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 (NPS) Merking av stamceller og monocytter <ol><li> Kultur mesenchymale stamceller (MSC) i Dulbeccos modifiserte Eagles medium (DMEM) supplementert med 10% føtalt bovint serum (FBS) og antibiotika til ≥80% konfluens før merking. Tilsvarende kultur THP-1-celler (ATCC) i Roswell Park Memorial Institute (RPMI) 1640-medium supplert med 10% FBS til en tetthet på ~ 10 6 celler / ml. </li><li> Load silika NPs (~ 500 mikrometer) med calcein fargeløsning (200 mm) ved hjelp av natten omrøring. ...

<ol>
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Den DFF celle rensemetoder som er beskrevet her gjør det mulig hurtig og kontinuerlig separering av merkede celler i en høy gjennomstrømning måte. Denne separering tilnærmingen er ideell for stort prøvevolum eller høy cellekonsentrasjonen prøvebehandling, og er bedre enn konvensjonelle membran-baserte filtrering som er utsatt for tilstopping etter lang tids bruk. Tilsvarende krever affinitets-baserte magnetisk separasjon ytterligere celle merketrinn som er arbeidskrevende og kostbart. De rensede celler er vist ?...

Ingeniør celler ved nestleder lastet mikro / nanopartikler (NPS) er en enkel, genomisk integrering fritt, og allsidig metode for å forbedre Bioimaging evne og styrke / supplere sin opprinnelige terapeutiske egenskaper i regenerativ medisin. 1-3 Cellular modifikasjoner oppnås ved å merke plasma membran eller cytoplasma med et overskudd konsentrasjon av nestleder belastede NPs å mette bindingssteder. Imidlertid er en stor ulempe ved denne metode er de store mengder av ubundne partikler som er igjen i oppløsningen etter cellemerkeprosesser, noe som kan forvirre presis identifisering av partikkel-konstruerte celler eller komplisere terapeutiske resultater. 4,5 I tillegg, eksponering for NPS inneholdende transformerende agenter (vekstfaktorer, kortikosteroider, etc.) ved for høy konsentrasjon kan føre cytotoksisitet og irrelevante eksponering kan forårsake utilsiktede konsekvenser på ikke-målceller. Selv partikkel bærere bestående of &quot;biokompatible&quot; materialer [f.eks poly (melkesyre co-glykolsyre), PLGA] kan egge potente immuncellere under visse forhold også. 6 Dette er spesielt risikabelt i personer med nedsatt immunforsvar (for eksempel reumatoid artritt) som potensielt forsinkelser systemisk clearance nanopartikkel. 7 således effektiv fjerning av frie partikler før innføring av partikkel konstruerte celler som er av stor viktighet for å minimalisere toksikologisk profil og redusere feilsendt eksponering til agenten belastet partiklene in vivo. Konvensjonell gradient sentrifugering blir ofte brukt for å separere modifiserte cellene fra frie partikler, men er arbeidskrevende og drives i batch-modus. Videre skjærspenninger oppleves av celler under høyhastighetssentrifugering og bestanddelene av tettshetsgradient medium kan kompromittere celle integritet og / eller innflytelse celle atferd. 8 Microfluidics er et attraktivt alternativ med sevtater separasjonsteknologier, inkludert determinissideveis forskyvning (DLD) 9, dieletrophoresis 10,11 og acoustophoresis 12 utviklet for små partikler separasjon og buffer børs applikasjoner. Imidlertid er disse fremgangsmåter lider av lav gjennomstrømning (1-10 ul x min - 1) og er utsatt for tilstopping problemer. Aktive separasjoner som dielectrophoresis-baserte metoder krever også forskjeller i indre dielectrophoretic celle fenotyper eller ekstra celle merking skritt for å oppnå separasjon. En mer lovende tilnærming innebærer treghet MicroFluidics - lateral migrasjon av partikler eller celler over strømlinje å fokusere på forskjellige posisjoner på grunn av dominerende løftekraft (F L) ved høy Reynolds tall (Re) 13 På grunn av sin høye strømningsforhold og overlegen størrelse oppløsning. har det ofte vært utnyttet til størrelse basert celleseparasjon 14,15 og buffer børs applikasjoner. 16-18 However, forblir bufferbytte ytelsen dårlig med ~10-30% forurensningsoppløsning som de separerte celler som vanligvis er nær grensen mellom opprinnelige og nye bufferløsninger. 16-18 Viktigere er det at størrelsesfordelingen av målceller må være lik for å oppnå nøyaktig treghets fokusering og separasjon fra den opprinnelige bufferoppløsning som utgjør et problem, spesielt i behandlingen av heterogene størrelse celletyper slik som stamceller (MSC). 19 Vi har tidligere utviklet en ny treghets MicroFluidics cellesortering teknikk betegnes Dean Flow Fraksjonering (DFF) for å isolere sirkulerende tumorceller (CTCs) 20 og bakterier 21 fra helt blod ved hjelp av en to-innløp, 2-utløp spiral microchannel enhet. I denne video-protokollen, vil vi beskrive prosessen for merking av THP-1 (human akutt monocyttisk leukemicellelinje) suspensjon monocyttiske celler (~ 15 nm) og MSC (10-30 um) med calcein-loaded NPs, etterfulgt av fremstilling og drift av DFF spiral microdevice for en effektiv utvinning av merkede celler og fjerning av ubundne NPS. 22 Denne enkelttrinnsrensestrategi muliggjør kontinuerlig utvinning av merket suspensjon og adherente celler suspendert i frisk bufferløsning uten sentrifugering. Videre kan det behandle opptil 10 millioner celler · ml -1, en ​​celle tetthet mottagelig for regenerativ medisin applikasjoner.

