Published: June 16th, 2016
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.
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.
Currently placing single cells individually in a culture space is commonly achieved by using limiting dilution or fluorescence-activated cell sorting (FACS). For many laboratories, limiting dilution is a convenient method, as it only requires a pipette and tissue culture plates, which are readily available. In this case, a cell suspension is serially diluted to an appropriate cell density, and then placed into culture wells by using a manual pipette. These compartmented single cells are then used for cell analysis, such as genetic heterogeneity screening1 and colony formation2. However, this method is low-throughput and labor-intensive, without u....
Note: The photomask designs for our microfluidic device fabrication were drawn by using a computer aided design (CAD) software. The designs were then utilized to fabricate chrome photomasks using a commercial service. The PDMS devices were made using soft lithography techniques.11
1. Fabrication of Master Molds by Lithography
The microfluidic platform for single-cell isolation and culture comprises a microchannel (200 µm in height) with two sets of microwell arrays (Figure 2A). The two sets of microwell arrays are termed as capture-well (25 µm in diameter and 27 µm in depth) and culture-well (285 µm in diameter and 300 µm in depth) for single-cell isolation and culture, respectively, and each capture-well is positioned at the center of a culture-well when seen from the.......
Microwell-based device systems6,14 have been utilized for single-cell manipulation and analysis, such as large-scale single cell trapping6 and single hematopoietic stem cell proliferation15. Although well size, number, and shape can be adjusted for specific applications, the single-cell isolation efficiency is always compromised when the size of the well is increased.9,15
To overcome this limitation, Park et al. reported a microfluidic chip .......
|AutoCAD LT 2011
|Part No. 057C1-74A111-1001
|Bench-Top Plasma Treatment System
|SU-8 50 negative photoresist
|SU-8 100 negative photoresist
|Synrex Co., Ltd.
|SC-HMI 2" ~ 6"
|Deya Optronic CO.
|Grand Chemical Companies
|Propylene glycol monomethyl ether acetate
|Scanning laser profilometer
|TRIDECAFLUORO-1,1,2,2-TETRAHYDROOCTYL. Hazardous. Corrosive to the respiratory tract., reacts violently with water.
|SPACE SAVER VACUUM DESICCATOR 190MM WHITE BASE
|Polydimethylsiloxane (PDMS) kit
|Harris Uni-Core puncher
|Ted Pella Inc.
|with 0.75 mm inner-diameter
|Scotch Removable Tape 811
|Bovine serum albumin (BSA)
|DMEM basal medium
|Fetal bovine serum
|Recombinant enzyme mixture
|Innovative cell technology
|DiIC12(3) cell membrane dye
|Used as a cell tracker
|Plastic syringe (1 mL)
|23 gauge blunt needles
|Ever Sharp Technology, Inc.
|Poly-tetrafluoroethene (PTFE) tubing
|Ever Sharp Technology, Inc.
|inner diameter, 0.51 mm; outer diameter, 0.82 mm
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