Name: a microfluidic platform for high-throughput single-cell isolation and culture
Uploaded: 2016-06-16T15:00:00
Description: Watch this Scientific Journal Video about A Microfluidic Platform for High-throughput Single-cell Isolation and Culture at JoVE.com

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 1...

<ol>
	<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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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+column:+microwell+arrays+for+mammalian+cell+culture.">Integration column: microwell arrays for mammalian cell culture.</a> Integr. Biol. 1 (11-12), 11-12 (2009).</li><li>Lindstrom, S., 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=High-density+microwell+chip+for+culture+and+analysis+of+stem+cells.">High-density microwell chip for culture and analysis of stem cells.</a> PloS one. 4 (9), e6997 (2009).</li><li>Lin, C. H., 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=A+microfluidic+dual-well+device+for+high-throughput+single-cell+capture+and+culture.">A microfluidic dual-well device for high-throughput single-cell capture and culture.</a> Lab Chip. 15 (14), 2928-2938 (2015).</li><li>Xia, Y. N., Whitesides, G. 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Appendix 3 (Appendix 3B), (2001).</li><li>Lindstrom, S., Andersson-Svahn, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Miniaturization+of+biological+assays+-+Overview+on+microwell+devices+for+single-cell+analyses.">Miniaturization of biological assays - Overview on microwell devices for single-cell analyses.</a> Bba-Gen Subjects. 1810 (3), 308-316 (2011).</li><li>Lecault, V., 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=High-throughput+analysis+of+single+hematopoietic+stem+cell+proliferation+in+microfluidic+cell+culture+arrays.">High-throughput analysis of single hematopoietic stem cell proliferation in microfluidic cell culture arrays.</a> Nat Methods. 8 (7), 581-593 (2011).</li><li>Park, J. Y., 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+trapping+in+larger+microwells+capable+of+supporting+cell+spreading+and+proliferation.">Single cell trapping in larger microwells capable of supporting cell spreading and proliferation.</a> Microfluid Nanofluid. 8 (2), 263-268 (2010).</li><li>Tirino, V., 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=Cancer+stem+cells+in+solid+tumors:+an+overview+and+new+approaches+for+their+isolation+and+characterization.">Cancer stem cells in solid tumors: an overview and new approaches for their isolation and characterization.</a> FASEB J. 27 (1), 13-24 (2013).</li><li>Chen, P. C., Huang, Y. Y., Juang, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MEMS+microwell+and+microcolumn+arrays:+novel+methods+for+high-throughput+cell-based+assays.">MEMS microwell and microcolumn arrays: novel methods for high-throughput cell-based assays.</a> Lab Chip. 11 (21), 3619-3625 (2011).</li><li>Liang, P., 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=Drug+Screening+Using+a+Library+of+Human+Induced+Pluripotent+Stem+Cell-Derived+Cardiomyocytes+Reveals+Disease-Specific+Patterns+of+Cardiotoxicity.">Drug Screening Using a Library of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Reveals Disease-Specific Patterns of Cardiotoxicity.</a> Circulation. 127 (16), 1677-1691 (2013).</li></ol>

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.

