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Longitudinal Bioluminescent Quantification of Three Dimensional Cell Growth
利用生物发光技术持续定量监测细胞的三维生长   

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Abstract

The use of three-dimensional (3D) cell culture systems is widely accepted as representing a more physiologically relevant means to propagate mammary epithelial and breast cancer cells. However, 3D cultures systems are plagued by several experimental and technical limitations as compared to their traditional 2D counterparts. For instance, quantifying the growth of mammary epithelial or breast cancer organoids longitudinally is particularly troublesome using standard [3H]thymidine or MTT assay systems, or using computer-assisted area calculations. Likewise, the nature of the multicellular aggregates and organoids formed by breast cancer cells under 3D conditions precludes efficient recovery of the cells from 3D matrices, an event that is time consuming and leads to spurious results. The assay described here utilizes stable expression of firefly luciferase as means to quantify the longitudinal outgrowth of cells propagated within a 3D matrices. The major advantages of this technique include its high-throughput nature and ability to longitudinally track single wells over a defined period of time, thereby decreasing the costs associated with assay performance. Finally, this technique can be readily combined with drug treatments and/or genetic manipulations to assay their effects on the growth of 3D organoids.

Keywords: Proliferation(增殖), Bioluminescence(生物发光), 3D-cultures(三维培养), In vitro outgrowth(在体外生长)

Materials and Reagents

  1. Murine 4T1 mammary carcinoma cells (ATCC, catalog number: CRL-2539 ) or any cell line of interest engineered to stably express firefly luciferase under the control of a constitutively-active promoter such cytomegalovirus.
    Note: Several Luciferase encoding plasmids are commercially available and typically employ pcDNA3.1- or pBabe-based backbones to deliver firefly or renilla luciferases. In either scenario, antibiotic administration is used to select and maintain stable expression of luciferase in reporter cells.
  2. Cultrex: Reduced growth factor (RGF) basement membrane extract (BME) (Trevigen, catalog number: 3433-005-01 )
  3. Ice
  4. Dulbecco’s Modified Eagle Medium (DMEM) (Life Technologies, catalog number: 10313-021 )
  5. Penicillin/Streptomycin (Pen/Strep) (Gibco®, catalog number: 15140 )
  6. Fetal bovine serum (Sigma-Aldrich, catalog number: F1051 )
  7. D-luciferin, Potassium Salt (15 mg/ml) in sterile H2O (Gold Bio, catalog number: LUCK-1G )

Equipment

  1. 2D culture dishes
  2. White walled, clear bottom 96-well culture dishes (Corning, Costar®, catalog number: 3610 )
  3. Luminometer capable of reading 96-well plate format (Promega GloMax-Multi Detection System or similar bioluminescent plate reader).
  4. Hemocytometer or other means of cell counting
  5. 37 °C/5% CO2 cell incubator

Procedure

  1. Cells are grown in DMEM supplemented with 10% FBS and Pen/Strep (full growth media).
  2. Cells are harvested from actively proliferating, sub-confluent 2D culture dishes.
  3. Cells are trypsinized, washed in excess full-growth media, and pelleted by gentle centrifugation. Afterward, the resulting cell pellets are resuspended and allowed to recover in full growth media for 2 h.
  4. During this time, coat the 96-well dish with 50 μl of 100% Cultrex per well and allow to gel at 37 °C.
    Note: Cultrex must be maintained on ice at all times to prevent solidification of the matrices that transpires as the gel warms to >4 °C.
  5. Count the cells using the hemocytometer and dilute them to 1,000 cells in 150 μl of full growth media, which is supplemented with 4% Cultrex. Thoroughly mix cells and media/4% Cultrex solution and subsequently plate the cells on top of the solidified Cultrex cushions.
    Note: The 4% Cultrex solutions do not solidify, and as such, cells contained within these mixtures will readily attach to solidified Cultrex cushions, thereby permitting top layer media changes and/or replacements throughout the experiment.
  6. Prior to plating the cells, other compounds such as growth factors or chemical inhibitors may be mixed with the cells and 4% Cultrex solutions. Each experimental condition should be plated in triplicate.
    Note: All test compounds must be screened initially by short-term exposure to verify that these agents do not directly impact the expression of CMV-driven luciferase and/or the activity of luciferase:luciferin reactions.
  7. Two hours after plating the cells, obtain initial time zero (T0) luminescence readings by adding 2 μl of D-luciferin (15 mg/ml) and gently tap the side of the plate to mix.
    Note: Culture lid is open while in the plate reader, and as such, it is imperative that the plate reader remain clean and well-sanitized, and potentially be located in a laminar flow hood to prevent unwanted cell contamination. Culture medium does not need to be replaced at this time.
  8. Place culture in a 37 °C incubator with 5% CO2.
  9. Four days after plating, multicellular organoids will have begun to form. Obtain a second luminescence reading as described in step 7. Afterward, change and refresh all media and experimental test components, being extremely careful not to disrupt the cells and solidified Cultrex cushion.
    Note: At this point, therapeutic protocols can be initiated to monitor the anticancer activities of various chemotherapies against established organoids.
  10. For all experimental conditions, repeat steps 7-9 at days 8 and 11 post-plating.
    Note: These time points may vary depending dramatically based on the relative growth characteristics of the cell line under study. As such, longitudinal luciferase readings, dosing regiments, and cell plating densities need to be determined empirically for each cell line to be studied to maximize signal-to-noise ratios.
  11. Use Excel to normalize each luminescence value to the initial T0 value derived after plating cells.

