Human ovarian granulosa cell culture: determination of blood cell contamination and evaluation of possible culture purification steps

Beckmann, Matthias W.; Polacek, Denise C.; Seung, Lisa P.; Schreiber, James R.

doi:10.1016/s0015-0282(16)54659-7

Cited by 49 publications

(25 citation statements)

References 21 publications

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“…In order to investigate the secretion of these substances in an in vitro study, granulosa cells were separated from FF by a slightly modified procedure that was previously described for IVF-ET [16,17]. Briefly, in order to remove the erythrocytes, the cell suspension was gently treated with lymphocyte separation medium (LSM), then centrifuged at 3000 rpm for 20 min.…”

Section: Granulosa Cell Culturementioning

confidence: 99%

The clinical role of interleukin-6 and interleukin-6 soluble receptor in human follicular fluids

Kawasaki¹,

Kawano²,

Hasan³

et al. 2003

Clinical and Experimental Medicine

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In order to investigate the role of interleukin-6 (IL-6) and interleukin-6 soluble receptor (sR) in human ovulation, we evaluated the concentrations in human follicular fluid and analyzed the correlation of IL-6 and IL-6 sR with oocyte maturation. The oocytes were obtained from the follicular fluid of 45 women undergoing in vitro fertilization and embryo transfer. The concentrations of IL-6 and IL-6 sR in follicular fluid were measured by ELISA. In addition, granulosa cells obtained from the follicular fluid were cultured and treated with forskolin and 12- o-tetradecanoylphorbol 13-acetate for 24-48 h. The concentration of IL-6 was significantly higher in the follicular fluid than in the serum (P<0.01). In contrast, the concentration of IL-6 sR was significantly lower in the follicular fluid than in the serum (P<0.001). The concentrations of IL-6 and IL-6 sR were significantly higher in the follicular fluid containing mature oocytes than in fluid containing immature oocytes (P<0.05). The production of IL-6 was markedly increased over the basal level after 24 h of treatment with forskolin (P<0.001) and 48 h of treatment (P<0.01) with cultured granulosa cells. Our data suggest that IL-6 and IL-6 sR may play an important role in follicular growth and development in human preovulatory processes. It is possible that IL-6 in particular may be regulated by cAMP. IL-6 and IL-6 sR might also be valuable biochemical markers in the evaluation of oocyte maturation.

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Section: Granulosa Cell Culturementioning

confidence: 99%

The clinical role of interleukin-6 and interleukin-6 soluble receptor in human follicular fluids

Kawasaki¹,

Kawano²,

Hasan³

et al. 2003

Clinical and Experimental Medicine

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“…In working with granulosa-luteal cell cultures, one must contend with a modest amount of cell contamination, as absolutely pure preparations are unobtainable (9). Granulosa-luteal cell cultures prepared by either of the methods discussed contain a small percentage of other nucleated cells such as leukocytes and resident ovarian macrophages.…”

Section: Discussionmentioning

confidence: 99%

“…This method varies slightly from lab to lab but in general it involves an initial 300-500 × g centrifugation for up to 15-20 min to concentrate the cells. The resuspended cell pellet is then gently layered over a 40-50% Percoll cushion (4)(5)(6) or similarly effective concentrations of Ficoll (7)(8)(9) or in some laboratories, a two-step 40 and 60% discontinuous Percoll gradient is used (10). This is followed by centrifugation at generally around 500 × g for 20 min.…”

Section: Introductionmentioning

confidence: 99%

Ontario, Canada

Lobb

Younglai²

2006

J Assist Reprod Genet

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“…A high rate of leukocyte contamination has been described in primary cultures of lutein cells enriched by the density gradients Ficoll™ and Percoll™ (5,6). These density gradient media are not totally inert and have a potential to elicit the cellular activation in leukocytes during separation procedure (15).…”

Section: The Cultivation Of Human Granulosa Cellsmentioning

confidence: 99%

The Cultivation of Human Granulosa Cells

Brůčková

Soukup

Moos

et al. 2008

Acta Med. (Hradec Kralove, Czech Repub.)

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Summary:The major functions of granulosa cells (GCs) include the production of steroids, as well as a myriad of growth factors to interact with the oocyte during its development within the ovarian follicle. Also FSH stimulates GCs to convert androgens (coming from the thecal cells) to estradiol by aromatase. However, after ovulation the GCs produce progesterone that may maintain a potential pregnancy. Experiments with human GCs are mainly focused on the purification of GCs from ovarian follicular fluid followed by FACS analysis or short-term cultivation. The aim of our study was to cultivate GCs for a long period, to characterize their morphology and phenotype. Moreover, we have cultivated GCs under gonadotropin stimulation in order to simulate different pathological mechanisms during folliculogenesis (e.g. ovarian hyperstimulation syndrome). GCs were harvested from women undergoing in vitro fertilization. Complex oocyte-cumulus oophorus was dissociated by hyaluronidase. The best condition for transport of GCs was optimized as short transport in follicular fluid at 37 °C. GCs expansion medium consisted of DMEM/F12, 2 % FCS, ascorbic acid, dexamethasone, L-glutamine, gentamycine, penicillin, streptomycin and growth factors (EGF, bFGF). GCs transported in follicular fluid and cultivated in 2 % FCS containing DMEM/F12 medium supplemented with follicular fluid presented increased adhesion, proliferation, viability and decreased doubling time. Cell viability was 92 % and mean cell doubling time was 52 hrs. We have optimized transport and cultivation protocols for long-term cultivation of GCs.

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Human ovarian granulosa cell culture: determination of blood cell contamination and evaluation of possible culture purification steps

Cited by 49 publications

References 21 publications

The clinical role of interleukin-6 and interleukin-6 soluble receptor in human follicular fluids

The clinical role of interleukin-6 and interleukin-6 soluble receptor in human follicular fluids

Ontario, Canada

The Cultivation of Human Granulosa Cells

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