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Bioengineering

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Published: June 16th, 2016

DOI:

10.3791/54105

1Institute of Biomedical Engineering and Nanomedicine, National Health Research Institutes, Taiwan, 2Tissue Engineering and Regenerative Medicine, National Chung Hsing University, 3Institute of NanoEngineering and MicroSystems, National Tsing Hua University

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....

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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

  1. Before the photolithography process12, use the 4-inch silicon wafers as a substrate and dehydrate the wafers in a conventional oven at 1.......

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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.......

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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 .......

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This work was supported by a grant from the National Health Research Institutes (03-A1 BNMP11-014).

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Name Company Catalog Number Comments
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

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