Acknowledgments

We thank the members of the Schiemann laboratory for their helpful comments and suggestions. Bioluminescent 3D-organotypic longitudinal growth assays were adapted from Wendt et al. (2011) and Wendt et al. (2013). Research support was provided in part by the National Institutes of Health to M.K.W. (CA166140), and to W.P.S. (CA129359 and CA177069).

References

  1. Wendt, M. K., Schiemann, B. J., Parvani, J. G., Lee, Y. H., Kang, Y. and Schiemann, W. P. (2013). TGF-beta stimulates Pyk2 expression as part of an epithelial-mesenchymal transition program required for metastatic outgrowth of breast cancer. Oncogene 32(16): 2005-2015.
  2. Wendt, M. K., Taylor, M. A., Schiemann, B. J. and Schiemann, W. P. (2011). Down-regulation of epithelial cadherin is required to initiate metastatic outgrowth of breast cancer. Mol Biol Cell 22(14): 2423-2435.

简介

使用三维(3D)细胞培养系统被广泛接受为代表更多生理相关手段以增殖乳腺上皮和乳腺癌细胞。然而,与其传统的2D对应物相比,3D培养系统受到若干实验和技术限制的困扰。例如,使用标准的[3 H]胸苷或MTT测定系统或使用计算机辅助面积计算,纵向量化乳腺上皮或乳腺癌组织的生长是特别麻烦的。同样,在3D条件下由乳腺癌细胞形成的多细胞聚集体和类器体的性质阻止了细胞从3D基质的有效回收,这是耗时的并且导致假的结果的事件。这里描述的测定利用萤火虫荧光素酶的稳定表达作为定量在3D矩阵内传播的细胞的纵向生长的手段。该技术的主要优点包括其高通量性质和在规定的时间段内纵向跟踪单个孔的能力,从而降低与测定性能相关的成本。最后,该技术可以容易地与药物治疗和/或遗传操作组合以测定它们对3D类器官生长的影响。

关键字:增殖, 生物发光, 三维培养, 在体外生长

材料和试剂

  1. 鼠4T1乳腺癌细胞(ATCC,目录号:CRL-2539)或在组成型活性启动子如巨细胞病毒控制下经工程改造以稳定表达萤火虫荧光素酶的任何目的细胞系。
    注意:几种荧光素酶编码质粒是可商购的,并且通常使用基于pcDNA3.1或pBabe的主链来递送萤火虫或海肾萤光素酶。 在任一情况下,使用抗生素施用来选择和维持荧光素酶在报道细胞中的稳定表达。
  2. Cultrex:减少生长因子(RGF)基底膜提取物(BME)(Trevigen,目录号:3433-005-01)

  3. Dulbecco改良的Eagle培养基(DMEM)(Life Technologies,目录号:10313-021)
  4. 青霉素/链霉素(Pen/Strep)(Gibco ,目录号:15140)
  5. 胎牛血清(Sigma-Aldrich,目录号:F1051)
  6. D-luciferin,钾盐(15mg/ml)在无菌H 2 O中(Gold Bio,目录号:LUCK-1G)

设备

  1. 二维培养皿
  2. 白色壁,透明底96孔培养皿(Corning,Costar ,目录号:3610)
  3. 能够读取96孔板格式(Promega GloMax多检测系统或类似的生物发光板读数器)的照度计。
  4. 血细胞计数器或其他细胞计数方法
  5. 37℃/5%CO 2细胞培养箱

程序

  1. 细胞在补充有10%FBS和Pen/Strep(完全生长培养基)的DMEM中生长。
  2. 从活跃增殖,亚汇合的2D培养皿收获细胞
  3. 将细胞胰蛋白酶化,在过量的完全生长培养基中洗涤,并通过 温和离心沉淀细胞。然后,将所得细胞沉淀物重悬浮并使其在完全生长培养基中恢复2小时
  4. 在此期间,用50μl100%Cultrex /孔包被96孔板,并允许在37℃凝胶。
    注意:Cultrex必须始终保持在冰上,以防止随着凝胶温度升至> 4℃时出现的基质凝固。
  5. 使用血细胞计数器计数细胞,并将其稀释至1,000细胞在150μl的完全生长培养基中,其补充有4%Cultrex。彻底混合细胞和培养基/4%Cultrex溶液,然后将细胞铺在固化的Cultrex垫子顶部。
    注意:4%Cultrex溶液不固化,因此,这些混合物中所含的细胞将容易附着于固化的Cultrex垫子,从而允许在整个实验中顶层培养基更换和/或替换。 br />
  6. 在铺板细胞之前,可以将其它化合物如生长因子或化学抑制剂与细胞和4%Cultrex溶液混合。每个实验条件应一式三份。
    注意:所有测试化合物必须首先通过短期暴露进行筛选,以验证这些试剂不直接影响CMV驱动的荧光素酶的表达和/或荧光素酶:荧光素反应的活性。
    />
  7. 在接种细胞两小时后,通过加入2μlD-荧光素(15mg/ml)获得初始时间零(T0)发光读数,轻轻敲打板的侧面混合。
    注意:在读板器中培养盖是打开的,因此,读板器必须保持清洁和消毒良好,并且可能位于层流罩中以防止不想要的细胞污染。此时无需更换培养基。
  8. 将培养物置于具有5%CO 2的37℃培养箱中
  9. 接种后4天,多细胞类器官将开始形成。获得第二个发光读数,如步骤7所述。之后,更换和刷新所有媒体和实验测试组件,非常小心不要破坏细胞和固化Cultrex坐垫。
    注意:在这一点上,可以启动治疗方案以监测针对已建立的类固醇的各种化疗的抗癌活性。
  10. 对于所有实验条件,在接种后第8和11天重复步骤7-9。
    注意:这些时间点可以根据所研究的细胞系的相对生长特性显着变化。因此,对于要研究的每个细胞系,需要根据经验确定纵向萤光素酶读数,给药方案和细胞铺板密度,以最大化信噪比。
  11. 使用Excel将每个发光值归一化为电镀后的初始T0值。

致谢

我们感谢Schiemann实验室的成员提出的有益的意见和建议。 生物发光3D-器官型纵向生长测定从Wendt等人(2011)和Wendt等人(2013)改编。 研究支持部分由国家卫生研究院提供给M.K.W. (CA166140),和W.P.S. (CA129359和CA177069)。

参考文献

  1. Wendt,M.K.,Schiemann,B.J.,Parvani,J.G.,Lee,Y.H.,Kang,Y。和Schiemann,W.P。(2013)。 TGF-β刺激Pyk2表达,作为乳腺转移性生长所需的上皮 - 间质转化程序的一部分 癌症。癌基因 32(16):2005-2015。
  2. Wendt,M.K.,Taylor,M.A.,Schiemann,B.J.and Schiemann,W.P。(2011)。 需要下调上皮钙粘蛋白以启动乳腺癌的转移性生长。 em> Mol Biol Cell 22(14):2423-2435。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wendt, M. K. and Schiemann, W. P. (2013). Longitudinal Bioluminescent Quantification of Three Dimensional Cell Growth. Bio-protocol 3(23): e993. DOI: 10.21769/BioProtoc.993.
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