Name: bioparticle microarrays for chemotactic and molecular analysis of human neutrophil swarming in vitro
Uploaded: 2020-02-16T14:00:00
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Detta arbete stöddes av finansiering från William G. Lowrie Institutionen för kemi och biomolekylär teknik och Comprehensive Cancer Center vid Ohio State University. Data som presenteras i denna rapport kom från bilder som bearbetas med Imaris x64 (ver. 9.3.0 Bitplane) som finns på Campus Microscopy och Imaging Facility, Ohio State University. Denna anläggning stöds delvis av grant P30 CA016058, National Cancer Institute, Bethesda, MD.

Författarna erkänner friska frivilliga som vänligt donerade sitt blod. Blodprover erhölls efter informerat frivilligt samtycke enligt institutionella granskningsnämnden (IRB) protokoll #2018H0268 granskas av Biomedical Sciences kommittén vid Ohio State University.
1. Mikrotillverkning av biopartikelmikroarray
<ol><li>Med hjälp av vanliga fotolitografiförfaranden, generera befälhavaren kisel rån.<ol>
<li>Generera ett bevis på önskad design med hjälp av en datorstödd design (CAD)...

<ol>
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Vi utvecklade en mikrostämplingsplattform för att generera enhetliga matriser av biopartiklar för att stimulera in vitro neutrophil swarming. In vitro karaktär vår plattform tillåter oss att kringgå de komplikationer som uppstår med in vivo sväring experiment, nämligen den dåliga förmågan att analysera medlare som släppts av svärmar neutrofiler5. Dessutom utförs in vivo-modeller vanligtvis hos gnagare11,12,<sup c...

Neutrofiler, den mest rikliga vita blodkroppar i blodomloppet1, får uppmärksamhet som potentiella diagnostiska och terapeutiska mål2,3 eftersom de kan vara inblandade i en mängd olika medicinska tillstånd inklusive gout4, sepsis3, trauma5,6, cancer1,7,8, och olika autoimmuna sjukdomar5,9. Neutrofil swarming är en flerstegs, tätt reglerad process med en komplexitet som gör det till ett särskilt intressant fokus för studie5,10,11. Under svärma, neutrofiler isolera en plats för inflammation från den omgivande friskavävnaden 5,10,11. Korrekt reglering av neutrofil swarming är viktigt att främja sårläkning och i slutändan inflammation upplösning5,12. Neutrofil swarming har främst studerats in vivo hos gnagare12,13,14,15 och zebrafiskar10,11,12,15 modeller. Men arten av dessa in vivo djurmodeller ger upphov till begränsningar5. Till exempel är de medlare som släppts av neutrofiler under svärmar inte lättillgängliga för analys5. Dessutom finns det många potentiella källor för en viss medlare in vivo, så ett in vivo-experiment måste införa en genetisk brist för att hämma cellproduktionen och/eller interaktionen för att undersöka medlarens roll i en viss process13. Ett in vitro-experiment kringgår denna komplikation genom att möjliggöra neutrofil observation utan samband med ytterligare celler. Dessutom är forskning som beskriver human neutrophil samordnad migration begränsad16. På en in vitro-svärmande plattform kan mänskliga neutrofiler analyseras direkt. En in vitro-svärmande plattform skulle kunna utveckla den kunskap som erhållits från in vivo-studier genom att ge möjligheter att fylla de luckor som finns kvar av begränsningarna i in vivo-studier.
För att ta itu med behovet av en in vitro-plattform som efterliknar in vivo neutrophil swarming, utvecklade vi en microstamping plattform som gör det möjligt för oss att mönster biopartikel mikroarrayer som stimulerar neutrofil svärmar på ett rumsligt kontrollerat sätt. Vi genererar biopartikelmikroarrayer på glasrutschbanor i en tvåstegsprocess. Först använder vi mikrostämpling för att generera en mikroarray av katjoniska polyelektrolyt (CP) fläckar. För det andra lägger vi till en lösning av biopartiklar som följer CP-fläckarna via elektrostatisk interaktion. Genom att först mönstra CP-skiktet kan vi selektivt mönster negativt laddade biopartiklar för att generera önskat neutrofils sväringsmönster. Det positivt laddade skiktet håller de negativt laddade biopartiklarna genom det kraftfulla tvättsteget som tar bort biopartiklarna från områdena på glasbilden som inte har CP. Dessutom är den CP som används här, en sampolymer av akrylamid och pittorerad katjonisk monomer, biokompatibel, så det framkallar inte ett svar från neutrofiler. Den har en mycket hög ytladdning som immobiliserar mikronstora biopartiklar till glasbilden, vilket hämmar neutrofiler från att ta bort partiklarna från den mönstrade positionen på glasbilden. Detta resulterar i biopartikelkluster ordnade i en mikroarray. När vi lade till neutrofiler i mikroarrayen bildade de stabila svärmar runt biopartikelhoparna. Genom att spåra neutrofilmigration fann vi att svärmar neutrofiler aktivt migrerar mot biopartikelklustren. Dessutom använde vi denna plattform för att analysera vissa medlare som neutrofiler släpper under svärmande. Vi hittade 16 medlare som uttrycks i differentially under uppvärmningen. Deras koncentrationer följer tre allmänna trender över tiden: ökning, minskning eller spik. Vår in vitro neutrofil swarming plattform underlättar analysen av rumsligt kontrollerad mänsklig neutrophil swarming, liksom insamling och analys av medlare släpptes av neutrofil swarming. I en tidigare publikation visade vi att patienter med vissa medicinska tillstånd (trauma, autoimmun sjukdom och sepsis), hade neutrofiler som fungerade annorlunda än de från friska donatorer5. I framtida forskningsstudier kan vår plattform användas för att analysera neutrofilfunktion bland en mängd olika patientpopulationer. Denna plattform kan kvantitativt analysera den komplexa samordning en svärmning. Ytterligare studier kan göras för att ge insikt om neutrofil funktion av en viss patientpopulation eller neutrofil svar på en patogen av intresse.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#34;The Big Easy&#34; EasySep Magnet</td>
			<td>STEMCELL Technologies</td>
			<td>18001</td>
			<td>Magnet to use with neutrophil isolation kit</td>
		</tr>
		<tr>
			<td>Cell Incubator</td>
			<td>Okolab</td>
			<td>777057437 / 77057343</td>
