In vitro Tumorsphere Formation Assays

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A tumorsphere is a solid, spherical formation developed from the proliferation of one cancer stem/progenitor cell. These tumorspheres (Figure 1a) are easily distinguishable from single or aggregated cells (Figure 1b) as the cells appear to become fused together and individual cells cannot be identified. Cells are grown in serum-free, non-adherent conditions in order to enrich the cancer stem/progenitor cell population as only cancer stem/progenitor cells can survive and proliferate in this environment. This assay can be used to estimate the percentage of cancer stem/progenitor cells present in a population of tumor cells. The size, which can vary from less than 50 micrometers to 250 micrometers, and number of tumorspheres formed can be used to characterize the cancer stem/progenitor cell population within a population of in vitro cultured cancer cells and within in vivo tumors (Lo et al., 2012; Liu et al., 2009). While several cell lines can be used for tumorsphere formation assay (e.g. primary mammary tumor cells from Her2/neu-transgenic mice, MCF7, BT474 and HCC1954), some cell lines may not form typical tumorsphere structures and may be difficult to count or classify definitively as tumorspheres.

Figure 1. Shows tumorspheres formed by HCC1954 cells after 7 day incubation. The solid, circular formations represent tumorspheres (a). The individual or aggregated cells are not considered tumorspheres (b).

Note: The tumorspheres should be solid, round structures, but the size varies greatly from less than 50 μm to around 250 μm. With aggregated cells, you can still see individual cells attached to one another. With tumorspheres, the cells appear fused together and it is difficult to distinguish them as individual cells.

Materials and Reagents
  1. 50x B27 Supplement (Life Technologies, InvitrogenTM, catalog number: 17504-044 )
  2. Basic Fibroblast Growth Factor (Sigma-Aldrich, catalog number: F0291 )
  3. Epidermal Growth Factor (Sigma-Aldrich, catalog number: E5036 )
  4. Insulin (Life Technologies, InvitrogenTM, catalog number: A11429IJ)
  5. Bovine Serum Albumin (Sigma-Aldrich, catalog number: A9576 )
  6. Dulbecco’s Modified Eagle Medium/F12 (Sigma-Aldrich, catalog number: D8437 )
  7. Sterile 1x Dulbecco’s Phosphate Buffered Saline (Sigma-Aldrich, catalog number: D8537 )
  8. Trypan Blue (Life Technologies, InvitrogenTM, catalog number: 15250-061 )
  9. Tumorsphere medium (500 ml) (see Recipes)


  1. Incubator with CO2 input
  2. Counting Chamber/Hemocytometer (Hausser Scientific, catalog number: 3200 )
  3. 96-well Ultra-low Attachment Plates (Corning Incorporated, catalog number: 3474 )


  1. Add B27 supplement (50x) to tumorsphere medium for making 1x concentration as needed for experiments. B27 should be freshly added before each experiment.
    Note: The addition of B27 has been shown to increase tumorsphere formation and sustain several passages of sphere cultures (Gu et al., 2011).
  2. Harvest cells and suspend the resulting pellet in 5 ml 1x PBS. Keep cells on ice while not in use for the duration of the experiment.
  3. Gently mix the suspension and pipette 20 μl of the suspension into an eppendorf tube.
  4. Add 20 μl (1:1 ratio) of Trypan Blue to the cells in the eppendorf tube and mix well.
  5. Pipette approximately 10 μl of the mixture onto the hemocytometer.
  6. Count only the bright cells within the four corner quadrants of the counting chamber for viable cell count.
  7. Take the needed number of cells and add the appropriate volume of tumorsphere medium to make the cell concentration at 1 cell/L. Keep this suspension on ice and mixed well for plating.
  8. Add 1x PBS to the first and last columns (column 1 and 12) of the 96-well plate to help minimize medium evaporation. This will leave 10 wells available for each row.
  9. Seed 200 μl of the cells suspended in tumorsphere medium into each well (200 cells per well).
    Note: The number of cells used for tumorsphere formation assays may vary with the cell type.
  10. For each cell line or treatment, seed cells into the wells of 2 rows for a total of 20 wells. This will equal a total of 4,000 cells.
  11. Seal the upper and lower edges of the 96-well plate with laboratory tape to avoid evaporation of medium and place the cells in an incubator set to 37 °C and supply the cells with 5% CO2 for one week.
    Note:The medium is not changed or added so as to not disturb the formation of the tumorspheres.
  12. After one-week incubation, tumorsphere numbers are counted under a phase-contrast microscope using the 40x magnification lens (See Figure 1).
  13. Results can be presented as a percentage of the number of tumorspheres present divided by the initial number of cells seeded (4,000 cells).


