三维球体制作一个高效，灵活细胞聚集方法
Summary
Here, we describe a rapid and flexible protocol for the formation of 3D cell spheroids through cell aggregation. This is easily adapted to multiple cell types and is suitable for use in a variety of applications including cell migration, invasion, or anoikis assays, and for imaging and quantifying cell-matrix interactions.
Abstract
单层细胞培养不充分模拟组织的体内行为，这涉及复杂的细胞-细胞和细胞-基质相互作用。三维（3D）细胞培养技术是最近开发用于解决贴壁细胞培养的缺点一个创新。而在体外产生组织类似物几种技术已经开发，这些方法往往是复杂，以建立价格昂贵，需要专门的设备，并且通常由兼容性只有某些细胞类型的限制。在这里，我们描述了用于凝集细胞成一致的大小是与多种肿瘤和正常细胞系的生长兼容的多细胞三维球状体一个快速和灵活的协议。我们利用不同浓度的血清和甲基纤维素（MC），以促进依赖贴壁球体的产生和防止在一个高度可再生的方式的细胞单层的形成。逐张为最佳条件idual细胞系可以通过在球体形成介质调整MC或血清浓度来实现。生成的3D球体可被收集用于在广泛的应用范围，包括细胞信号传导或基因表达研究中，候选药物筛选，或在细胞过程如肿瘤细胞侵袭和迁移的研究中使用。该协议也容易地适用于产生从单个细胞克隆的球状体，并可以适于评估贴壁依赖性生长和失巢凋亡抗性。总的来说，我们的协议提供了用于产生和利用为了概括组织的微环境的3D模型和正常和肿瘤细胞的体内生长三维细胞球状体的容易修改的方法。
Introduction
的肿瘤细胞的行为生物相关的评估是使用传统的二维（2D）细胞培养的方法，部分是因为这些不充分地反映在体内发现的细胞微环境挑战。结合细胞外基质成分进入文化（ 如 Boyden小室测定）的替代方法是体内组织的环境更生理代表。然而，它们可被限定在单个细胞行为的评估，并且不概括在有助于组织或肿瘤生长1，2，3细胞-基质和细胞-细胞相互作用的体内组合的复合物。
使用多细胞球体是最近的做法更准确地再现体内细胞生长1的结构紧凑，4。球状体可以用于研究正常细胞的细胞-基质相互作用，而且还可以作为肿瘤类似物肿瘤进展的特征，如转移性生长或药物抗性4建模。
球状体可以通过嵌入在基体5，或更快速的单细胞的增殖而形成，通过促进多种细胞的聚集，以形成一单细胞群（ 例如 ，悬滴，离心方法）6,7。现有的细胞聚集技术可能需要昂贵的材料或专门设备。此外，这些球状体具有宽范围的尺寸和形态，并且可以是难以生产大批量，使得生长条件或处理困难之间的比较。最后，通过这些方法产生的球状体可能难以从蛋白质extracel隔离细胞性矩阵，其中它们嵌入在其他应用中使用。
在这里，我们描述了用于快速形成使用市售U型底细胞斥板和惰性粘合促进基质，甲基纤维素一致大小细胞球体的一个强大的和易于修改细胞聚集方法。一旦形成，这些多细胞球状体可容易地分离出一种用于在广泛的应用中使用。该协议也容易适于产生通过细胞增殖，其可用于评估其他细胞过程球状体。这里，我们表明细胞侵袭测定法，免疫荧光染色定量，一个失巢凋亡测定中，由于这两种不同的球体形成的协议的应用实例。
Protocol
Representative Results
Discussion
我们提出了生产3D细胞球体使用廉价的和广泛使用的试剂在体内组织的架构模型快速，灵活的方法。我们的协议利用MC 8,9的非细胞毒性和粘合促进性质介导的细胞聚集和降低细胞单层的形成。不像从动物来源分离的基于蛋白质的基质，MC是惰性的，不包含生长因子，并且很容易通过洗涤除去，允许球状体在各种应用中使用的隔离无残余基质的存在?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
The authors thank M. Gordon of the Queen’s University Biomedical Imaging Centre for assistance. This work was supported by operating grants from the Cancer Research Society of Canada (19439) and the Canadian Institutes for Health Research (MOP-142303) (LMM), and by Ontario Graduate Scholarships and studentships from the Terry Fox Research Institute Training Program in Transdisciplinary Cancer Research (SMM, EYL), and by a Craig Jury Summer Studentship (SMM).