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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

Ingeniør celler med aktiv ingrediens lastet mikro / nanopartikler (NPS) blir en stadig mer populær metode for å forbedre innfødte terapeutiske egenskaper, gjør at bio bildebehandling og kontrollere cellefenotyp. En kritisk, men en tilfredsstill problemet er betydelig antall av partikler som forblir ubundet etter cellemerking som ikke lett kan fjernes ved vanlig sentrifugering. Dette fører til en økning i biologisk avbildning bakgrunnsstøy, og kan formidle transformerende effekter bort på nærliggende ikke-målceller. I denne protokollen, presenterer vi en treghets MicroFluidics basert buffer utveksling strategi betegnes som Dean Flow Fraksjone (DFF) for å effektivt skille merkede celler fra frie NPs i en høy gjennomstrømning måte. Den utviklede spiral microdevice letter sammenhengende samling (&gt; 90% celle gjenvinning) av rensede celler (THP-1 og MSC) suspendert i ny buffer-oppløsning, samtidig som man oppnår&gt; 95% reduksjon av ubundet fluorescerende fargestoff eller fargestoff belastede NPS (silica eller PLGA). Dette enkelt-trinn, gjør det mulig størrelse basert celle rensestrategi høy celle behandling gjennomløp (10 6 celler / min), og er meget nyttig for stor-volum celle rensing av mikro / nanopartikkel konstruert for celler for å oppnå støyfri klinisk anvendelse.

Etter merking av cellene med bio avbildningsmiddel belastede NPS over natten, blir de merkede celler (inneholdende frie partikler) høstet og renset ved DFF spiral microdevice for å fjerne frie NPS i en enkelttrinnsprosess (figur 1A). Den 2-innløp, 2-uttak spiral microchannel er designet av engineering programvare og microfabricated bruker SU-8 fotoresist. Den mønstrede silikonplaten blir deretter anvendt som et templat for PDMS replika støping ved bruk av myke litog...

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 protokollen beskriver bruken av en treghets MicroFluidics basert bufferbytte strategi for å rense mikro / nanopartikkel modifiserte cellene med effektiv uttømming av ubundne partikler.

Microfluidic Buffer Exchange for Forstyrrelsesfri Micro / partikler 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

Stedsspesifikk Bakteriell Chromosome 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 av<em&gt; In Vitro</em&gt; Cell Rolling Ved hjelp av en multi-brønn 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

Cell klemme som en robust, Microfluidic Intracellulær Levering 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 Technique å sondere Cell Fleksibilitet

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

Kontrollert mikrofluid Miljø for Dynamic Gransking av røde blodceller 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

Ved hjelp av lim Mønstring å konstruere 3D Paper microfluidic enheter

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 plattform for High-throughput encellede 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 Mønstring for Gransker Kavitasjon Bubble (e)-celle Samhandling og den resulterende bioeffekter på Single-cellenivå

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-trinns variabel Høyde Fotolitografi for Valved Multilayer microfluidic enheter

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