restore tissue functions. Biomaterials and bioreactor systems provide both the technological means to reveal fundamental mechanisms of cell function in a 3D environment, and the potential to improve the quality of engineered tissues. Mesenchymal stem cells (MSC) have a large capacity for self-renewal while maintaining their multipotency and represent a promising tool for regenerative medicine. Our studies showed the possibility to derive MSC from the human ovarian follicular fluid (FF cells) that picked-up during human assisted reproduction techniques and usually wasted during IVF procedures. FF cells were collected from healthy women undergone an IVF treatment during oocytes retrieval and cultured with DMEM + 10% FBS in in vitro minimal medium conditions, without any growth factor, including leukemia-inhibiting factor (LIF), previously considered essential. FF cells showed typical mesenchymal stemness markers (including CD-90, CD-44, CD-105, CD-73) and multipotent differentiation capacity in osteogenic, chondrogenic and adipogenic lineages. Now we also tested capability of FF cells to grow on gelatin cryogel scaffolds, promising new biomaterials owing to their biocompatibility, compared to MSC derived from human bone marrow. Data collected demonstrated that FF cells could grow on biomaterial not only on the top but also in the layers below till 60 μm of deepness. Results suggested that cells observed were mesenchymal ones due to their positive immunostained for vimentin and CD-44, typical markers for MSC. Finally we evaluated if ultrasound treatment on FF cells cultures have positive effects on proliferation activity. To study effects of mechanical conditioning, we tested two different times of ultrasound stimulus (LIPUS), 2 or 5 minutes, corresponding respectively to 17.88 and 44.70 joule per day on cell culture and compared data to control without any kind of mechanical stimulation. The percentage number of cells showing intensive proliferative activity was 11.26 ± 5.19% in non-stimulated control cultures, 15.60 ± 1.70% in LIPUS stimulated cultures for 2 min, and 46.43 ± 15.04% in LIPUS stimulated cultures for 5 min. Comparisons with Bonferroni test showed statistically significant differences between control and LIPUS stimulation for 5 min (p=0.003) and between the two LIPUS stimulations (p=0.021); on the other hand, the proliferation did not increase with the shorter ultrasound treatment. Our results suggest that cells provided by mesenchymal plasticity can be easily isolated by waste follicular fluid, avoiding scraping of human ovaries. Moreover, successfully growth of putative MSC derived from follicular fluid on three-dimensional cryogel scaffold and positive effect on proliferation activity induced by ultrasound stimulus, open potential developments in biotechnological or medical applications. Therefore, FF cells may be introduced as a valuable model system with which to study the mesenchymal lineages for basic research and tissue engineering.