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Self-renewing cells from adult tissue (such as bone) that represent a progenitor population can be grown in suspension cultures in the presence of defined serum-free medium. Progenitor cells can be identified by this property of anchorage-independent growth in suspension cultures. These spherical clusters of progenitor bone cells growing under non-adherent conditions are called osteospheres. Such progenitor populations often possess characteristics of multipotency and can differentiate into multiple mesenchymal lineages. Cancer cells capable of growing in suspension have also been reported in osteosarcomas, tumors of the bone tissue. These spherical colonies formed from single cells (clonal) in non-adherent conditions are generally considered to represent self-renewing, stem-like cells and can be employed for other assays such as multipotency and limiting dilution analysis (LDA).

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Sphere Formation (Osteosphere/Sarcopshere) Assay
球体形成(骨球体/肌球体)试验

癌症生物学 > 癌症干细胞 > 细胞生物学试验 > 自我更新
作者: Upal Basu-Roy
Upal Basu-RoyAffiliation: Department of Microbiology, NYU School of Medicine, New York, USA
For correspondence: upal.basuroy@nyumc.org
Bio-protocol author page: a190
Claudio Basilico
Claudio BasilicoAffiliation: Department of Microbiology, NYU School of Medicine, New York, USA
Bio-protocol author page: a191
 and Alka Mansukhani
Alka MansukhaniAffiliation: Department of Microbiology, NYU School of Medicine, New York, USA
For correspondence: alka.mansukhani@med.nyu.edu
Bio-protocol author page: a192
Vol 2, Iss 24, 12/20/2012, 9481 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.307

[Abstract] Self-renewing cells from adult tissue (such as bone) that represent a progenitor population can be grown in suspension cultures in the presence of defined serum-free medium. Progenitor cells can be identified by this property of anchorage-independent growth in suspension cultures. These spherical clusters of progenitor bone cells growing under non-adherent conditions are called osteospheres. Such progenitor populations often possess characteristics of multipotency and can differentiate into multiple mesenchymal lineages. Cancer cells capable of growing in suspension have also been reported in osteosarcomas, tumors of the bone tissue. These spherical colonies formed from single cells (clonal) in non-adherent conditions are generally considered to represent self-renewing, stem-like cells and can be employed for other assays such as multipotency and limiting dilution analysis (LDA).

[Abstract] 该实验方案的中文版正在准备中...

Materials and Reagents

  1. Osteosarcoma cells (Basu-Roy et al., 2012)
  2. Trypsin
  3. Trypan blue from Invitrogen (Life Technologies, Invitrogen™, catalog number: 15250-061 )
  4. N2B27-defined serum free medium
    1. DMEM/F-12 1:1 from Invitrogen (Life Technologies, Invitrogen™, catalog number: 11330-057 )
    2. Neurobasal medium from Invitrogen (Life Technologies, Invitrogen™, catalog number: 21103-049 )
    3. Glutamax – from Invitrogen (Life Technologies, Invitrogen™, catalog number: 35050-061 )
    4. 55 mM Beta-mercaptoethanol – from Invitrogen (Life Technologies, Invitrogen™, catalog number: 21985-023 )
    5. B27 serum-free supplement – from Invitrogen (Life Technologies, Invitrogen™, catalog number: 17504-044 )
    6. Ndiff Neuro-2-medium supplement from Millipore (EMD Millipore, catalog number: SCM012 )
  5. Complete N2B27-medium (see Recipes)

Equipment

  1. Standard tissue culture equipment
  2. Ultra-low attachment tissue culture plastic ware from Corning (Corning Incorporated, catalog number: 3473 for 24 well plate; catalog number: 3474 for 96 well plate )
  3. 40 μM cell trainer from BD Falcon (BD Biosciences, Falcon®, catalog number: 352340 )
  4. Dissecting microscope
  5. Accumax (Innovative Cell Technologies, model: AM 105 )
  6. Hemocytometer
  7. 10-cm plate (Corning Incorporated, catalog number: 3262 )

Procedure

  1. Plating cells for sphere formation assay
    1. Trypsinize log-phase (actively growing) osteosarcoma cells (or cell of interest) growing under adherent conditions, and neutralize trypsin with serum-containing medium and count live cells after Trypan Blue staining, using a hemocytometer.
    2. Take required amount of cell suspension, spin down and resuspend cells at 1,000 live cells ml in N2B27-defined medium. Plate 1 ml cell suspension (1,000 cells) per well in triplicate in 24-well Corning ultra-low attachment plates for 7-10 days in N2B27 defined serum free medium.
      Note: Cell density is critical since too many cells will lead to clumping (and false positive results) and too few cells will not lead to sphere formation. We recommend seeding multiple densities as a pilot experiment to empirically determine best plating cell density before proceeding with the actual experiment. Cell numbers presented here have worked well in our osteosarcoma system. Also make sure that cells are single-cell suspension before plating by checking under microscope.
    3. Feed cells every 3 days by adding 50-100 μl medium per well. Do not aspirate medium since medium change is usually not required. Medium lost through evaporation is replenished during feeding. Also, be careful while feeding spheres since vigorous pipetting tends to disassociate the spheres.
    4. Check for sphere formation and count spheres in each well after 7-10 days.
      Note: Some cells especially non-transformed cells may require longer incubation of 2-3 weeks to form countable spheres. Spheres can be distinguished from clumps since spheres are uniformly circular whereas clumps look like aggregates that are not uniform in appearance.


