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.

1. Fabrication of a wafer mold by soft lithography
NOTE: Mask pattern data is available in our previous publication14.
<ol>
	<li>Dehydrate a 4-inch silicon wafer in a 120 &#176;C oven for 15 min.</li>
	<li>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 &#181;m thick layer (layer #1).</li>
	<li>Soft bake layer #1 on a 65 &#176;C hotplate for 1 min and then transfer layer #1 to a 95 &#176;C hotplate for 3 min.</li>
	<li>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).</li>
	<li>Expose layer #1 with 365 nm UV light at a dose of 150 mJ/cm2.</li>
	<li>Remove layer #1 from the aligner and post-bake on a 65 &#176;C hotplate for 1 min. Transfer layer #1 to a 95 &#176;C hotplate for 1 min.</li>
	<li>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.</li>
	<li>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 &#181;m thick layer #2.</li>
	<li>Remove the tape, soft bake layer #2 on a 65 &#176;C hotplate for 3 min, and then transfer layer #2 to a 95 &#176;C hotplate for 7 min.</li>
	<li>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.</li>
	<li>Expose layer #2 with 365 nm UV light at a dose of 200 mJ/cm2.</li>
	<li>Remove layer #2 from the aligner and post-bake on a 65 &#176;C hotplate for 1 min and transfer layer #2 to a 95 &#176;C hotplate for 3 min.</li>
	<li>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 &#181;m thick layer #3.</li>
	<li>Remove the tape, soft bake layer #3 on a 65 &#176;C hotplate for 5 min, and then transfer layer #3 to a 95 &#176;C hotplate for 20 min.</li>
	<li>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.</li>
	<li>Expose layer #3 with 365 nm UV light at a dose of 240 mJ/cm2.</li>
	<li>Remove layer #3 from the aligner and post-bake on a 65 &#176;C hotplate for 5 min. Transfer layer #3 to a 95 &#176;C hotplate for 10 min.</li>
	<li>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.</li>
</ol>
2. PDMS device preparation for multiple single cell capture
<ol>
	<li>Place the wafer mold and the weighing dish containing 100 &#181;L 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.</li>
	<li>Stop the vacuum, and then silanize the wafer mold in the desiccator (only for silanization) at 37 &#176;C for at least 1 h.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Place the 15 cm dish in an oven at 65 &#176;C for 2-4 h to cure PDMS.</li>
	<li>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).</li>
	<li>Clean the PDMS replica and the slide surface with removable tape and then treat the surface with oxygen plasma (100 W for 14 s).</li>
	<li>Manually align the PDMS replicas with the slide and bring them into contact with each other.</li>
	<li>Place the PDMS slide in a 65 &#176;C oven for 1 day. 
	NOTE: Permanent bonding between the slide and the PDMS replica is achieved to form the device.</li>
	<li>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.</li>
	<li>Place the PDMS device in a cell culture hood and sterilize the device with UV light (light wavelength: 254 nm) for 30 min.</li>
	<li>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 &#176;C for 1 day. This prevents cells from adhering to the PDMS surface.</li>
</ol>
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.
<ol>
	<li>Remove the culture medium when the cells achieve 70-80% confluence. Then gently wash the cells with 5 mL of sterile PBS three times.</li>
	<li>Add 1 mL of DMEM-F12 medium containing 1 &#181;M 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.</li>
	<li>Gently wash the cells with 5 mL of sterile PBS three times.</li>
	<li>Remove the PBS and add 2 mL of 0.25% Trypsin-EDTA (0.05% Trypsin-EDTA for LECs).</li>
	<li>Incubate the cells for 4 min at room temperature and then gently tap the tissue culture dish to promote cells detachment.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
4. Multiple single-cell capture and triple single-cell culture
<ol>
	<li>Connect a poly-tetrafluoroethene (PTFE) tube between the outlet of the device and syringe pump. Remove the medium and add 1 &#181;L of cell suspension at a concentration of 3 x 105 cells/mL into the inlet of the PDMS device.</li>
	<li>Load the cell suspension into the device by a syringe pump at a flow rate of 0.3 &#181;L/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.</li>
	<li>Add 1 &#181;L 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 &#181;L/min (Figure 2B). Flow direction was flowing from the inlet to the outlet.</li>
	<li>Load 0.3 &#181;L of DMEM-F12 medium into the device by a syringe pump at a flow rate of 10 &#181;L/min (Figure 2C). Flow direction was flowing from the outlet to the inlet.</li>
	<li>Repeat steps 4.1 to 4.4 to load other cell types into the device.</li>
	<li>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.</li>
	<li>Remove the PTFE tube and seal the inlet and the outlet with polyolefin tape to create a closed culture system.</li>
	<li>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.</li>
	<li>Transfer the culture dish to an incubator (37 &#176; C, 5% CO2 and 95% humidity) for triple single-cell culture.</li>
	<li>Microscopically observe and photograph cell growth every 12 h.</li>
</ol>

