3D 乳腺集落形成细胞培养方法

Giusy  Tornillo; Sara  Cabodi

doi:10.21769/BioProtoc.1087

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Peer-reviewed

3D Mammary Colony-Forming Cell Assay

3D 乳腺集落形成细胞培养方法

Giusy Tornillo email

Sara Cabodi

发布: 2014年04月05日第4卷第7期 DOI: 10.21769/BioProtoc.1087 浏览次数: 16612

评审: Vanesa Olivares-IllanaAnonymous reviewer(s)

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参见作者原研究论文

The authors used this protocol in:

Cover of Stem Cells, featuring study using the protocol.

Jul 2013

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Abstract

The mammary epithelium consists of multiple phenotypically and functionally distinct cell populations, which are organized as a hierarchy of stem cells, progenitors and terminally differentiated cells.

Identification of the mechanisms regulating the growth and differentiation of mammary stem and progenitor cells is of great interest not only to better understand the mammary gland development but also to clarify the origins of breast cancer, as these cells seem to be the likely targets of malignant transformation within the mammary epithelium. Hence, a variety of approaches have been developed for quantifying and studying these specific mammary cell subsets.

Given their high proliferative capacity, mammary progenitor cells are able to form colonies in vitro in low-density cultures. Here we describe how to perform a three dimensional (3D) Mammary Colony-Forming Cell (Ma-CFC) Assay, an in vitro functional assay suitable for the detection and analysis of mammary progenitor cells in feeder-free culture conditions.

Briefly, this protocol involves the seeding of mammary single cells, at clonal density, onto a semi-solid matrix (Matrigel), thus allowing mammary progenitors to proliferate and give rise to discrete 3D colonies. The number and the cell composition of the resulting colonies will vary according to the frequency and the differentiation potential of the progenitors, respectively.

Keywords: Mammary (乳腺)

Materials and Reagents

Single-cell suspension of primary mouse mammary cells [see Debnath et al. (2003) for details about the dissociation of mouse mammary glands into single cells]
EpiCult^®-B Basal Medium Mouse (STEMCELL Technologies, catalog number: 05611 )
EpiCult^®-B Proliferation Supplements (STEMCELL Technologies, catalog number: 05612 )
Trypan blue
Recombinant human Epidermal Growth Factor (EGF) (Sigma-Aldrich, catalog number: E9644 )
Recombinant human basic Fibroblast Growth Factor (bFGF) (Life Technologies, catalog number: PHG0023 )
Heparin sodium salt (Sigma-Aldrich, catalog number: H3149 )
Fetal Bovine Serum (FBS) (heat-inactivated) (Life Technologies, catalog number: 10270-106 )
Penicillin-Streptomycin (Pen/Strep) (Life Technologies, catalog number: 15070-063 )
Growth Factor Reduced (GFR) BD Matrigel^TM Matrix [protein concentration > 8 mg/ml, endotoxin levels < 2 Endotoxin Units (EU)/ml] (BD Biosciences, catalog number: 354230 )
Note: BD Matrigel^TM Matrix is supplied as a frozen solution. Thaw it on ice overnight and stored as 1 ml aliquots at -20 °C. Leave Matrigel aliquots to thaw on ice for 2 h before use.
Complete EpiCult-B Medium (see Recipes)