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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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-efficiently prepare single cells3. However, FACS may not be accessible to some laboratories due to expensive instrumentation and maintenance cost. Microfabricated devices have been recently developed for single cell trapping4, single cell pairing5, and single cell culture applications. These devices are advantageous based on their ability to accurately manipulate single cells6, perform high-throughput experiments, or reduce sample and reagent consumption. However, performing single-cell co-culture experiments with multiple cell types with the current microfluidic devices is still challenging due the limitation of Poisson distribution1,7,8, or inability of the devices to capture more than two types of single cells4,5,6,9,10.

For example, Yoon et al. reported a microfluidic device for cell-cell interaction study11. This device uses the probabilistic method to pair cells in one chamber. However, it can only achieve the pairing of two different cell types due to geometric restrictions in the device structure. Another report from Lee et al. demonstrated a deterministic method to capture and pair single cells12. This device is increases pairing efficiency by the deterministic method but it is limited by the prolonged operation time required to pair cells. Specifically, the second cell capture can only be performed after the first captured cell is attached to the surface after 24 h. Zhao et al. reported a droplet-based microfluidic device to capture two types of a single cell13. We can found that the droplet-based microfluidic device is still limited to the Poisson distribution and can only be used on non-attached cells, and it is not possible to change the culture solution during the cultivation process.

Previously, we have developed a microfluidic "hydrodynamic shuttling chip" that utilizes deterministic hydrodynamic forces to capture multiple types of single-cell into the culture chamber and can subsequently perform cell co-culture experiment to analyze individual cell migration behavior under cell-cell interactions14. The hydrodynamic shuttling chip comprises an arrayed sets of units that each contains a serpentine by-pass channel, a capture-site, and a culture chamber. By using the difference in flow resistance between the serpentine by-pass channel and the culture chamber, and a specially designed operation procedure, different types of single cells can be repeatedly captured into the culture chamber. Notably, the ample space of the culture chamber can not only prevent the cell from being flushed during cell capture out but also provide sufficient space for the cells to spread, proliferate and migrate, allowing for observing of live single-cell interactions. In this article, we focus on the production of this device and detailed protocol steps.

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 photomask (capture-site layer).
  5. Expose layer #1 with 365 nm UV light at a dose of 150 mJ/cm2.
  6. Remove layer #1 from the aligner and post-bake on a 65 °C hotplate for 1 min. Transfer layer #1 to a 95 °C hotplate for 1 min.
  7. Cool layer #1 to room temperature. Immerse in a propylene glycol monomethyl ether acetate solution to wash away the uncrosslinked photoresist for 2 min. Gently dry with nitrogen gas to reveal a layer #1 alignment mark.
  8. Cover the layer #1 alignment mark by an adhesive tape, spin coat 4 g of SU-8 10 negative photoresist onto the layer #1 at 1,230 rpm for 30 s to create a 25 µm thick layer #2.
  9. Remove the tape, soft bake layer #2 on a 65 °C hotplate for 3 min, and then transfer layer #2 to a 95 °C hotplate for 7 min.
  10. Cool layer #2 to room temperature, place layer #2 onto the holder of the semi-automatic mask aligner, and align the layer #2 chrome-plated photomask (bypass channel layer) to the layer #1 alignment mark.
  11. Expose layer #2 with 365 nm UV light at a dose of 200 mJ/cm2.
  12. Remove layer #2 from the aligner and post-bake on a 65 °C hotplate for 1 min and transfer layer #2 to a 95 °C hotplate for 3 min.
  13. Cool layer #2 to room temperature, and cover the layer #1 alignment mark by adhesive tape. Spin coat 4 g of SU-8 2050 negative photoresist onto layer #2 at 1,630 rpm for 30 s to create a 100 µm thick layer #3.
  14. Remove the tape, soft bake layer #3 on a 65 °C hotplate for 5 min, and then transfer layer #3 to a 95 °C hotplate for 20 min.
  15. Cool layer #3 to room temperature, place layer #3 onto the holder of the semi-automatic mask aligner, and align the layer #3 chrome-plated photomask (culture chamber layer) to the layer #1 alignment mark.
  16. Expose layer #3 with 365 nm UV light at a dose of 240 mJ/cm2.
  17. Remove layer #3 from the aligner and post-bake on a 65 °C hotplate for 5 min. Transfer layer #3 to a 95 °C hotplate for 10 min.
  18. Cool layer #3 to room temperature. Immerse in a propylene glycol monomethyl ether acetate solution to washed away the uncrosslinked photoresist for 10 min, and gently dry with nitrogen gas.

