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

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
<ol>
	<li>Before the photolithography process12, use the 4-inch silicon wafers as a substrate and dehydrate the wafers in a conventional oven at 120 &#176;C for 10 min.</li>
	<li>Clean the dehydrated silicon wafers by using oxygen plasma treatment at 100 watts for 30 sec in a plasma cleaner.</li>
	<li>Preheat two hotplates at 65 &#176;C and 95 &#176;C, respectively, for the following baking process.</li>
	<li>Coat 5 g of negative photoresist (PR) on the cleaned silicon wafers by a spin coater; spin at 1,200 rpm (SU-8 50) for 30 sec to produce the microchannel layer.</li>
	<li>Place the PR coated wafer on a preheated hotplate at 65 &#176;C for 12 min and transfer it to another preheated hotplate at 95 &#176;C for 33 min (for 100 &#181;m thick patterns) to perform a soft bake process.</li>
	<li>After baking, place the PR coated silicon wafer on the holder of a semi-automated mask aligner and align it to a 25,400 dpi resolution transparency photomask.</li>
	<li>Expose the PR coated silicon wafer to UV light (365 nm) at a dose of 500 mJ/cm2 to create the PR pattern on the silicon wafer.</li>
	<li>Remove the wafer from the aligner and place it on a hotplate for post-baking at 95 &#176;C for 12 min.</li>
	<li>Soak the wafers in SU-8 developer (propylene glycol monomethyl ether acetate, PGMEA) solution to wash off uncrosslinked PR for 12 min and gently dry with nitrogen gas to expose the alignment marks.</li>
	<li>Again, coat 5 g of negative photoresist on the wafers by a spin coater; spin at 700 rpm (SU-8 100) for 30 sec and 1,200 rpm (SU-8 10) for 30 sec for 300 &#181;m thick pattern and 27 &#181;m thick pattern respectively to make the microwell layer.</li>
	<li>Place the PR coated wafer on a hotplate at 65 &#176;C for 4 min and at 95 &#176;C for 8 min (for 27 &#181;m deep capture-well layer); and at 65 &#176;C for 40 min and at 95 &#176;C for 110 min (for 300 &#181;m deep culture-well layer).</li>
	<li>After cooling, place the PR coated silicon wafer on the mask aligner equipped with UV light.</li>
	<li>Expose the PR coated silicon wafer to the UV light (365 nm) at a dose of 250 mJ/cm2 (for 27 &#181;m thick pattern) and 700 mJ/cm2 (for 300 &#181;m thick pattern).</li>
	<li>Bake the wafers at 95 &#176;C for 5 min (27 &#181;m thick pattern) and 30 min (300 &#181;m thick pattern), respectively.</li>
	<li>Wash off the uncrosslinked PR by the PGMEA for 6 min (27 &#181;m thick pattern) and 25 min (300 &#181;m thick pattern), respectively, and then dry them with nitrogen gas.</li>
	<li>Measure the height of pattern features on the wafer with a scanning laser profilometer by placing the wafer on the xy-stage of a scanning laser profilometer.
	<ol>
		<li>Adjust the focal plane to clearly show the pattern features on the wafer by using the &#34;camera view&#34; observation mode under a 20X objective lens.</li>
		<li>Switch the observation mode from &#34;camera view&#34; to &#34;laser view&#34;, and set the upper and lower positions of the features.</li>
		<li>Set the measurement mode, area, quality, and z-pitch up to transparent (top), 1 line (1,024 x 1), high-accuracy and 0.5 &#181;m, respectively, and then press the start bottom to begin the measurement.</li>
	</ol>
	</li>
</ol>
2. Preparation of PDMS Devices for Single-cell Isolation
<ol>
