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. Nanopartikler (NP) Mærkning af mesenchymale stamceller og monocytter <ol><li> Kultur mesenchymstamceller (MSC&#39;er) i Dulbeccos modificerede Eagles medium (DMEM) suppleret med 10% føtalt bovint serum (FBS) og antibiotika til ≥80% sammenflydning før mærkning. Tilsvarende kultur THP-1-celler (ATCC) i Roswell Park Memorial Institute (RPMI) 1640 medium suppleret med 10% FBS til en densitet på ~ 10 6 celler / ml. </li><li> Load silica NP (~ 500 um) med calcein farveopløsning (200 uM) ved anvendelse ...

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DFF celle rensning teknologi beskrevet heri muliggør hurtig og kontinuerlig adskillelse af mærkede celler i et højt gennemløb måde. Denne adskillelse tilgang er ideel til store prøvevolumen eller høj koncentration celle prøve behandling, og er bedre end konventionel membran-baserede filtrering, som er tilbøjelige til at tilstopning efter længere tids brug. Tilsvarende affinitet-baserede magnetisk separation kræver yderligere trin mærkning celle, som er besværlig og dyr. De oprensede celler er vist at bevare...

Engineering celler ved agent-loaded mikro- / nanopartikler (NPS) er en enkel, genomisk integration-fri, og alsidig metode til at forbedre bioimaging kapacitet og forøge / supplere sine indfødte terapeutiske egenskaber i regenerativ medicin. 1-3 Cellular modifikationer opnås ved mærkning af plasmamembranen eller cytoplasmaet med et overskud koncentrationen af ​​midlet-loaded NP&#39;er at mætte bindingsstederne. Men en stor ulempe ved denne metode er de betydelige mængder af ubundne partikler tilbage i opløsning efter mærkning celle processer, som potentielt kan forvirre præcis identifikation af partikel-celler eller komplicere terapeutiske resultater. 4,5 Desuden, eksponering for nationale parlamenter, der indeholder transformative midler (vækstfaktorer, kortikosteroider, etc.) ved for høj koncentration kan forårsage cytotoksicitet og fejlrettet eksponering kan fremkalde utilsigtede konsekvenser på ikke-målceller. Selv partikelformige bærere, der omfatter of &quot;biokompatible&quot; materialer [f.eks, poly (mælkesyre co-glycolsyre), PLGA] kan opildne potente immun celle responser visse betingelser samt. 6 Dette er især risikabelt hos personer med svækket immunitet (fx leddegigt) som potentielt forsinkelser systemisk nanopartikel clearance. 7 således effektiv fjernelse af frie partikler forud for indførelsen af partikel-modificerede celler er af stor betydning at minimere toksicitetsprofil og reducere fejldirigeret eksponering for agent-loaded partikler in vivo. Konventionel gradientcentrifugering anvendes ofte til at adskille manipulerede celler fra frie partikler, men er besværlig og drives i batch-mode. Desuden forskydningsspændinger opleves af celler under høj hastighed centrifugering og bestanddele af densitetsgradient medium kan kompromittere celle integritet og / eller indflydelse celle adfærd. 8 Microfluidics er et attraktivt alternativ med sevrale teknologier separation herunder deterministisk sideforskydning (DLD) 9, dieletrophoresis 10,11 og akustoforese 12 udviklet til små partikler separation og bufferudskiftning applikationer. Imidlertid lider disse fremgangsmåder fra lille produktion (1-10 pi · min - 1) og er tilbøjelige til tilstopning problemer. Aktive separationer såsom dielektroforese-baserede metoder kræver også forskelle i iboende dielektroforetisk celle fænotyper eller yderligere skridt mærkning celle for at opnå separation. En mere lovende tilgang involverer inerti mikrofluidik - den laterale vandring af partikler eller celler på tværs strømliner at fokusere på forskellige positioner på grund af dominerende lift kræfter (F L) ved høje Reynolds tal (Re) 13 På grund af sin høje strømningsforhold og overlegen størrelse opløsning. det er ofte blevet udnyttet til størrelse-baserede celleseparation 14,15 og buffer udvekslingsprogrammer. 16-18 However, buffer udveksling ydeevne forbliver fattige med ~10-30% forurenende løsning, da de adskilte celler normalt forbliver tæt på grænsen mellem originale og nye bufferopløsninger. 16-18 Vigtigere, størrelsesfordelingen af target-celler skal være ens for at opnå præcis inertial fokusering og adskillelse fra den oprindelige pufferopløsning, der udgør et problem, især i behandlingen af heterogene størrelse celletyper såsom mesenkymale stamceller (MSC&#39;er). 19 Vi har tidligere udviklet en ny inertial mikrofluidik cellesortering teknik betegnet Dean Flow Fraktionering (DFF) til isolering af cirkulerende tumorceller (CTCs) 20 og bakterier 21 fra fuldblod under anvendelse af en 2-indløb, 2-udløb spiral mikrokanal enhed. I denne video protokol, vil vi beskrive processen med mærkning THP-1 (human akut monocytisk leukæmi cellelinje) suspension monocytiske celler (~ 15 pm) og MSC&#39;er (10-30 um) med calcein-loaded nationale parlamenter, efterfulgt af fremstilling og drift af DFF spiral microdevice for effektiv inddrivelse af mærkede celler og fjernelse af ubundne NP&#39;er. 22 Denne enkelt trin rensning strategi muliggør kontinuerlig genvinding af mærket suspension og adhærente celler suspenderet i frisk bufferopløsning uden centrifugering. Det kan desuden behandle op til 10 millioner celler · ml-1, en ​​celletæthed medgørlige til regenerativ medicin applikationer.

