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Generation of Tumour-stroma Minispheroids for Drug Efficacy Testing
用于检测药物疗效的肿瘤基质小球体的生成   

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参见作者原研究论文

本实验方案简略版
Oncogene
Mar 2016

Abstract

The three-dimensional organisation of cells in a tissue and their interaction with adjacent cells and extracellular matrix is a key determinant of cellular responses, including how tumour cells respond to stress conditions or therapeutic drugs (Elliott and Yuan, 2011). In vivo, tumour cells are embedded in a stroma formed primarily by fibroblasts that produce an extracellular matrix and enwoven with blood vessels. The 3D mixed cell type spheroid model described here incorporates these key features of the tissue microenvironment that in vivo tumours exist in; namely the three-dimensional organisation, the most abundant stromal cell types (fibroblasts and endothelial cells), and extracellular matrix. This method combined with confocal microscopy can be a powerful tool to carry out drug sensitivity, angiogenesis and cell migration/invasion assays of different tumour types.

Keywords: Mixed cell type 3-dimensional (3D) culture (混合细胞类型三维(3D)培养), Tumour sphere (肿瘤球), Breast cancer (乳腺癌), TRAIL (TRAIL), Drug resistance (耐药性)

Background

The traditional monolayer cell culture (2-dimensional) enforces an artificial environment, which is vastly different from the tissues cells exists in vivo. One of the most critical differences is that in monolayer cultures the cells are polarised, i.e., the surface of the cells facing the culture-plastic and the upper cell surface exposed to the culture medium receive completely different, often opposing signals (Fitzgerald et al., 2015). To address the problem of cell polarization, tumour spheroid cultures are increasingly used in cancer research. Tumour spheroids can replicate the 3-dimensional cell-cell interactions present in a tissue and to some extent paracrine signaling via cytokines and chemokines by reducing their diffusion and dilution by the growth medium that typically occurs in monolayer cultures (Lawlor et al., 2002; Barrera-Rodríguez and Fuentes, 2015). The current tumour-stroma minispheroid protocol is one such method. Compared to the other tumour-spheroid protocols, this method also incorporates additional, key features of the tissue environment, namely stromal cells and extracellular matrix in the spheroid and thus provides a model that replicates the in vivo tumour microenvironment more faithfully.

Materials and Reagents

  1. 96 U-shaped well plate for suspension cells (Greiner Bio One, catalog number: 650161 )
  2. FiltopurTM syringe filters (SARSTEDT, catalog number: 83.1826.001 )
  3. 50 ml syringes (TERUMO, catalog number: SS+50ES )
  4. 12 well dishes with 10 mm diameter glass bottom (MATTEK, catalog number: P12G-0-10-F )
  5. 1.5 ml sterile Eppendorf tubes (SARSTEDT, catalog number: 72.690.001 )
  6. 50 ml sterile centrifuge tubes (Corning, catalog number: 430829 )
  7. 35 mm glass bottom dish, 14 mm diameter (MATTEK, catalog number: P35G-0.170-14-C )
  8. T75 flasks for adherent cells (SARSTEDT, catalog number: 83.3911 )
  9. Serological pipettes (5 ml, 10 ml) (CORNING, catalog numbers: 4051 and 4101 , respectively)
  10. Pipette Tips (10 μl, 200 μl, 1,000 μl) (SARSTEDT, catalog numbers: 70.1130.100 , 70.760.002 and 70.762.100 , respectively)
  11. Cell lines: MDA-MB-231 breast cancer epithelial cells (ATCC, HTB-26TM, catalog number: MDA-MB-231); human umbilical vein endothelial cells (HUVEC) (ATCC, CRL-1730TM, catalog number: HUV-EC-C ); normal human dermal fibroblasts (NHDF) (Lonza, catalog number: CC-2509 )
  12. Recombinant human tumour necrosis factor-related apoptosis-inducing ligand (rhTRAIL) (purified in-house), receptor-selective TRAIL mutant, TRAIL-45 (O’Leary et al., 2016; van der Sloot et al., 2006)
  13. Dulbecco’s modified Eagle medium (DMEM)-low glucose concentration (Sigma-Aldrich, catalog number: D6046 )
  14. Fetal bovine serum (Sigma-Aldrich, catalog number: F7524 )
  15. L-glutamine solution, 200 Mm stock (Sigma-Aldrich, catalog number: G7513 )
  16. 1x trypsin-EDTA buffer in HBSS
  17. CellTrackerTM CM-Dil Dye (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: C7001 ) or CMTPX red cell tracker dye (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: C34552 )
  18. Rat tail collagen type I (Corning, catalog number: 354236 )
  19. Hoechst33342 – 10 mg/ml solution in water (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: H3570 )
  20. SYTOX Green nucleic acid dye (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: S7020 )
  21. Endothelial cell growth medium-2 (EGM-2) prepared by adding EGMTM-2 SingleQuotsTM Kit (Lonza, catalog number: CC-4176 ) to EBM-2 basal Medium (Lonza, catalog number: CC-3156 )
  22. Hanks’ balanced salt solution (HBSS) (Thermo Fisher Scientific, GibcoTM catalog number: 24020117 )
  23. 1 N NaOH solution
  24. Methylcellulose solution (see Recipes)