	<li>Before PDMS casting, silanize the master molds with trichlorosilane to create a hydrophobic surface, which makes it easier to peel off the PDMS replicas from the master molds.
	<ol>
		<li>Place the master molds and a weighting boat containing 200 &#181;l of trichlorosilane in a desiccator, and apply vacuum (-85 kPa) for 15 min.</li>
		<li>Stop the vacuum and then leave the master molds in the desiccator to silanize the master molds at room temperature for at least 1 hr.</li>
		<li>Remove the master molds from the desiccator and place each master mold in a 10 cm Petri dish.</li>
	</ol>
	</li>
	<li>Mix a total amount of 17.6 g PDMS polymer kit containing a base and a curing agent at a ratio of 10:1, and then pour the PDMS onto the master mold in the Petri dish.</li>
	<li>Place the Petri dish in a desiccator and apply vacuum (-85 kPa) for 1 hr to remove air bubbles in the PDMS.</li>
	<li>Remove the Petri dish from the desiccator and place it in a conventional oven at 65 &#176;C for 3 - 6 hr to cure the PDMS.</li>
	<li>Remove the cured PDMS replicas from the master molds and punch two holes as an inlet and an outlet at the two ends of the microchannel on the capture-well array PDMS replica by a puncher with inner-diameter of 0.75 mm for the fluidic channel (Figure 2A).</li>
	<li>Use tape to clean the surface of the PDMS replicas, and then place the PDMS replicas in a plasma cleaner for brief oxygen plasma treatment (100 watts for 14 sec).</li>
	<li>Remove the PDMS replicas from the oxygen plasma machine.</li>
	<li>Align a top (containing the capture microwells) and a bottom PDMS (containing the culture microwells) replica by hand under a stereomicroscope and bring them into contact.</li>
	<li>Place the aligned PDMS replicas in an oven at 65 &#176;C for 24 hr to achieve permanent bonding between the PDMS replicas to form the final device.</li>
	<li>Soak the PDMS device in a deionized (DI)-water filled container and place the container in a desiccator under vacuum (-85 kPa) for 15 min to remove air from the microchannel of the PDMS device.</li>
	<li>Place the DI water-filled PDMS device in a tissue culture hood and use UV light (wavelength of light: 254 nm) to sterilize of the device for 30 min.</li>
	<li>Replace the DI water in the PDMS device with a 5% bovine serum albumin (BSA) in 1x PBS solution and incubate at 37 &#176;C for 30 min to prevent cells from sticking to the PDMS surface. 
	Note: The BSA coating is critical to improve the transfer efficiency of isolated cells from capture-well to culture-well.</li>
	<li>Replace the 5% BSA solution in the PDMS device with sterilized 1x PBS solution.</li>
</ol>
3. Preparation of Single-cell Suspension
<ol>
	<li>Culture Neural stem cell KT98, and human carcinoma cell A549 and MDA-MB-435 we in Petri dish in conventional cell culture incubator (37 &#176;C, 5% CO2, and 95% humidity) to prepare cells for the PDMS device cell experiment.</li>
	<li>Remove and discard the spent culture medium (DMEM basal medium supplied with 10% fetal bovine serum and 1% antibiotics) from the culture dish with cells grown to 70 - 80% confluence.</li>
	<li>Gently wash the cells with sterilized PBS three times.</li>
	<li>Remove and discard the PBS, and add 2 ml of a recombinant enzyme mixture with proteolytic, collagenolytic, and DNase activities (please see the Materials List for the detailed information).</li>
	<li>Incubate the culture dish at room temperature for 5 min, and then tap the culture dish to facilitate cell detachment.</li>
