Name: microfluidic buffer exchange for interference-free micro/nanoparticle cell engineering
Uploaded: 2016-07-10T15:00:00
Description: Watch this Scientific Journal Video about Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering at JoVE.com

Kind gift of THP-1 cells from Dr. Mark Chong and assistance in microfabrication from Dr. Yuejun Kang and Dr. Nishanth V. Menon (School of Chemical and Biomedical Engineering, Nanyang Technological University) were greatly acknowledged. This project was funded by NTU-Northwestern Institute of Nanomedicine (Nanyang Technological University). H.W.H. was supported by Lee Kong Chian School of Medicine (LKCMedicine) postdoctoral fellowship.

1. Nanoparticles (NPs) Labeling of Mesenchymal Stem Cells and Monocytes
<ol>
	<li>Culture mesenchymal stem cells (MSCs) in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics to &#8805;80% confluency prior to labeling. Similarly, culture THP-1 cells (ATCC) in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS to a density of ~106 cells/ml.</li>
	<li>Load silica NPs (~500 &#181;m) with calcein dye solution (200 &#181;M) usi...

<ol>
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G., Toner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+inertial+focusing,+ordering,+and+separation+of+particles+in+microchannels.">Continuous inertial focusing, ordering, and separation of particles in microchannels.</a> Proc. Natl. Acad. of Sci USA. 104, 18892-18897 (2007).</li><li>Hur, S. C., Henderson-MacLennan, N. K., McCabe, E. R. B., Di Carlo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deformability-based+cell+classification+and+enrichment+using+inertial+microfluidics.">Deformability-based cell classification and enrichment using inertial microfluidics.</a> Lab Chip. 11, 912-920 (2011).</li><li>Mach, A. J., Di Carlo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+scalable+blood+filtration+device+using+inertial+microfluidics.">Continuous scalable blood filtration device using inertial microfluidics.</a> Biotechnol. Bioengin. 107, 302-311 (2010).</li><li>Gossett, D. R., 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=Inertial+manipulation+and+transfer+of+microparticles+across+laminar+fluid+streams.">Inertial manipulation and transfer of microparticles across laminar fluid streams.</a> Small. 8, 2757-2764 (2012).</li><li>Amini, 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=Engineering+fluid+flow+using+sequenced+microstructures.">Engineering fluid flow using sequenced microstructures.</a> Nat. 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W., 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=Isolation+and+retrieval+of+circulating+tumor+cells+using+centrifugal+forces.">Isolation and retrieval of circulating tumor cells using centrifugal forces.</a> Sci. Rep. 3, 1259 (2013).</li><li>Hou, H. W., Bhattacharyya, R. P., Hung, D. T., Han, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+detection+and+drug-resistance+profiling+of+bacteremias+using+inertial+microfluidics.">Direct detection and drug-resistance profiling of bacteremias using inertial microfluidics.</a> Lab Chip. 15, 2297-2307 (2015).</li><li>Yeo, D. C., 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=Interference-free+Micro/nanoparticle+Cell+Engineering+by+Use+of+High-Throughput+Microfluidic+Separation.">Interference-free Micro/nanoparticle Cell Engineering by Use of High-Throughput Microfluidic Separation.</a> ACS Appl. Mater. Inter. 7, 20855-20864 (2015).</li><li>Xia, Y., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+Lithography.">Soft Lithography.</a> Angew. Chem. Int. Edit. 37, 550-575 (1998).</li><li>Lee, W. 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The DFF cell purification technology described herein enables rapid and continuous separation of labeled cells in a high throughput manner. This separation approach is ideal for large sample volume or high cell concentration sample processing, and is better than conventional membrane-based filtration which is prone to clogging after extended use. Similarly, affinity-based magnetic separation requires additional cell labelling steps which are laborious and expensive. The purified cells are shown to retain their labeled ag...

