癌症细胞球体机器人生产与药物测试的双水相系统
Summary
A protocol for robotic printing of cancer cell spheroids in a high throughput 96-well plate format using an aqueous two-phase system is presented.
Abstract
Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing both mono-culture and co-culture spheroids in a high throughput 96-well plate format is described in this work. This approach utilizes an aqueous two-phase system to confine cells into a drop of the denser aqueous phase immersed within the second aqueous phase. The drop rests on the well surface and keeps cells in close proximity to form a single spheroid. This technology has been adapted to a robotic liquid handler to produce size-controlled spheroids and expedite the process of spheroid production for compound screening applications. Spheroids treated with a clinically-used drug show reduced cell viability with increase in the drug dose. The use of a standard micro-well plate for spheroid generation makes it straightforward to analyze viability of cancer cells of drug-treated spheroids with a micro-plate reader. This technology is straightforward to implement both robotically and with other liquid handling tools such as manual pipettes.
Introduction
基于细胞的测定提供用于开发和发现新的抗癌药物的重要工具。1,2-历史上，癌细胞的单层培养物已被用于研究的候选化合物对特定类型的癌细胞的功效。维护在标准培养板单层培养的易用性，标准板与用于加入试剂的商业机器人工具的兼容性，并用筛分设备对细胞反应的化学化合物下游分析是渲染2D培养一个有吸引力的工具的主要好处用于药物测试。3不幸的是，单层细胞测定往往不能预测在体内化合物的功效，使得药物开发和发现极其昂贵的过程。4,5-尽管显著投资和努力由制药公司和学术单位，只有约1％的抗癌药物在临床试验中被批准由FDA在过去的二十年。2D培养和肿瘤细胞在体内的复杂的三维环境之间差距6是单层培养系统的一个主要缺点。7因此，候选化合物对肿瘤细胞中的一个设置的筛选即更类似于三维肿瘤环境可能会加快新型化疗药物的发展。8
癌细胞球体呈现一个相关的三维肿瘤模型体外 9,10球体是通过癌细胞上非粘附表面或在悬浮液中使用诸如旋转烧瓶，液体覆盖，微加工技术的微自发或诱导的组件，其形成致密簇。井阵列，微流体，及悬滴11-16球体模仿实体肿瘤，包括几何和氧，营养物，和药物化合物有限传输到中央区的关键特征;因此，它们更紧密地再生药物responSE相比单层培养实体肿瘤的17-19尽管这样明显的好处，球体是不经常使用的抗肿瘤细胞的化学化合物的筛选。在一个标准的高吞吐量的设置，与市售的机器人和筛选/成像工具兼容产生均匀大小的球体的困难阻碍球体文化纳入药品开发过程。尽管定制的材料和板最近已成为市售于满足这一需求，成本考虑阻止它们的广泛使用。
两个主要的技术以生产高通量一致尺寸的球状体用一个新的悬滴平台和微制造微井的能力。13,16,20然而，这两种方法都需要特殊的板，并且是昂贵的制造和不方便端点用户设备核心研究中心和医药等行业，其中最为主要的EF堡垒为新的抗癌药物的发现进行的。尽管在含细胞滴与最近悬滴板的设计的稳定性的一些改进，仍培养过程中使用的板的仅每隔一个孔，以避免滴扩频/合并。16此显著降低实验吞吐量。药物添加和更新很难与手工或机器人移液和球状体需要被转移到一个标准的板，用于生化分析，因为该板配置不与常规的筛选设备容易地兼容诸如板的读者。21微井使用软光刻还制作可以控制大小的球体的生产。13,20然而，这个平台与标准的移液工具的不兼容性防止治疗与不同的药物化合物/浓度个体球体，揭露所有的球体，以单一的治疗条件。因此，这种方法不适合于高通量化合物筛选，需要多个化合物/浓度的同时进行测试。
为了克服这些障碍，已经开发了高通量生产的标准96孔板一致尺寸的癌细胞球体的新技术。22,23的方法是基于一种聚合物的水性两相体系（ATPS）与聚乙二醇（上PEG）和葡聚糖（DEX），为相形成聚合物。24 ATPSs最近已应用在各种新的细胞生物学应用，使细胞图案化和局部递送生物试剂对细胞的高含水介质。25-32为了形成球体，癌细胞与含水DEX相和所得悬浮液的一个子微升滴混合的吸移到孔含有所述浸渍水的PEG相溶液。落仍然从浸没相和局限的细胞不混溶的，以便形成一个球状体。恶魔ortantly，高含水浸渍阶段提供营养物质的球状体的细胞和减少的介质蒸发的众所周知的问题通用的一些其它测定法，导致在介质渗透压变化和药物浓度的波动。这项技术使球体的生产和仅使用市售的试剂和移液工具在标准的96孔板的药物治疗。重要的是，在同一个板块使用标准生化分析和板块读者进行的球体细胞反应的分析。与ATPS的方法来机器人液体处理的适应性和加工的便利性，使高通量产生两个单培养和共培养球形体一个简单的实验室技术。这种新方法将是向前迈进一大步走向融合的肿瘤细胞球体与提高测试吞吐量和成本效益（增加测试的化合物和热度数量药物开发和发现过程CED试剂消耗）和效率（降低手工操作时间）。
机器人的生产癌细胞球体在96孔板使用ATPS方法的一个详细的协议如下所述。此外，所得的球状体和使用商业生物化学测定细胞应答下游分析的药物治疗的信息呈现。
Protocol
Representative Results
Discussion
球体呈现一个现实的模型，以更好地理解肿瘤生理学和药效，并为抗癌药物开发的有用工具。这样的应用将极大地从简单的球体生成和维护技术，只需要标准的实验室器具，液体处理工具和筛分设备中受益。使用的含水两相体系中滴相中自发地聚集癌细胞允许高效生产和维护球体与机器人液体处理程序，并在原位药物治疗和商业试剂和工具的细胞反应终点分析。因此，该技术的一大优势在?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors acknowledge funding from the National Institutes of Health R21CA182333.