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			<td height="21" style="height:21px;width:401px;">AutoCAD software</td>
			<td style="width:225px;">Autodesk</td>
			<td style="width:235px;">AutoCAD LT 2011</td>
			<td style="width:463px;">Part No. 057C1-74A111-1001</td>
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			<td height="21" style="height:21px;">Silicon wafer&#160;</td>
			<td>Eltech corperation</td>
			<td style="width:235px;">SPE0039</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Conventional oven</td>
			<td>YEONG-SHIN company</td>
			<td style="width:235px;">ovp45</td>
			<td></td>
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			<td height="21" style="height:21px;">Plasma cleaner</td>
			<td>Nordson</td>
			<td style="width:235px;">AP-300</td>
			<td>Bench-Top Plasma Treatment System</td>
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			<td height="20" style="height:20px;">SU-8 50 negative photoresist</td>
			<td>MicroChem</td>
			<td>Y131269</td>
			<td style="width:463px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 100 negative photoresist</td>
			<td>MicroChem</td>
			<td>Y131273</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Spin coater</td>
			<td>Synrex Co., Ltd.</td>
			<td>SC-HMI 2&#34; ~ 6&#34;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hotplate</td>
			<td>YOTEC company</td>
			<td>YS-300S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Msak aligner</td>
			<td>Deya Optronic CO.</td>
			<td>A1K-5-MDA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 developer</td>
			<td>Grand Chemical Companies</td>
			<td>GP5002-000000-72GC</td>
			<td>Propylene glycol monomethyl ether acetate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scanning laser profilometer</td>
			<td>KEYENCE</td>
			<td>VK-X 100</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Trichlorosilane</td>
			<td>Gelest, Inc</td>
			<td>SIT8174.0</td>
			<td style="width:463px;">TRIDECAFLUORO-1,1,2,2-TETRAHYDROOCTYL.&#160; Hazardous. Corrosive to the respiratory tract., reacts violently with water.</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Desiccator</td>
			<td>Bel-Art Products&#160;</td>
			<td>F42020-0000</td>
			<td>SPACE SAVER VACUUM DESICCATOR 190MM WHITE BASE</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Polydimethylsiloxane (PDMS) kit</td>
			<td>Dow corning</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Harris Uni-Core puncher</td>
			<td>Ted Pella Inc.</td>
			<td>15072</td>
			<td>with 0.75 mm inner-diameter</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Removable tape</td>
			<td>3M Company</td>
			<td>Scotch Removable Tape 811</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Stereomicroscope</td>
			<td>Leica Microsystems</td>
			<td>Leica E24</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin (BSA)</td>
			<td>Bersing Technology</td>
			<td>ALB001.500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM basal medium</td>
			<td>Gibco</td>
			<td style="width:235px;">12800-017</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal bovine serum</td>
			<td>Thermo Hyclone</td>
			<td>SH30071.03HI</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibiotics</td>
			<td>Biowest</td>
			<td>L0014-100</td>
			<td>Glutamine-Penicillin-Streptomycin</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Recombinant enzyme mixture</td>
			<td>Innovative cell technology</td>
			<td style="width:235px;">AM-105</td>
			<td>Accumax</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DiIC12(3) cell membrane dye</td>
			<td>BD Biosciences</td>
			<td style="width:235px;">354218</td>
			<td>Used as a cell tracker</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td style="width:235px;">703007</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plastic syringe (1 mL)</td>
			<td>BD Biosciences</td>
			<td style="width:235px;">309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">23 gauge blunt needles</td>
			<td>Ever Sharp Technology, Inc.</td>
			<td style="width:235px;">TD21</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Poly-tetrafluoroethene (PTFE) tubing</td>
			<td>Ever Sharp Technology, Inc.</td>
			<td style="width:235px;">TFT-23T</td>
			<td>&#160;inner diameter, 0.51 mm; outer diameter, 0.82 mm</td>
		</tr>
	</tbody>
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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...

Watch this Scientific Journal Video about A Microfluidic Platform for High-throughput Single-cell Isolation and Culture at JoVE.com

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 ...

