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Isolation and culture of ovarian tumour cells, cytological and cell survival evaluation
Umeå University, Faculty of Medicine, Department of Clinical Sciences, Obstetrics and Gynaecology.
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Histology and Cell Biology.
Umeå University, Faculty of Medicine, Department of Clinical Sciences, Obstetrics and Gynaecology.
Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology.
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1999 (English)In: Anticancer Research, ISSN 0250-7005, Vol. 19, no 6B, 5069-5073 p.Article in journal (Refereed) Published
Abstract [en]

The purpose of this study was to evaluate the reproducibility and reliability of the fluorometric microculture cytotoxicity assay (FMCA). Emphasis was placed on obtaining pure tumour cell cultures which were subjected to careful cytological evaluation. Preparations of 39 ovarian tumours, malignant, borderline and benign were made, of which 37 were successfully cultured. In 34 of the 37 tumour cell cultures, the epithelial cell fraction was > 90%, and in 30 of 39 cultures the epithelial cell fraction was > 95%. Transportation within 24 h and the 72 h incubation did not change the yield or epithelial cell fraction. There was a linear relationship between fluorescence and the number of viable cells. The fluorescence increased with time, making only comparisons within each assay plate possible. The sensitivity of the method makes it possible to perform many analyses on a small amount of material. The method also makes it possible to study cells derived from all stages of the disease, including benign tumours.

Place, publisher, year, edition, pages
1999. Vol. 19, no 6B, 5069-5073 p.
URN: urn:nbn:se:umu:diva-44794PubMedID: 10697512OAI: diva2:421936
Available from: 2011-06-10 Created: 2011-06-10 Last updated: 2011-06-10Bibliographically approved
In thesis
1. Ovarian tumours, cell isolation, cell survival and sex steroids
Open this publication in new window or tab >>Ovarian tumours, cell isolation, cell survival and sex steroids
2002 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Background: Ovarian cancer is the most deadly malignancy of the female reproductive tract. Ovarian epithelial tumours are considered to be endocrine related, but the factors that govern their genesis and progression remain unclear. Radical surgery and recent advances in chemotherapy have only moderately improved survival rates. Biological and endocrine approaches may help to satisfy the pressing need for alternative therapies.

Aims: The aims of the studies included in this thesis were to isolate and culture ovarian tumour cells with a high epithelial cell fraction (ECF) and to evaluate the fluorometric microculture cytotoxity assay (FMCA) as a tool in the development of alternative therapies. We also aimed to measure the effects of bispecific antibodies (BsAbs) directed against three ovarian tumour markers, separately and in combination, and to study the effects of sex steroids on tumour cell survival, on autonomous steroid secretion and on the expression of estrogen (ER) and progesterone receptors (PR).

Methods: Tumour cells from a total of 75 patient were isolated and cultured in the experiment described by the four papers. The cells were incubated in the presence of BsAbs and peripheral blood mononuclear cells (PBMCs), and at varying concentrations of sex steroids. The FMCA and the neutral red cytotoxity assay were used to measure tumour cell survival. The time-resolved fluoroimmunoassay technique - DELFIA was used to measure steroid concentrations and immunohistochemistry was used to detect tumour markers, ER and PR.

Results and conclusions: It was shown that it is possible to isolate tumour cells from benign as well as malignant tumours, and that the fraction of malignant and atypical cells from carcinoma and borderline tumours can be determined in the cell preparation. Reliable predictive tests such as the FMCA performed on primary cell cultures can make it possible to create individual patinet profiles, thus optimising drug treatment.

Analysis of the BsAbs cultures showed that a combination of the three BsAbs used in our study was the most efficient in tumour cell elimination. In some cases however, the effect of one single BsAb was better than the combination treatment, and this suggests that an optimal therapeutic approach based on BsAbs in a clinical setting requires a panel of BsAbs directed against the commonly expressed ovarian tumour markers.

Tumour cells cultured in 17 β-estradiol and testosterone showed a reduced cell survival and this reduction was inversely proportional to hormone concentrations within the range studied. It was found that 17 β-estradiol was secreted from the primary cell cultures and, interestingly, the amount of 17 β-estradiol secreted increased with increasing levels of 17 β-estradiol in the environment. The majority of the isolated cells analysed for ER and PR expressed both of these receptors to some degree, i.e. the combination ER+/PR+ was the most common. Both ER and PR expression decreased after 72 h culture, revealing an unexpectedly dynamic system. Lowering levels of 17 β-estradiol in cell cultures reduced cell survival, but it appears that this observation depends on factors other than ER. The survival rates of cells cultured in progesterone seemed to be inversely related to their PR expression. It is believed that 17 β-estradiol has an antiapoptotic effect on ovarian surface epithelial (OSE) cells. Reduction of 17 β-estradiol in the environment may inhibit this effect, resulting in reduced cell survival. The ovarian epithelial tumour cell's ability to secrete 17 β-estradiol  suggests that epithelial ovarian tumours play an active role in altering their own hormonal environment, promoting tumour progression.

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2002. 54 p.
Umeå University medical dissertations, ISSN 0346-6612 ; 799
National Category
Obstetrics, Gynecology and Reproductive Medicine
Research subject
Obstetrics and Gynaecology
urn:nbn:se:umu:diva-44796 (URN)91-7305-271-X (ISBN)
Public defence
2002-09-13, Hörsal E04, byggnad 6E, Norrlands universitetssjukhus, Umeå, 09:00 (English)
Available from: 2011-06-10 Created: 2011-06-10 Last updated: 2011-06-10Bibliographically approved

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