Materials
| Reagents and Consumables | |||
| Polyethylene glycol, Mw: 35,000 | Sigma-Aldrich | 94646 | |
| Dextran, Mw: 500,000 | Pharmacosmos | 5510 0500 9007 | |
| Dulbecco's Modified Eagle Medium (DMEM) | Sigma-Aldrich | D6429 | |
| Fetal Bovine Serum | Sigma-Aldrich | 12306C | |
| Glutamine | Life Technologies | 35050-061 | |
| Antibiotic | Life Technologies | 15240-062 | |
| Clacein AM | Life Technologies | C3100MP | |
| Hoechst | Life Technologies | 33342 | |
| Cisplatin | Spectrum Chemicals | 15663-27-1 | |
| PrestoBlue | Life Technologies | A-13261 | |
| Pluronic F-108 | Sigma Aldrich | 542342 | |
| Disposable Tips (10 µl) | Fluotics | C-P10V11.ST | |
| Disposable Tips (70 µl) | Fluotics | C-P70V11.ST | |
| Round-bottom 96-well plates | Corning | 7007 | |
| Equipment | |||
| Liquid Handler | Agilent Technologies | SRT Bravo | |
| Microplate Reader | Biotek Instruments | Synergy H1M |
References
- Butcher, E. C., Berg, E. L., Kunkel, E. J. Systems biology in drug discovery. Nat. Biotechnol. 22, 1253-1259 (2004).
- Gonzalez-Nicolini, V., Fussenegger, M. In vitro assays for anticancer drug discovery–a novel approach based on engineered mammalian cell lines. Anticancer Drugs. 16, 223-228 (2005).
- Castel, D., Pitaval, A., Debily, M. A., Gidrol, X. Cell microarrays in drug discovery. Drug Discov. Today. 11, 616-622 (2006).
- Gidrol, X., et al. 2D and 3D cell microarrays in pharmacology. Current Opin. Pharmacol. 9, 664-668 (2009).
- LaBarbera, D. V., Reid, B. G., Yoo, B. H. The multicellular tumor spheroid model for high-throughput cancer drug discovery. Expert Opin. Drug Discov. 7, 819-830 (2012).
- Ward, D. J., Martino, O. I., Simpson, S., Stevens, A. J. Decline in new drug launches: myth or reality? Retrospective observational study using 30 years of data from the UK. BMJ Open. 3, (2013).
- Yamada, K. M., Cukierman, E. Modeling tissue morphogenesis and cancer in 3D. Cell. 130, 601-610 (2007).
- Hirschhaeuser, F., et al. Multicellular tumor spheroids: An underestimated tool is catching up again. J. Biotechnol. 148, 3-15 (2010).
- Ghajar, C. M., Bissell, M. J. Tumor engineering: the other face of tissue engineering. Tissue Eng. A. 16, 2153-2156 (2010).
- Kenny, P. A., et al. The morphologies of breast cancer cell lines in three-dimensional assays correlate with their profiles of gene expression. Mol. Oncol. 1, 84-96 (2007).
- Carlsson, J., Yuhas, J. M. Liquid-overlay culture of cellular spheroids. Rec. Results Cancer Res. 95, 1-23 (1984).
- Wartenberg, M., et al. Tumor-induced angiogenesis studied in confrontation cultures of multicellular tumor spheroids and embryoid bodies grown from pluripotent embryonic stem cells. FASEB J. 15, 995-1005 (2001).
- Tekin, H., et al. Stimuli-responsive microwells for formation and retrieval of cell aggregates. Lab chip. 10, 2411-2418 (2010).
- Patra, B., et al. A microfluidic device for uniform-sized cell spheroids formation, culture, harvesting and flow cytometry analysis. Biomicrofluidics. 7, 54114 (2013).
- Frey, O., Misun, P. M., Fluri, D. A., Hengstler, J. G., Hierlemann, A. Reconfigurable microfluidic hanging drop network for multi-tissue interaction and analysis. Nat. Commun. 5, 4250 (2014).