Equipment

  1. HeraeusTM MegafugeTM centrifuge (15 ml, 50 ml tube) (Thermo Fisher Scientific, Thermo ScientificTM, model: 16 Centrifuge Series )
  2. Mammalian cell culture incubator (37 °C, 5% CO2) (Thermo Fisher Scientific, Thermo ScientificTM, model: FormaTM Steri-CycleTM )
  3. Hemocytometer
  4. Pipettes (10 μl, 200 μl, 1,000 μl)
  5. Pipette aid
  6. Confocal microscopy system (AndorTM, Revolution Spinning Disk Confocal systemTM)
    1. High-resolution EMCCD camera (Andor iXon EM+)
    2. Olympus IX81 motorised inverted microscope, fitted with a variable temperature/CO2 humidified incubation chamber for live cell experiments
    3. Yokagawa CSU22 spinning disk confocal unit
  7. Magnetic stirrer
  8. Orbital shaker

Software

  1. VolocityTM software (PerkinElmer)

Procedure

  1. Production of multicellular, mixed cell type spheroids
    1. Culture MDA-MB-231 cells, HUVEC cells in EGM-2 medium and NHDF in low-glucose DMEM supplemented with 10% fetal calf serum and 2 mM L-glutamine in T-75 flasks to reach near confluency.
    2. Harvest MDA-MB-231 cells by trypsinization. Collect 1 x 105 cells by centrifuging at 300 x g for 5 min and resuspend them in 0.1 ml fresh growth medium (obtaining a cell concentration of 1 x 106 cells/ml).
    3. Label MDA-MB-231 cells with the cell tracker dye CM-Dil or CMTPX by incubating the 0.1 ml of cell suspension with 2 μM of either of the two dyes for 30 min at 37 °C in the dark, shaking every 5 to 10 min.
      Note: Depending on the needs of the specific assay, you may leave the tumour cells unstained and label the non-malignant cell components using the same procedure.
    4. Harvest HUVEC and NHDF cells by trypsinisation. Collect 1 x 105 HUVEC cells and 0.5 x 105 NHDF cells by centrifuging them at 300 x g for 5 min and resuspend the cell pellets in 0.2 ml fresh growth medium (obtaining a cell concentration of 0.5 x 106 and 0.25 x 106 cells/ml, respectively).
    5. Add 150 μl of HUVEC cells (7.5 x 104 cells), 150 μl of NHDF (3.75 x 104 cells) and 75 μl of MDA-MB-231 cells (7.5 x 104 cells) into 15 ml of EGM-2 medium containing 20% methylcellulose solution (see Recipes).
    6. Add 150 μl of the above solution to each well of a 96 U-shaped well suspension plate and incubate for 24 h at 37 °C to allow for spheroid formation.
      Note: Figures 1A and 1B show the transmission light microscopic image of the single cell suspension in the U-shape well and the forming sphere (with still primarily round-shaped cells) 24 h after seeding the cells.


      Figure 1. Formation and morphology of mixed cell type tumour minispheres. A, B and D. Transmission light microscopic images of (A) the cell constituents of the tumour sphere immediately after seeding in U-shaped wells, (B) assembled tumour sphere after 24 h of incubation in U-shaped well, and (D) fully formed tumour sphere embedded in collagen matrix at 72 h after seeding. C. Collection of formed spheres from U-shaped wells using a 5 ml serological pipette. 
      Note: The pipette is gently placed to the bottom of the well and the content collected. The pipette is then moved to the next well without releasing the collected medium. The medium with the tumour spheres collected in the pipette from several wells is then transferred into a 15 ml centrifuge tube (not shown). 