The overall goal of this protocol is to demonstrate the fabrication and operation of a microfluidic chip, which allows for high-efficiency single-cell isolation and culture. This method provides a useful tool for any area that would benefit from single-cell isolation and culture. The main advantage of this technique is that it is highly efficient at loading single cells into large microwell arrays.Additionally, only gentle flow and the gravitational force are used to operate the device, which minimizes the cell damage. The implication of this technique is turned towards the end diagnosis of human disease such as cancer, in which cellular heterogeneity affects the response of cell, so We came up with the idea of developing this method when we were establishing microwell cell line, because limit dilution method was very time-consuming and inefficient. Now this method can provide insight into individual cell analysis.It can also be applied to other applications such as the establishment of and time-ness of observation of individual cell course and behavior. To begin, fabricate silanized master molds for both the single cell isolation layer and the microwell culture layer, using standard photolithography as described in the accompanying text protocol. For each master mold, combine 16 grams of PDMS base with 1.6 grams of a curing agent in a disposable cup, and stir the mixtures until combined.Then, place the master molds into 150 millimeter petri dishes, and pour the PDMS on top of the molds. Place the petri dishes into a desiccator and apply a vacuum for one hour to remove air bubbles from the PDMS. After one hour, remove the dishes from the desiccator and place them in a conventional oven at 65 degrees Celsius for three to six hours to cure the PDMS.Then, remove the dishes from the oven and let them cool down to room temperature. Once cool, peel the cured PDMS capture-well array and microwell culture array from the master molds and cut out the devices using a razor blade. Next, use a 0.75 millimeter punch to create one hole at each end of the microchannel on the capture-well array layer.Use tape to clean what will become the inner surface of the device, and then place the individual layers with the inner surfaces facing up into a plasma cleaner for a brief oxygen plasma treatment. When finished, remove the PDMS layers from the plasma cleaner and use a stereo microscope to align and connect the top and bottom layers of the device. Place the aligned device in an oven at 65 degrees Celsius for 24 hours to create a permanent bond between the PDMS layers.Next, place the bonded PDMS device in deionized water and place the container in a desiccator under vacuum for 15 minutes, in order to remove the air from the microchannel of the device. With all of the air now removed from the device, place the PDMS device in a tissue culture hood and use UV light to sterilize the surface of the device for 30 minutes. Next, replace the deionized water in the device with 5%bovine serum albumin in PBS and incubate the device at 37 degrees Celsius for 30 minutes.This will block the surface and improve the transfer efficiency of isolated cells from capture-wells to the culture-wells. After 30 minutes, replace the blocking solution in the device with sterile PBS. Prepare a single-cell suspension with 2.5 million cells per milliliter and load a pipette with 50 microliters of the suspension.Then, transfer the cells into the device through the device's outlet hole. Load another 50 microliters of the cell suspension and transfer it into the device through the inlet hole to evenly fill up the entire microchannel with cells. Once loaded, use a sterile plug made from one millimeter diameter nylon fishing line to seal the outlet hole of the device.Then, fill a one milliliter sterile syringe with culture medium, eject any residual air bubbles, and set it into the syringe pump. Connect a 23 gauge blunt needle to the syringe and use polytetrafluoroethylene tubing to connect the needle to the inlet of the device. After connecting the tube, remove the plug from the outlet hole and wait two minutes while the cells settle into the single cell capture-wells by gravitational force.Next, set the syringe pump to 600 microliters per minute. Remove the outlet plug and wash away the uncaptured cells for 30 seconds. Wait two minutes for the pressure to stabilize.Then remove the tube from the inlet of the device and seal both the inlet and outlet holes with plugs to form a closed culture system. Now flip the device by hand to transfer the captured single cells to the culture microwells on the other side of the device. Then, place the device in a 100 millimeter tissue culture dish, add 10 milliliters of sterilized PBS around the device, and place the dish into an incubator.To replace the culture medium, first place two droplets of culture medium on top of the PDMS device near the inlet and outlet to avoid introducing air bubbles into the microchannel. Then, use a 0.75 millimeter biopsy puncher to punch two holes from the top of the PDMS device near the ends of the microchannel. Be sure to only punch through the top layer of the device.Next, fill a one milliliter plastic syringe containing fresh prewarmed medium, and use a 23 gauge blunt needle and PTFE tubing to connect it to the newly created inlet port. Slowly flow approximately 120 microliters of fresh medium into the device over five minutes to replace the old medium. When finished, seal the inlet and outlet ports using two plugs, and return the device to the cell culture incubator.The number of cells that end up in the capture-wells ranges from about 68%to 85%depending on the type of cell. In addition, most of the capture-wells contain only a single cell, for all three of the cell types. Following the transfer of captured KT98 cells to the culture-wells, about 77%of the wells had only a single cell, 16%had no cells, 6%had two cells, and less than 1%of the wells had three or more cells.Over the course of seven days, isolated single cells exhibited heterogeneous growth patterns. While some of the cells multiplied, others did not. Once mastered, this technique can be done in approximately 20 minutes if it is performed properly.After watching this video, you should have a good understanding of how to use this high-throughput single cell isolation endocrinal culture platform to boost the productivity of your single cell experiments. While attempting this procedure, it's important to remember to avoid air bubbles getting into the microchannel and to prevent cells from aggregating together as time goes on.