- Hsiao, A. Y., et al. Micro-ring structures stabilize microdroplets to enable long term spheroid culture in 384 hanging drop array plates. Biomed. Microdev. 14, 313-323 (2012).
- Mehta, G., Hsiao, A. Y., Ingram, M., Luker, G. D., Takayama, S. Opportunities and challenges for use of tumor spheroids as models to test drug delivery and efficacy. J. Control. Release. 164, 192-204 (2012).
- Mueller-Klieser, W. Three-dimensional cell cultures: from molecular mechanisms to clinical applications. Am. J. Physiol. 273, C1109-C1123 (1997).
- Minchinton, A., Tannock, I. F. Drug penetration in solid tumors. Nat. Rev. Cancer. 6, 583-592 (2006).
- Jeong, G. S., et al. Surface tension-mediated, concave-microwell arrays for large-scale, simultaneous production of homogeneously sized embryoid bodies. Adv. Healthc. Mater. 2, 119-125 (2013).
- Cavnar, S. P., Salomonsson, E., Luker, K. E., Luker, G. D., Takayama, S. Transfer imaging, and analysis plate for facile handling of 384 hanging drop 3D tissue spheroids. JALA. 19, 208-214 (2014).
- Atefi, E., Lemmo, S., Fyffe, D., Luker, G. D., Tavana, H. High throughput, polymeric aqueous two-phase printing of tumor spheroids. Adv. Func. Mater. 24, 6509-6515 (2014).
- Lemmo, S., Atefi, E., Luker, G. D., Tavana, H. Optimization of aqueous biphasic tumor spheroid microtechnology for anti-cancer drug testing in 3D culture. Cell. Mol. Bioeng. 7, 344-354 (2014).
- Albertsson, P. -. A., Tjerneld, F. Phase diagrams. Methods Enzym. 228, 3-13 (1994).
- Frampton, J. P., White, J. B., Abraham, A. T., Takayama, S. Cell co-culture patterning using aqueous two-phase systems. J. Vis. Exp. (73), (2013).
- Frampton, J. P., et al. Aqueous two-phase system patterning of detection antibody solutions for cross-reaction-free multiplex ELISA. Sci. Rep. 4, 4878 (2014).
- Lai, D., Frampton, J. P., Tsuei, M., Kao, A., Takayama, S. Label-free direct visual analysis of hydrolytic enzyme activity using aqueous two-phase system droplet phase transitions. Anal. Chem. 86, 4052-4057 (2014).
- Petrak, D., Atefi, E., Yin, L., Chilian, W., Tavana, H. Automated spatio-temporally controlled cell microprinting with polymeric aqueous biphasic system. Biotech. Bioeng. 11, 404-412 (2014).
- Tavana, H., et al. Nanolitre liquid patterning in aqueous environments for spatially defined reagent delivery to mammalian cells. Nat. Mater. 8, 736-741 (2009).
- Tavana, H., et al. Polymeric aqueous biphasic system rehydration facilitates high throughput cell exclusion patterning for cell migration studies. Adv. Func. Mater. 21, 2920-2926 (2011).
- Tavana, H., Mosadegh, B., Takayama, S. Polymeric aqueous biphasic systems for non-contact cell printing on cells: engineering heterocellular embryonic stem cell niches. Adv. Mater. 22, 2628-2631 (2010).
- Tavana, H., Mosadegh, B., Zamankhan, P., Grotberg, J. B., Takayama, S. Microprinted feeder cells guide embryonic stem cell fate. Biotechnol. Bioeng. 108, 2509-2516 (2011).
- Kalluri, R., Zeisberg, M. . Nat Rev Cancer. 6, 392-401 (2006).
- Olsen, C. J., Moreira, J., Lukanidin, E. M., Ambartsumian, N. S. Human mammary fibroblasts stimulate invasion of breast cancer cells in a three-dimensional culture and increase stroma development in mouse xenografts. BMC Cancer. 10, 444 (2010).
- Ozdemir, B. C., et al. Depletion of carcinoma-associated fibroblasts and fibrosis induces immunosuppression and accelerates pancreas cancer with reduced survival. Cancer Cell. 25, 719-734 (2014).
- Hsiao, A. Y., et al. Microfluidic system for formation of PC-3 prostate cancer co-culture spheroids. Biomaterials. 30, 3020-3027 (2009).
- Friedrich, J., Seidel, C., Ebner, R., Kunz-Schughart, L. A. Spheroid-based drug screen: considerations and practical approach. Nat. Protoc. 4, 309-324 (2009).
- Weigelt, B., Bissell, M. J. Unraveling the microenvironmental influences on the normal mammary gland and breast cancer. Semin. Cancer Biol. 18, 311-321 (2008).
- Mueller, M. M., Fusenig, N. E. Friends or foes – bipolar effects of the tumour stroma in cancer. Nature Reviews Cance. 4, 839-849 (2004).
- Bissell, M. J., Radisky, D. Putting tumours in context. Nat. Rev. Cancer. 1, 46-54 (2001).