  2. Drug efficacy testing on multicellular tumour spheroids
    1. Prepare a 1.5 mg/ml collagen type-I stock solution by diluting rat tail collagen type-I into an appropriate volume of EGM-2 medium. Neutralize the pH to 7.0 by drop-wise addition of 1 N NaOH. Filter-sterilise the final solution using a syringe and syringe filter. Prepare this solution fresh each time.
    2. Add 100 μl of collagen stock solution to the bottom of wells of a glass-bottom 12 well dish which has been previously warmed to 37 °C in the incubator. Incubate dish at 37 °C for 30 min to allow the collagen gel to set.
    3. At the end of the 24 h incubation, gently harvest the formed spheroids from the 96 U-shaped well plate into a 15 ml centrifuge tube using a 5 ml or a 10 ml serological pipette and collect the spheroids by centrifuging the solution at 300 x g for 5 min.
      Note: An image of the harvesting step is shown in Figure 1C. Typically, one sphere forms per well, though it may vary for different cell types.
    4. Remove the medium, taking care not to disturb the spheroid pellet. Add 1,400 μl freshly prepared 1.5 mg/ml collagen stock solution and carefully resuspend the spheroid pellet by tapping the bottom of the tube.
    5. Add 100 μl of spheroid suspension on top of each collagen gel in the 12 well plate to generate between 5-8 spheroids per well. Place the plate in incubator at 37 °C for 1 h to allow this second collagen gel layer to set.
      Note: It may take a bit longer for the gel to set. Proceed with the protocol only after the gel has set, but not later than 2.5 h.
    6. Add 1.5 ml of EGM-2 medium to each well and incubate the spheroids for 24-48 h.
      Note: A transmission light microscopic image of the fully formed spheroid embedded in collagen is shown in Figure 1D.
    7. Treat cells with a drug/drugs of choice at appropriate concentration.
    8. 2 h before analysis, add 1 μg/ml Hoechst33342 nuclear dye and SYTOX Green viability dye at a final concentration of 1 μM to the media and complete the incubation time at 37 °C (for 2 h).
    9. Analyse induction of cell death by monitoring the number of SYTOX Green positive cells both in the CM-DiI/CMTPX-labelled tumour cell population and the CM-DiI/CMTPX-negative non-malignant cellular components using confocal microscopy (e.g., Taking images in 0.5 μm Z-stack planes without fixing the spheroids beforehand).

Data analysis

This protocol describes the generation of tumour spheres that can be used to assess efficacy of cytotoxic, anti-angiogenic, cytostatic as well as other drugs. Data analysis and statistics depends on the downstream applications, such as methods of detection of cell death. The above example we show is for the assessment of cytotoxicity based on microscopic detection of dying cells. The steps of the analysis are summarized below:

  1. 40 μl of the collagen-I solution was added in 35 mm Petri dishes with 14 mm glass slide bottom for microscopy (MatTek Corporation), which were previously warmed to 37 °C.
  2. The dishes were then incubated at 37 °C for 45 min to allow for the collagen gel to set.
  3. 4-6 spheroids harvested from the U-shape 96-well plates in a volume of 50 µl collagen-I were added in each dish on top of the collagen gel.
  4. The dishes were placed in the incubator at 37 °C for an additional 1-2 h to allow the second collagen gel embedding the tumour spheres to set.
  5. 2 ml of EGM-2 medium was then added to each well and the spheres were incubated for 48 h.
  6. The spheres were treated with a receptor-selective mutant of the death ligand cytokine, TRAIL, named TRAIL45a at a concentration of 250 ng/ml for 24 h.
  7. 2 h before analysis 1 µg/ml of Hoechst33342 and 1 µM of SYTOX Green was added to the culture media to label nuclei and dying cells, respectively.
  8. Induction of cell death was determined by counting the SYTOX Green positive dying/dead cells with confocal microscopy without any processing of the spheres. The steps of the detection are detailed below.
    Note: Images of the spheres were taken in situ, in the glass-bottom dishes without digestion of the collagen matrix, enzymatic (or other) dissociation of the spheres or fixation with formaldehyde or other fixatives.
  9. Images were taken from unfixed spheres using an AndorTM Revolution Spinning Disk Confocal systemTM with the following components: Yokagawa CSU22 spinning disk confocal system, four solid state laser lines: 405 nm, 488 nm, 561 nm and 640 nm; high-resolution EMCCD camera (Andor iXon EM+), Olympus IX81 motorised inverted microscope fitted with a variable temperature/CO2 humidified incubation chamber for live cell experiments. The following filters were used for the detection of the fluorescent signals: 360-390 nm/420-460 nm excitation/emission filter for Hoecsht33342, 470-495 nm/510-550 nm excitation/emission filter for SYTOX Green and 540-550 nm/575-625 nm excitation/emission filter for CMTPX.
  10. Z-stack images of the middle section of the spheroids were taken as 0.5 μm slices at an overall magnification of 600x (with a 60x immersion oil objective), which were then used to generate the 3D composite images shown in Figure 2 with the VolocityTM software (Perkin-Elmer).