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The authors declare that they have no competing financial interests.

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 culture chamber by a deterministic method, without being limited by the Poisson distribution limitation in the dilution method and the microwell platform. By providing a high triple single cell capture efficiency (greater than 45%, the Poisson distribution method is 5%) and demonstrating that in the culture chamber, space is sufficient for cell growth and proliferation (Figure 4). Due to its ability to efficiently perform multiple single-cell captures and live cell culture observations with simple setup and protocol, we envision these microfluidic devices as useful tools for an extensive range of applications, including cell-cell interactions between multiple cells18, drug screening19, and cancer biology20. On the other hand, the device structure is moldable, and the structure and size of the capture-site can be changed and applied to other fields such as microorganisms and plant cells. In theory, our method is also adaptable to establish and track microbial co-cultures (e.g., bacteria, yeasts, etc.).
The main limitation of this approach is that the precision level required for the device fabrication is high. This is mainly because the flow resist of the smallest channel can be dramatically changed if its fabricated dimensions are slightly offset. The control of resistances of the microchannels are crucial for the high-efficiency single-cell capture of the device. On the other hand, during cell culture, there is no closure between the chambers. Therefore, paracrine secretion of cells in the chamber may spread into other chambers to affect other cells. Finally, attention must be paid to the cleanliness of the channel and tubing during the preparation of the device. The culture medium and any buffer used in the experiment need to be filtered to prevent particles and debris from blocking the channel.

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 &#34;hydrodynamic shuttling chip&#34; 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.

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

establishing single-cell based co-cultures in a deterministic manner with a microfluidic chip

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&#39; 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.

