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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.

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3D Mammary Colony-Forming Cell Assay
3D 乳腺集落形成细胞培养方法

干细胞 > 成体干细胞 > 维持和分化
作者: Giusy Tornillo
Giusy TornilloAffiliation: Cardiff School of Biosciences, European Cancer Stem Cell Research Institute, Cardiff University, Cardiff, UK
For correspondence: tornillog@cardiff.ac.uk
Bio-protocol author page: a1281
 and Sara Cabodi
Sara CabodiAffiliation: Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy
Bio-protocol author page: a1282
Vol 4, Iss 7, 4/5/2014, 5290 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1087

[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), Progenitor(祖), 3D(3d), Matrigel(Matrigel), Colony(殖民地)

[Abstract]

Materials and Reagents

  1. 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]
  2. EpiCult®-B Basal Medium Mouse (STEMCELL Technologies, catalog number: 05611 )
  3. EpiCult®-B Proliferation Supplements (STEMCELL Technologies, catalog number: 05612 )
  4. Trypan blue
  5. Recombinant human Epidermal Growth Factor (EGF) (Sigma-Aldrich, catalog number: E9644 )
  6. Recombinant human basic Fibroblast Growth Factor (bFGF) (Life Technologies, catalog number: PHG0023 )
  7. Heparin sodium salt (Sigma-Aldrich, catalog number: H3149 )
  8. Fetal Bovine Serum (FBS) (heat-inactivated) (Life Technologies, catalog number: 10270-106 )
  9. Penicillin-Streptomycin (Pen/Strep) (Life Technologies, catalog number: 15070-063 )
  10. Growth Factor Reduced (GFR) BD MatrigelTM Matrix [protein concentration > 8 mg/ml, endotoxin levels < 2 Endotoxin Units (EU)/ml] (BD Biosciences, catalog number: 354230 )
    Note: BD MatrigelTM 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.
  11. Complete EpiCult-B Medium (see Recipes)

Equipment

  1. BD Falcon 8-well culture slides (BD Biosciences, catalog number: 354108 )
  2. Refrigerated centrifuge with swinging bucket rotor
  3. Humidified 37 °C, 5% CO2 cell culture incubator
  4. Inverted tissue culture microscope
  5. Zeiss Observer Z.1 microscope (5x/0.12 objective)

Software

  1. AxioVision Rel 4.8 software
  2. ImageJ

Procedure

  1. The 8-well chamber slide is placed on ice and loaded with 80 µl of Matrigel per well. The Matrigel drop is positioned in the centre of each well, taking care to avoid air-bubble formation.
    Note: To prevent gelation, it is recommended to handle Matrigel on ice.
  2. Next, the chamber slide is secured onto a pre-cooled adapter for swing-bucket rotor and centrifuged at 300 x g (with a slow acceleration ramp) for 10 min at 4 °C. The Matrigel layer is now flat and uniformly distributed onto the bottom of the chamber wells.
    Note: It is convenient to use some plastic paraffin film to keep the lid of the culture slide in place during step 2.
  3. The chamber slide is then transferred to a cell culture incubator to allow Matrigel solidification for at least 15 min.
  4. Meanwhile, freshly prepared single mouse mammary cells are resuspended in Complete EpiCult-B Medium and counted.
    Note: Trypan Blue stain is one of several methods recommended for viable cell counting.
  5. The cell suspension is diluted in Complete EpiCult-B Medium to obtain a final concentration of 25,000 viable cells/ml.
  6. A stock of Complete EpiCult-B Medium supplemented with 5% Matrigel is prepared by adding 50 µl of Matrigel per ml of Complete EpiCult-B Medium and mixing by pipetting repeatedly.
  7. The cell suspension from step 5 is mixed with the medium from step 6 in a 1:1 ratio. 400 µl of this mixture, which correspond to 5,000 cells in Complete EpiCult-B Medium 2.5% Matrigel, are gently plated per well onto the solidified Matrigel from step 3 (Figure 1).
  8. The chamber slide is placed in the cell culture incubator. The cells will settle onto the solid Matrigel layer.
  9. The overlay liquid medium is replaced with fresh Complete EpiCult-B Medium supplemented with 2.5% Matrigel every 3 days. To avoid damaging the bottom gel layer, the medium is aspirated from the corner of each well and the fresh medium is gently dispensed along the walls.
  10. Colonies will be generated after 10-14 days of culture. Representative images are shown in Figure 2.
  11. The number of colonies can be determined, using an inverted microscope, either by manual counting or with the support of image analysis programs, such as Image J software.