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	<tbody>
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			<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 celler med aktiv-ingrediens-loaded mikro- / nanopartikler (NPS) bliver en stadig mere populær metode til at forbedre indfødte terapeutiske egenskaber, aktivere bio imaging og styre celle fænotype. En kritisk endnu utilstrækkeligt behandlet problem er det store antal partikler, der forbliver ubundet efter cellemærkning, som ikke let kan fjernes ved konventionel centrifugering. Dette fører til en stigning i bio imaging baggrundsstøj og kan give transformative virkninger onto nabolande uden for målgruppen celler. I denne protokol, præsenterer vi en inerti mikrofluidik-baserede buffer udveksling strategi betegnes som Dean Flow Fraktionering (DFF) til effektivt at separere mærkede celler fra frie nationale parlamenter på en high throughput måde. Den udviklede spiral microdevice letter kontinuerlig samling (&gt; 90% celleindvinding) renset celler (THP-1 og MSC) suspenderet i ny bufferopløsning, og samtidig opfylde&gt; 95% depletering af ubundet fluorescerende farvestof eller Dye-loaded NP&#39;er (silICA eller PLGA). Denne enkelt trin, størrelse-baserede celle rensning strategi giver høj celle behandling gennemløb (10 6 celler / min) og er yderst nyttigt for store mængder celle rensning af mikro- / nanopartikel manipuleret celler for at opnå fejlfri klinisk anvendelse.

Efter mærkning af cellerne med bio billeddannende middel-loaded NP&#39;er natten de mærkede celler (indeholdende frie partikler) høstes og oprenses ved DFF spiral microdevice at fjerne frie NP&#39;er i et enkelt trin proces (Figur 1A). Den 2-indløb, 2-stikkontakt spiral mikrokanalplade er designet af engineering-software og mikrofabrikerede hjælp SU-8 fotoresist. Den mønstrede siliciumskive anvendes derefter som template til PDMS replika støbning med bløde litogr...

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

Denne protokol beskriver anvendelsen af ​​et inertial mikrofluidik-baserede bufferskift strategi til oprensning mikro / nanopartikel manipulerede celler med effektiv udtømning af ubundne partikler.

Mikrofluid Buffer Exchange til Interferensfri Micro / Nanopartikel Cell Engineering

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

Research

JoVE Journal

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

Site-specifik bakteriekromosomet Engineering: ΦC31 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

Systematisk analyse af<em&gt; In Vitro</em&gt; Cell Rolling Ved hjælp af en multi-brønds plade Mikrofluid 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

Cell Klemme som en robust, Mikrofluid Intracellulær 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

En Mikrofluid Teknik til sonde Cell deformerbarhed

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

Kontrolleret Mikrofluid Miljø for Dynamic Undersøgelse af røde blodlegemer Sammenlægning

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

Brug Adhesive mønstret Konstruér 3D Paper mikrofluidenheder

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

En Mikrofluid platform for High-throughput Single-celle Isolering og Kultur

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

En mikrofluidsystem med Surface mønster for Undersøgelse Kavitation Bubble (r)-Cell Interaktion og de resulterende bioeffekter på Single-celle niveau

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

Multi-trins Variabel Højde Fotolitografi for med ventiler Multilayer mikrofluidenheder

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