BACKGROUND: Several protocols for the isolation of luteinized granulosa cells (LGCs) contained in follicular fluid have been described but no previously published study has compared the relative efficiency of these protocols. Our objective is to obtain conclusive scientific evidence for the superiority of one method over another. METHODS: Different purification methods for LGCs based on the recognition of specific cell markers, aggregates, differential adhesion and LGC size were evaluated. We compared the levels of CD45 cell contamination and the percentage of total cell viability in paired aliquots of cells (before and after purification) derived from the follicular fluid obtained from women who were donating oocytes (n = 72). Each of the six purification methods was performed six times using pooled follicular fluids from two women. RESULTS: Samples processed by means of recognition of specific cell markers were characterized by their greater purity (0.1-1.33% CD45+) but low rate of LGC recovery (17.13-25.4%) when compared with the other methods (3.29-12% CD45+, P < 0.05 and 51.67-73.20% LGC, P < 0.05). It is noteworthy that the filter method, which is based on the LGC size, combined one of the highest rates of LGC recovery (∼70%) with acceptable low levels of contamination (<5%). CONCLUSIONS: There is currently no gold standard method for the isolation of LGCs, and protocols should be chosen depending on the purpose in question. We conclude that fluorescence-activated cell sorting is the best protocol for isolating LGCs when purity is the principal criterion, and magnetic separation when both purity and viability are essential. However, cell straining (filter) is probably the least laborious and, overall, the most efficient method to isolate LGCs.
BACKGROUND: Several protocols for the isolation of luteinized granulosa cells (LGCs) contained in follicular fluid have been described but no previously published study has compared the relative efficiency of these protocols. Our objective is to obtain conclusive scientific evidence for the superiority of one method over another. METHODS: Different purification methods for LGCs based on the recognition of specific cell markers, aggregates, differential adhesion and LGC size were evaluated. We compared the levels of CD45 cell contamination and the percentage of total cell viability in paired aliquots of cells (before and after purification) derived from the follicular fluid obtained from women who were donating oocytes (n = 72). Each of the six purification methods was performed six times using pooled follicular fluids from two women. RESULTS: Samples processed by means of recognition of specific cell markers were characterized by their greater purity (0.1-1.33% CD45+) but low rate of LGC recovery (17.13-25.4%) when compared with the other methods (3.29-12% CD45+, P < 0.05 and 51.67-73.20% LGC, P < 0.05). It is noteworthy that the filter method, which is based on the LGC size, combined one of the highest rates of LGC recovery (∼70%) with acceptable low levels of contamination (<5%). CONCLUSIONS: There is currently no gold standard method for the isolation of LGCs, and protocols should be chosen depending on the purpose in question. We conclude that fluorescence-activated cell sorting is the best protocol for isolating LGCs when purity is the principal criterion, and magnetic separation when both purity and viability are essential. However, cell straining (filter) is probably the least laborious and, overall, the most efficient method to isolate LGCs.
Authors: Prapti Singh; Marli Amin; Erica Keller; Ariel Simerman; Paul Aguilera; Christine Briton-Jones; David L Hill; David H Abbott; Gregorio Chazenbalk; Daniel A Dumesic Journal: J Assist Reprod Genet Date: 2013-03-20 Impact factor: 3.412
Authors: Irma Virant-Klun; S Omejec; M Stimpfel; P Skerl; S Novakovic; N Jancar; E Vrtacnik-Bokal Journal: Stem Cell Rev Rep Date: 2019-08 Impact factor: 5.739