微机械动态细胞培养平台
Summary
在这个协议中，我们展示的微驱动器阵列的垂直流离失所该技术是基于职位的制造，以及如何可以修改此基础技术，进行二维和三维培养高吞吐量的机械动态细胞培养范例。
Abstract
在体外细胞组织工程，药物发现或基本的细胞生物学研究的mechanobiological刺激组合系统探测能力由目前的生物反应器技术，它不能同时适用于各种培养细胞的机械性刺激是有限的。为了解决这个问题，我们已经制定了一系列旨在为机械刺激的影响画面在高通量格式的微型平台。在这个协议中，我们展示了微驱动器阵列的垂直流离失所的技术为基础，进一步展示如何可以修改此基础技术进行高通量的机械动态细胞培养两种维和职位的制造三个立体养殖的范例。
Protocol
A.设备的描述和操作设备都采用多层软光刻技术1，在聚二甲基硅氧烷（PDMS），能够同时产生一系列机械在单个细胞的微阵列跨文化地点条件。在这个协议中，首先描述的步骤来制作气动驱动microposts的阵列，步骤来修改设备，使机械动态二维（2D）和三维（3D）的文化范式文化。概述了微加工方法增加吞吐量比现有的宏观系统，适用于各种机械条件的影响，是最好?…
Discussion
虽然概念上很简单，设备制造并采取了一定的实验技能和实践。设备中的多层次的对齐，特别是在2D细胞培养的情况下，可以是具有挑战性的的，尤其是在一个大面积的数组。实事求是地讲，我们可以可靠地实现了100％对齐的成功率，使用50微米的公差，在间距之间多层次的相邻功能的设备。我们还成功地展示了低至15微米的公差对齐，但对齐所需时间大大提高，并实现在一个较低的成功率。对准?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们承认，从自然科学和工程研究理事会，加拿大和加拿大健康研究机构（CHRPJ 323533-06），安大略研究生奖学金计划到CM，和加拿大研究主席在微型和纳米工程系统的财政支持黏， Mechanobiology到CAS。
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Sylgard 184 PDMS Monomer and Crosslinker Kit | Dow Corning | |||
| SU-8 masters | ||||
| Silanization agent: (tridecafluoro-1, 1, 2, 2-tetrahydrooctyl)-1-trichlorosilane | United Chemical Technologies | |||
| Foam pads | Craft supply stores, 1-2 mm thick | |||
| Overhead inkjet transparencies | Grand & Toy | |||
| Plexiglass plates | ||||
| C-clamp | hardware store | |||
| Micromanipulator system | Siskiyou | |||
| Custom-made vaccum mount | ||||
| Vision system, Navitar 12x zoom | Navitar | |||
| Connecting tubes | VWR International | Clay Adam Intramedic PE190 | ||
| Blunt 18G needles | Smallparts.com / Amazon Inc. | |||
| Eccentric diaphragm micropump | Schwarzer Precision | SP 500 EC-LC4.5V DC | ||
| Solenoid valves | Pneumadyne | S10MM-30-12-3 | ||
| Solenoid manifold | Pneumadyne | MSV10-1 | ||
| Function generator | ||||
| 3-(trimethoxysilyl) propyl methacrylate | Sigma | |||
| Polyethylene glycol diacrylate 3.4 kDa | Laysan Bio | |||
| Polyethylene glycol 8 kDa | Sigma | |||
| Irgacure 2959 | Ciba Specialty Chemicals | |||
| 1-vinyl-2-pyrolidinone | Sigma | |||
| Standard cell culture reagents |
Referências
- Unger, M. A., Chou, H. P., Thorsen, T., Scherer, A., Quake, S. R. Monolithic microfabricated valves and pumps by multilayer soft lithography. Science. 288, 113-116 (2000).
- Schaffer, J. L. Device for the application of a dynamic biaxially uniform and isotropic strain to a flexible cell culture membrane. J Orthop Res. 12, 709-719 (1994).
- Jo, B., Lerberghe, L. V. a. n., Motsegood, K., Beebe, D. Three-dimensional micro-channel fabrication in polydimethylsiloxane (PDMS) elastomer. Journal of Microelectromechanical Systems. 9, 76-81 (2000).
- Moraes, C., Sun, Y., Simmons, C. A. Solving the shrinkage-induced PDMS alignment registration issue in multilayer soft lithography. J. Micromech. Microeng. 19, 065015-065015 (2009).
- Haubert, K., Drier, T., Beebe, D. PDMS bonding by means of a portable, low-cost corona system. Lab on a chip. 6, 1548-1549 (2006).
- Bongaerts, J., Fourtouni, K., Stokes, J. Soft-tribology: Lubrication in compliant PDMS-PDMS contact. Tribology International. 40, 1531-1542 (2007).
- Moraes, C., Chen, J. H., Sun, Y., Simmons, C. A. Microfabricated arrays for high-throughput screening of cellular response to cyclic substrate deformation. Lab on a chip. 10, 227-234 (2010).
- Liu, V. A., Bhatia, S. N. Three-dimensional photopatterning of hydrogels containing living cells. Biomedical Microdevices. 4, 257-266 (2002).
- Hahn, M. S. Photolithographic patterning of polyethylene glycol hydrogels. Biomaterials. 27, 2519-2524 (2006).
- Moraes, C., Wang, G., Sun, Y., Simmons, C. A. A microfabricated platform for high-throughput unconfined compression of micropatterned biomaterial arrays. Biomaterials. 31, 577-584 (2010).
- Wipff, P. J. The covalent attachment of adhesion molecules to silicone membranes for cell stretching applications. Biomaterials. 30, 1781-1789 (2009).
- Moraes, C. Integrating polyurethane culture substrates into poly(dimethylsiloxane) microdevices. Biomaterials. 30, 5241-5250 (2009).
- Ifkovits, J. L., Burdick, J. A. Review: photopolymerizable and degradable biomaterials for tissue engineering applications. Tissue engineering. 13, 2369-2385 (2007).
Citar este artigo
Moraes, C., Sun, Y., Simmons, C. A. Microfabricated Platforms for Mechanically Dynamic Cell Culture. J. Vis. Exp. (46), e2224, doi:10.3791/2224 (2010).