	<li>Add 4 ml of sterilized PBS to disperse cells, and then transfer the cell suspension to a 15 ml conical tube.</li>
	<li>Centrifuge the tube at 300 x g for 3 min and remove the supernatant.</li>
	<li>Gently resuspend the cell pellet in 1 ml sterilized PBS and count the live cell number using the standard Trypan Blue exclusion method13. 
	Note: This resuspension step is critical for preparing well-dissociated single-cell suspension to improve the single-cell isolation efficiency.</li>
</ol>
4. Single-cell Isolation and Clonal Culture
<ol>
	<li>Load 50 &#181;l of cell suspension at a concentration of 2.2 - 2.5 x106 cells/ml into the microchannel of the PDMS device via the device&#39;s outlet hole with a handheld pipette. 
	Note: The cell suspension loading pipette needs to be stopped and held at the first stop position to avoid introducing air bubbles into the microchannel.</li>
	<li>Load another 50 &#181;l of cell suspension through the inlet hole to evenly fill up the whole microchannel with cells.</li>
	<li>Seal the outlet hole with a plug (a 3 mm long, 1 mm diameter cut nylon fishing line) to avoid flow induced by hydrostatic pressure from the droplets on inlet and outlet holes. 
	Note: The plugs were UV sterilized inside a tissue culture hood prior to use.</li>
	<li>Fill up a 1 ml sterile syringe with culture medium, eject air bubbles from it, and set it up on a syringe pump.</li>
	<li>Connect the medium loaded syringe to the inlet hole of the PDMS device via a 23 G blunt needle and a poly-tetrafluoroethene (PTFE) tubing.</li>
	<li>Remove the plug from outlet hole and allow a 2-min time interval to allow the cells to settle into the single-cell capture-wells by gravitational force.</li>
	<li>Wash away the uncaptured cells with 300 &#181;l of culture medium at a flow rate of 600 &#181;l/min driven by the syringe pump.</li>
	<li>Wait for 2 min to stabilize the device, and seal the inlet and outlet holes with plugs to form a closed culture system.</li>
	<li>Flip the device by hand to transfer the captured single-cells to the culture microwells.</li>
	<li>Place the PDMS device in a 100-mm tissue culture dish, and add 10 ml of sterilized PBS around the device to avoid culture medium evaporation from the microchannel.</li>
	<li>Move the culture dish to a conventional cell culture incubator (37 &#176;C, 5% CO2, and 95% humidity) for clonal culture of the single-cells.</li>
</ol>
5. Culture Medium Replenishment
<ol>
	<li>After 1 d of culture, replace the culture medium in the PDMS device with fresh medium to improve cell proliferation.</li>
	<li>Place two droplets of culture medium on top of the PDMS device near the inlet and outlet holes areas to avoid introducing air bubbles into the microchannel while performing the next step.</li>
	<li>Punch two holes from the top of the PDMS device near the two ends of the microchannel as an inlet and an outlet for the microchannel. 
	Note: Do not punch through the whole two layers of the PDMS device; instead, just punch through the top layer of the PDMS.</li>
	<li>Connect a 1 ml plastic syringe containing fresh medium to the inlet via a 23 G blunt needle and PTFE tubing.</li>
	<li>Flow 120 &#181;l fresh medium into the device for 5 min to replace the old medium.</li>
	<li>Insert two plugs to seal the inlet and outlet holes, and return the device to the cell culture incubator.</li>
	<li>Refresh the culture medium every 2 d during the period of culture by removing the sealing plugs, followed by repeating steps 5.4 to 5.5.</li>
</ol>

