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

Abstract

Background3D 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 FindingsWe 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.ConclusionsBecause 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.

Morphometrical, Morphological, and Immunocytochemical Characterization of a Tool for Cytotoxicity Research: 3D Cultures of Breast Cell Lines Grown in Ultra-Low Attachment Plates

“…Compared to the typical schematic portrayal of 3D cultures, these changes in the distribution of caspase-3 and Ki67 positive cells may be attributed to the ellipsoid form of the MCAs. However, a similar random distribution of Ki67 positive cells was observed in MCF7 [ 22 ] (≈300–400 µm of diameter) and MDA-MB-231 spheroids (≈500–600 µm) [ 94 ], and other small spheroids from breast cell lines (≈300 µm) [ 95 ]. Thus, according to our results, the classical three-layered spheroid structure is too simplistic and does not represent the real structure of all the 3D cell cultures.…”

Analysis of Anticancer Activity and Chemical Sensitization Effects of Dendropanax morbifera and Commersonia bartramia Extracts

“…A 3D culture is more similar to the in vivo environment than a 2D culture, and therefore can yield results that are closer to the drug responses seen in clinical trials (30). 3D-HDRA is a type of 3D culture that is being used to determine the efficacy of chemotherapeutic agents in individuals by identifying their chemical sensitization profiles (31).…”

Advances in the development of improved animal-free models for use in breast cancer biomedical research

“…The availability of reconstituted basement membrane, Matrigel™, allowed 3D culture of normal and tumourous human breast cell lines (Wang et al 2002 ; Debnath et al 2003 ; Ivascu and Kubbies 2007 ) and, in the case of normal, MCF-10A mammary epithelial cells, formed acini-like spheroids that recapitulated facets of the native mammary gland (Debnath et al 2003 ). Collagen matrix was also adopted as a means of offering greater physiological relevance (Holliday et al 2009 ; Roberts et al 2016 ), and a range of natural and synthetic matrices have since been used for 3D culture of breast cancer cells (Bissell and Bilder 2003 ; Lee et al 2007 ; Russ et al 2012 ; Nash et al 2015 ). Other techniques include the liquid overlay technique (Ivascu and Kubbies 2007 ) and hanging drop method (Nagelkerke et al 2013 ).…”

“…Other techniques include the liquid overlay technique (Ivascu and Kubbies 2007 ) and hanging drop method (Nagelkerke et al 2013 ). More recently, our group has used a fully humanised cell culture medium, which encouraged spheroid formation in the absence of supporting matrix (Roberts et al 2016 ).…”

“…Nevertheless, spheroids do have their limitations. Different cell types have varying abilities to form spheroids; for example, the BT-474 HER2 overexpressing cell line forms tightly packed rounded spheroids, whereas the SKBR3 HER2 overexpressing cell line forms loose, grape-like aggregates (Froehlich et al 2016 ; Roberts et al 2016 ). Also, it can be challenging to control the size of spheroids formed and, therefore, the reproducibility of experiments for high-throughput drug screening is limited.…”