    Figure 1. Schematic diagram depicting the procedure described above as step 7


    Figure 2. 3D mammary colony-forming cell assay. Phase-contrast image of colonies derived from 3D culture (day 10) of mouse mammary single cells. This image was obtained by using a Zeiss Observer Z.1 microscope (5x/0.12 objective) and the AxioVision Rel 4.8 software. Scale bar, 100 µm

Recipes

  1. Complete EpiCult-B Medium (50 ml)
    EpiCult-B Basal Medium: 42 ml
    EpiCult®-B Proliferation Supplements: 5 ml
    FBS: 2.5 ml
    Pen/Strep: 0.5 ml
    EGF (0.2 mg/ml): 2.5 µl
    bFGF (0.2 mg/ml): 2.5 µl
    Heparin (50 mg/ml): 4 µl
    Filter-sterilize through a 0.2 µm filter
    Stored at 4 °C and use within one week
    Complete EpiCult-B Medium must be supplemented with Matrigel immediately before use

Acknowledgments

We thank Dr Agata Tinnirello and Prof Senthil Muthuswamy for technical advice on the 3D overlay cell-culture method (previously described by Debnath et al. (2003). This work was funded by AIRC (Grant IG2008 and IG2011), MIUR (FIRB Giovani 2008), PiSTEM and Progetto Sanità Finalizzata. G. Tornillo was supported by a FIRC fellowship.

References

  1. Debnath, J., Muthuswamy, S. K. and Brugge, J. S. (2003). Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. Methods 30(3): 256-268.
  2. Tornillo, G., Elia, A. R., Castellano, I., Spadaro, M., Bernabei, P., Bisaro, B., Camacho-Leal Mdel, P., Pincini, A., Provero, P., Sapino, A., Turco, E., Defilippi, P. and Cabodi, S. (2013). p130Cas alters the differentiation potential of mammary luminal progenitors by deregulating c-Kit activity. Stem Cells 31(7): 1422-1433.

材料和试剂

  1. 原代小鼠乳腺细胞的单细胞悬浮[参见Debnath等人(2003)关于小鼠乳腺解离成单个细胞的细节]
  2. EpiCult -B Basal Medium Mouse(STEMCELL Technologies,目录号:05611)
  3. EpiCult -B增殖补充剂(STEMCELL Technologies,目录号:05612)
  4. 台盼蓝
  5. 重组人表皮生长因子(EGF)(Sigma-Aldrich,目录号:E9644)
  6. 重组人碱性成纤维细胞生长因子(bFGF)(Life Technologies,目录号:PHG0023)
  7. 肝素钠盐(Sigma-Aldrich,目录号:H3149)
  8. 胎牛血清(FBS)(热灭活)(Life Technologies,目录号:10270-106)
  9. 青霉素 - 链霉素(Pen/Strep)(Life Technologies,目录号:15070-063)
  10. 生长因子减少(GFR)BD Matrigel TM 基质[蛋白质浓度> 8mg/ml,内毒素水平< 2内毒素单位(EU)/ml](BD Biosciences,目录号:354230) Matrix是以冻结的方式提供的。 解冻 在冰上过夜,并以1ml等分试样在-20℃下储存。 离开 Matrigel等分试样在冰上解冻2小时后使用。
  11. 完成EpiCult-B Medium(见配方)

设备

  1. BD Falcon 8孔培养载玻片(BD Biosciences,目录号:354108)
  2. 带旋转叶轮的冷冻离心机
  3. 加湿37℃,5%CO 2细胞培养箱
  4. 倒置组织培养显微镜
  5. Zeiss Observer Z.1显微镜(5x/0.12目标)

