Name: Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering (Video) | JoVE Journal
Uploaded: 2016-07-10T15:00:00.000+00:00
Duration: 10 min 27 s
Description: Learn microfluidic buffer exchange for interference-free cell engineering using inertial focusing in spiral micro-devices. This high-throughput method efficiently separates free nanoparticles from engineered cells for regenerative medicine applications.

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5 minutes

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) using overnight stirring. Fabricate PLGA-calcein AM (CAM) using a protocol described previously.22
	<ol>
		<li>Dissolve 250 &#181;g CAM and 100 mg PLGA (50:50) in chloroform at 4 &#176;C.</li>
		<li>Generate single emulsion NPs using a high speed homogenizer (13,600 x g, 60 sec) at room temperature. Evaporate the chloroform in a chemical hood (&#8805;3 hr) before collection using centrifugation (3,400 x g for 5 min), washing (double distilled water), freeze-drying and storage in -20 &#176;C.</li>
	</ol>
	</li>
	<li>Incubate CAM-PLGA NPs (1 mg) or calcein silica NPs (150 &#181;g) in 0.01% poly-L-lysine (PLL) solution at room temperature for 15 - 20 min.</li>
	<li>Centrifuge at 3,400 x g for 5 min to remove excess PLL supernatant before NPs resuspension in 1 ml of respective culture medium.</li>
	<li>Incubate the NPs with cells (MSCs or THP-1, ~1 - 2 x 106 cells in total) for approximately 24 hr (0.1 mg&#183;ml&#8722;1 labeling concentration).</li>
	<li>Dissociate and harvest the labeled adherent MSCs using 2 ml of 0.25% trypsin (5 min, 37 &#176;C) and quench with 6 ml of DMEM. Spin down the cells at (1,000 x g, 4 min) and resuspend to a concentration of 105 - 106 cells/ml for microfluidic processing. Use labeled THP-1 cells (105 - 106 cells/ml) directly for microfluidics purification.</li>
</ol>
2. Microfluidic Device Preparation
<ol>
	<li>Device Fabrication
	<ol>
		<li>Fabricate the microfluidic spiral device (500 &#181;m (w) &#215; 115 &#181;m (h)) with polydimethylsiloxane (PDMS) from a commercial kit using standard soft lithography steps.23</li>
		<li>Mix 30 g of base prepolymer and 3 g curing agent thoroughly in a weighing boat. De-gas the mixture in a desiccator for 60 min to remove any air bubbles.</li>
		<li>Pour the PDMS mixture onto the silicon wafer master mold patterned with the spiral channel design carefully to a height of ~5 - 10 mm.</li>
		<li>De-gas the mixture in a desiccator vacuum for 60 min again to remove any air bubbles. Repeat the process until all bubbles are eliminated.</li>
		<li>Cure the PDMS mixture in an 80 &#176;C oven for 2 hr until the PDMS is set. Ensure the wafer is not tilted during curing to have a constant device height.</li>
		<li>Cut out the PDMS spiral device using a scalpel and carefully peel the PDMS slab from the master mold.</li>
		<li>Trim the edges of the device with a scalpel to ensure smooth surface for bonding.</li>
		<li>Punch two holes (1.5 mm) for the inlets and two holes (1.5 mm) for the outlets on the PDMS device using a 1.5 mm biopsy puncher.</li>
		<li>Wash the device with isopropanol (IPA) to remove any debris and dry the device in an 80 &#176;C oven for 5 min.</li>
		<li>Clean the bottom surface of the PDMS device (with channel features) using masking tape.</li>
		<li>Clean one side of a glass slide (2&#34; by 3&#34;) using masking tape.</li>
