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

Objective Aspirated ovarian follicular fluids (FF) contain luteal granulosa cells (LGC) and other contaminating cell types. Several strategies such as the antibody binding methods, the flask method, the cell strainer, and the positive selection of granulosa aggregates after density gradient centrifugation, were tested as LGCs purification methods. Each of these strategies has its own advantages and disadvantages. The positive selection of granulosa aggregates after density gradient centrifugation is simple, rapid, and efficient in terms of LGC recovery. However, it results in a low purity. Here we aimed to test if modifying the traditional protocol by collecting the aggregates, from the follicular fluid, before the density gradient centrifugation could decrease the percentage of contaminating cells. MaterialsAndMethods In the present prospective study, 32 FF, from 32 women,were randomly assigned into one of the two purification techniques: The positive selection of granulosa aggregates after density gradient centrifugation (DG/Agg) (n=16)or the positive selection of granulosa aggregates from the FF before density gradient centrifugation (Agg/DG) (n=16). At the end of each procedure the cell count, vitality, morphology, and the purity of the cell suspension were evaluated. Results No significant difference was detected in the total number of granulosa cells between DG/Agg and Agg/DG (p>0.05). However, a higher percentage of granulosa cells with normal morphology was detected in Agg/DG compared to DG/Agg (p < 0.001). Moreover, lower percentages of white blood cells (p