黑色素瘤干细胞球体形成测定

Alessandra  Tuccitto; Valeria  Beretta; Francesca  Rini; Chiara Castelli; Michela  Perego

doi:10.21769/BioProtoc.2233

Improve Research Reproducibility A Bio-protocol resource

提交稿件
订阅
CN
- EN - English
- CN - 中文

Peer-reviewed

Melanoma Stem Cell Sphere Formation Assay

黑色素瘤干细胞球体形成测定

Alessandra Tuccitto

VB Valeria Beretta

FR Francesca Rini

CC Chiara Castelli email

MP Michela Perego email

发布: 2017年04月20日第7卷第8期 DOI: 10.21769/BioProtoc.2233 浏览次数: 14259

评审: Anonymous reviewer(s)

PDF

Q&A

引用

Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Stem Cells, featuring study using the protocol.

Oct 2016

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

相关实验方案

利用极端有限稀释分析法（ELDA）检测神经干细胞和胶质瘤干细胞的自我更新能力

Hong PT Nguyen [...] Theo Mantamadiotis

2018年09月05日 10676 阅读

来自骨髓增生性肿瘤患者的造血祖细胞的血小板生成素不依赖性巨核细胞分化

Chloe A. L. Thompson-Peach [...] Daniel Thomas

2023年01月20日 1461 阅读

基于外植体培养法从宫颈癌原发瘤中分离并鉴定癌相关间充质干细胞

Surbhi Singla [...] Shalmoli Bhattacharyya

2025年06月20日 1844 阅读

Abstract

Self-renewal is the ability of cells to replicate themselves at every cell cycle. Throughout self-renewal in normal tissue homeostasis, stem cell number is maintained constant throughout life. Cancer stem cells (CSCs) share this ability with normal tissue stem cells and the sphere formation assay (SFA) is the gold standard assay to assess stem cells (or cancer stem cells) self-renewal potential in vitro. When single cells are plated at low density in stem cell culture medium, only the cells endowed with self-renewal are able to grow in tridimensional clusters usually named spheres. In the recent years, SFA has also been used also to test the effect of several drugs, chemical and natural compounds or microenviromental components on stem cells self-renewal capacity. Here we will illustrate a detailed protocol to assess self-renewal of human melanoma stem cells, growing as melanospheres.

Keywords: Melanoma (黑色素瘤)

Cancer stem cells (癌症干细胞)

Melanospheres (黑色素瘤细胞球)

Self-renewal (自我更新)

Stem cell medium (干细胞培养基)

Background

Cancer stem cells (CSCs) were first found in acute myeloma leukemia (Lapidot et al., 1994) and then were identified in many solid tumors including melanoma. CSCs are defined as cells strongly endowed with self-renewal and tumor initiating capacity, being able to regenerate the whole tumor heterogeneity in vivo. CSCs can be isolated from the tumor mass with different approaches based on phenotypic characteristic or biological properties, then their properties have to be tested in vitro (self-renewal) and in vivo (tumorigenic potential). Melanoma CSCs were isolated using a combination of cell surface markers, (Fang et al., 2005; Monzani et al., 2007; Schatton et al., 2008; Boiko et al., 2010; Boonyaratanakornkit et al., 2010) or through culture in specific stem cell media (Perego et al., 2010; Santini et al., 2012). To validate melanoma CSC self-renewal, and to study the effect of tumor microenvironmental factors on it (Tuccitto et al., 2016), we used the sphere formation assay (SFA) in vitro. Melanoma CSCs are plated at low density in stem cell culture medium and they grow in anchorage-independent, three-dimensional spherical structures, called melanospheres (tumorspheres, in general). Spheres forming efficiency is directly proportional to the number of melanoma CSCs present in the culture (one CSC corresponds to one melanosphere), thus giving a direct quantification of CSC amount in culture. This relatively simple method is useful to study the ability of any exogenous factors (growth factors, cytokines and chemokines, drugs) in perturbing CSC self-renewal (Tsuyada et al., 2012; Tuccitto et al., 2016). Here we provide detailed information about the SFA protocol we optimized in our laboratory for melanoma SFA.

Materials and Reagents

Cell culture flask, area 150 cm² (Corning, catalog number: 430823 )
15 ml Falcon tube (Greiner Bio One International, catalog number: 188261 )
Micropipette P200 tips (Corning, catalog number: 4823 )
24-wells plates flat bottom (Corning, Costar^®, catalog number: 3527 )
70 μm cell strainer (Corning, Falcon^®, catalog number: 352350 )
15 ml polystyrene serological pipets (Corning, Falcon^®, catalog number: 357551 )
Melanospheres are obtained as described by Perego et al., starting from cell suspension obtained from melanoma surgical specimens or from previously established melanoma cell lines after culturing in stem cell medium (SCM) (Perego et al., 2010)
RPMI
10% FBS
Trypan blue solution, 0.4% (Sigma-Aldrich, catalog number: T8154 )
SCM (see Recipes)

DMEM:F-12, 1:1 mixture’ (Lonza, catalog number: BE12-719F )
Epidermal growth factor (EGF) (PeproTech, catalog number: AF-100-15 )
Basic fibroblast growth factor (bFGF) (PeproTech, catalog number: 100-18B )
D-glucose (Sigma-Aldrich, catalog number: G7021 )
Insulin (Sigma-Aldrich, catalog number: I6634 )
Putrescine dihydrochloride (Sigma-Aldrich, catalog number: P5780 )
Sodium selenite (Sigma-Aldrich, catalog number: S9133 )
Progesterone (Sigma-Aldrich, catalog number: P6149 )
Transferrin (Sigma-Aldrich, catalog number: T8158 )
Sodium bicarbonate (NaHCO₃) (Sigma-Aldrich, catalog number: S8761 )
1x phosphate buffered saline (PBS) (Lonza, catalog number: 17-516 )
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A1933 )
HEPES buffer (Lonza, catalog number: BE17-737 )
L-glutamine (Lonza, catalog number: BE17-605E )
Penicillin-streptomycin (Lonza, catalog number: 17-602E )

Equipment

Automatic pipettor (PBI, catalog number: 857075 )
Tissue culture incubator with CO₂ input (Thermo Fisher Scientific, Thermo Scientific^TM, model: Forma^TM Series II 3110 )
Centrifuge (Eppendorf, catalog number: 5810 )
Hemocytometer (Marienfeld-Superior, Bürker, catalog number: 0640211 )
Optical microscope (Carl Zeiss, model: Axiovert 25 )

Procedure

English

中文翻译

文章信息

版权信息

如何引用

Tuccitto, A., Beretta, V., Rini, F., Castelli, C. and Perego, M. (2017). Melanoma Stem Cell Sphere Formation Assay. Bio-protocol 7(8): e2233. DOI: 10.21769/BioProtoc.2233.