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	<li>Meacham, C. E., Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour+heterogeneity+and+cancer+cell+plasticity.">Tumour heterogeneity and cancer cell plasticity.</a> Nature. 501 (7467), 328-337 (2013).</li><li>Vermeulen, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+cloning+of+colon+cancer+stem+cells+reveals+a+multi-lineage+differentiation+capacity.">Single-cell cloning of colon cancer stem cells reveals a multi-lineage differentiation capacity.</a> P Natl Acad Sci USA. 105 (36), 13427-13432 (2008).</li><li>Shapiro, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Practical flow cytometry. , (2005).</li><li>Leong, K. G., Wang, B. E., Johnson, L., Gao, W. 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The authors declare that they have no competing financial interests.

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 ...

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 utilizing a robotic arm for assistance, because the Poisson distribution nature of the limiting dilution method restricts single-cell events to a maximum probability of 37%3. FACS machines with an integrated robotic arm can overcome the limitation of Poisson distribution by accurately placing one single-cell in a culture well at a time4. However, the high mechanical shear stress (thus, lowered cell viability)5 and machine purchase and operational costs have limited its usage in many laboratories.
To overcome the above limitations, microscale devices have been developed to highly efficiently load single cells into microwells6. However, the microwells do not provide adequate space for the loaded cells to proliferate, due to the need of making the size of each microwell close to that of a single cell to maximize the single-cell loading probability. As culture assays are required in many cell-based applications (e.g., clonogenic assay7), larger microwells (from 90 - 650 &#181;m in diameter or in side length) have also been utilized to allow for extended cell cultures. However, like the limiting dilution method, they also possess low single cell loading efficiencies, ranging from 10 - 30%.8,9
Previously, we have developed a high-throughput microfluidic platform to isolate single cells in individual microwells and demonstrate its application in clonogenic assay of the isolated cells.10 The device was made with poly-dimethylsiloxane (PDMS), and comprises two sets of microwell arrays with different microwell sizes, which can largely improve the efficiency in loading a single cell in a microwell whose size is significantly larger than the cell. Notably, this &#34;dual-well&#34; concept allows the size of the culture area to be flexibly adjusted without affecting the single-cell capture efficiency, making it straightforward to adjust the design of the device to suit different cell types and applications. This high-efficiency method should be useful for long-term cell culture experiments for cell heterogeneity studies and monoclonal cell line establishment.

<table><tbody><tr><td>AutoCAD software</td><td>Autodesk</td><td>AutoCAD LT 2011</td><td>Part No. 057C1-74A111-1001</td></tr><tr><td>Silicon wafer&amp;nbsp;</td><td>Eltech corperation</td><td>SPE0039</td><td></td></tr><tr><td>Conventional oven</td><td>YEONG-SHIN company</td><td>ovp45</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>SU-8 50 negative photoresist</td><td>MicroChem</td><td>Y131269</td><td></td></tr><tr><td>SU-8 100 negative photoresist</td><td>MicroChem</td><td>Y131273</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>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>SU-8 developer</td><td>Grand Chemical Companies</td><td>GP5002-000000-72GC</td><td>Propylene glycol monomethyl ether acetate</td></tr><tr><td>Scanning laser profilometer</td><td>KEYENCE</td><td>VK-X 100</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>Desiccator</td><td>Bel-Art Products</td><td>F42020-0000</td><td>Space saver vacuum desiccator 190&amp;nbsp;mm white base</td></tr><tr><td>Polydimethylsiloxane (PDMS) kit</td><td>Dow corning</td><td>Sylgard 184</td><td></td></tr><tr><td>Harris Uni-Core puncher</td><td>Ted Pella Inc.</td><td>15072</td><td>with 0.75 mm inner-diameter</td></tr><tr><td>Removable tape</td><td>3M Company</td><td>Scotch Removable Tape 811</td><td></td></tr><tr><td>Stereomicroscope</td><td>Leica Microsystems</td><td>Leica E24</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Bersing Technology</td><td>ALB001.500</td><td></td></tr><tr><td>DMEM basal medium</td><td>Gibco</td><td>12800-017</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Thermo Hyclone</td><td>SH30071.03HI</td><td></td></tr><tr><td>Antibiotics</td><td>Biowest</td><td>L0014-100</td><td>Glutamine-Penicillin-Streptomycin</td></tr><tr><td>Recombinant enzyme mixture</td><td>Innovative cell technology</td><td>AM-105</td><td>Accumax</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>Syringe pump</td><td>Harvard Apparatus</td><td>703007</td><td></td></tr><tr><td>Plastic syringe (1 ml)</td><td>BD Biosciences</td><td>309659</td><td></td></tr><tr><td>23 gauge blunt needles</td><td>Ever Sharp Technology, Inc.</td><td>TD21</td><td></td></tr><tr><td>Poly-tetrafluoroethene (PTFE) tubing</td><td>Ever Sharp Technology, Inc.</td><td>TFT-23T</td><td>inner diameter, 0.51&amp;nbsp;mm; outer diameter, 0.82&amp;nbsp;mm</td></tr></tbody></table>

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.