2. PDMS device preparation for multiple single cell capture

  1. Place the wafer mold and the weighing dish containing 100 µL of trichlorosilane in a desiccator (only for silanization) and apply a vacuum (-85 kPa) for 15 min.
    NOTE: Silanize the wafer surface with trichlorosilane to create hydrophobic surface properties before PDMS castingso that it can effortlessly be peeled off from the wafer PDMS mold.
  2. Stop the vacuum, and then silanize the wafer mold in the desiccator (only for silanization) at 37 °C for at least 1 h.
  3. Mix PDMS base and PDMS curing agent in a ratio of 10:1. Pour a total of 20 g of mixed PDMS onto the wafer mold in a 15 cm dish.
  4. Place the 15 cm dish into a desiccator and apply vacuum (-85 kPa) for 1.5 min. Then remove the 15 cm dish from the desiccator. Keep for 20 min at room temperature. Finally, remove residual air bubbles in PDMS with nitrogen gas.
  5. Place the 15 cm dish in an oven at 65 °C for 2-4 h to cure PDMS.
  6. Remove the PDMS replica from the wafer mold, and then punch a 1.5 mm inlet and a 0.5 mm outlet on the PDMS using a 1.5 mm inner diameter and a 0.5 mm inner diameter puncher (Figure 1C).
  7. Clean the PDMS replica and the slide surface with removable tape and then treat the surface with oxygen plasma (100 W for 14 s).
  8. Manually align the PDMS replicas with the slide and bring them into contact with each other.
  9. Place the PDMS slide in a 65 °C oven for 1 day.
    NOTE: Permanent bonding between the slide and the PDMS replica is achieved to form the device.
  10. Immerse the PDMS device in a container filled with phosphate buffered saline and place into a desiccator. Then apply vacuum (-85 kPa) for 15 min to remove air bubbles.
  11. Place the PDMS device in a cell culture hood and sterilize the device with UV light (light wavelength: 254 nm) for 30 min.
  12. Replace the PDMS device buffer with medium (DMEM-F12 basal medium containing 1% antibiotic and 10% fetal bovine serum) and incubate the PDMS device at 4 °C for 1 day. This prevents cells from adhering to the PDMS surface.

3. Preparation of a single-cell suspension

NOTE: Cell types include human lymphatic endothelial cells (LECs), human OSCC TW2.6 cells expressing WNT5B-specific shRNA (WNT5B sh4) and vector control (pLKO-GFP) which were obtained from our previous study15. Please refer to our previous publication for detailed cultivation steps.

  1. Remove the culture medium when the cells achieve 70-80% confluence. Then gently wash the cells with 5 mL of sterile PBS three times.
  2. Add 1 mL of DMEM-F12 medium containing 1 µM fluorescent dye into WNT5B sh4 and pLKO-GFP cells (use MV2 medium for LECs) and then incubate the cells for 30 min at room temperature.
    NOTE: LECs were stained with green chloromethylfluorescein diacetate (CMFDA) Dye, WNT5B sh4 cells were stained with blue 7-amino-4-chloromethylcoumarin (CMAC) Dye and pLKO-GFP cells were stained with red Dil fluorescent dye.
  3. Gently wash the cells with 5 mL of sterile PBS three times.
  4. Remove the PBS and add 2 mL of 0.25% Trypsin-EDTA (0.05% Trypsin-EDTA for LECs).
  5. Incubate the cells for 4 min at room temperature and then gently tap the tissue culture dish to promote cells detachment.
  6. Add 4 mL of DMEM-F12 medium to disperse WNT5B sh4 and pLKO-GFP cells (For LECs use 3 mL of MV2 medium and 1 mL of trypsin neutralizer solution). Then transfer the cells into a 15 mL tube, and centrifuge at 300 x g for 3 min.
  7. Remove the supernatant, and resuspend the cell pellet in 1 mL of DMEM-F12 medium gently. Count the number of live cells in a hemocytometer by using the standard Trypan Blue exclusion method16. Prepare 1 mL of cell suspension at 3 x 105 cells/mL concentration in DMEM-F12 medium, and then keep cells on ice to prevent cell aggregation.
    NOTE: In order to improve single-cell capture efficiency, careful preparation of the single cell suspension with well-dissociated is required.