a-microfluidic-platform-for-high-throughput-single-cell-isolation

Research

JoVE Journal

Bioengineering

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

High Throughput Single-cell and Multiple-cell Micro-encapsulation

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 &mu;m polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency Pk=1 of 83.7% and a double-particle encapsulation efficiency Pk=2 of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. 
Introduction 
Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies.1,2 The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes.3 Encapsulation of cells in drops has been utilized to improve detection of protein expression,4 antibodies,5,6 enzymes,7 and metabolic activity8 for high throughput screening, and could be used to improve high throughput cytometry.9 Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry10 and targeted surface cell coatings.11 Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation12 which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells.
To achieve monodisperse drop generation, microfluidic "flow focusing" enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge.13 For a given nozzle geometry, the drop generation frequency f and drop size can be altered by adjusting oil and aqueous flow rates Qoil and Qaq. As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle.14 
When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops Dk containing k cells is dictated by Poisson statistics, where Dk = &lambda;k exp(-&lambda;)/(k!) and &lambda; is the average number of cells per drop. The fraction of cells which end up in the "correctly" encapsulated drops is calculated using Pk = (k x Dk)/&Sigma;(k' x Dk'). The subtle difference between the two metrics is that Dk relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and Pk relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low &lambda;) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric Pk would be high, the majority of drops would be empty (low Dk), thus requiring a sorting mechanism to remove empty drops, also reducing throughput.15
Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg16 and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number).17,18 Here, the Reynolds number Re =uDh/&nu; and particle Reynolds number Rep =Re(a/Dh)2, where u is a characteristic flow velocity, Dh [=2wh/(w+h)] is the hydraulic diameter, &nu; is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Rep increase. Note that the high Re and Rep requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity.12 However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells.
Because very few studies address inter-particle train spacing,19,20 determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz,12 a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz.4,15 In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average.12
In previous controlled encapsulation work,12 the average number of particles per drop &lambda; was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when &lambda; is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.

Combining monodisperse drop generation with inertial ordering of cells and particles, we describe a method to encapsulate a desired number of cells or particles in a single drop at kHz rates. We demonstrate efficiencies twice exceeding those of unordered encapsulation for single- and double-particle drops.

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement ...

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The &#8216;CometChip&#8217; is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.

We describe here a platform that allows comet assay detection of DNA damage with unprecedented throughput. The device patterns mammalian cells into a microarray and enables parallel processing of 96 samples. The approach facilitates analysis of base level DNA damage, exposure-induced DNA damage and DNA repair kinetics.

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular ...

High Throughput Microfluidic Rapid and Low Cost Prototyping Packaging Methods

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging techniques.
The one-time use packaging technique employs UV-based and temperature curing epoxies to connect microtubes to access holes, wire-bonding for integrated circuit connections, and silver epoxy for electrical connections. This method is based on a robust assembly technique that can support relatively high pressure close to 1 psi and does not need any support to strengthen the microfluidic architecture.
Reusable packaging techniques consist of PDMS-based microtube interconnectors and anisotropic adhesive films for electrical connections. These devices are more sensitive and fragile. Consequently, Plexiglas support is added to the microfluidic structure to improve the electrical contact when anisotropic adhesive films are used, and also to strengthen the microfluidic architecture. In addition, a micromanipulator is needed to maintain tubes while using a thin PDMS layer to connect them to the access holes. Different PDMS layer thicknesses, ranging from 0.45-3 mm, are tested to compare the best adherence versus injection rates. Applied injection rates are varied from 50-300 &#956;l/hr for 0.45-3 mm PDMS layers, respectively. These techniques are mainly applicable for low-pressure applications. However, they can be extended for high-pressure ones through plasma-oxygen process to permanently seal the PDMS to glass substrates. The main advantage of this technique, besides the fact that it is reusable, consists of keeping the device observable when the microchannel length is very short (in the range of 3 mm or lower).

In this article we describe different techniques for microfluidic rapid prototyping platforms. The proposed techniques are based on ultraviolet (UV) sensitive and temperature curing epoxies, polydimethylsiloxane (PDMS) based tubing, wire-bonding, and anisotropic adhesive films. The assembling procedures presented are developed for both one-time use devices as well as reusable microfluidic systems.

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved useful as a convenient live imaging platform providing capabilities for precise control of experimental conditions in real time. In this article, we demonstrate live imaging of individual worms employing WormSpa, a previously-published custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. To illustrate the capability, we performed proof-of-principle experiments in which worms were infected in the device with pathogenic bacteria, and the dynamics of expression of immune response genes and egg laying were monitored continuously in individual animals. The simple design and operation of this device make it suitable for users with no previous experience with microfluidic-based experiments. We propose that this approach will be useful for many researchers interested in longitudinal observations of biological processes under well-defined conditions.

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes.

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...

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 ...

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.

This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth ...