细胞的浸入式印刷到改性表面采用连续流动Microspotter
Summary
This 3D microfluidic printing technology prints arrays of cells onto submerged surfaces. We describe how arrays of cells are delivered microfluidically in 3D flow cells onto submerged surfaces. By printing onto submerged surfaces, cell microarrays were produced that allow for drug screening and cytotoxicity assessment in a multitude of areas.
Abstract
The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell phenotype after printing, poor organization and distribution of desired cells, and the inability to deliver drugs and/or nutrients to targeted areas in the array. Our 3D microfluidic printing technology is uniquely capable of sealing and printing arrays of cells onto submerged surfaces in an automated and multiplexed manner. The design of the microfluidic cell array (MFCA) 3D fluidics enables the printhead tip to be lowered into a liquid-filled well or dish and compressed against a surface to form a seal. The soft silicone tip of the printhead behaves like a gasket and is able to form a reversible seal by applying pressure or backing away. Other cells printing technologies such as pin or ink-jet printers are unable to print in submerged applications. Submerged surface printing is essential to maintain phenotypes of cells and to monitor these cells on a surface without disturbing the material surface characteristics. By printing onto submerged surfaces, cell microarrays are produced that allow for drug screening and cytotoxicity assessment in a multitude of areas including cancer, diabetes, inflammation, infections, and cardiovascular disease.
Introduction
在医药工业中的最新进展已导致使用移动芯片在药物发现过程用于药物筛选和cytotoxicological分析1,2,3兴趣增加。利用细胞微阵列体外高通量检测和筛选方法的发展将有利于候选药物的快速和具有成本效益的发展，以及提前1,4单元的基本认识。传统的方法来筛查细胞采用常规孔板平台;然而，这种方法由于成本高，产量有限，以及对细胞功能1,5定量信息有限，能力有限。由于这些限制，研究在细胞微阵列技术是新兴的分子生物学特性，组织工程和药物筛选1,6。细胞微阵列的优点包括更小的样品使用，影响最小细胞表型异质性掩蔽的信息，最重要的自动化测定法更高通量应用1,7,8的能力。
制药行业目前采用高通量细胞系筛选测定与2D细胞单层培养物在微量滴定孔板9的药物筛选。复细胞在微量滴定板的孔中提供了独特的实验方案更高的吞吐量的潜力。另外，在目前的技 术用于蜂窝芯片使细胞干这可能显着地从改变细胞的表型在体内 10,11。为了克服这些问题，MFCA被设计并且被示于图1。的MFCA三维流体的设计使打印头前端在图1中被降低到一个浴和压靠在一个表面上以形成一个密封。打印头的软硅胶尖的行为就像一个垫片，形成了一个可逆的密封。该MFCA技术是唯一适合用潜面，这都需要细胞培养和组织切片系统的接口，并且是很难或不可能与大多数其他方法。销或喷墨印刷将无法正常工作，和2D的微流体装置不适合于沉积或离散点的大型阵列接口。另外，通过小型化和定位的实验 – 细胞微阵列 – 的MFCA克服了与高通量细胞系筛选测定法相关的主要问题。
该CFM采用3D频道网络周期小体积流体样品在表面上的微观12,13点的位置。通过用流量进行打印时，生物分子，细胞和其它试剂均保持在一个液体环境中在整个印刷过程中，使敏感的生物分子和细胞的印刷不暴露于空气中，这妨碍了当前小区的印刷技术。另外，也可以直接从粗物质，如杂交瘤或提供上清液有该阵列表面上的捕获机构进行打印。该原稿的目的是详细浸没印刷两种细胞类型的解释在表面上。
Protocol
Representative Results
Discussion
这里所描述的3D微流体印刷技术是唯一能够细胞microfluidically印刷阵列放入盛好液体， 即一个水下表面。通过印刷在潜面，细胞微阵列可以制造维持细胞的生理相关的细胞表型，以及多路复用单元中的单个孔图4底部的能力。本研究的结果表明，microfluidically印刷细胞的结果在细胞附着与细胞相媲美的形态和活力;然而，印刷单元可被更密集地附着在导致较短的总研究时间定义的位?…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者要感谢克里斯·莫罗的技术援助。经费是由美国国立卫生研究院SBIR（R43）提供给予1R43GM101859-01（MPI）GRANT10940803。
Materials
| Continuous Flow Microspotter | Wasatch Microfluidics | ||
| NIH/3T3 cells | ATCC | CRL-1658 | |
| Dubbleco's Modified Eagle Medium | Invitrogen | 11965-092 | base media for cells |
| HEPES buffer | Invitrogen | 15630-080 | cell media additive (control pH) |
| Sodium pyruvate | Invitrogen | 11360-070 | cell media additive |
| Penicillin-Streptomycin | Invitrogen | cell media additive | |
| Trypan blue | Invitrogen | 15250-061 | stain cell sfor counting |
| Haemocytometer | Fisher | 267110 | cell chamber to count cells |
| Nikon Eclipse TS100 | Nikon | Used to check on cells | |
| Nikon Eclipse TE2000-U | Nikon | Used for collecting images | |
| Phosphate Buffered Saline (with calcium and magnesium) | Invitrogen | 14040-133 | rinsing cells before passaging and before staining with PI |
| TrypLE Express | Invitrogen | A12177-01 | used to remove cells from surface |
Referências
- Fernandes, T. G., Diogo, M. M., Clark, D. S., Dordick, J. S., Cabral, J. High-throughput cellular microarray platforms: applications in drug discovery, toxicology and stem cell research. Trends in Biotechnology. 27, 342-349 (2009).