			<td>Okolab cage incubator for temperature and CO2 control</td>
		</tr>
		<tr>
			<td>EasySep Human Neutrophil Isolation Kit</td>
			<td>STEMCELL Technologies</td>
			<td>17957</td>
			<td>Kit for immunomagnetic negative selection of human neutrophils</td>
		</tr>
		<tr>
			<td>Eclipse Ti2</td>
			<td>Nikon Instruments</td>
			<td>MEA54010 / MEF55037</td>
			<td>Inverted research microscope</td>
		</tr>
		<tr>
			<td>Escherichia coli (K-12 strain) BioParticles Texas Red conjugate</td>
			<td>Invitrogen</td>
			<td>E2863</td>
			<td>Bioparticle powder, dissolve in water prior to addition to Zetag&#174; array</td>
		</tr>
		<tr>
			<td>Harris Uni-Core 8-mm biopsy punch</td>
			<td>Sigma Aldrich</td>
			<td>Z708925</td>
			<td>To cut PDMS stamps</td>
		</tr>
		<tr>
			<td>HetaSep</td>
			<td>STEMCELL Technologies</td>
			<td>7906</td>
			<td>Erythrocyte aggregation agent for separating buffy coat from red blood cells in fresh human blood</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Life Technologies</td>
			<td>H3570</td>
			<td>Nucleus fluorescent stain</td>
		</tr>
		<tr>
			<td>Human L1000 Array</td>
			<td>Raybiotech Inc.</td>
			<td>AAH-BLG-1000-4</td>
			<td>High density array to detect 1000 human proteins</td>
		</tr>
		<tr>
			<td>Human Serum Albumin (HSA)</td>
			<td>Sigma Aldrich</td>
			<td>A5843</td>
			<td>Low endotoxin HSA, to prepare 2 % solutions in IMDM for isolated neutrophils</td>
		</tr>
		<tr>
			<td>Iscove&#39;s Modified Dulbeccos&#39; Medium (IMDM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>12440053</td>
			<td>To resuspend isolated neutrophils</td>
		</tr>
		<tr>
			<td>K2-EDTA tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>02-657-32</td>
			<td>Tubes for blood collection</td>
		</tr>
		<tr>
			<td>Low Reflective Chrome Photomask</td>
			<td>Front Range Photomask</td>
			<td>N/A</td>
			<td>Dimensions 5&#34; x 5&#34; x 0.09&#34; (L x W x D)</td>
		</tr>
		<tr>
			<td>Microarray Scanner</td>
			<td>Perkin Elmer</td>
			<td>ASCNGX00</td>
			<td>Fluorescence reader of protein patterned microdomains</td>
		</tr>
		<tr>
			<td>Microscopy Image Analsysis Software - Imaris</td>
			<td>Bitplane</td>
			<td>9.3.0</td>
			<td>Software for automatic cell tracking analysis</td>
		</tr>
		<tr>
			<td>NiS Elements Advanced Research Software Package</td>
			<td>Nikon Instruments</td>
			<td>MQS31100</td>
			<td>Software for automatic live cell imaging and swarm size calculation</td>
		</tr>
		<tr>
			<td>Poly-L-lysine fluorescein isothiocyanate (PLL-FITC)</td>
			<td>Sigma Aldrich</td>
			<td>P3069-10MG</td>
			<td>30,000 - 70,000 MW PLL labeled with FITC, used to fluorescently label CP solution</td>
		</tr>
		<tr>
			<td>SecureSeal 8-well Imaging Spacer</td>
			<td>Grace Bio-Labs</td>
			<td>654008</td>
			<td>8-well, 9-mm diameter, adhesive imaging spacer</td>
		</tr>
		<tr>
			<td>Silicon Wafer</td>
			<td>University Wafer</td>
			<td>590</td>
			<td>Silicon 100 mm N/P (100) 0- 100 ohm-cm 500 &#956;m SSP test</td>
		</tr>
		<tr>
			<td>Spin Coater</td>
			<td>Laurell</td>
			<td>WS-650MZ-23NPPB</td>
			<td>Used to spincoat a 40-&#181;m layer of photoresist onto silicon wafer</td>
		</tr>
		<tr>
			<td>SU-8 2025</td>
			<td>MicroChem</td>
			<td>2025</td>
			<td>Negative photoresist to make silicon master wafer</td>
		</tr>
		<tr>
			<td>SU-8 Developer</td>
			<td>MicroChem</td>
			<td>Y020100</td>
			<td>Photoresist developer. Remove non-crosslinked SU-8 2025 from silicon wafer</td>
		</tr>
		<tr>
			<td>Sylgard 184 (polydimethylsiloxane, PDMS)</td>
			<td>Dow</td>
			<td>1673921</td>
			<td>2-part silicone elastomer kit for making microstamps and PDMS wells</td>
		</tr>
		<tr>
			<td>UV Exposure Masking System</td>
			<td>Klo&#233;</td>
			<td>UV-KUB 2</td>
			<td>Used to crosslink photoresist on silicon wafer through chrome mask with UV light</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1287303</td>
			<td>High quality water to dilute bioparticles</td>
		</tr>
		<tr>
			<td>Zetag 8185</td>
			<td>BASF</td>
			<td>8185</td>
			<td>Cationic polyelectrolyte (CP), powder, Copolymer of acrylamide and quaternized cationic monomer, forms &#34;inking solution&#34; for microstamping when dissolved in water</td>
		</tr>
		<tr>
			<td>Zymosan A S. cerevisiae BioParticles Texas Red conjugate</td>
			<td>Invitrogen</td>
			<td>Z2843</td>
			<td>Bioparticle powder, dissolve in water prior to addition to Zetag array</td>
		</tr>
	</tbody>
</table>

