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Hanging Drop Aggregation Assay of Breast Cancer Cells

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Jan 2014



Hanging drop assay can be used to investigate cell-cell cohesion and cell-substratum adhesion through generation of 3D spheroids under physiological conditions. It also can be used to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between two (or more) different cell populations. This simple method requires no specialized equipment and provides a means of generating tissue-like cellular aggregates for measurement of biomechanical properties for molecular and biochemical analysis in a physiologically relevant model.

Keywords: Hanging drop (悬滴), Cancer (癌症), Aggregation (聚集), Migration (迁移)

Materials and Reagents

  1. Human breast cancer cell lines T-47D (ATCC, catalog number: ATCC® HTB-133TM ), MCF7 (ATCC, catalog number: ATCC® HTB-22TM )
  2. Trypsin-EDTA (0.05%) (phenol red) (Life Technologies, catalog number: 25300062 )
  3. Gibco® RPMI media 1640 (Life Technologies, catalog number: 11875-085 )
  4. Gibco® PBS (Life Technologies, catalog number: 10010023 )
  5. Fetal bovine serum (Life Technologies, catalog number: 16000-044 )
  6. Gibco® penicillin-streptomycin-glutamine (100x) (Life Technologies, catalog number: 10378-016 )
  7. Complete medium (see Recipes)


  1. CorningTM culture dishes (Corning, catalog number: 430591 )
  2. Inverted microscope (ZEISS)
  3. Haemocytometer (Thermo Fisher Scientific, catalog number: 02-671-52 )


  1. Plate T-47D and MCF7 cells in complete medium and let them grow to reach a confluency of 80%.
  2. Remove the complete medium from culture dish and add 0.05% trypsin for 3-5 min after PBS washing. As soon as cells have detached, collect cells by adding complete medium.
  3. After spinning down (1,000 x g for 5 min), count the cells using a haemocytometer and adjust cell concentration to 2.0 x 106 cells/ml# in complete culture medium.
  4. Carefully deposit cells in 30 μl drops on the inner side of a 100 mm dish lid. It is possible to place at least 30 drops per lid (Figure 1A).
  5. Turn the lid of the plate upside down and place on top of the plate filled with 10 ml PBS (to humidify the culture chamber after distribution of the drops) (Figure 1B). Cells under the force of gravity will be aggregated at the bottom of the hanging drop. Make sure avoid placing the drops close to the plate edges and drops are placed sufficiently apart so as to not touch.
  6. Monitor the drops daily and incubate until either cell sheets or aggregates have formed, and photograph cell clusters at the endpoint of experiment* by a microscopy within 20 min. Adjust the lighting and focus to provide the most contrast between the 3D structure and background**.
  7. Collect cell clusters by wide end of tips (100 µl pipette tip cut widely 5-6 mm from the end), and disburse them by pipetting up and down at least 7 times following with a wash of PBS supplemented with 10% fetal bovine serum.
  8. Count single cells released from the clusters using a haemocytometer.

    Figure 1. A. 30 μl drops on the inner side of a 100 mm dish lid; B. lid with drops upside down and on top of the plate filled with 10 ml PBS.

Representative data

Figure 2. Photographs of representative spheroids from T-47D and MCF7 breast cancer cell lines. T-47D and MCF7 cells were cultured in hanging drops and formation of spheroids was observed at 2 and 4 days after culture.


  1. This protocol works equally well with other types of cancer cells or genetic modified cancer cells (Defresne et al., 2011; Yip and Cho, 2013; Kumar et al., 2014; Teng et al., 2013).
  2. #Cell concentration may need to be adjusted up- or down depending on cell size.
  3. *The assay may be conducted longer than 6 days if desired.
  4. **The use of fluorescence microscopy for cells that forced express fluorescent protein (such as GFP, RFP) or have a fluorescent label may improve contrast and subsequent analysis.


  1. Complete medium
    90% RPMI media 1640
    10% fetal bovine serum
    1% penicillin-streptomycin-glutamine


This study was supported, in part, by the National Institutes of Health Grant CA120510.


  1. Defresne, F., Bouzin, C., Grandjean, M., Dieu, M., Raes, M., Hatzopoulos, A. K., Kupatt, C. and Feron, O. (2011). Preconditioned endothelial progenitor cells reduce formation of melanoma metastases through SPARC-driven cell-cell interactions and endocytosis. Cancer Res 71(14): 4748-4757.
  2. Kumar, A., Fan, D., Dipette, D. J. and Singh, U. S. (2014). Sparstolonin B, a novel plant derived compound, arrests cell cycle and induces apoptosis in N-myc amplified and N-myc nonamplified neuroblastoma cells. PLoS One 9(5): e96343.
  3. Teng, Y., Mei, Y., Hawthorn, L. and Cowell, J. (2013). WASF3 regulates miR-200 inactivation by ZEB1 through suppression of KISS1 leading to increased invasiveness in breast cancer cells. Oncogene 33(2): 203-211.
  4. Yip, D. and Cho, C. H. (2013). A multicellular 3D heterospheroid model of liver tumor and stromal cells in collagen gel for anti-cancer drug testing. Biochem Biophys Res Commun 433(3): 327-332.


