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Proliferative potential and phenotypic analysis of long-term cultivated human granulosa cells initiated by addition of follicular fluid

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Abstract

Purpose

The aim of this study was to develop and optimize a strategy for long-term cultivation of luteinizing human granulosa cells (GCs).

Methods

GCs were cultivated in DMEM/F12 medium supplemented with 2% fetal calf serum. In vitro proliferation of GCs was supported by follicular fluid as well as FSH and growth factors.

Results

The cultured GCs were maintained for 45 days with a doubling time of 159 ± 24 h. GCs initiated by the addition of follicular fluid and cultivated under low serum conditions reached 10 ± 0.7 population doublings. GCs maintain the typical phenotypic expression and the telomere length according to specific culture conditions.

Conclusion

Our present study has demonstrated that GCs can be maintained in vitro for at least 45 days and this cell model can be beneficial when studying hormonal regulation associated with follicular maturation and preparation of oocytes for fertilization.

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Abbreviations

bFGF:

basic fibroblast growth factor

COV434:

cell line derived from a human granulosa cell tumor (Sigma-Aldrich)

DMEM/F12:

dulbecco’s modified eagle’s medium

DT:

doubling time

EGF:

epidermal growth factor

FCS:

fetal calf serum

FF:

follicular fluid

FSH:

follicle stimulating hormone

GCs:

luteinizing human granulosa cells

GC8:

one of the isolated GCs from patient with different characters

IVF:

in vitro fertilization

LH:

luteinizing hormone

PD:

the number of population doublings

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Acknowledgments

The authors wish to acknowledge MSMT 0021620820, MSMT 0021627502 and IGA MZ NS/9781-3 for financial support of research program. The authors would like to acknowledge Ms. Rückerova for help in the tissue cultures laboratory.

Author information

Authors and Affiliations

  1. Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic

    Lenka Bruckova

  2. Department of Histology and Embryology, Charles University in Prague, Medical Faculty in Hradec Kralove, Simkova 870, 500 38, Hradec Kralove, Czech Republic

    Lenka Bruckova, Tomas Soukup, Benjamin Visek & Jaroslav Mokry

  3. Department of Obstetrics and Gynecology, Charles University in Prague, 1st Medical Faculty and General Teaching Hospital in Prague, Prague, Czech Republic

    Jiri Moos, Martina Moosova, Jana Pavelkova & Karel Rezabek

  4. Department of Clinical Genetics, Teaching Hospital in Hradec Kralove, Hradec Kralove, Czech Republic

    Lenka Kucerova

  5. Department of Pharmacology, Charles University in Prague, Medical Faculty in Hradec Kralove, Prague, Czech Republic

    Stanislav Micuda & Eva Brcakova

Authors
  1. Lenka Bruckova
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  2. Tomas Soukup
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  3. Benjamin Visek
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Corresponding author

Correspondence to Tomas Soukup.

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Capsule

This paper is especially focused on the improvement of the cultivation protocol enabling granulosa cells to proliferate and maintain the typical phenotypic expression.

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Bruckova, L., Soukup, T., Visek, B. et al. Proliferative potential and phenotypic analysis of long-term cultivated human granulosa cells initiated by addition of follicular fluid. J Assist Reprod Genet 28, 939–950 (2011). https://doi.org/10.1007/s10815-011-9617-6

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  • DOI: https://doi.org/10.1007/s10815-011-9617-6

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