The microfluidic platform for single-cell isolation and culture comprises a microchannel (200 &#181;m in height) with two sets of microwell arrays (Figure 2A). The two sets of microwell arrays are termed as capture-well (25 &#181;m in diameter and 27 &#181;m in depth) and culture-well (285 &#181;m in diameter and 300 &#181;m 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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A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

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 ...

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11.3K Views.  National Health Research Institutes, Taiwan. 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. 

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

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 Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

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 Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15&#8211;1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing.

This protocol describes a system architecture for performing automated small volume (0.15&#8211;1.5 ml) particle separations using a microfluidic device, and discusses methods to optimize acoustofluidic device performance and operation.

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

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.

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 ...

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 ...

Clinical Microfluidic Chip Platform for the Isolation of Versatile Circulating Tumor Cells

Circulating tumor cells (CTCs) are significant in cancer prognosis, diagnosis, and anti-cancer therapy. CTC enumeration is vital in determining patient disease since CTCs are rare and heterogeneous. CTCs are detached from the primary tumor, enter the blood circulation system, and potentially grow at distant sites, thus metastasizing the tumor. Since CTCs carry similar information to the primary tumor, CTC isolation and subsequent characterization can be critical in monitoring and diagnosing cancer. The enumeration, affinity modification, and clinical immunofluorescence staining of rare CTCs are powerful methods for CTC isolation because they provide the necessary elements with high sensitivity. Microfluidic chips offer a liquid biopsy method that is free of any pain for the patients. In this work, we present a list of protocols for clinical microfluidic chips, a versatile CTC isolating platform, that incorporate a set of functionalities and services required for CTC separation, analysis, and early diagnosis, thus facilitating biomolecular analysis and cancer treatment. The program includes rare tumor cell counting, clinical patient blood preprocessing, which includes red blood cell lysis, and the isolation and recognition of CTCs in situ on microfluidic chips. The program allows the precise enumeration of tumor cells or CTCs. Additionally, the program includes a tool that incorporates CTC isolation with versatile microfluidic chips and immunofluorescence identification in situ on the chips, followed by biomolecular analysis.

The clinical microfluidic chip is an important biomedical analysis technique that simplifies clinical patient blood sample preprocessing and immunofluorescently stains circulating tumor cells (CTCs) in situ on the chip, allowing the rapid detection and identification of a single CTC.

Circulating tumor cells (CTCs) are significant in cancer prognosis, diagnosis, and anti-cancer therapy. CTC enumeration is vital in determining patient ...

Automated and High-throughput Microbial Monoclonal Cultivation and Picking Using The Single-cell Microliter-droplet Culture Omics System

Pure bacterial cultures are essential for the study of microbial culturomics. Traditional methods based on solid plates, well plates, and micro-reactors are hindered by cumbersome procedures and low throughput, impeding the rapid progress of microbial culturomics research. To address these challenges, we had successfully developed the Single-cell Microliter-droplet Culture Omics System (MISS cell), an automated high-throughput platform that utilizes droplet microfluidic technology for microbial monoclonal isolation, cultivation, and screening. This system can generate a large number of single-cell droplets and cultivate, screen, and collect monoclonal colonies in a short time, facilitating an integrated process from microbial isolation to picking. In this protocol, we demonstrated its application using the isolation and cultivation of human gut microbiota as an example and compared the microbial isolation efficiency, monoclonal culture performance, and screening throughput using the solid-plate culture method. The experimental workflow was simple, and reagent consumption was very low. Compared to solid-plate culture methods, the MISS cell could cultivate a greater diversity of gut microbiota species, offering significant potential and value for microbial culturomics research.

This protocol describes how to use the Single-cell Microliter-droplet Culture Omics System (MISS cell) to perform microbial monoclonal isolation, cultivation, and picking. The MISS cell achieves an integrated workflow based on droplet microfluidic technology, which offers excellent droplet monodispersity, high parallel cultivation, and high-throughput biomass detection.

Pure bacterial cultures are essential for the study of microbial culturomics. Traditional methods based on solid plates, well plates, and micro-reactors ...

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

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