		<li>Carefully place and expose the cleaned surfaces of the PDMS device and glass slide in the chamber of a plasma cleaner and subject them to vacuum for 60 sec. Next, switch on the plasma power to maximum and lower the chamber pressure until the chamber turns pink in color.
		<ol>
			<li>Expose the surfaces to air plasma for 60 sec. The plasma creates reactive species on the exposed surfaces of the PDMS and glass which enables tight bonding when brought into physical contact. Turn off the plasma power and release the pressure from the plasma cleaner to retrieve the device and glass slide.</li>
		</ol>
		</li>
		<li>Bond the PDMS device and glass slide together by pressing the plasma-exposed surfaces tightly and ensuring that no bubbles are trapped between the two surfaces.</li>
		<li>Heat the bonded device using a hotplate set at 80 &#176;C for 2 hr to strengthen the bonding.</li>
	</ol>
	</li>
	<li>Device Operation
	<ol>
		<li>Cut two pieces of tubing (1.52 mm OD) of ~15 - 20 cm for the inlet syringes and attach a syringe tip (gauge 23) on one end of each tubing.</li>
		<li>Cut two pieces of tubing (1.52 mm OD) of ~5 - 10 cm for the outlets and attach to the outlet holes of the PDMS device.</li>
		<li>Prior to sample running, manually prime the device with a syringe containing 70% ethanol until it flows out of the outlet tubing. Allow the ethanol sit for 30 sec to 1 min to sterilize the device.</li>
		<li>Load 30 ml of filtered (0.2 &#181;m pore) sheath buffer (Phosphate-Buffered Saline (PBS) with 0.1% Bovine Serum Albumin (BSA)) into the 60 ml syringe and secure the syringe on a syringe pump. Set the pump to the correct settings (Syringe size: 60 ml, Volume: 60,000 &#181;l). 
		Note: The addition of BSA to PBS is to minimize cell-cell binding and non-specific binding between cells and the PDMS device.</li>
		<li>Load 3 ml of labeled cells into the 3 ml syringe and secure the syringe on a separate syringe pump. Set the pump to the correct settings (Syringe size: 3 ml, Volume: 3,000 &#181;l).</li>
		<li>Check that no air bubbles are trapped in the syringes to ensure a stable flow. Remove any air bubbles by gently ejecting few drops of liquid out of the nozzle.</li>
		<li>Connect the inlet syringe tips and tubing to the syringes, and insert them into the respective inlets of the device (sheath and sample inlet). Ensure that there are no bubbles along the tubing.</li>
		<li>Mount the devices onto an inverted phase-contrast microscope for real time imaging during cell sorting process.</li>
		<li>Secure a small waste beaker and two 15 ml tubes close to the device on the microscope stage using adhesives.</li>
		<li>Place the outlet tubings into the waste beaker.</li>
		<li>Set the flow ratio for sample to sheath buffer to 1:10 and start both syringe pumps to initiate the sorting process (Example: 120 &#181;l/min for sample syringe and 1,200 &#181;l/min for sheath syringe; channel flow velocity ~0.38 m/sec).</li>
		<li>Run the device for 1.5 min for the flow rate to stabilize. This can be confirmed by presence of inertially focused cells near the channel inner wall under bright-field with phase contrast using a high-speed camera (~5,000 - 10,000 frames per second (fps), exposure time: 10-50 &#181;sec).</li>
		<li>Position the outlet tubings into different tubes to collect the eluents from the cell outlet and waste outlet.</li>
	</ol>
	</li>
</ol>