bioparticle microarrays for chemotactic and molecular analysis of human neutrophil swarming in vitro

Neutrofil swarming är en samarbetsprocess genom vilken neutrofiler spärra av en infektionsplats och främja vävnad omorganisation. Swarming har klassiskt studerats in vivo i djurmodeller som visar karakteristiska mönster av cellmigration. Men in vivo modeller har flera begränsningar, inklusive intercellulära medlare som är svåra att komma åt och analysera, liksom oförmåga att direkt analysera mänskliga neutrofiler. På grund av dessa begränsningar finns det ett behov av en in vitro-plattform som studerar med mänskliga neutrofiler och ger enkel tillgång till de molekylära signaler som genereras under svärmande. Här används en multistegs mikrostämplingsprocess för att generera en biopartikelmikroarray som stimulerar svärmande genom att härma en in vivoinfektion. Biopartikelmikroarrayen inducerar neutrofiler att svära på ett kontrollerat och stabilt sätt. På mikroarrayen ökar neutrofiler i hastighet och bildar stabila svärmar runt biopartikelkluster. Dessutom analyserades supernatant som genererades av neutrofiler och 16 proteiner upptäcktes ha uttryckts differentially under svärmande. Denna in vitro-svärmande plattform underlättar direkt analys av neutrofilmigration och proteinutsläpp på ett reproducerbart, rumsligt kontrollerat sätt.

När neutrofiler läggs till i biopartikelmikroarrayen aktiveras neutrofiler som kontaktar biopartikelklustren och initierar svärmande svar. Biopartikelmikroarrayen validerades med tidsförfall fluorescerande mikroskopi för att spåra neutrofilmig migration mot biopartikelkluster (Video S1). Migreringen av enskilda neutrofilkärnor spåras när de migrerar mot biopartikelklustret. När neutrofiler når biopartikelklustret överlappar deras kärnor med andra kärnor i klustret. Det är således inte mö...

Watch this Scientific Journal Video about Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro at JoVE.com

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

Detta protokoll genererar biopartikelmikroarrayer som ger rumsligt kontrollerad neutrofilssvärmar. Det ger enkel tillgång till medlare som neutrofiler släpper under migration och möjliggör kvantitativ bildanalys.

Biopartikel Mikroarrayer för chemotaktisk och molekylär analys av human neutrophil svärma in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...

