Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

Summary

This report describes a microfluidic chip-based method to set up a single cell culture experiment in which high-efficiency pairing and microscopic analysis of multiple single cells can be achieved.

Abstract

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases including cancer. However, it is challenging to clarify the complex mechanism of intercellular interactions in highly heterogeneous cell populations using conventional co-culture systems because the heterogeneity of the cell subpopulation is obscured by the average values; the conventional co-culture systems can only be used to describe the population signal, but are incapable of tracking individual cells behavior. Furthermore, conventional single-cell experimental methods have low efficiency in cell manipulation because of the Poisson distribution. Microfabricated devices are an emerging technology for single-cell studies because they can accurately manipulate single cells at high-throughput and can reduce sample and reagent consumption. Here, we describe the concept and application of a microfluidic chip for multiple single-cell co-cultures. The chip can efficiently capture multiple types of single cells in a culture chamber (~46%) and has a sufficient culture space useful to study the cells' behavior (e.g., migration, proliferation, etc.) under cell-cell interaction at the single-cell level. Lymphatic endothelial cells and oral squamous cell carcinoma were used to perform a single-cell co-culture experiment on the microfluidic platform for live multiple single-cell interaction studies.

Introduction

Efficient capture of different types of single cells and providing sufficient culture space are needed for single cell co-culture experiments of multiple types of single cells¹. Limiting dilution is the most commonly used method to prepare the single cells for such experiments, due to the low cost of equipment required. However, due to the Poisson distribution limitation, the maximum single cell acquisition probability is only 37%, making the experimental operation laborious and time-consuming². In contrast, using fluorescence activated cell sorting (FACS) can overcome the Poisson distribution limitation to high-efficien....

Protocol

1. Fabrication of a wafer mold by soft lithography

NOTE: Mask pattern data is available in our previous publication¹⁴.

1. Dehydrate a 4-inch silicon wafer in a 120 °C oven for 15 min.
2. Spin coat 4 g of SU-8 2 negative photoresist onto a 4-inch silicon wafer at 1,000 rpm for 30 s to create a 5 µm thick layer (layer #1).
3. Soft bake layer #1 on a 65 °C hotplate for 1 min and then transfer layer #1 to a 95 °C hotplate for 3 min.
4. Cool layer #1 to room temperature, place it onto the holder of the semi-automatic mask aligner, and align with the layer #1 chrome-plated ....

Results

The device has a three-layer structure as shown by the cross-section photograph of a cut PDMS device (Figure 1A). The first layer contains a capture-site (6.0 µm in width and 4.6 µm in height) that connects the culture chamber and the by-pass channel. The difference in flow resistance between the culture chamber and the by-pass channel causes the cells to flow into the capture position and fill the entrance of the small path. After .......

Discussion

The intercellular interactions of various cells in the tumor microenvironment play an important role in the progression of the tumor¹⁷. In order to understand the mechanism of cell-cell interactions, co-culture systems are used as a common analytical method. However, multiple cell types and the heterogeneity of the cells themselves have led to experimental complexity and analytical difficulties.

The hydrodynamic shuttling chip allows multiple single-cell loading in the .......

Materials

Name	Company	Catalog Number	Comments
3M Advanced Polyolefin Diagnostic Microfluidic Medical Tape	3M Company	9795R
Antibiotics	Biowest	L0014-100	Glutamine-Penicillin-Streptomycin
AutoCAD software	Autodesk	AutoCAD LT 2011	Part No. 057C1-74A111-1001
CellTracke Blue CMAC Dye	Invitrogen	C2110
CellTracker Green CMFDA Dye	Invitrogen	C7025
Conventional oven	YEONG-SHIN company	ovp45
Desiccator	Bel-Art Products	F42020-0000	Space saver vacuum desiccator 190 mm white base
DiIC12(3) cell membrane dye	BD Biosciences	354218	Used as a cell tracker
DMEM-F12 medium	Gibco	11320-082
Endothelial Cell Growth Medium MV 2	PromoCell	C-22022
Fetal bovine serum Hyclone	Thermo	SH30071.03HI
Hamilton 700 series Glass syringe ( 0.1 ml )	Hamilton	80630	100 µL, Model 710 RN SYR, Small Removable NDL, 22s ga, 2 in, point style 2
Harris Uni-Core puncher	Ted Pella Inc.	15075	with 1.5mm inner-diameter
Harris Uni-Core puncher	Ted Pella Inc.	15071	with 0.5mm inner-diameter
Hotplate	YOTEC company	YS-300S
Msak aligner	Deya Optronic CO.	A1K-5-MDA
Oxygen plasma	NORDSON MARCH	AP-300
Plasma cleaner	Nordson	AP-300	Bench-Top Plasma Treatment System
Polydimethylsiloxane (PDMS) kit	Dow corning	Sylgard 184
Poly-tetrafluoroethene (PTFE)	Ever Sharp Technology, Inc.	TFT-23T	inner diameter, 0.51 mm; outer diameter, 0.82 mm
Removable tape	3M Company	Scotch Removable Tape 811
Silicon wafer	Eltech corperation	SPE0039
Spin coater	Synrex Co., Ltd.	SC-HMI 2" ~ 6"
Stereomicroscope	Leica Microsystems	Leica E24
SU-8 10 negative photoresist	MicroChem	Y131259
SU-8 2 negative photoresist	MicroChem	Y131240
SU-8 2050 negative photoresist	MicroChem	Y111072
SU-8 developer	Grand Chemical Companies	GP5002-000000-72GC	Propylene glycol monomethyl ether acetate
Syringe pump	Harvard Apparatus	703007
Trichlorosilane	Gelest, Inc	SIT8174.0	Tridecafluoro-1,1,2,2-tetrahydrooctyl. Hazardous. Corrosive to the respiratory tract, reacts violently with water.
Trypsin Neutralizer Solution	Gibco	R-002-100