  1. Tumorsphere medium (500 ml)
    20 ng/ml epidermal growth factor
    10 ng/ml basic fibroblast growth factor
    5 μg/ml insulin
    0.4% Bovine Serum Albumin
    500 ml Dulbecco’s Modified Eagle Medium/F12


This protocol was first described in Lo et al. (2012). This work was supported by the Elsa U. Pardee Cancer Foundation grant (B94AFFAA), the American Cancer Society Research Award (RSG-10-067-01-TBE) to HC and NIH grant (3P20RR017698-08) to HC and QW.


  1. Gu, Y., Fu, J., Lo, P., Wang, S., Wang, Q., Chen, H. (2011). The effect of B27 supplement on promoting in vitro propagation of Her2/neutransformed mammary tumorspheres. J Biotech Res 3: 7-18.
  2. Liu, J. C., T. Deng, R. S. Lehal, J. Kim and E. Zacksenhaus (2007). Identification of tumorsphere- and tumor-initiating cells in HER2/Neu-induced mammary tumors. Cancer Res 67(18): 8671-8681.
  3. Lo, P. K., D. Kanojia, X. Liu, U. P. Singh, F. G. Berger, Q. Wang and H. Chen (2012). CD49f and CD61 identify Her2/neu-induced mammary tumor-initiating cells that are potentially derived from luminal progenitors and maintained by the integrin-TGFbeta signaling. Oncogene 31(21): 2614-2626.


肿瘤球是从一个癌干细胞/祖细胞的增殖形成的固体,球形形式。这些肿瘤球(图1a)易于与单个或聚集的细胞(图1b)区分开,因为细胞看起来融合在一起并且不能鉴定个体细胞。细胞在无血清,非粘附条件下生长以富集癌干细胞/祖细胞群,因为只有癌干/祖细胞可在这种环境中存活和增殖。该测定可用于估计存在于肿瘤细胞群中的癌干细胞/祖细胞的百分比。可以从小于50微米至250微米变化的大小和形成的肿瘤球的数目可以用于表征体外培养的癌细胞群内的癌干/祖细胞群,以及在体内肿瘤(Lo等人,2012; Liu等人,2009)。虽然几种细胞系可用于肿瘤球形成测定(例如来自Her2/neu转基因小鼠,MCF7,BT474和HCC1954的原发性乳腺肿瘤细胞),但一些细胞系可能不形成典型的肿瘤球结构,并且可能难以计数或最终分类为肿瘤球。

图1 。 S 如何形成肿瘤球 HCC1954细胞孵育7天后。固体圆形形式代表肿瘤球(a)。个人或 不考虑聚合细胞 肿瘤球(b)。

注意:肿瘤球应该是实心的圆形结构,但尺寸 i> 到约250 μ span> = span> 使用聚合单元格 仍然可以看到彼此附着的单个细胞。肿瘤球 细胞出现融合在一起,很难将它们区分为 个别单元格

  1. 50x B27补充物(Life Technologies,Invitrogen TM ,目录号:17504-044)
  2. 碱性成纤维细胞生长因子(Sigma-Aldrich,目录号:F0291)
  3. 表皮生长因子(Sigma-Aldrich,目录号:E5036)
  4. 胰岛素(Life Technologies,Invitrogen TM ,目录号:A11429IJ)
  5. 牛血清白蛋白(Sigma-Aldrich,目录号:A9576)
  6. Dulbecco's Modified Eagle Medium/F12(Sigma-Aldrich,目录号:D8437)
  7. 无菌1×Dulbecco's磷酸盐缓冲盐水(Sigma-Aldrich,目录号:D8537)
  8. 台盼蓝(Life Technologies,Invitrogen TM,目录号:15250-061)
  9. 肿瘤球培养基(500ml)(见配方)


  1. 带CO 2 输入的培养箱
  2. 计数室/血细胞计数器(Hausser Scientific,目录号:3200)
  3. 96孔超低附着板(Corning Incorporated,目录号:3474)


  1. 添加B27补充(50x)肿瘤球培养基进行1x浓度为实验所需。 B27应在每次实验前新添加 注意:添加B27已显示增加肿瘤球形成并维持球形培养物的几次传代(Gu等人,2011)。
  2. 收获细胞并将所得沉淀悬浮于5ml 1x PBS中。 保持细胞在冰上,而不是在实验期间使用。
  3. 轻轻混合悬浮液和吸管20微升的悬浮液到eppendorf管
  4. 向eppendorf管中的细胞中加入20μl(1:1比例)的台盼蓝,并混匀。
  5. 吸取大约10微升的混合物到血细胞计数器
  6. 只计数活细胞计数的计数室的四个角象限内的亮细胞
  7. 取所需数量的细胞,加入适当体积的肿瘤球培养基,使细胞浓度为1个细胞/L。 将此悬浮液保持在冰上,混匀后进行电镀
  8. 向96孔板的第一列和最后一列(第1列和第12列)加入1 x PBS,以帮助最小化培养基蒸发。 这将为每一行留出10口井
  9. 种子200微升悬浮在肿瘤球培养基中的细胞(每孔200个细胞) 注意:用于肿瘤球形成测定的细胞数可以随细胞类型而变化。
  10. 对于每个细胞系或处理,将种子细胞接种到2行的孔中,总共20个孔。 这将等于4,000个单元格。
  11. 用实验室胶带密封96孔板的上边缘和下边缘,以避免培养基的蒸发,并将细胞置于设定为37℃的培养箱中,并向细胞提供5%CO 2 周。
  12. 在一周孵育后,使用40倍放大透镜在相差显微镜下计数肿瘤数目(参见图1)。
  13. 结果可以表示为存在的肿瘤球的数目除以接种的细胞的初始数目(4,000个细胞)的百分比。


