Optimization of The Cell Aggregates Method for Isolation and Purification of Human Granulosa Cells from Follicular Fluid

BACKGROUND: Aspirated ovarian follicular fluids (FF) contain luteal granulosa cells (LGCs) and other contaminating cell types. Several strategies, such as the antibody binding methods, the flask method, the cell strainer and positive selection of granulosa aggregates after density gradient (DG) centrifugation, were tested as LGC purification methods. Each of these strategies has its own advantages and disadvantages. Positive selection of granulosa aggregates after DG centrifugation is simple, rapid and efficient in terms of LGC recovery. However, it results in a low purity. Here, we aimed to test whether modifying the traditional protocol by collecting the aggregates from the FF, before the DG centrifugation could decrease the percentage of contaminating cells. MATERIALS AND METHODS: In the present prospective study, 32 FF, from 32 women,were randomly assigned into one of the two purification techniques: positive selection of granulosa aggregates from the FF, after DG centrifugation (DG/ Agg, n=16) or positive selection of granulosa aggregates from the FF, before DG centrifugation (Agg/DG, n=16). At the end of each procedure cell count, vitality, morphology and purity of the cell suspension were evaluated. RESULTS: No significant difference was detected in the total number of GCs between DG/Agg and Agg/DG (P>0.05). However, higher percentage of GCs with normal morphology was detected in Agg/DG compared to DG/Agg (P<0.001). Moreover, lower percentages of white blood cells (P<0.01), red blood cells (P<0.001) and epithelial cells (P<0.01) were identified in Agg/DG compared to DG/Agg. CONCLUSION: Here we showed that positive selection of granulosa aggregates from the FF prior to DG technique had a higher purity compared to the traditional protocol. Thus, it could be a method of choice to prepare GCs for research purposes in clinical in vitro fertilization settings.