<ol><li>Wiraja, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aptamer+technology+for+tracking+cells%27+status+%26+function%5BTitle%5D)+AND+%22Mol.+Cell.+Ther%22%5BJournal%5D)">Aptamer technology for tracking cells' status & function.</a> Mol. Cell. Ther. 2, 33(2014).</li>
<li>Yeo, D. C., Wiraja, C., Mantalaris, A., Xu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nanosensors+for+Regenerative+Medicine%5BTitle%5D)+AND+%22J.+Biomed.+Nanotech%22%5BJournal%5D)">Nanosensors for Regenerative Medicine.</a> J. Biomed. Nanotech. 10, 2722-2746 (2014).</li>
<li>Naumova, A. V., Modo, M., Moore, A., Murry, C. E., Frank, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinical+imaging+in+regenerative+medicine%5BTitle%5D)+AND+%22Nat.+Biotechnol%22%5BJournal%5D)">Clinical imaging in regenerative medicine.</a> Nat. Biotechnol. 32, 804-818 (2014).</li>
<li>Soenen, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+toxicity+of+inorganic+nanoparticles%3A+Common+aspects+and+guidelines+for+improved+nanotoxicity+evaluation%5BTitle%5D)+AND+%22Nano+Today%22%5BJournal%5D)">Cellular toxicity of inorganic nanoparticles: Common aspects and guidelines for improved nanotoxicity evaluation.</a> Nano Today. 6, 446-465 (2011).</li>
<li>Donaldson, K., Poland, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nanotoxicity%3A+challenging+the+myth+of+nano-specific+toxicity%5BTitle%5D)+AND+%22Curr.+Opin.+Biotechnol%22%5BJournal%5D)">Nanotoxicity: challenging the myth of nano-specific toxicity.</a> Curr. Opin. Biotechnol. 24, 724-734 (2013).</li>
<li>Veiseh, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Size-+and+shape-dependent+foreign+body+immune+response+to+materials+implanted+in+rodents+and+non-human+primates%5BTitle%5D)+AND+%22Nat.+Mater%22%5BJournal%5D)">Size- and shape-dependent foreign body immune response to materials implanted in rodents and non-human primates.</a> Nat. Mater. 14, 643-651 (2015).</li>
<li>Jones, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nanoparticle+clearance+is+governed+by+Th1%2FTh2+immunity+and+strain+background%5BTitle%5D)+AND+%22J.+Clin.+Invest%22%5BJournal%5D)">Nanoparticle clearance is governed by Th1/Th2 immunity and strain background.</a> J. Clin. Invest. 123, 3061-3073 (2013).</li>
<li>Marchi, L. F., Sesti-Costa, R., Chedraoui-Silva, S., Mantovani, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+four+methods+for+the+isolation+of+murine+blood+neutrophils+with+respect+to+the+release+of+reactive+oxygen+and+nitrogen+species+and+the+expression+of+immunological+receptors%5BTitle%5D)+AND+%22Comp.+Clin.+Pathol%22%5BJournal%5D)">Comparison of four methods for the isolation of murine blood neutrophils with respect to the release of reactive oxygen and nitrogen species and the expression of immunological receptors.</a> Comp. Clin. Pathol. 23, 1469-1476 (2014).</li>
<li>Morton, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hydrodynamic+metamaterials%3A+Microfabricated+arrays+to+steer%2C+refract%2C+and+focus+streams+of+biomaterials%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+of+Sci+USA%22%5BJournal%5D)">Hydrodynamic metamaterials: Microfabricated arrays to steer, refract, and focus streams of biomaterials.</a> Proc. Natl. Acad. of Sci USA. 105, 7434-7438 (2008).</li>
<li>Hu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Marker-specific+sorting+of+rare+cells+using+dielectrophoresis%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+of+Sci+USA%22%5BJournal%5D)">Marker-specific sorting of rare cells using dielectrophoresis.</a> Proc. Natl. Acad. of Sci USA. 102, 15757-15761 (2005).</li>
<li>Cummings, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Streaming+dielectrophoresis+for+continuous-flow+microfluidic+devices%5BTitle%5D)+AND+%22IEEE+Eng.+Med.+Biol.+Mag%22%5BJournal%5D)">Streaming dielectrophoresis for continuous-flow microfluidic devices.</a> IEEE Eng. Med. Biol. Mag. 22, 75-84 (2003).</li>