Materials
| Buffers | |||
| 10x Phosphate buffered saline | Thermo Fisher Scientific | AM9625 | |
| Calcium Chloride Solution | Sigma-Aldrich | 21114 | Used for PBS* wash buffer; Do not autoclave PBS* wash buffer upon addition of calcium chloride |
| Magnesium Chloride Solution | Sigma-Aldrich | M1028 | Used for PBS* wash buffer; Do not autoclave PBS* wash buffer upon addition of magnesium chloride |
| Name | Company | Catalog number | Comments |
| For Spheroid Formation | |||
| 96-well U-bottom Cell-Repellent Plate | Greiner Bio-One | 650970 | |
| Dulbecco's Modified Eagle's Medium | Sigma-Aldrich | D5546 | For culturing SH-SY5Y, PANC-1, TPC-1 cell lines |
| F12K Medium | Thermo Fisher Scientific | 2112722 | For culturing TT cell line |
| Fetal Bovine Serum | Sigma-Aldrich | F1051 | Filter prior to use to remove particulate contaminants |
| Methyl cellulose | Sigma-Aldrich | M7027 | Prepare in water to 100 mg/mL |
| Roswell Park Memorial Institute Medium | Sigma-Aldrich | R8758 | For culturing HCT-116, BxPC-3 cell lines |
| TrypLE Express | Thermo Fisher Scientific | 12605028 | Dissociation buffer |
| Name | Company | Catalog number | Comments |
| For Invasion Assay | |||
| Bovine Type I Collagen | Corning Incorporated | 354231 | Stock 3.1mg/ml; Maintain on ice when in use |
| DMEM Phenol Red Free Low Glucose | Thermo Fisher Scientific | 11054-20 | Less background fluorescence compared to Phenol Red supplemented medium |
| Glial Cell Line Derived Neurotrophic Factor | Peprotech | 450-10 | Chemoattractant |
| Name | Company | Catalog number | Comments |
| For Immunofluorescence Microscopy | |||
| #1.5 Coverglass | Electron Microscopy Sciences | 72225-01 | For mounting excised spheroids |
| Alexa-Fluor 488 Phalloidin | Thermo Fisher Scientific | A12379 | Used to stain actin at 1:200 |
| Bovine Serum Albumin | Bioshop Canada Incorporated | ALB001 | Used in BSA blocking buffer |
| Dabco 33-LV | Sigma-Aldrich | 290734 | Antifade |
| Glycerol | Bioshop Canada Incorporated | GLY001 | Used in MOWIOL mounting medium |
| ImageJ Software | Freeware, NIH | – | Used for image analysis |
| Microslides | VWR International | 48312-024 | For mounting excised spheroids |
| MOWIOL 4-88 | EMD-Millipore | 475904 | Used in MOWIOL mounting medium |
| Paraformaldehyde | EMD-Millipore | PX0055-3 | Used in fixation buffer |
| Triton X-100 | Bioshop Canada Incorporated | TRX777 | Used in permeabilization buffer |
References
- Zimmermann, M., Box, C., Eccles, S. A. Two-dimensional vs. three-dimensional in vitro tumor migration and invasion assays. Methods Mol Biol. 986 (986), 227-252 (2013).
- Albini, A., Noonan, D. M. The ‘chemoinvasion’ assay, 25 years and still going strong: the use of reconstituted basement membranes to study cell invasion and angiogenesis. Curr Opin Cell Biol. 22 (5), 677-689 (2010).
- Pampaloni, F., Reynaud, E. G., Stelzer, E. H. K. The third dimension bridges the gap between cell culture and live tissue. Nat. Rev. Mol. Cell Biol. 8 (10), 839-845 (2007).
- Fennema, E., Rivron, N., Rouwkema, J., van Blitterswijk, C., de Boer, J. Spheroid culture as a tool for creating 3D complex tissues. Trends Biotechnol. 31 (2), 108-115 (2013).
- Carletti, E., Motta, A., Migliaresi, C. Scaffolds for tissue engineering and 3D cell culture. Methods Mol Biol. 695, 17-39 (2011).
- Foty, R. A simple hanging drop cell culture protocol for generation of 3D spheroids. J Vis Exp. (51), (2011).
- Handschel, J. G., et al. Prospects of micromass culture technology in tissue engineering. Head Face Med. 3, 4 (2007).
- Stuckhoff, A. P., DelValle, L., Amini, S., White, M. K. . Neuronal Cell Culture Methods and Protocols Vol. 1078 Methods in Molecular Biology. 1078, 119-132 (2013).
- Stewart, G. J., Wang, Y., Niewiarowski, S. Methylcellulose protects the ability of anchorage-dependent cells to adhere following isolation and holding in suspension. Biotechniques. 19 (4), 598-604 (1995).
- Vinci, M., et al. Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation. BMC Biol. 10, 29 (2012).
- Nagelkerke, A., Bussink, J., Sweep, F. C. G. J., Span, P. N. Generation of multicellular tumor spheroids of breast cancer cells: How to go three-dimensional. Anal. Biochem. 437 (1), 17-19 (2013).
