高通量单细胞的分离和文化的微流体平台
Summary
Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.
Abstract
Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.
Introduction
目前将单个细胞单独在培养空间通常是通过使用有限稀释或荧光激活细胞分选（FACS）来实现的。对于许多实验室，有限稀释是一个方便的方法，因为它仅需要一个吸管和组织培养板中，这是容易获得的。在这种情况下，细胞悬浮液连续稀释至适当的细胞密度，然后通过使用手动移液管放置到培养孔。然后，这些隔间单细胞被用于细胞分析，如遗传异质性筛选1和集落形成2。然而，该方法是低通量和劳动密集的，不利用用于协助一机械臂，因为有限稀释法的泊松分布性质限制了单细胞事件的37％3的最大概率。 FACS机，集成机械臂可以通过准确PLAC克服泊松分布的限制ING在文化井一单细胞在同一时间4。然而，高机械剪切应力（因此，降低细胞活力）5和机购买和操作成本在许多实验室已经限制了它的使用。
为了克服上述限制，微型装置已经发展到高效的单细胞装入微孔6。然而，微孔不提供足够的空间用于装载的细胞增殖，由于需要使每个的尺寸微孔关闭到单个细胞的最大化单细胞加载的概率。作为培养测定在许多基于细胞的应用（ 例如，克隆形成实验7），较大微孔需要（从90 – 650微米的直径或边长）也被用于允许延长细胞培养物。然而，像有限稀释法，它们也具有低的单细胞负载效率，范围从10 – 30％的8，9
以前，我们已经开发出一种高通量微流体平台，单个细胞中分离出在个体微孔，并在分离的细胞的克隆形成实验证明其应用10的装置，用聚二甲基硅氧烷（PDMS）制成，并包括两组微孔阵列具有不同微孔尺寸，从而可以大大提高在微孔的大小加载单个细胞的效率是比电池显著大。值得注意的是，这种“双阱”的概念允许在不影响单细胞捕获效率，使得它直接地调整该装置的设计，以适应不同的细胞类型和应用程序灵活调整培养面积的大小。这种高效率的方法应该是对于长期的细胞培养实验对于细胞的异质性的研究和单克隆细胞系建立有用的。
Protocol
Representative Results
Discussion
基于微孔的设备系统6,14已用于单细胞操纵和分析，例如大型单细胞俘获6和单造血干细胞增殖15。虽然井大小，数量和形状可以调节为特定的应用中，当孔的大小增加时，单细胞分离效率总是受到损害。9,15
为了克服此限制，Park等人报告了具有高的单细胞的捕集率（58.34％）的三角形的微孔的微流体芯片，而微孔尺寸放大以允许细胞扩散?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This work was supported by a grant from the National Health Research Institutes (03-A1 BNMP11-014).
Materials
| AutoCAD software | Autodesk | AutoCAD LT 2011 | Part No. 057C1-74A111-1001 |
| Silicon wafer | Eltech corperation | SPE0039 | |
| Conventional oven | YEONG-SHIN company | ovp45 | |
| Plasma cleaner | Nordson | AP-300 | Bench-Top Plasma Treatment System |
| SU-8 50 negative photoresist | MicroChem | Y131269 | |
| SU-8 100 negative photoresist | MicroChem | Y131273 | |
| Spin coater | Synrex Co., Ltd. | SC-HMI 2" ~ 6" | |
| Hotplate | YOTEC company | YS-300S | |
| Msak aligner | Deya Optronic CO. | A1K-5-MDA | |
| SU-8 developer | Grand Chemical Companies | GP5002-000000-72GC | Propylene glycol monomethyl ether acetate |
| Scanning laser profilometer | KEYENCE | VK-X 100 | |
| Trichlorosilane | Gelest, Inc | SIT8174.0 | TRIDECAFLUORO-1,1,2,2-TETRAHYDROOCTYL. Hazardous. Corrosive to the respiratory tract., reacts violently with water. |
| Desiccator | Bel-Art Products | F42020-0000 | SPACE SAVER VACUUM DESICCATOR 190MM WHITE BASE |
| Polydimethylsiloxane (PDMS) kit | Dow corning | Sylgard 184 | |
| Harris Uni-Core puncher | Ted Pella Inc. | 15072 | with 0.75 mm inner-diameter |
| Removable tape | 3M Company | Scotch Removable Tape 811 | |
| Stereomicroscope | Leica Microsystems | Leica E24 | |
| Bovine serum albumin (BSA) | Bersing Technology | ALB001.500 | |
| DMEM basal medium | Gibco | 12800-017 | |
| Fetal bovine serum | Thermo Hyclone | SH30071.03HI | |
| Antibiotics | Biowest | L0014-100 | Glutamine-Penicillin-Streptomycin |
| Recombinant enzyme mixture | Innovative cell technology | AM-105 | Accumax |
| DiIC12(3) cell membrane dye | BD Biosciences | 354218 | Used as a cell tracker |
| Syringe pump | Harvard Apparatus | 703007 | |
| Plastic syringe (1 mL) | BD Biosciences | 309659 | |
| 23 gauge blunt needles | Ever Sharp Technology, Inc. | TD21 | |
| Poly-tetrafluoroethene (PTFE) tubing | Ever Sharp Technology, Inc. | TFT-23T | inner diameter, 0.51 mm; outer diameter, 0.82 mm |
Riferimenti
- Meacham, C. E., Morrison, S. J. Tumour heterogeneity and cancer cell plasticity. Nature. 501 (7467), 328-337 (2013).