<li>Augustsson, P., Persson, J., Ekstrom, S., Ohlin, M., Laurell, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Decomplexing+biofluids+using+microchip+based+acoustophoresis%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">Decomplexing biofluids using microchip based acoustophoresis.</a> Lab Chip. 9, 810-818 (2009).</li>
<li>Di Carlo, D., Irimia, D., Tompkins, R. G., Toner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Continuous+inertial+focusing%2C+ordering%2C+and+separation+of+particles+in+microchannels%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+of+Sci+USA%22%5BJournal%5D)">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/pubmed?term=((Deformability-based+cell+classification+and+enrichment+using+inertial+microfluidics%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">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/pubmed?term=((Continuous+scalable+blood+filtration+device+using+inertial+microfluidics%5BTitle%5D)+AND+%22Biotechnol.+Bioengin%22%5BJournal%5D)">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/pubmed?term=((Inertial+manipulation+and+transfer+of+microparticles+across+laminar+fluid+streams%5BTitle%5D)+AND+%22Small%22%5BJournal%5D)">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/pubmed?term=((Engineering+fluid+flow+using+sequenced+microstructures%5BTitle%5D)+AND+%22Nat.+Commun%22%5BJournal%5D)">Engineering fluid flow using sequenced microstructures.</a> Nat. Commun. 4, 1826(2013).</li>
<li>Sollier, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inertial+microfluidic+programming+of+microparticle-laden+flows+for+solution+transfer+around+cells+and+particles%5BTitle%5D)+AND+%22Microfluid.+Nanofluid%22%5BJournal%5D)">Inertial microfluidic programming of microparticle-laden flows for solution transfer around cells and particles.</a> Microfluid. Nanofluid. 19, 53-65 (2015).</li>
<li>Lee, W. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multivariate+biophysical+markers+predictive+of+mesenchymal+stromal+cell+multipotency%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+of+Sci+USA%22%5BJournal%5D)">Multivariate biophysical markers predictive of mesenchymal stromal cell multipotency.</a> Proc. Natl. Acad. of Sci USA. 111, E4409-E4418 (2014).</li>
<li>Hou, H. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+retrieval+of+circulating+tumor+cells+using+centrifugal+forces%5BTitle%5D)+AND+%22Sci.+Rep%22%5BJournal%5D)">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/pubmed?term=((Direct+detection+and+drug-resistance+profiling+of+bacteremias+using+inertial+microfluidics%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">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/pubmed?term=((Interference-free+Micro%2Fnanoparticle+Cell+Engineering+by+Use+of+High-Throughput+Microfluidic+Separation%5BTitle%5D)+AND+%22ACS+Appl.+Mater.+Inter%22%5BJournal%5D)">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/pubmed?term=((Soft+Lithography%5BTitle%5D)+AND+%22Angew.+Chem.+Int.+Edit%22%5BJournal%5D)">Soft Lithography.</a> Angew. Chem. Int. Edit. 37, 550-575 (1998).</li>
<li>Lee, W. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+cell+cycle+synchronization+using+inertial+forces+in+spiral+microchannels%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">High-throughput cell cycle synchronization using inertial forces in spiral microchannels.</a> Lab Chip. 11, 1359-1367 (2011).</li>
<li>Kuntaegowdanahalli, S. S., Bhagat, A. A., Kumar, G., Papautsky, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inertial+microfluidics+for+continuous+particle+separation+in+spiral+microchannels%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">Inertial microfluidics for continuous particle separation in spiral microchannels.</a> Lab Chip. 9, 2973-2980 (2009).</li>
</ol>