软件

  1. AxioVision Rel 4.8软件
  2. ImageJ

程序

  1. 将8孔腔室载玻片置于冰上,并每孔加载80μlMatrigel。将基质胶滴定位在每个孔的中心,小心避免气泡形成。
    注意:为了防止凝胶化,建议在冰上处理Matrigel。
  2. 接下来,将室滑板固定到用于回转桶式转子的预冷却适配器上,并且在4℃下以300×g离心(具有缓慢的加速斜坡)10分钟。 Matrigel层现在是平的并且均匀地分布在腔室的底部 注意:在步骤2中使用一些塑料石蜡膜保持培养皿的盖子很方便。
  3. 然后将室玻片转移至细胞培养培养箱中,以使基质胶固化至少15分钟。
  4. 同时,将新鲜制备的单小鼠乳腺细胞重悬于Complete EpiCult-B培养基中并计数。
    注意:台盼蓝染色是推荐用于活细胞计数的几种方法之一。
  5. 将细胞悬浮液在Complete EpiCult-B培养基中稀释,以获得25,000个活细胞/ml的最终浓度。
  6. 通过加入50μlMatrigel/ml完全EpiCult-B培养基并通过重复吹打混合来制备补充有5%Matrigel的Complete EpiCult-B培养基的储备物。
  7. 将来自步骤5的细胞悬浮液与来自步骤6的培养基以1:1的比例混合。将400μl的该混合物(其相当于Complete EpiCult-B Medium2.5%Matrigel中的5,000个细胞)逐步地平铺在来自步骤3(图1)的固化基质胶上。
  8. 将室玻片置于细胞培养箱中。细胞将沉积在固体Matrigel层上。
  9. 将覆盖液体培养基用每3天补充2.5%Matrigel的新鲜Complete EpiCult-B培养基替换。为了避免损坏底部凝胶层,从每个孔的角落吸出培养基,并沿着壁轻轻地分配新鲜培养基。
  10. 菌落将在培养10-14天后产生。代表图像如图2所示。
  11. 可以使用倒置显微镜,通过手动计数或者利用图像分析程序(例如Image J软件)的支持来确定集落数目。


    图1.描述上述步骤7
    的示意图

    图2. 3D乳腺集落形成细胞测定。来自小鼠乳腺单细胞的3D培养物(第10天)的集落的相差图像。 该图像通过使用Zeiss Observer Z.1显微镜(5x/0.12物镜)和AxioVision Rel 4.8软件获得。 比例尺,100μm,

食谱

  1. 完成EpiCult-B培养基(50ml)
    EpiCult-B基础培养基:42ml
    EpiCult ® -B增殖补充剂:5ml
    FBS:2.5ml
    Pen/Strep:0.5ml
    EGF(0.2mg/ml):2.5μl
    肝素(50mg/ml):4μl
    通过0.2μm过滤器过滤灭菌
    储存在4°C,并在一周内使用
    完成EpiCult-B Medium必须在使用前立即用Matrigel补充

致谢

我们感谢Agata Tinnirello博士和Senthil Muthuswamy教授关于3D叠加细胞培养方法的技术建议(以前由Debnath等人(2003)描述),这项工作由AIRC资助(Grant IG2008和IG2011 ),MIUR(FIRB Giovani 2008),PiSTEM和ProgettoSanitàFinalizzata。G. Tornillo得到了FIRC研究金的支持。

参考文献

  1. Debnath,J.,Muthuswamy,S.K.and Brugge,J.S。(2003)。 在三维基底膜培养物中生长的MCF-10A乳腺上皮腺泡的形态发生和肿瘤形成。 a> 方法 30(3):256-268
  2. Tornillo,G.,Elia,AR,Castellano,I.,Spadaro,M.,Bernabei,P.,Bisaro,B.,Camacho-Leal Mdel,P.,Pincini,A.,Provero,P.,Sapino,A 。,Turco,E.,Defilippi,P。和Cabodi,S。(2013)。 p130Cas通过解除c-Kit活性来改变乳腺祖细胞的分化潜能。 em> Stem Cells 31(7):1422-1433。
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How to cite this protocol: Tornillo, G. and Cabodi, S. (2014). 3D Mammary Colony-Forming Cell Assay. Bio-protocol 4(7): e1087. DOI: 10.21769/BioProtoc.1087; Full Text



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