- Vermeulen, L., et al. Single-cell cloning of colon cancer stem cells reveals a multi-lineage differentiation capacity. P Natl Acad Sci USA. 105 (36), 13427-13432 (2008).
- Shapiro, H. M. . Practical flow cytometry. , (2005).
- Leong, K. G., Wang, B. E., Johnson, L., Gao, W. Q. Generation of a prostate from a single adult stem cell. Nature. 456 (7223), 804-808 (2008).
- Shapiro, E., Biezuner, T., Linnarsson, S. Single-cell sequencing-based technologies will revolutionize whole-organism science. Nat Rev Genet. 14 (9), 618-630 (2013).
- Rettig, J. R., Folch, A. Large-scale single-cell trapping and imaging using microwell arrays. Anal. Chem. 77 (17), 5628-5634 (2005).
- Liu, J., et al. Soft fibrin gels promote selection and growth of tumorigenic cells. Nat Mater. 11 (8), 734-741 (2012).
- Charnley, M., Textor, M., Khademhosseini, A., Lutolf, M. P. Integration column: microwell arrays for mammalian cell culture. Integr. Biol. 1 (11-12), 11-12 (2009).
- Lindstrom, S., et al. High-density microwell chip for culture and analysis of stem cells. PloS one. 4 (9), e6997 (2009).
- Lin, C. H., et al. A microfluidic dual-well device for high-throughput single-cell capture and culture. Lab Chip. 15 (14), 2928-2938 (2015).
- Xia, Y. N., Whitesides, G. M. Soft lithography. Angew Chem Int Edit. 37 (5), 550-575 (1998).
- Shin, Y., et al. Microfluidic assay for simultaneous culture of multiple cell types on surfaces or within hydrogels. Nat Protoc. 7 (7), 1247-1259 (2012).
- Strober, W. Trypan blue exclusion test of cell viability. Curr. Protoc. Immunol. Appendix 3 (Appendix 3B), (2001).
- Lindstrom, S., Andersson-Svahn, H. Miniaturization of biological assays – Overview on microwell devices for single-cell analyses. Bba-Gen Subjects. 1810 (3), 308-316 (2011).
- Lecault, V., et al. High-throughput analysis of single hematopoietic stem cell proliferation in microfluidic cell culture arrays. Nat Methods. 8 (7), 581-593 (2011).
- Park, J. Y., et al. Single cell trapping in larger microwells capable of supporting cell spreading and proliferation. Microfluid Nanofluid. 8 (2), 263-268 (2010).
- Tirino, V., et al. Cancer stem cells in solid tumors: an overview and new approaches for their isolation and characterization. FASEB J. 27 (1), 13-24 (2013).
- Chen, P. C., Huang, Y. Y., Juang, J. L. MEMS microwell and microcolumn arrays: novel methods for high-throughput cell-based assays. Lab Chip. 11 (21), 3619-3625 (2011).
- Liang, P., et al. Drug Screening Using a Library of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Reveals Disease-Specific Patterns of Cardiotoxicity. Circulation. 127 (16), 1677-1691 (2013).