悬滴测定可用于通过在生理条件下产生3D球体来研究细胞 - 细胞内聚性和细胞 - 基质粘附。 它还可以用于阐明细胞 - 细胞或细胞-ECM相互作用在指定两种(或更多种)不同细胞群体之间的空间关系中的作用。 这种简单的方法不需要专门的设备,并且提供了在生理相关模型中产生用于测量生物力学性质的组织样细胞聚集体用于分子和生物化学分析的方法。

关键字:悬滴, 癌症, 聚集, 迁移


  1. 人乳腺癌细胞系T-47D(ATCC,目录号:ATCC,HTB-133),MCF7(ATCC,目录号:ATCC sup> HTB-22 TM
  2. 胰蛋白酶-EDTA(0.05%)(酚红)(Life Technologies,目录号:25300062)
  3. Gibco RMI RPMI培养基1640(Life Technologies,目录号:11875-085)
  4. Gibco PBS(Life Technologies,目录号:10010023)
  5. 胎牛血清(Life Technologies,目录号:16000-044)
  6. 青霉素 - 链霉素 - 谷氨酰胺(100x)(Life Technologies,目录号:10378-016)
  7. 完整介质(见配方)


  1. Corning TM 培养皿(Corning,目录号:430591)
  2. 倒置显微镜(ZEISS)
  3. 血细胞计数器(Thermo Fisher Scientific,目录号:02-671-52)


  1. 平板T-47D和MCF7细胞在完全培养基中,让它们生长达到80%的汇合。
  2. 从培养皿中取出完全培养基,并在PBS洗涤后加入0.05%胰蛋白酶3-5分钟。 一旦细胞分离,通过添加完全培养基收集细胞。
  3. 在离心(1,000xg,5分钟)后,使用血细胞计数器计数细胞,并将细胞浓度调节至2.0×10 6个细胞/ml >在完全培养基中。
  4. 小心地沉积细胞在30微升滴在100毫米的餐具盖的内侧。 每个盖子可以放置至少30滴(图1A)。
  5. 将板的盖子倒置,并放置在填充有10ml PBS的板的顶部(在分配液滴后润湿培养室)(图1B)。细胞在重力作用下会聚集在悬滴的底部。确保避免将液滴靠近板边缘放置,液滴应放在足够远的地方,以免接触。
  6. 每天监测滴,孵化直到细胞片或聚集体形成,并在实验的终点通过显微镜在20分钟内的照片细胞簇。调整照明和聚焦以提供3D结构和背景之间的最大对比**。
  7. 通过广泛的末端(100微升移液器尖端切割广泛5-6毫米从末端)收集细胞群,并通过吹吸向上和向下至少7次,随后用PBS洗涤补充10%胎牛血清的洗涤。
  8. 计数使用血细胞计数器从簇释放的单个细胞

    图1. 。在100mm皿盖的内侧上滴30μl; B.将盖子倒下,并在装有10ml PBS的板的顶部




  1. 该方案同样适用于其他类型的癌细胞或遗传修饰的癌细胞(Defresne等人,2011; Yip和Cho,2013; Kumar等人,2014年, ; Teng等人,2013)。
  2. 细胞浓度可能需要根据细胞大小向上或向下调整。
  3. *如果需要,测定可以进行6天以上
  4. **对于强制表达荧光蛋白(如GFP,RFP)或具有荧光标记的细胞使用荧光显微镜可以改善对比度并随后进行分析。


  1. 完成媒介
    10%胎牛血清 1%青霉素 - 链霉素 - 谷氨酰胺




  1. Defresne,F.,Bouzin,C.,Grandjean,M.,Dieu,M.,Raes,M.,Hatzopoulos,A.K.,Kupatt,C.and Feron, 预处理的内皮祖细胞通过SPARC驱动的细胞 - 细胞相互作用和内吞作用减少黑素瘤转移的形成。/a> Cancer Res 71(14):4748-4757
  2. Kumar,A.,Fan,D.,Dipette,D.J.and Singh,U.S.(2014)。 Sparstolonin B,一种新型植物衍生化合物,可抑制细胞周期并在N-myc扩增中诱导凋亡N-myc非扩增的神经母细胞瘤细胞。 9(5):e96343。
  3. Teng,Y.,Mei,Y.,Hawthorn,L。和Cowell,J。(2013)。 WASF3通过抑制KISS1调节ZEB1的miR-200失活,导致增加乳腺癌细胞的侵袭性。癌基因 33(2):203-211。
  4. Yip,D.和Cho,C.H。(2013)。 用于抗癌药物测试的胶原凝胶中肝肿瘤和基质细胞的多细胞3D异形体模型。 Biochem Biophys Res Commun 433(3):327-332。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Teng, Y. (2015). Hanging Drop Aggregation Assay of Breast Cancer Cells. Bio-protocol 5(3): e1393. DOI: 10.21769/BioProtoc.1393.