  1. 肿瘤球培养基(500ml)
    20ng/ml表皮生长因子 10ng/ml碱性成纤维细胞生长因子 5μg/ml胰岛素
    500ml Dulbecco's Modified Eagle Medium/F12


该方案首先在Lo等人(2012)中描述。 这项工作得到了Elsa U. Pardee癌症基金会资助(B94AFFAA),美国癌症协会研究奖(RSG-10-067-01-TBE)到HC和NIH资助(3P20RR017698-08)到HC和QW的支持。


  1. Gu,Y.,Fu,J.,Lo,P.,Wang,S.,Wang,Q.,Chen,H。(2011)。 B27补充对在vitr中促进的效果 o Her2/neutransformed mammary tumorspheres的繁殖。生物技术研究3:7-18。
  2. Liu,J.C.,T.Deng,R.S.Lahal,J.Kim和E.Zacksenhaus(2007)。 在HER2/Neu诱导的乳腺肿瘤中鉴定肿瘤球和肿瘤起始细胞 。 Cancer Res 67(18):8671-8681。
  3. Lo,PK,D. Kanojia,X. Liu,UP Singh,FG Berger,Q.Wang和H.Chen(2012)。 CD49f和CD61鉴定了可能源自鲁米那祖细胞并由整联蛋白-TGFβ信号传导维持的Her2/neu诱导的乳腺肿瘤起始细胞。 em> Oncogene 31(21):2614-2626。
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引用:Johnson, S., Chen, H. and Lo, P. (2013). In vitro Tumorsphere Formation Assays. Bio-protocol 3(3): e325. DOI: 10.21769/BioProtoc.325.

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Colm Rowan
University college Dublin
Hi there,

I ran this experiment and I managed to form tumorspheres. However the tumorspheres seem to be adherent. I used ultra low attachment 96 well plates but the spheres don't seem to move under the microscope when the plate is moved? Has this happened to anyone else?
11/5/2014 1:30:16 AM Reply
Sara Johnson
Biological Sciences Department, University of South Carolina, USA


I have had this happen once before with tumorspheres kept in culture for over one week. It is possible that the tumorspheres have remained in culture too long. We generally counted spheres after one week, so a lengthier culture can lead to attachment. From my understanding, some cells respond better to different types of non-tissue culture petri dishes and low attachment plates as well.

I hope this helps.

11/11/2014 1:02:15 PM

tara fani
joundishapour university
I have seen Tumorspheres with invert-microscopy,but i can't determine their size,I was wondering if you'd mind helping me,what method or device can i use to determine their size(50-200µm)?
12/20/2013 9:56:17 PM Reply
Sara Johnson
Biological Sciences Department, University of South Carolina, USA

Dear Tara,

When counting and photographing the spheres, we use the imaging software that accompanied our Olympus inverted scope (DP2-BSW software). This program has a 100 micrometer scale bar that allows us to estimate the size of the tumorspheres.

I hope that this helps to answer your question,

12/30/2013 12:09:21 PM

Ting Yu
Sichuan University

Dear Sara,
How to identify the range of diameter for tumorspheres? 50-250 micrometers?
Ting Yu

6/19/2016 12:54:53 AM

melanie weiss melanie
Dear Sara, Hexin and Pang-Kuo,

I just have a technical question concerning the tumorsphere formation assay.
Are you using 96-well Ultra-low Attachment Plates with flat or round bottom ? Because, I tried out the one's with a round bottom, and I think that all my cells, are attracted to the middle of the well (lowest point of the well), and this leads to the formation of a sphere-like structure, which is in my mind more the result of a cell cluster.

I am looking forward to hear from you.
Kind regards,

9/30/2013 5:28:56 AM Reply
Hexin Chen
Department of Biological Science, University of South Carolina, USA

Dear Melanine,

We are using the flat-bottom plate. The cells are likely to be attacked to the edge but they should be able to form sphere well.



10/1/2013 11:27:05 PM