The authors have nothing to disclose.

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><tbody><tr><td>&lt;strong&gt;Cell lines &amp;amp; Media&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Mesenchymal Stem Cells (MSCs)</td><td>Lonza</td><td>PT-2501</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium (DMEM)</td><td>Lonza</td><td>12-614F</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco</td><td>10270-106</td><td></td></tr><tr><td>THP-1 monocyte cells (THP-1)</td><td>ATCC</td><td>TIB-202</td><td></td></tr><tr><td>Roswell Park Memorial Institute (RPMI) 1640 media</td><td>Lonza</td><td>12-702F</td><td></td></tr><tr><td>&lt;strong&gt;Reagents &amp;amp; Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.01% poly-L-lysine (PLL)</td><td>Sigma-Aldrich</td><td>P8920</td><td></td></tr><tr><td>3 ml Syringe</td><td>BD</td><td>302113</td><td>Syringe 3 ml Luer-Lock</td></tr><tr><td>60 ml Syringe</td><td>BD</td><td>309653</td><td>Syringe 60 ml Luer-Lock</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Biowest</td><td>P6154-100GR</td><td></td></tr><tr><td>Calcein, AM (CAM)</td><td>Life Technologies</td><td>C1430</td><td></td></tr><tr><td>Calcein</td><td>Sigma-Aldrich</td><td>C0875</td><td></td></tr><tr><td>Isopropanol</td><td>Fisher Chemical</td><td>#P/7507/17</td><td>HPLC Grade 2.5 L</td></tr><tr><td>Phosphate-Buffered Saline (PBS)</td><td>Lonza</td><td>17-516Q/12</td><td></td></tr><tr><td>Plain Microscope Slides</td><td>Fisher Scientific</td><td>FIS#12-550D</td><td>75 x 25 x 1 mm&lt;sup&gt;3&lt;/sup&gt;</td></tr><tr><td>Polydimethylsiloxane (PDMS)</td><td>Dow Corning</td><td>SYLGARD&amp;reg; 184</td><td></td></tr><tr><td>Scotch tape</td><td>3M</td><td>21200702044</td><td>18 mm x 25 m</td></tr><tr><td>Silica NPs (&amp;sim;200 &amp;mu;m)</td><td>Sigma-Aldrich</td><td>748161</td><td>Pore size 4 nm</td></tr><tr><td>Syringe Tip</td><td>JEC Technology</td><td>7018302</td><td>23 G 0.013 x 0.25</td></tr><tr><td>Trypsin-EDTA (0.25%)</td><td>Life Technologies</td><td>25200-056</td><td></td></tr><tr><td>Tygon Tubing</td><td>Spectra-Teknik</td><td>06419-01</td><td>0.02 x 0.06&quot; 100</td></tr><tr><td>Poly (D,L-lactide-co-glycolide) (PLGA; 50:50)</td><td>Sigma-Aldrich</td><td>P2191</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Biopsy punch</td><td>Harris Uni-Core</td><td>69036-15</td><td>1.50 mm</td></tr><tr><td>Dessicator</td><td>Scienceware</td><td>111/4 IN OD</td><td></td></tr><tr><td>High-speed Camera</td><td>Phantom&amp;nbsp;</td><td>V9.1</td><td></td></tr><tr><td>Inverted phase-contrast microscope&amp;nbsp;</td><td>Nikon</td><td>Eclipse Ti</td><td></td></tr><tr><td>Plasma cleaner</td><td>Harrick Plasma</td><td>PDC-002</td><td></td></tr><tr><td>Syringe Pump</td><td>Chemyx</td><td>CX Fusion 200</td><td></td></tr></tbody></table>