Engineering cells by agent-loaded micro/nanoparticles (NPs) is a simple, genomic integration-free, and versatile method to enhance bioimaging capability and augment/supplement its native therapeutic properties in regenerative medicine.1-3 Cellular modifications are achieved by labeling the plasma membrane or cytoplasm with an excess concentration of agent-loaded NPs to saturate the binding sites. However, a major drawback of this method is the significant quantities of unbound particles remaining in solution after cell labeling processes, which can potentially confound precise identification of particle-engineered cells or complicate therapeutic outcomes.4,5 In addition, exposure to NPs containing transformative agents (growth factors, corticosteroids, etc.) at excessively high concentration can cause cytotoxicity and misdirected exposure may induce unintended consequences on non-target cells. Even particulate carriers comprising of &#34;biocompatible&#34; materials [e.g., poly(lactic co-glycolic acid), PLGA] can incite potent immune cell responses under certain conditions as well.6 This is especially risky in individuals with impaired immunity (e.g., rheumatoid arthritis) which potentially delays systemic nanoparticle clearance.7 Thus, efficient removal of free particles prior to the introduction of particle-engineered cells is of great importance to minimize toxicity profile and reduce misdirected exposure to agent-loaded particles in vivo.
Conventional gradient centrifugation is often used to separate engineered cells from free particles but is laborious and operated in batch mode. Moreover, shear stresses experienced by cells during high-speed centrifugation and the constituents of the density gradient medium may compromise cell integrity and/or influence cell behavior.8 Microfluidics is an attractive alternative with several separation technologies including deterministic lateral displacement (DLD)9, dieletrophoresis10,11 and acoustophoresis12 developed for small particles separation and buffer exchange applications. However, these methods suffer from low throughput (1-10 &#181;l&#183;min&#8722;1) and are prone to clogging issues. Active separations such as dielectrophoresis-based methods also require differences in intrinsic dielectrophoretic cell phenotypes or additional cell labeling steps to achieve separation. A more promising approach involves inertial microfluidics &#8212; the lateral migration of particles or cells across streamlines to focus at distinct positions due to dominant lift forces (FL) at high Reynolds number (Re).13 Due to its high flow conditions and superior size resolution, it has often been exploited for size-based cell separation14,15 and buffer exchange applications.16-18 However, buffer exchange performance remains poor with &#8764;10&#8722;30% contaminant solution as the separated cells usually remain close to the boundary between original and new buffer solutions.16-18 More importantly, the size distribution of target cells has to be similar to achieve precise inertial focusing and separation from the original buffer solution which poses an issue especially in the processing of heterogeneous-sized cell types such as mesenchymal stem cells (MSCs).19
We have previously developed a new inertial microfluidics cell sorting technique termed Dean Flow Fractionation (DFF) for isolating circulating tumor cells (CTCs)20 and bacteria21 from whole blood using a 2-inlet, 2-outlet spiral microchannel device. In this video protocol, we will describe the process of labeling THP-1 (human acute monocytic leukemia cell line) suspension monocytic cells (~15 &#181;m) and MSCs (10-30 &#181;m) with calcein-loaded NPs, followed by fabrication and operation of the DFF spiral microdevice for efficient recovery of labeled cells and removal of unbound NPs.22 This single step purification strategy enables continuous recovery of labeled suspension and adherent cells suspended in fresh buffer solution without centrifugation. Moreover, it can process up to 10 million cells&#183;ml&#8722;1, a cell density amenable for regenerative medicine applications.