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 &#181;m in width and 4.6 &#181;m 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 a cell is captured at the capture position, the flow resistance of the small path is increased, causing the next incoming cell to go toward and through the by-pass channel to the next downstream capture-site. The serpentine design of the by-pass channel (25.0 &#181;m in width and 26.4 &#181;m in height) of the second layer is used to increase its flow resistance. The dimensions of the culture chamber in the third layer (500.3 &#181;m in diameter and 111.3 &#181;m in height) are designed to provide sufficient space for cell culture experiment, and to reduce the flow rate in the chamber to keep the cells from being flushed out of the chamber.
The tools required for the operation procedure (Figure 2) are common in general laboratories, including microscope, syringe pump, glass syringe, centrifuge tube, tubing, and pipette. The device is approximately 1/5 of a coverslip with a 2 mm thickness and is suitable for observing under a microscope with 4x to 20x lens. With this method, one type for single cells can be captured in the culture chambers under 7 min, so the total operation time for triple single cells was less than 21 min. The single cell capture efficiencies of the demonstrated three cells were 70.83% &#177; 15.42% (LECs), 73.96% &#177; 14.09% (WNT5B sh4) and 78.13% &#177; 3.13% (pLKO-GFP), respectively. The triple single cell capture efficiency was 47.92% &#177; 7.86% (Figure 3B). The reflux operation step is used to release the cells from the capture sites simultaneously and flow the cells into the culture chambers (Figure 2C). During this process, if a cell is not released from its capture-site, the released cell at its downstream capture-site will not enter the culture chamber due to by-pass channel having a lower flow resistance than the culture chamber. This is the major reason why the HSC has a triple single cell capture efficiency of only 47.92 &#177; 7.86%, which is still significantly greater than the probability of a Poisson distribution (~5%).
The cell culture results showed that multiple single-cell co-cultures of lymphatic endothelial cells and squamous cancer cells can be performed for 24 h, and the cells&#39; proliferation and morphology can be observed under microscope (Figure 4, Supplementary Videos 1-3). In the presence of pLKO-GFP cells, WNT5B sh4 cells and LECs showed better proliferative capacity and showed that the morphology approached lamellipodia. These results demonstrate the ability of this device to high-efficiently capture multiple types of single cells in a culture chamber and provide a sufficient culture space useful for studying multiple single cell type interactions.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60202/60202fig1.jpg" /> 
Figure 1: Photograph and microscope Images of hydrodynamic shuttling chip. (A) Microscope image of the PDMS sectional view. (B) A single cell trapping unit of magnified view. (C) Appearance of the chip containing 48 units. Scale bar: 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60202/60202fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60202/60202fig2.jpg" /> 
Figure 2: Hydrodynamic shuttling chip operation procedure. (A) Due to the high hydrodynamic resistance of the by-pass channel, a single cell is trapped in the capture-site. (B) After the first cell is trapped and has occluded the capture-site, the following cells flow toward the by-pass channel due to the increased flow resistance. Use medium to wash remaining cells in channel. (C) Reflux the cell into culture chamber. Scale bar: 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60202/60202fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60202/60202fig3.jpg" /> 
Figure 3: Single cell capture efficiency of LECs, WNT5B sh4 and pLKO-GFP in the hydrodynamic shuttling chip. (A) Microscope image of captured triple single cells in culture chamber. (B) The green, blue, and red bars represent the capture efficiency of individual cells, respectively, and the yellow bars represent the capture efficiency of triple single-cell. Scale bar: 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60202/60202fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60202/60202fig4.jpg" /> 
Figure 4: Paired single cell co-culture and triple single cell co-culture in the hydrodynamic shuttling chip for 24 h. (A) Microscope image of triple single cells co-culture for 0, 12 and 24 h. (B) Microscope image of pLKO-GFP and WNT5B sh4 cells co-culture for 0, 12 and 24 h. LECs were stained with green CMFDA Dye, WNT5B sh4 cells were stained with blue CMAC Dye and pLKO-GFP cells were stained with red DiI fluorescent dye. Scale bar: 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60202/60202fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Video 1" src="/files/ftp_upload/60202/60202vid1.jpg" /> 
Supplementary Video 1: Cell Loading. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60202/s1_cell_loading.mp4" target="_blank">Please click here to view this video (Right click to download).</a>
<img alt="Video 2" src="/files/ftp_upload/60202/60202vid2.jpg" /> 
Supplementary Video 2: Cell Refluxing. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60202/s2_cell_refluxing.mp4" target="_blank">Please click here to view this video (Right click to download).</a>
<img alt="Video 3" src="/files/ftp_upload/60202/60202vid3.jpg" /> 
Supplementary Video 3: Second cell type reflux into the culture chamber. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60202/s3_second_cell_type.mp4" target="_blank">Please click here to view this video (Right click to download).</a>

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

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.

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases ...

establishing-single-cell-based-co-cultures-deterministic-manner-with

Nanyang Technological University

Research

JoVE Journal

Bioengineering

6.0K Views.  National Health Research Institutes. Het is van cruciaal belang om effectief te combineren capture efficiëntie met dynamische live cel beeld observatie om het effect van cel-cel interactie op celgedrag te begrijpen. Deze buis kan zeer efficiënt meerdere enkele cellen in een grotere kamer vastleggen en een voldoende cultuurbasis bieden, voor dynamische tracking van meerdere eencellige interactiegedrag. Begin met het voorbereiden van het PDMS-apparaat.Plaats een wafer mal in de weegschaal met 100 microliters van trichloro...

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

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from non-biodegradable polymers, e.g., polycarbonate or polymethyl methacrylate by micro injection molding, micro hot embossing or micro thermoforming. But, it can also be manufactured from bio-degradable polymers. Its overall dimensions are 0.7 1 x 20 x 20 x 0.7 1 mm (h x w x l). The main features of the chips used are either a grid of up to 1156 cubic micro-containers (cf-chip) each the size of 120-300 x 300 x 300 &mu; (h x w x l) or round recesses with diameters of 300 &mu; and a depth of 300 &mu; (r-chip). The scaffold can house 10 Mio. cells in a three-dimensional configuration. For an optimal nutrient and gas supply, the chip is inserted in a bioreactor housing. The bioreactor is part of a closed steril circulation loop that, in the simplest configuration, is additionaly comprised of a roller pump and a medium reservoir with a gas supply. The bioreactor can be run in perfusion, superfusion, or even a mixed operation mode. We have successfully cultivated cell lines as well as primary cells over periods of several weeks. For rat primary liver cells we could show a preservation of organotypic functions for more than 2 weeks. For hepatocellular carcinoma cell lines we could show the induction of liver specific genes not or only slightly expressed in standard monolayer culture. The system might also be useful as a stem cell cultivation system since first differentiation experiments with stem cell lines were promising.

We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from ...

Biology

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

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

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 ...

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

Neuroscience

A Microfluidics Approach for the Functional Investigation of Signaling Oscillations Governing Somitogenesis

Periodic segmentation of the presomitic mesoderm of a developing mouse embryo is controlled by a network of signaling pathways. Signaling oscillations and gradients are thought to control the timing and spacing of segment formation, respectively. While the involved signaling pathways have been studied extensively over the last decades, direct evidence for the function of signaling oscillations in controlling somitogenesis has been lacking. To enable the functional investigation of signaling dynamics, microfluidics is a previously established tool for the subtle modulation of these dynamics. With this microfluidics-based entrainment approach endogenous signaling oscillations are synchronized by pulses of pathway modulators. This enables modulation of, for instance, the oscillation period or the phase-relationship between two oscillating pathways. Furthermore, spatial gradients of pathway modulators can be established along the tissue to study how specific changes in the signaling gradients affect somitogenesis.
The present protocol is meant to help establish microfluidic approaches for the first-time users of microfluidics. The basic principles and equipment needed to set up a microfluidic system are described, and a chip design is provided, with which a mold for chip generation can conveniently be prepared using a 3D printer. Finally, how to culture primary mouse tissue on a microfluidic chip and how to entrain signaling oscillations to external pulses of pathway modulators are discussed.
This microfluidic system can also be adapted to harbor other in vivo and in vitro model systems such as gastruloids and organoids for functional investigation of signaling dynamics and morphogen gradients in other contexts.

A protocol for the culture and manipulation of mouse embryonic tissue on a microfluidic chip is provided. By applying pulses of pathway modulators, this system can be used to externally control signaling oscillations for the functional investigation of mouse somitogenesis.

Periodic segmentation of the presomitic mesoderm of a developing mouse embryo is controlled by a network of signaling pathways. Signaling oscillations and ...

Developmental Biology

Generation of Heterogeneous Drug Gradients Across Cancer Populations on a Microfluidic Evolution Accelerator for Real-Time Observation

Conventional cell culture remains the most frequently used preclinical model, despite its proven limited ability to predict clinical results in cancer. Microfluidic cancer-on-chip models have been proposed to bridge the gap between the oversimplified conventional 2D cultures and more complicated animal models, which have limited ability to produce reliable and reproducible quantitative results. Here, we present a microfluidic cancer-on-chip model that reproduces key components of a complex tumor microenvironment in a comprehensive manner, yet is simple enough to provide robust quantitative descriptions of cancer dynamics. This microfluidic cancer-on-chip model, the &#34;Evolution Accelerator,&#34; breaks down a large population of cancer cells into an interconnected array of tumor microenvironments while generating a heterogeneous chemotherapeutic stress landscape. The progression and the evolutionary dynamics of cancer in response to drug gradient can be monitored for weeks in real time, and numerous downstream experiments can be performed complementary to the time-lapse images taken through the course of the experiments.

We present a microfluidic cancer-on-chip model, the &#34;Evolution Accelerator&#34; technology, which provides a controllable platform for long-term real-time quantitative studies of cancer dynamics within well-defined environmental conditions at the single-cell level. This technology is expected to work as an in vitro model for fundamental research or pre-clinical drug development.

Conventional cell culture remains the most frequently used preclinical model, despite its proven limited ability to predict clinical results in cancer. ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

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

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