Representative data



Figure 2. Detection of drug efficacy in mixed cell type tumour minispheres. 3D reconstituted confocal microscopic image of the middle 10 μm section of an (A) untreated tumour minisphere and (B) a sphere treated with an engineered, receptor-specific mutant of recombinant human TRAIL (DR4 and DR5-bispecific; TRAIL-45a). Blue: all nuclei (Hoechst33342), red: non-malignant cells: HUVEC and NHDF (CMTPX), green: dead cells (SYTOX Green). The scale bar represents 10 μM. Tumour minispheres were grown for 48 h before exposure to 250 ng/ml of TRAIL-45a for 24 h. The treated samples were stained with Hoechst33342 and SYTOX Green for the final 2 h of the treatment. Images were taken from unfixed samples using an AndorTM Revolution Spinning Disk Confocal systemTM with images taken as 0.5 μm Z-stacks at 600x magnification.

Notes

Tumour cell types other than MDA-MB-231 cells may be used, but their ability/tendency to form mixed-cell type spheroids can vary significantly and thus requires testing. Other cell types used commonly in minispheroid generation include MCF-7 and BT474 human breast cancer cells (Monazzam et al., 2006). The generation of these single-cell type spheroids uses a similar method substituting the collagen solution for agarose. Other mixed cell type spheroids involve the use of colonic adenocarcinoma cell lines, namely COLO320HSR and SNU-C1 (Park et al., 2016) mixed with fibroblasts. This protocol in particular used rotating conditions via orbital shaker to induce the generation of spheroids.
The spheroids described in this protocol can also be used to monitor angiogenesis (Correa de Sampaio et al., 2012) or cell migration/invasion.

Recipes

  1. Methylcellulose solution
    1. Autoclave 6 g of methylcellulose in a 500 ml flask with a magnetic stirrer
    2. Add 250 ml of EGM-2 medium previously heated to 60 °C to the autoclaved methylcellulose 
    3. Stir to facilitate dissolving the methylcellulose for 20 min
    4. Add an additional 250 ml of EGM-2 medium warmed to room temperature
    5. Stir at 4 °C for 2 h to fully dissolve methylcellulose
    6. Centrifuge the final solution at 5,000 x g for 2 h at room temperature to remove undissolved methylcellulose
    7. Transfer the supernatant into a clean bottle

Acknowledgments

Research in the ES laboratory is supported by Science Foundation Ireland and the Irish Cancer Society (BCNI, 14/ICS/B3042). The authors thank Prof. Gillian Murphy (Cambridge University) for sharing their minitumour protocol that this current protocol is based on.

References

  1. Barrera-Rodríguez, R. and Fuentes, J. M. (2015). Multidrug resistance characterization in multicellular tumour spheroids from two human lung cancer cell lines. Cancer Cell Int 15: 47.
  2. Correa de Sampaio, P., Auslaender, D., Krubasik, D., Failla, A. V., Skepper, J. N., Murphy, G. and English, W. R. (2012). A heterogeneous in vitro three dimensional model of tumour-stroma interactions regulating sprouting angiogenesis. PLoS One 7(2): e30753.
  3. Elliott, N. T. and Yuan, F. (2011). A review of three-dimensional in vitro tissue models for drug discovery and transport studies. J Pharm Sci 100(1): 59-74.
  4. Fitzgerald, K. A., Malhotra, M., Curtin, C. M., O'Brien, F. J. and O’Driscoll, C. M. (2015). Life in 3D is never flat: 3D models to optimise drug delivery. J Controll Release 215: 39-54.
  5. Lawlor, E. R., Scheel, C., Irving, J. and Sorensen, P. H. (2002). Anchorage-independent multi-cellular spheroids as an in vitro model of growth signaling in Ewing tumors. Oncogene 21(2): 307-318.
  6. O’Leary, L., van der Sloot, A. M., Reis, C. R., Deegan, S., Ryan, A. E., Dhami, S. P., Murillo, L. S., Cool, R. H., Correa de Sampaio, P., Thompson, K., Murphy, G., Quax, W. J., Serrano, L., Samali, A. and Szegezdi, E. (2016). Decoy receptors block TRAIL sensitivity at a supracellular level: the role of stromal cells in controlling tumour TRAIL sensitivity. Oncogene 35(10): 1261-1270.
  7. van der Sloot, A. M., Tur, V., Szegezdi, E., Mullally, M. M., Cool, R. H., Samali, A., Serrano, L. and Quax, W. J. (2006). Designed tumor necrosis factor-related apoptosis-inducing ligand variants initiating apoptosis exclusively via the DR5 receptor. Proc Natl Acad Sci U S A 103(23): 8634-8639.
  8. Monazzam, A., Razifar, P., Simonsson, M., Qvarnstrom, F., Josephsson, R., Blomqvist, C., Langstrom, B. and Bergstrom, M. (2006). Multicellular tumour spheroid as a model for evaluation of [18F]FDG as biomarker for breast cancer treatment monitoring. Cancer Cell Int 6: 6.
  9. Park, J. I., Lee, J., Kwon, J. L., Park, H. B., Lee, S. Y., Kim, J. Y., Sung, J., Kim, J. M., Song, K. S. and Kim, K. H. (2016). Scaffold-free coculture spheroids of human colonic adenocarcinoma cells and normal colonic fibroblasts promote tumorigenicity in nude mice. Transl Oncol 9(1): 79-88.

