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) ce...
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| Format: | Article |
| Language: | English |
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Royan Institute (ACECR), Tehran
2020-01-01
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| Series: | International Journal of Fertility and Sterility |
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| Online Access: | http://www.ijfs.ir/article_45568_7b25708df568ab1e32f0a0526661071f.pdf |
| _version_ | 1818218494564499456 |
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| author | Georges Raad Marwa Bazzi Judy Tanios Youmna Mourad Joan Azouri Joseph Azouri Chadi Fakih |
| author_facet | Georges Raad Marwa Bazzi Judy Tanios Youmna Mourad Joan Azouri Joseph Azouri Chadi Fakih |
| author_sort | Georges Raad |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-12T07:24:39Z |
| format | Article |
| id | doaj.art-3b418978916a4e6a83ceb6d6c6e3cc2f |
| institution | Directory Open Access Journal |
| issn | 2008-076X 2008-0778 |
| language | English |
| last_indexed | 2024-12-12T07:24:39Z |
| publishDate | 2020-01-01 |
| publisher | Royan Institute (ACECR), Tehran |
| record_format | Article |
| series | International Journal of Fertility and Sterility |
| spelling | doaj.art-3b418978916a4e6a83ceb6d6c6e3cc2f2022-12-22T00:33:11ZengRoyan Institute (ACECR), TehranInternational Journal of Fertility and Sterility2008-076X2008-07782020-01-0113433934510.22074/ijfs.2020.579045568Optimization of The Cell Aggregates Method for Isolation and Purification of Human Granulosa Cells from Follicular FluidGeorges Raad0Marwa Bazzi1Judy Tanios2Youmna Mourad3Joan Azouri4Joseph Azouri5Chadi Fakih6Azoury IVF Clinic, Mount Lebanon Hospital, Camille Chamoun Bvd, Beirut, LebanonAl-Hadi Laboratory and Medical Center, Beirut, LebanonLebanese University, Faculty of Sciences, Section II, Fanar, Lebanon and Azoury IVF Clinic, Mount Lebanon Hospital, Camille Chamoun Bvd, Beirut, LebanonAl-Hadi Laboratory and Medical Center, Beirut, Lebanon4OB-GYN Department, Inova Fairfax Hospital, Falls Church, VirginiaAzoury IVF Clinic, Mount Lebanon Hospital, Camille Chamoun Bvd, Beirut, LebanonAl-Hadi Laboratory and Medical Center, Beirut, LebanonBackground 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.http://www.ijfs.ir/article_45568_7b25708df568ab1e32f0a0526661071f.pdfdensity gradientfollicular fluidgranulosa cellsisolation and purification |
| spellingShingle | Georges Raad Marwa Bazzi Judy Tanios Youmna Mourad Joan Azouri Joseph Azouri Chadi Fakih Optimization of The Cell Aggregates Method for Isolation and Purification of Human Granulosa Cells from Follicular Fluid International Journal of Fertility and Sterility density gradient follicular fluid granulosa cells isolation and purification |
| title | Optimization of The Cell Aggregates Method for Isolation and Purification of Human Granulosa Cells from Follicular Fluid |
| title_full | Optimization of The Cell Aggregates Method for Isolation and Purification of Human Granulosa Cells from Follicular Fluid |
| title_fullStr | Optimization of The Cell Aggregates Method for Isolation and Purification of Human Granulosa Cells from Follicular Fluid |
| title_full_unstemmed | Optimization of The Cell Aggregates Method for Isolation and Purification of Human Granulosa Cells from Follicular Fluid |
| title_short | Optimization of The Cell Aggregates Method for Isolation and Purification of Human Granulosa Cells from Follicular Fluid |
| title_sort | optimization of the cell aggregates method for isolation and purification of human granulosa cells from follicular fluid |
| topic | density gradient follicular fluid granulosa cells isolation and purification |
| url | http://www.ijfs.ir/article_45568_7b25708df568ab1e32f0a0526661071f.pdf |
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