      Figure 1. A typical sphere generated from osteosarcoma cells

    5. Count spheres under a dissecting microscope. These spheres are called primary spheres.
      Note: Typically, we see 0.1-0.5% of primary cells plated form spheres and 1-5% of transformed cells plated form spheres. Sphere forming frequency is determined by dividing the total number of spheres by the number of cells plated.
    6. After counting spheres under dissecting microscope, collect primary spheres by passing through a 40 μm cell strainer. Wash the plate once with PBS and pass the wash through the strainer to collect residual spheres. Do not forget to mention that you have collected spheres whose size is > 40 μm in your methods section.
      Note: Alternatively, you can plate more cells (10,000 cells/ml) - 10 ml cell suspension/10-cm plate or use the same cell density but plate higher volumes, and collect the spheres if you need more spheres. It should be stated here that you might need to increase the number of cells/ml if you plate higher volumes since autocrine and paracrine factors that are secreted by cells get diluted in higher volumes and hence, higher cell numbers might need to be used to compensate for this dilution.
    7. After collection of spheres, they can be used for various downstream applications such as protein extraction, RNA isolation, colony assay and re-plating for secondary sphere formation.
    8. For re-plating for colony or secondary sphere formation assay, primary spheres can be dissociated into single cells using Accumax according to the manufacturer's protocol. Before re-plating for colony or secondary sphere formation assay, check viability of cells by doing Trypan blue dye exclusion and counting cells using a hemocytometer.

Recipes

  1. Complete N2B27-medium
    To make complete N2B27-medium, mix
    200 ml DMEM/F-12
    200 ml Neurobasal medium
    1 ml Ndiff Neuro-2-medium supplement
    2 ml Glutamax
    4 ml B27 supplement
    4 ml Penicillin/Streptomycin
    728 μl of Beta-mercaptoethanol

Acknowledgments

This investigation was supported by PHS Grants AR051358 from the NIAMS and DE013745 from the NIDCR, and by an NCI UO1 award (to SHO). UBR is a recipient of a fellowship from The Children's Cancer Research Fund in memory of Dr A Rausen. AM is a recipient of a research grant from St Baldrick's Foundation. JAP is a postdoctoral fellow of the American Cancer Society. SHO is an Investigator of the Howard Hughes Medical Institute. This protocol is associated with the manuscript Basu-Roy et al. (2012).

References

  1. Basu-Roy, U., Ambrosetti, D., Favaro, R., Nicolis, S. K., Mansukhani, A. and Basilico, C. (2010). The transcription factor Sox2 is required for osteoblast self-renewal. Cell Death Differ 17(8): 1345-1353.
  2. Basu-Roy, U., Seo, E., Ramanathapuram, L., Rapp, T. B., Perry, J. A., Orkin, S. H., Mansukhani, A. and Basilico, C. (2012). Sox2 maintains self renewal of tumor-initiating cells in osteosarcomas. Oncogene 31(18): 2270-2282.

材料和试剂

  1. 骨肉瘤细胞(Basu-Roy等人,2012)
  2. 胰蛋白酶
  3. 来自Invitrogen(Life Technologies,Invitrogen TM,目录号:15250-061)的台盼蓝
  4. N2B27定义的无血清培养基
    1. 来自Invitrogen(Life Technologies,Invitrogen TM,目录号:11330-057)的DMEM/F-12 1:1
    2. 来自Invitrogen(Life Technologies,Invitrogen TM,目录号:21103-049)的神经基础培养基
    3. Glutamax - 来自Invitrogen(Life Technologies,Invitrogen TM,目录号:35050-061)
    4. 55mMβ-巯基乙醇 - 来自Invitrogen(Life Technologies,Invitrogen TM,目录号:21985-023)
    5. B27无血清补充物 - 来自Invitrogen(Life Technologies,Invitrogen TM,目录号:17504-044)
    6. 来自Millipore(EMD Millipore,目录号:SCM012)的Ndiff Neuro-2-培养基补充物
  5. 完成N2B27-培养基(参见配方)

设备

  1. 标准组织培养设备
  2. 来自Corning的超低附着组织培养塑料制品(Corning Incorporated,目录号:3473,用于24孔板;目录号:3474,用于96孔板)
  3. 来自BD Falcon的40μM细胞培养器(BD Biosciences,Falcon ,目录号:352340)
  4. 解剖显微镜
  5. Accumax(Innovative Cell Technologies,型号:AM 105)
  6. 血细胞计数器
  7. 10cm板(Corning Incorporated,目录号:3262)