- Michelini, E., Cevenini, L., Mezzanotte, L., Coppa, A., Roda, A. Cell-based assays: fuelling drug discovery. Anal Bioanal Chem. 398, 227-238 (2010).
- Gao, D., et al. Recent developments in microfluidic devices for in vitro cell culture for cell-biology research. TrAC Trends in Analytical Chemistry. 35, 150-164 (2012).
- Geysen, H. M., Schoenen, F., Wagner, D., Wagner, R. Combinatorial compound libraries for drug discovery: an ongoing challenge. Nature Reviews Drug Discovery. 2, 222-230 (2003).
- Xu, Y., Shi, Y., Ding, S. A chemical approach to stem-cell biology and regenerative medicine. Nature. 453, 338-344 (2008).
- Khademhosseini, A., Langer, R., Borenstein, J., Vacanti, J. P. Microscale technologies for tissue engineering and biology. Proceedings of the National Academy of Sciences of the United States of America. 103, 2480-2487 (2006).
- Bhadriraju, K., Chen, C. S. Engineering cellular microenvironments to improve cell-based drug testing. Drug Discovery Today. 7, 612-620 (2002).
- Castel, D., Pitaval, A., Debily, M. -. A., Gidrol, X. Cell microarrays in drug discovery. Drug Discov. Today. 11, 616-622 (2006).
- Tsui, J. H., Lee, W., Pun, S. H., Kim, J., Kim, D. -. H. Microfluidics-assisted in vitro drug screening and carrier production. Advanced Drug Delivery Reviews. , (2013).
- Gottwald, E., et al. A chip-based platform for the in vitro generation of tissues in three-dimensional organization. Lab on a Chip. 7, 777-785 (2007).
- Liu, R., Lee, A. P. . Integrated Biochips for DNA Analysis. , (2008).
- Natarajan, S., et al. Continuous-flow microfluidic printing of proteins for array-based applications including surface plasmon resonance imaging. Analytical Biochemistry. 373, 141-146 (2008).
- Natarajan, S., Hatch, A., Myszka, D. G., Gale, B. K. Optimal Conditions for Protein Array Deposition Using Continuous Flow. Anal. Chem. 80, 8561-8567 (2008).
- Keighley, W. The need for high throughput kinetics early in the drug discovery process. , (2011).
- Xu, F., et al. A three-dimensional in vitro ovarian cancer coculture model using a high-throughput cell patterning platform. Biotechnol J. 6, 204-212 (2011).
- Tuckwell, D. S., Weston, S. A., Humphries, M. J. Integrins: a review of their structure and mechanisms of ligand binding. Symposia of the Society for Experimental Biology. 47, 107 (1993).
- Roberts, C., et al. Using mixed self-assembled monolayers presenting RGD and (EG) 3OH groups to characterize long-term attachment of bovine capillary endothelial cells to surfaces. Journal of the American Chemical Society. 120, 6548-6555 (1998).
- Chandra, R. A., Douglas, E. S., Mathies, R. A., Bertozzi, C. R., Francis, M. B. Programmable cell adhesion encoded by DNA hybridization. Angewandte Chemie. 118, 910-915 (2006).
- Hsiao, S. C., et al. Direct cell surface modification with DNA for the capture of primary cells and the investigation of myotube formation on defined patterns. Langmuir. 25, 6985-6991 (2009).
- Black, C. B., Duensing, T. D., Trinkle, L. S., Dunlay, R. T. Cell-Based Screening Using High-Throughput Flow Cytometry. ASSAY and Drug Development Technologies. 9, 13-20 (2011).
- Derby, B. Bioprinting: inkjet printing proteins and hybrid cell-containing materials and structures. J. Mater. Chem. 18, 5717-5721 (2008).
- Onoe, H., et al. Cellular Microfabrication: Observing Intercellular Interactions Using Lithographically-Defined DNA Capture Sequences. Langmuir. 28, 8120-8126 (2012).
- Hsiao, S. C., et al. DNA-Coated AFM Cantilevers for the Investigation of Cell Adhesion and the Patterning of Live Cells. Angewandte Chemie International Edition. 47, 8473-8477 (2008).