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

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...

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 ...

The overall goal of this procedure, is to demonstrate a convenient, efficient, and high throughput microfluidic purification technology, to separate free nanoparticles from cells, following cell engineering with the nanoparticles. The major want of this technology is to allow the quick and efficient removal of unbound nanoparticles After the cell laboring procedure, we gather up all our ses-fa-tion. This microfluidic cell purification technology achieves efficient size based separation due to the differential inertial focusing effects of cells and nanoparticles in a spiral micro-device.It can process up to 10 million cells per mil concentration which is very useful for regenerative medicine, as well as latch volume cell processing. This technology is high desired for cell engineering field, which often using nanoparticle to labor cells, for imaging purpose or function control. Demonstrating the procedure will be Hui Min Tay, a research assistant from my laboratory, as well as Doctor David Yeo, a post doctor research fellow from Professor Xi's laboratory.Culture mesenchymal stem cells to greater than 80%confluency on a six well plate prior to labeling the cells with the nanoparticles. Similarly culture THP-1 cells to a density of one million cells per milliliter. Next, dissolve one milligram of silica nanoparticles with one milliliter of two hundred micromolar calcium dye solution.And stir the particles overnight. Also, fabricate PLGA calcium AM nanoparticles using methods described in the reference shown here. Mix either one milligram of the PLGA calcium AM nanoparticles or 150 micrograms of the calcium silica nanoparticles in 01%poly-l-lysine solution in water by pipetting the solution up and down for one minute.And then let it incubate at room temperature for 15 to 20 minutes. Next centrifuge the nanoparticles. Remove the excess poly-l-lysine supernatant and re-suspend the nanoparticles in one milliliter of the appropriate culture medium for the cell type to be labelled.Add 1 milligrams of the labelled nanoparticles to one milliliter of medium for every one million cells in culture. Pipette the mixture of cells, nanoparticles, and medium thoroughly for one minute. And then incubate the mixture for approximately 24 hours.Once labelled, dissociate and harvest the stem cells, and re-suspend them to a concentration of between 100, 000 and 1, 000, 000 cells per milliliter for microfluidic processing. Use labelled THP-1 cells at the same concentration. In order to fabricate the microfluidic spiral device, first prepare a silicone wafer master mold using standard microfabrication techniques.Then, mix 30 grams of the base PDMS prepolymer and three grams of a curing agent thoroughly in a weighing boat. Degas the mixture under vacuum for 60 minutes to remove any air bubbles. Pour the PDMS mixture onto the master mold, patterned with the spiral channel design, carefully to a height of five to 10 millimeters.Then degas the mixture for 60 minutes to remove any air bubbles. Repeat this process until all bubbles are eliminated. Next, place the wafer in an oven, and ensure that the wafer is not tilted during curing, to ensure a constant device height.Cure the PDMS at 80 degrees Celsius for two hours. Once cool, cut out the PDMS spiral device using a scalpel and carefully peel the PDMS slab from the master mold. Then, use a scalpel to trim the edges of the device to ensure a smooth surface for bonding.Next, use a 1.5 millimeter biopsy punch to create two holes for the inlets, and two holes for the outlets. Wash the device with isopropanol to remove any debris. And dry the device in an 80 degree Celsius oven for five minutes.Then, use masking tape to clean the bottom surface of the PDMS device, and one side of a glass slide. Carefully place the glass slide and PDMS device into a plasma chamber with the clean surfaces exposed. Switch on the plasma power to maximum, lower the chamber pressure until the chamber turns pink in color, and expose the components for 60 seconds.Then, remove the slide and PDMS device from the chamber. And bond them together by pressing the plasma exposed surfaces tightly together. Ensure no bubbles are trapped between the two surfaces.Heat the bonded device on a hot plate set at 80 degrees celsius for two hours to strengthen the bond. Cut two pieces of tubing 15 to 20 centimeters in length for the inlets, and attach a 23 gauge needle on one end of each tube. Then, cut two pieces of the same tubing that are five to 10 centimeters in length for the outlets, and attach them to the outlet holes of the device.Prior to running the sample, manually prime the device with a syringe containing 70%ethanol until it flows from the outlet tubing. Allow the ethanol to sit in the device for at least 30 seconds to sterilize it. Then, load 30 milliliters of filtered PBS with 1%BSA into a 60 milliliter syringe.Also, load three milliliters of labelled cells into a three milliliter syringe and check that no air bubbles are trapped in either syringe. Remove any air bubbles by gently ejecting a few drops of liquid out of the nozzle. Connect the inlet syringe tips and tubing to the syringes and secure the syringes to the syringe pumps.Insert the tubes into their respective inlets of the device, and ensure that there are no bubbles along the tubing. With the fluidics now set up, mount the device onto an inverted phase contrast microscope for real time imaging during the cell sorting process. Secure a small waste beaker and two 15 milliliter tubes close to the device on the microscope stage.Place the outlet tubing from both of the device's outlets into the waste beaker. Now, set the flow ratio for sample to sheath buffer to one to 10 by setting the flow rate of the sample syringe to 120 microliters per minute and the sheath syringe to 1, 200 microliters per minute. Run the device for one and a half minutes to give the flow rate a chance to stabilize.Use a high speed camera to confirm the presence of inertially focused cells near the channel inner wall under bright field with phase contrast. Once steady state flow is achieved, and the device is running correctly, position the outlet tubes into different vials to collect separate eluents from the cell outlet and waste outlet. This device is able to correctly sort a labelled suspension of THP-1 monocytic cells from fluorescent silica nanoparticles.High separation efficiencies were obtained as confirmed by both high speed imaging and flow cytometry analysis. The single step purification strategy enables continuous recovery of a labelled suspension and adherent cells that are suspended in fresh buffer solution without the need for centrifugation. The device is capable of high efficiency separation with both silica and PLGA nanoparticles and can separate up to 10 million cells per milliliter with the same high efficiency.After watching this video, you should have a good understanding of how to label cells with nanoparticles. And how to purify the labelled cells by removing excess unbound particles using our specialized microfluidic device.

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Learn microfluidic buffer exchange for interference-free cell engineering using inertial focusing in spiral micro-devices. This high-throughput method efficiently separates free nanoparticles from engineered cells for regenerative medicine applications.

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

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