程序

  1. 电镀细胞用于球形成测定
    1. 胰蛋白酶处理在粘附条件下生长的对数期(活跃生长的)骨肉瘤细胞(或目标细胞),并用含血清的培养基中和胰蛋白酶,并使用血细胞计数器在台盼蓝染色后计数活细胞。
    2. 取需要量的细胞悬液,离心和重悬细胞在1,000个活细胞ml在N2B27确定培养基。在24孔康宁超低附着板中,在N2B27定义的无血清培养基中,将1ml细胞悬浮液(1000个细胞)每孔一式三份铺板在培养基中。 注意:细胞密度是至关重要的,因为太多的细胞会导致结块(和假阳性结果),太少的细胞不会导致球形成。我们建议种植多重密度作为试验实验,以在进行实际实验之前根据经验确定最佳铺板细胞密度。这里介绍的细胞数量在我们的骨肉瘤系统中工作良好。还要确保细胞在通过在显微镜下检查进行电镀之前是单细胞悬浮液。
    3. 通过每孔加入50-100μl培养基每3天饲养细胞。不要吸入介质,因为通常不需要更换介质。在进食期间补充通过蒸发损失的介质。另外,喂养球时要小心,因为剧烈的移液会使球脱离
    4. 检查球体形成,并在7-10天后计数每个孔中的球体 注意:一些细胞,特别是非转化细胞可能需要更长时间孵育2-3周以形成可数球。球体可以区别于团块,因为球体是均匀的圆形,而团块看起来像外观不一致的聚集体。


      图1.由骨肉瘤细胞产生的典型球体

    5. 在解剖显微镜下计数球体。这些球体称为初级球体。
      注意:通常,我们看到0.1-0.5%的原始细胞电镀形成球体和1-5%的转化细胞电镀形成球体。球体形成频率由球体总数除以所镀细胞数来确定。
    6. 在解剖显微镜下计数球体后,通过通过40μm细胞过滤器收集初级球体。用PBS洗涤板一次,将洗涤液通过过滤器收集残余球体。不要忘了提到你收集的球体大小>方法部分中为40μm。
      注意:或者,您可以铺板更多的细胞(10,000个细胞/毫升) - 10毫升细胞悬浮液/10厘米板或使用相同的细胞密度,但板更高的体积,收集球体,如果你需要更多的球体。这里应该说明的是,如果平板更高的体积,您可能需要增加细胞数/ml,因为细胞分泌的自分泌和旁分泌因子被稀释到更高的体积,因此,可能需要使用更高的细胞数来补偿对于此稀释。
    7. 收集球体后,它们可用于各种下游应用,例如蛋白质提取,RNA分离,菌落测定和二次球形成的再镀覆。
    8. 对于用于集落或二次球形成测定的重新铺板,可以使用Accumax根据制造商的方案将初级球解离成单个细胞。在重新铺板为殖民地或 二级球形成试验,通过使用血细胞计数器进行台盼蓝染料排除和计数细胞来检查细胞的存活力。

食谱

  1. 完成N2B27培养基
    要制备完全N2B27-培养基,混合
    200 ml DMEM/F-12
    200 ml Neurobasal培养基
    1ml Ndiff Neuro-2-培养基补充剂
    2ml Glutamax
    4 ml B27补充剂
    4ml青霉素/链霉素 728μlβ-巯基乙醇

致谢

该调查由来自NIAMS的PHS授权AR051358和来自NIDCR的DE013745以及通过NCI UO1奖(授予SHO)支持。 UBR是来自儿童癌症研究基金的一个研究金的收件人,纪念Dr Rausen博士。 AM是来自圣巴德里克基金会的研究资助的收件人。 JAP是美国癌症协会的博士后研究员。 SHO是霍华德休斯医学研究所的研究员。 该协议与Basu-Roy等人的论文(2012)相关联。

参考文献

  1. Basu-Roy,U.,Ambrosetti,D.,Favaro,R.,Nicolis,S.K.,Mansukhani,A.and Basilico,C。(2010)。 转录因子Sox2是成骨细胞自我更新所必需的。细胞死亡 Differ 17(8):1345-1353。
  2. Basu-Roy,U.,Seo,E.,Ramanathapuram,L.,Rapp,T.B.,Perry,J.A.,Orkin,S.H.,Mansukhani,A.and Basilico,C。(2012)。 Sox2维持骨肉瘤肿瘤起始细胞的自我更新。癌基因 31(18):2270-2282。
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How to cite this protocol: Basu-Roy, U., Basilico, C. and Mansukhani, A. (2012). Sphere Formation (Osteosphere/Sarcopshere) Assay. Bio-protocol 2(24): e307. DOI: 10.21769/BioProtoc.307; Full Text



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