Current Issue

Volume 13, Number 4, Jan-Mar 2020 Pages: 339-345

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


Georges Raad, Ph.D, 1, *, Marwa Bazzi, M.Sc, 2, Judy Tanios, M.Sc, 3, Youmna Mourad, M.D, 2, Joan Azouri, M.D, 4, Joseph Azouri, M.D, 1, #, Chadi Fakih, M.D, 2, #,
Azoury IVF Clinic, Mount Lebanon Hospital, Camille Chamoun Bvd, Beirut, Lebanon
Al-Hadi Laboratory and Medical Center, Beirut, Lebanon
Lebanese University, Faculty of Sciences, Section II, Fanar, Lebanon and Azoury IVF Clinic, Mount Lebanon Hospital, Camille Chamoun Bvd, Beirut, Lebanon
OB-GYN Department, Inova Fairfax Hospital, Falls Church, Virginia
*Corresponding Address: Azoury IVF Clinic Mount Lebanon Hospital Camille Chamoun Bvd Beirut Lebanon

The last two authors equally contributed to this work. Email: georges.raad@live.com

Abstract

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.