<table border="0" cellpadding="0" cellspacing="0" width="781">
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	<tbody>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:780px;">Cell lines &#38; Media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Mesenchymal Stem Cells (MSCs)</td>
			<td style="width:127px;">Lonza</td>
			<td style="width:119px;">PT-2501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td style="width:127px;">Lonza</td>
			<td style="width:119px;">12-614F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:119px;">10270-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">THP-1 monocyte cells (THP-1)</td>
			<td style="width:127px;">ATCC</td>
			<td style="width:119px;">TIB-202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Roswell Park Memorial Institute (RPMI) 1640 media</td>
			<td style="width:127px;">Lonza</td>
			<td style="width:119px;">12-702F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Name</td>
			<td style="width:127px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:780px;">Reagents &#38; Materials</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">0.01% poly-L-lysine (PLL)</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;">P8920</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">3 mL Syringe</td>
			<td style="width:127px;">BD</td>
			<td style="width:119px;">302113</td>
			<td>Syringe 3ml Luer-Lock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">60 ml Syringe</td>
			<td style="width:127px;">BD</td>
			<td style="width:119px;">309653</td>
			<td>Syringe 60ml Luer-Lock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:127px;">Biowest</td>
			<td style="width:119px;">P6154-100GR</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Calcein, AM (CAM)</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;">C1430</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Calcein</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;">C0875</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Isopropanol</td>
			<td style="width:127px;">Fisher Chemical</td>
			<td style="width:119px;">#P/7507/17</td>
			<td>HPLC Grade 2.5 L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Phosphate-Buffered Saline (PBS)</td>
			<td style="width:127px;">Lonza</td>
			<td style="width:119px;">17-516Q/12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Plain Microscope Slides</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:119px;">FIS#12-550D</td>
			<td>75 X 25 X 1 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Polydimethylsiloxane (PDMS)</td>
			<td style="width:127px;">Dow Corning</td>
			<td style="width:119px;">SYLGARD&#174; 184</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Scotch tape</td>
			<td style="width:127px;">3M</td>
			<td style="width:119px;">21200702044</td>
			<td>18mm x 25m</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Silica NPs (&#8764;200 &#956;m)</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;">748161</td>
			<td>Pore size 4 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Syringe Tip</td>
			<td style="width:127px;">JEC Technology</td>
			<td style="width:119px;">7018302</td>
			<td style="width:167px;">23GA .013 X .25</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Trypsin-EDTA (0.25%)</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;">25200-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Tygon Tubing</td>
			<td style="width:127px;">Spectra-Teknik</td>
			<td style="width:119px;">06419-01</td>
			<td>.02X.06&#34; 100</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:368px;">Name</td>
			<td style="width:127px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:780px;">Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Biopsy punch</td>
			<td style="width:127px;">Harris Uni-Core</td>
			<td>69036-15</td>
			<td>1.50 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Dessicator</td>
			<td style="width:127px;">Scienceware</td>
			<td style="width:119px;">111/4 IN OD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">High-speed Camera</td>
			<td style="width:127px;">Phantom&#160;</td>
			<td style="width:119px;">V9.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Inverted phase-contrast microscope&#160;</td>
			<td style="width:127px;">Nikon</td>
			<td>Eclipse Ti</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Plasma cleaner</td>
			<td style="width:127px;">Harrick Plasma</td>
			<td style="width:119px;">PDC-002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Syringe Pump</td>
			<td style="width:127px;">Chemyx</td>
			<td style="width:119px;">CX Fusion 200</td>
			<td></td>
		</tr>
	</tbody>
</table>

microfluidic buffer exchange for interference-free micro/nanoparticle cell engineering

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic properties, enable bio imaging and control cell phenotype. A critical yet inadequately addressed issue is the significant number of particles that remain unbound after cell labeling which cannot be readily removed by conventional centrifugation. This leads to an increase in bio imaging background noise and can impart transformative effects onto neighboring non-target cells. In this protocol, we present an inertial microfluidics-based buffer exchange strategy termed as Dean Flow Fractionation (DFF) to efficiently separate labeled cells from free NPs in a high throughput manner. The developed spiral microdevice facilitates continuous collection (&#62;90% cell recovery) of purified cells (THP-1 and MSCs) suspended in new buffer solution, while achieving &#62;95% depletion of unbound fluorescent dye or dye-loaded NPs (silica or PLGA). This single-step, size-based cell purification strategy enables high cell processing throughput (106 cells/min) and is highly useful for large-volume cell purification of micro/nanoparticle engineered cells to achieve interference-free clinical application.

After labeling the cells with bio imaging agent-loaded NPs overnight, the labeled cells (containing free particles) are harvested and purified by DFF spiral microdevice to remove free NPs in a single step process (Figure 1A). The 2-inlet, 2-outlet spiral microchannel is designed by engineering software and microfabricated using SU-8 photoresist. The patterned silicon wafer is then used as a template for PDMS replica molding using soft lithography techniques (Figur...

Watch this Scientific Journal Video about Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering at JoVE.com

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

This protocol describes the use of an inertial microfluidics-based buffer exchange strategy to purify micro/nanoparticle engineered cells with efficient depletion of unbound particles.

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic ...

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Bioengineering

Site-specific Bacterial Chromosome Engineering: &#934;C31 Integrase Mediated Cassette Exchange (IMCE)

Site-specific Bacterial Chromosome Engineering: ÃƒÅ½Ã‚Â¦C31 Integrase Mediated Cassette Exchange IMCE

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as ...

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of ...

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 ...

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

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 ...

A Microfluidic System with Surface Patterning for Investigating Cavitation Bubble(s)&#8211;Cell Interaction and the Resultant Bioeffects at the Single-cell Level

A Microfluidic System with Surface Patterning for Investigating Cavitation BubblesÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã…â€œCell Interaction and the Resultant Bioeffects at the Single-cell Level

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...