简介

组织中细胞的三维组织及其与相邻细胞和细胞外基质的相互作用是细胞反应的关键决定因素,包括肿瘤细胞如何对应激条件或治疗药物的反应(Elliott和Yuan,2011)。在体内,肿瘤细胞被包埋在主要由成纤维细胞形成的基质中,所述成纤维细胞产生细胞外基质并用血管编织。这里描述的3D混合细胞类型球体模型包括了体内存在肿瘤的组织微环境的这些关键特征;即三维组织,最丰富的基质细胞类型(成纤维细胞和内皮细胞)和细胞外基质。该方法结合共聚焦显微镜可以成为不同肿瘤类型的药物敏感性,血管生成和细胞迁移/侵袭测定的有力工具。

背景 传统的单层细胞培养(二维)强化人造环境,其与体内存在的组织细胞大不相同。最重要的区别之一是在单层培养物中,细胞是极化的,即,面向培养物的细胞表面和暴露于培养基的上细胞表面完全不同,经常反对的信号(Fitzgerald等人,2015)。为了解决细胞极化的问题,肿瘤球状体培养越来越多地用于癌症研究。肿瘤球体可以通过减少其通常发生在单层培养物中的生长培养基的扩散和稀释,通过细胞因子和趋化因子复制存在于组织中的三维细胞 - 细胞相互作用和一定程度的旁分泌信号传导(Lawlor等,2002; Barrera-Rodríguez和Fuentes,2015)。目前的肿瘤基质微粒子方案是一种这样的方法。与其他肿瘤球体方案相比,该方法还包含组织环境的另外的关键特征,即球状体中的基质细胞和细胞外基质,因此提供了复制体内肿瘤微环境的模型更忠诚

关键字:混合细胞类型三维(3D)培养, 肿瘤球, 乳腺癌, TRAIL, 耐药性

材料和试剂

  1. 96型悬浮细胞U形孔板(Greiner Bio One,目录号:650161)
  2. Filtopur TM注射器过滤器(SARSTEDT,目录号:83.1826.001)
  3. 50 ml注射器(TERUMO,目录号:SS + 50ES)
  4. 12孔直径为10mm的玻璃底部(MATTEK,目录号:P12G-0-10-F)
  5. 1.5ml无菌Eppendorf管(SARSTEDT,目录号:72.690.001)
  6. 50ml无菌离心管(Corning,目录号:430829)
  7. 35毫米玻璃底盘,直径14毫米(MATTEK,目录号:P35G-0.170-14-C)
  8. T75烧瓶用于贴壁细胞(SARSTEDT,目录号:83.3911)
  9. 血清移液管(5ml,10ml)(CORNING,目录号:4051和4101)
  10. 移液管贴(10μl,200μl,1,000μl)(SARSTEDT,目录号:70.1130.100,70.760.002和70.762.100)
  11. 细胞系:MDA-MB-231乳腺癌上皮细胞(ATCC,HTB-26,目录号:MDA-MB-231);人脐静脉内皮细胞(HUVEC)(ATCC,CRL-1730 ,目录号:HUV-EC-C);正常人皮肤成纤维细胞(NHDF)(Lonza,目录号:CC-2509)
  12. 重组人肿瘤坏死因子相关凋亡诱导配体(rhTRAIL)(内部纯化),受体选择性TRAIL突变体TRAIL-45(O'Leary等人,2016; van der Sloot 等人,2006)
  13. Dulbecco改良Eagle培养基(DMEM) - 低浓度葡萄糖(Sigma-Aldrich,目录号:D6046)
  14. 胎牛血清(Sigma-Aldrich,目录号:F7524)
  15. L-谷氨酰胺溶液,200μm储备液(Sigma-Aldrich,目录号:G7513)
  16. 在HBSS中1倍胰蛋白酶-EDTA缓冲液
  17. CellTracker CM-Dil Dye(Thermo Fisher Scientific,Molecular Probes TM,目录号:C7001)或CMTPX红细胞跟踪染料(Thermo Fisher Scientific,Molecular Probes TM ,目录号:C34552)
  18. 大鼠尾巴胶原I型(康宁,目录号:354236)
  19. Hoechst33342-10mg/ml水溶液(Thermo Fisher Scientific,Molecular Probes TM,目录号:H3570)
  20. SYTOX绿色核酸染料(Thermo Fisher Scientific,Molecular Probes TM,目录号:S7020)
  21. 通过将EBM-2基础物质加入到EGM TM2单克隆试剂盒(Lonza,目录号:CC-4176)中制备的内皮细胞生长培养基-2(EGM-2)中等(Lonza,目录号:CC-3156)
  22. Hanks的平衡盐溶液(HBSS)(Thermo Fisher Scientific,Gibco TM目录号:24020117)
  23. 1N NaOH溶液
  24. 甲基纤维素溶液(见食谱)

设备

  1. Heraeus TM/sup> Megafuge TM离心机(15ml,50ml管)(Thermo Fisher Scientific,Thermo Scientific TM,型号:16离心机系列) br />
  2. 哺乳动物细胞培养箱(37℃,5%CO 2)(Thermo Fisher Scientific,Thermo Scientific TM,型号:Forma< S< S<循环 TM
  3. 血细胞计数器
  4. 移液管(10μl,200μl,1000μl)
  5. 移液管辅助装置
  6. 共焦显微镜系统(Andor TM ,革命旋转盘共焦系统 TM
    1. 高分辨率EMCCD相机(Andor iXon EM +)
    2. 奥林巴斯IX81电动倒置显微镜,配有可变温度/CO 2加湿孵化室,用于活细胞实验
    3. 横河CSU22旋转盘共焦单元
  7. 磁力搅拌器
  8. 轨道摇床

软件

  1. Volocity TM 软件(PerkinElmer)

程序

  1. 多细胞混合细胞型球体的生产
    1. 培养MDA-MB-231细胞,EGM-2培养基中的HUVEC细胞和在T-75烧瓶中补充有10%胎牛血清和2mM L-谷氨酰胺的低葡萄糖DMEM中的NHDF以达到接近汇合。
    2. 通过胰蛋白酶消化获得MDA-MB-231细胞。通过在300×g离心5分钟收集1×10 5个细胞,并将其重新悬浮在0.1ml新鲜生长培养基中(获得1×10 6个细胞浓度 cells/ml)。
    3. 使用细胞跟踪剂染料CM-Dil或CMTPX标记MDA-MB-231细胞,通过在暗处37℃下将0.1ml细胞悬浮液与2μM两种染料中的任一种孵育30分钟,每5至10次振荡min。
      注意:根据具体检测的需要,您可能会使肿瘤细胞未染色,并使用相同的程序标记非恶性细胞成分。

    4. 通过胰蛋白酶消化获得HUVEC和NHDF细胞。通过以300×g离心5分钟收集1×10 5个HUVEC细胞和0.5×10 5个NHDF细胞,并将细胞沉淀重新悬浮在0.2ml新鲜生长培养基(分别获得0.5×10 6个细胞/ml和0.25×10 6个细胞/ml)。
    5. 加入150μl的HUVEC细胞(7.5×10 4个细胞),150μl的NHDF(3.75×10 4个细胞)和75μl的MDA-MB-231细胞( 7.5×10 4个细胞)加入到含有20%甲基纤维素溶液的15ml EGM-2培养基中(参见食谱)。
    6. 向96 U形孔悬浮板的每个孔中加入150μl上述溶液,并在37℃下孵育24小时以允许球形成。
      注意:图1A和1B显示了播种细胞24小时后U形孔和成球(仍然主要是圆形细胞)的单细胞悬浮液的透射光学显微镜图像。


      图1.混合细胞型肿瘤微球的形成和形态.A,B和D.(A)在U型孔中接种之后(A)肿瘤球的细胞成分的透射光学显微镜图像, (B)在U形孔中孵育24小时后组装肿瘤球,以及(D)播种后72小时,完全形成的包埋在胶原基质中的肿瘤球。 C.使用5ml血清学移液管从U形孔收集成形的球体。 
      注意:将移液管轻轻地放在井的底部并收集内容物。然后将移液管移至下一个孔,而不释放收集的介质。将来自几个孔的移液管中收集的肿瘤球的培养基转移到15ml离心管中(未显示)。

  2. 多细胞肿瘤球状体的药物效力检测
    1. 通过将大鼠尾部胶原I型稀释成适当体积的EGM-2培养基来制备1.5mg/ml胶原I型原液。通过逐滴加入1N NaOH将pH中和至7.0。使用注射器和注射器过滤器对最终溶液进行过滤消毒。每次准备这个解决方案。
    2. 在玻璃底部12孔培养皿的孔的底部加入100μl胶原蛋白原液,其先前在培养箱中温热至37℃。在37℃下孵育30分钟,使胶原蛋白胶凝固。
    3. 在24小时孵育结束时,使用5ml或10ml血清移液管从96 U形孔板轻轻收获形成的球体至15ml离心管中,并通过在300℃离心溶液收集球体> xg 5分钟。
      注意:收集步骤的图像如图1C所示。通常,每个孔形成一个球体,尽管它可能因不同的细胞类型而异。
    4. 取出培养基,小心不要打扰球状颗粒。加入1,400μl新鲜制备的1.5mg/ml胶原蛋白原液,并通过敲击管的底部小心地重新悬浮球状颗粒。
    5. 在12孔板中的每个胶原凝胶上方加入100μl球状体悬浮液,每孔产生5-8个球体。将板置于37℃的培养箱中1小时,以使第二个胶原凝胶层固化。
      注意:凝胶可能需要更长时间才能设置。凝胶设定完毕后不得迟于2.5小时。
    6. 向每个孔中加入1.5ml EGM-2培养基,孵育球体24-48h。
      注意:嵌入胶原蛋白的完全形成的球体的透射光显微镜图像如图1D所示。
    7. 用适当浓度的药物/药物治疗细胞。
    8. 在分析前2小时,向培养基中加入终浓度为1μM的1μg/ml Hoechst33342核染料和SYTOX Green活性染料,并在37℃(2小时)下完成孵育时间。
    9. 通过使用共聚焦显微镜监测在CM-DiI/CMTPX标记的肿瘤细胞群体和CM-DiI/CMTPX阴性的非恶性细胞组分中监测SYTOX Green阳性细胞数目来分析细胞死亡的诱导(例如。在0.5μmZ-堆叠平面中拍摄图像,而不事先固定球体)。

数据分析

该方案描述了可用于评估细胞毒性,抗血管生成,细胞抑制以及其他药物功效的肿瘤球的产生。数据分析和统计取决于下游应用,如检测细胞死亡的方法。我们显示的上述例子是基于对死亡细胞的显微检测来评估细胞毒性。分析步骤总结如下:

  1. 将40μl胶原-I溶液加入到具有用于显微镜(MatTek Corporation)的14mm载玻片底板的35mm培养皿中,其预先温热至37℃。
  2. 然后将培养皿在37℃下孵育45分钟以使胶原凝胶凝固。
  3. 将从体积为50μl胶原-I的U形96孔板收获的4-6个球状体加入胶原凝胶顶部的每个培养皿中。
  4. 将培养皿置于37℃的培养箱中1-2小时以使第二胶原凝胶包埋肿瘤球。
  5. 然后将2ml EGM-2培养基加入每个孔中,将球体孵育48小时。
  6. 用250ng/ml浓度的命名为TRAIL45a的死亡配体细胞因子TRAIL的受体选择性突变体处理球24小时。
  7. 分析前2小时,将1μg/ml Hoechst33342和1μMSYTOX Green分别加入到培养基中以标记细胞核和染色细胞。
  8. 通过用共聚焦显微镜计数SYTOX Green阳性染色/死细胞,无任何球体处理,来测定细胞死亡的诱导。检测步骤如下:
    注意:球体的图像在玻璃底部的盘子中进行,不消化胶原基质,酶(或其他)解离球体或用甲醛或其他固定剂固定。 />
  9. 使用具有以下组件的Andor TM旋转旋转盘共焦系统从未固定的球体拍摄图像:Yokagawa CSU22旋转盘共焦系统,四个固态激光线:405nm ,488nm,561nm和640nm;高分辨率EMCCD相机(Andor iXon EM +),奥林巴斯IX81电动倒置显微镜,配有可变温度/CO 2加湿孵化室,用于活细胞实验。使用以下滤光片检测荧光信号:用于Hoecsht33342的360-390nm/420-460nm激发/发射滤光器,用于SYTOX Green的470-495nm/510-550nm激发/发射滤光片和540-550nm/575-625 nm激发/发射滤光片用于CMTPX。
  10. 将球形中间部分的Z-叠层图像以600x的总放大倍率(具有60x浸油目标)取为0.5μm切片,然后将其用于产生图2所示的3D复合图像,其中Volocity < sup> TM 软件(Perkin-Elmer)。

代表数据



图2.混合细胞型肿瘤微球中药物功效的检测
(A)未处理的肿瘤微球的中间10μm部分的3D重构共聚焦显微镜图像,(B)用工程化的受体 - 重组人TRAIL(DR4和DR5-双特异性; TRAIL-45a)的特异性突变体。蓝色:所有核(Hoechst33342),红色:非恶性细胞:HUVEC和NHDF(CMTPX),绿色:死细胞(SYTOX Green)。比例尺表示10μM。在暴露于250ng/ml TRAIL-45a 24小时之前,将肿瘤小球生长48小时。处理的样品用Hoechst33342和SYTOX Green染色处理的最后2小时。使用Andor TM Revolution旋转圆盘共焦系统 TM 从未固定的样品中取出图像,其中以600x放大倍率获取为0.5μmZ-堆叠的图像。

笔记

可以使用除MDA-MB-231细胞之外的肿瘤细胞类型,但是它们形成混合细胞型球体的能力/倾向可以显着变化,因此需要测试。通常用于微粒生成的其它细胞类型包括MCF-7和BT474人类乳腺癌细胞(Monazzam等人,2006)。这些单细胞类型球体的产生使用类似的方法将胶原溶液替代为琼脂糖。其他混合细胞型球体涉及使用与成纤维细胞混合的结肠腺癌细胞系,即COLO320HSR和SNU-C1(Park等人,2016)。该协议特别是通过轨道振荡器使用旋转条件来诱导球体的产生。
本协议中描述的球体也可用于监测血管发生(Correa de Sampaio et al。,2012)或细胞迁移/侵袭。

食谱

  1. 甲基纤维素溶液
    1. 在具有磁力搅拌器的500ml烧瓶中高压灭菌6g甲基纤维素
    2. 加入预先加热至60°C的250ml EGM-2培养基至高压灭菌甲基纤维素的
    3. 搅拌以促进甲基纤维素溶解20分钟
    4. 加入另外250毫升EGM-2培养基温至室温
    5. 在4℃下搅拌2小时以完全溶解甲基纤维素
    6. 在室温下将最终的溶液以5,000×g离心2小时以除去未溶解的甲基纤维素。
    7. 将上清液转移到干净的瓶子中

致谢

ES实验室的研究由爱尔兰科学基金会和爱尔兰癌症协会(BCNI,14/ICS/B3042)提供支持。作者感谢吉利安·墨菲(剑桥大学)教授分享了目前协议所基于的小型协议。

参考文献

  1. Barrera-Rodríguez,R.和Fuentes,JM(2015)。来自两个人肺癌细胞系的多细胞肿瘤球体中的多药耐药性表征。癌细胞Int 15:47.
  2. Correa de Sampaio,P.,Auslaender,D.,Krubasik,D.,Failla,AV,Skepper,JN,Murphy,G. and English,WR(2012)。  体外不均匀的肿瘤 - 基质相互作用的三维模型调节发芽血管生成 a> PLoS One 7(2):e30753。
  3. 新西兰Elliott和Yuan,F.(2011)。关于药物发现和运输研究的三维体外组织模型的综述.J Pharm Sci 100(1):59-74。 >
  4. Fitzgerald,KA,Malhotra,M.,Curtin,CM,O'Brien,FJ和O'Driscoll,CM(2015)。< a class ="ke-insertfile"href ="http://www.sciencedirect。 com/science/article/pii/S016836591530033X"target ="_ blank"> 3D中的生命永远不会平坦:3D模型来优化药物传递。控制发布 215:39-54。
  5. Lawlor,ER,Scheel,C.,Irving,J.and Sorensen,PH(2002)。  设计的肿瘤坏死因子相关凋亡诱导配体变体仅通过DR5受体启动凋亡。美国Proc Natl Acad Sci USA 103(23):8634-8639。
  6. Monazzam,A.,Razifar,P.,Simonsson,M.,Qvarnstrom,F.,Josephsson,R.,Blomqvist,C.,Langstrom,B.and Bergstrom,M。(2006)。< a class = ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/16556298"target ="_ blank">多细胞肿瘤球状体作为评估[18F] FDG作为乳腺癌治疗生物标志物的模型监测。癌症细胞Int 6:6.
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引用:Watters, M. and Szegezdi, E. (2017). Generation of Tumour-stroma Minispheroids for Drug Efficacy Testing. Bio-protocol 7(1): e2091. DOI: 10.21769/BioProtoc.2091.
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