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

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

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....

Protocol

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 120 °C for 10 min.
2. Clean the dehydrated silicon wafers by using oxygen plasma treatment at 100 watts for 30 sec in a plasma cleaner.

Results

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 top-view (Figure.......

Discussion

Microwell-based device systems^6,14 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 .......

Materials

List of materials used in this article

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 190 mm 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