Dataset Information

An Evaluation of Matrix-Containing and Humanised Matrix-Free 3-Dimensional Cell Culture Systems for Studying Breast Cancer.

ABSTRACT:

Background

3D cell cultures are emerging as more physiologically meaningful alternatives to monolayer cultures for many biological applications. They are attractive because they more closely mimic in vivo morphology, especially when co-cultured with stromal fibroblasts.

Methodology/principal findings

We compared the efficacy of 3 different 3D cell culture systems; collagen I, low attachment culture vessels and a modification of Fibrolife®, a specialised humanised cell culture medium devoid of animal-derived components, using breast cancer cell lines representative of the different molecular subtypes of breast cancer, cultured alone or with human mammary fibroblasts with a view to developing matrix-free humanised systems. 3D collagen I culture supported the growth of a range of breast cancer cell lines. By modifying the composition of Fibrolife® to epiFL, matrix-free cell culture was possible. During sequential transfer to epiFL breast cancer cells gradually detached from the flask, growing progressively as spheroids. Phenotype was stable and reversible with cells remaining actively proliferating and easily accessible throughout culture. They could also be revived from frozen stocks. To achieve co-culture with fibroblasts in epiFL required use of low attachment culture vessels instead of standard plastic as fibroblasts remained adherent in epiFL. Here, cancer cell spheroids were allowed to form before adding fibroblasts. Immunohistochemical examination showed fibroblasts scattered throughout the epithelial spheroid, not dissimilar to the relationship of tumour stroma in human breast cancer.

Conclusions

Because of its ease of handling, matrix-free 3D cell culture may be a useful model to study the influence of fibroblasts on breast cancer epithelial cells with use of epiFL culture medium taking this a step further towards a fully humanised 3D model. This methodology could be applied to other types of cancer cell lines, making this a versatile technique for cancer researchers wishing to use in vitro systems that better reflect cancer in vivo.

SUBMITTER: Roberts GC

PROVIDER: S-EPMC4907459 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Publications

An Evaluation of Matrix-Containing and Humanised Matrix-Free 3-Dimensional Cell Culture Systems for Studying Breast Cancer.

Roberts Grace C GC Morris Paul G PG Moss Marcus A MA Maltby Sarah L SL Palmer Chelsea A CA Nash Claire E CE Smart Emily E Holliday Deborah L DL Speirs Valerie V

PloS one 20160614 6

<h4>Background</h4>3D cell cultures are emerging as more physiologically meaningful alternatives to monolayer cultures for many biological applications. They are attractive because they more closely mimic in vivo morphology, especially when co-cultured with stromal fibroblasts.<h4>Methodology/principal findings</h4>We compared the efficacy of 3 different 3D cell culture systems; collagen I, low attachment culture vessels and a modification of Fibrolife®, a specialised humanised cell culture medi ...[more]

PMID: 27300768

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Project description:Disrupted/disordered ovarian steroidogenesis is associated with several fertility disorders such as polycystic ovary syndrome in humans and cystic ovarian disease in cattle. Methods to interrogate theca cell processes as part of follicular development are necessary to further research into treatments for these types of disorders. Multilayer follicles of dairy-breed cows were placed into culture in a novel matrix-free 3D system using round bottom low-attachment plates. Follicles were first cultured in the presence of two types of media previously used for in vitro follicle maturation (basal α-MEM and basal T-199). After the optimal media was identified, impact of supplementation of epidermal growth factor (EGF) on growth and survival of bovine secondary follicles to antral stage was evaluated. No differences were observed in growth and survival of follicles cultured in basal α-MEM media or basal T-199 media, although T-199 media's high phenol red content made assessment of follicles difficult. Further studies were then performed with α-MEM media. Three cohorts of follicles were observed based on time to antrum formation: ≤ 5 days (fast), 6-19 days (slow), or survived but did not form an antrum by 21 days (no). Supplementation of EGF to the basal α-MEM media dramatically improved follicle survival rates in culture (defined as follicles that either formed an antrum or did not form an antrum but did not die during 21 day culture period) from 29% to 95.7% (Chi-square p < 0.0001). However, in follicles that survived to form an antrum there were no differences in proportion of fast, slow and no antrum follicles after addition of EGF (Chi-square p > 0.7). Fast antrum follicles treated with EGF plateaued in size earlier in culture compared to controls (p = 0.013). Slow and no antrum follicles were larger in diameter during EGF culture than controls (p's < 0.0001). Many follicles cultured in this matrix-free system that formed an antrum approached 1.5-2 mm in size, an improvement from previous single follicle culture methods used for bovine pre-antral follicles in vitro. In addition, follicles displayed functional steroidogenesis in vitro producing measureable levels of estradiol and androstenedione. This matrix-free 3D culture system provides an excellent in vitro model to explore processes associated with folliculogenesis in cattle.

Dataset Information

An Evaluation of Matrix-Containing and Humanised Matrix-Free 3-Dimensional Cell Culture Systems for Studying Breast Cancer.

Background

Methodology/principal findings

Conclusions

Publications

An Evaluation of Matrix-Containing and Humanised Matrix-Free 3-Dimensional Cell Culture Systems for Studying Breast Cancer.

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