4. Multiple single-cell capture and triple single-cell culture

  1. Connect a poly-tetrafluoroethene (PTFE) tube between the outlet of the device and syringe pump. Remove the medium and add 1 µL of cell suspension at a concentration of 3 x 105 cells/mL into the inlet of the PDMS device.
  2. Load the cell suspension into the device by a syringe pump at a flow rate of 0.3 µL/min (Figure 2A). Flow direction is from the inlet to the outlet.
    NOTE: Load immediately after adding the cell suspension into the inlet to prevent cell sedimentation.
  3. Add 1 µL of DMEM-F12 medium into the inlet of the PDMS device after step 4.2.Load the DMEM-F12 medium into the device by a syringe pump at a flow rate of 0.3 µL/min (Figure 2B). Flow direction was flowing from the inlet to the outlet.
  4. Load 0.3 µL of DMEM-F12 medium into the device by a syringe pump at a flow rate of 10 µL/min (Figure 2C). Flow direction was flowing from the outlet to the inlet.
  5. Repeat steps 4.1 to 4.4 to load other cell types into the device.
  6. After completing the cell capture, use a microscope with 4x lens to image each culture chamber.
    NOTE: The fluorescence emissions of the cells was used to identify and count the number of individual cells in each culture chamber.
  7. Remove the PTFE tube and seal the inlet and the outlet with polyolefin tape to create a closed culture system.
  8. Move the PDMS device to a 10 cm culture dish and add 10 mL of sterile PBS around the PDMS device to avoid evaporation of the medium from the device.
  9. Transfer the culture dish to an incubator (37 ° C, 5% CO2 and 95% humidity) for triple single-cell culture.
  10. Microscopically observe and photograph cell growth every 12 h.

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 ...

Disclosures

The authors declare that they have no competing financial interests.

Acknowledgements

This work was supported by a grant from the Ministry of Science and Technology (105-2628-E-400-001-MY2), and the Ph.D. Program in Tissue Engineering and Regenerative Medicine, National Chung Hsing University and National Health Research Institutes.

Materials

NameCompanyCatalog NumberComments
3M Advanced Polyolefin Diagnostic Microfluidic Medical Tape3M Company9795R
AntibioticsBiowestL0014-100Glutamine-Penicillin-Streptomycin
AutoCAD softwareAutodeskAutoCAD LT 2011Part No. 057C1-74A111-1001
CellTracke Blue CMAC DyeInvitrogenC2110
CellTracker Green CMFDA DyeInvitrogenC7025
Conventional ovenYEONG-SHIN companyovp45
DesiccatorBel-Art ProductsF42020-0000Space saver vacuum desiccator 190 mm white base
DiIC12(3) cell membrane dyeBD Biosciences354218Used as a cell tracker
DMEM-F12 mediumGibco11320-082
Endothelial Cell Growth Medium MV 2PromoCellC-22022
Fetal bovine serum HycloneThermoSH30071.03HI
Hamilton 700 series Glass syringe ( 0.1 ml )Hamilton80630100 µL, Model 710 RN SYR, Small Removable NDL, 22s ga, 2 in, point style 2
Harris Uni-Core puncherTed Pella Inc.15075with 1.5mm inner-diameter
Harris Uni-Core puncherTed Pella Inc.15071with 0.5mm inner-diameter
HotplateYOTEC companyYS-300S
Msak alignerDeya Optronic CO.A1K-5-MDA
Oxygen plasmaNORDSON MARCHAP-300
Plasma cleanerNordsonAP-300Bench-Top Plasma Treatment System
Polydimethylsiloxane (PDMS) kitDow corningSylgard 184
Poly-tetrafluoroethene (PTFE)Ever Sharp Technology, Inc.TFT-23Tinner diameter, 0.51 mm; outer diameter, 0.82 mm
Removable tape3M CompanyScotch Removable Tape 811
Silicon waferEltech corperationSPE0039
Spin coaterSynrex Co., Ltd.SC-HMI 2" ~ 6"
StereomicroscopeLeica MicrosystemsLeica E24
SU-8 10 negative photoresistMicroChemY131259
SU-8 2 negative photoresistMicroChemY131240
SU-8 2050 negative photoresistMicroChemY111072
SU-8 developerGrand Chemical CompaniesGP5002-000000-72GCPropylene glycol monomethyl ether acetate
Syringe pumpHarvard Apparatus703007
TrichlorosilaneGelest, IncSIT8174.0Tridecafluoro-1,1,2,2-tetrahydrooctyl. Hazardous. Corrosive to the respiratory tract, reacts violently with water.
Trypsin Neutralizer SolutionGibcoR-002-100

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