Detta protokoll är betydande eftersom det möjliggör en in vitro-analys av neutrofil svärmning, som övervinner många begränsningar av vår nuvarande oförmåga att experimentera. Den största fördelen med denna teknik är att den ger enkel åtkomst till de utsöndrade cytokiner och lipidmeditorer som neutrofiler släpper under svärmning och möjliggör kvantitativ bildanalys. Medan vi tillämpat denna teknik på neutrofiler, det kan utvidgas för att analysera migration av andra vita blodkroppar som monocyter och T-celler.Börja med att förbereda en 1.6 milligram per milliliter katjonit polyelektrolite, eller CP lösning, genom att lägga till rätt mängd CP pulver till vatten och blanda den på rörplattan över natten eller tills alla fasta ämnen är upplösta. Om så önskas kan lösningen göras fluorescerande genom att poly-L-lysin märkt med FITC. Generera befälhavaren kisel wafer med hjälp av standard fotolitografi förfaranden.Den design som används här är en fyra med fyra milliliter rektangulär array av 30 mikrometer diameter fylld i cirklar, med en 150 mikrometer centrum till centrum avstånd, men det kan ändras som önskas för olika tillämpningar. Spin-coat en 40 mikrometer tjockt lager av negativa fotoresist på en kisel wafer. Sedan baka wafer på 65 grader Celsius i fem minuter.Och 95 grader Celsius i 10 minuter. Exponera wafer till UV-ljus genom en krom fotomask med 150 till 160 millijoule per centimeter i kvadrat. Grädda wafer igen vid 65 grader Celsius i fem minuter och 95 grader Celsius i 10 minuter.Sedan dränka den i fotoresist utvecklare i 10 minuter. Och skölj den med isopropylalkohol. Mönstret ska nu synas på rånet.Därefter blanda grundligt en 10 till ett förhållande av polydimetylsiloxan, eller PDMS, prepolymer och dess härdningsmedel. Och häll den ohärdade blandningen över master wafer i en petriskål. Vakuum behandla uncured PDMS blandningen tills inga luftbubblor är närvarande.Sedan bota det över natten vid 65 grader Celsius. På nästa dag, använd en skalpell för att skära runt utsidan av den mönstrade delen av rån. Och ta långsamt bort den härdade PDMS-plattan, placera den på en ren skärbräda med den mönstrade sidan uppåt.Stämpla ut enskilda stämplar från PDMS-plattan med en åtta millimeters biopsistans och placera varje stämpel nedåt på den självhäftande tejpen för att ta bort skräp. Med dessa stämplar vänd uppåt, prime varje stämpel med 100 mikroliter av den förberedda CP lösning, se till att inga luftbubblor bildas mellan lösningen och stämpeln. Sedan invertera stämplarna på ett lager av CP-lösning och ta bort dem efter en timme.Badda varje våt stämpel nedåt på en ren glasrutschkana sex till åtta gånger för att ta bort överflödig vätska. Då vakuum behandla frimärken i en till två minuter. Följ en åtta brunn, åtta millimeter diameter avbildning distans ovanpå en ren glasrutschbana som en guide för stämpel placering.Placera en stämpel med framsidan nedåt på glasrutschkanan i mitten av varje brunn av bildringsserymden. Placera sedan en 5,6 gram balanserad vikt ovanpå varje stämpel och låt 10 minuter för stämpling. Ta bort vikterna och frimärkena från objektglaset och låt CP-lagret torka i rumstemperatur i 24 timmar innan biopartiklar tillsätts.Om CP är taggad med FITC, kontrollera effektiviteten av stämpling med ett fluorescerande mikroskop. Skär en tom PDMS platta till storleken på bildframställning distans och använda åtta millimeter biopsi punch för att skapa brunnar i PDMS som överensstämmer med brunnarna i distansen. Följ sedan PDMS-plattan till glasglaset.Tina en lösning av biopartiklar och späd ut den till 500 mikrogram per milliliter i vatten för injektion. Tillsätt 100 mikroliter biopartikellösning till varje PDMS-brunn på glasglaset och gunga rutschkanan i 30 minuter. Skölj brunnarna noggrant med vatten och kontrollera mönstret på objektglaset med ett fluorescerande mikroskop.Biopartikelmikrorayen kan förvaras i en dammfri miljö vid fyra grader Celsius i upp till tre månader. Förbered cellerna genom att resu utgifterna dem på 7,5 gånger 10 till den femte celler per milliliter i IMDM med 0,4% humant serum albumen. Lägg till 100 mikroliter av suspensionen till en PDMS-brunn som innehåller biopartikelmikroarrayen, och se till att cellfjädringen är konvex över toppen av PDMS-brunnen och inte innehåller några bubblor.Täta brunnen med en 12 millimeters diameter täcka glida och tryck ner försiktigt med pincett så överskottet cell suspensionen flyr till kanten av brunnen, sedan använda en vävnad för att ta bort överskottet. För att avbilda cellerna, ladda mikropartikelarrayen med celler på den levande cellbildsstationen i ett mikroskop utrustat med en burinkubator inställd på 37 grader Celsius, 5%koldioxid och 90%relativ luftfuktighet. Använd tidsfördröjning, lysrör och ljus fältmikroskopi för att spela in bilder med 10X-förstoring var 10:e sekund vid 405 nanometer, 594 nanometer och ljust fält.Inkubera neutrofilerna i brunnarna med biopartiklar vid 37 grader Celsius och 5%koldioxid i tre timmar, med prover vid önskade tidpunkter. Använd ett ljust fältmikroskop för att verifiera att svärmar bildas på mikroarrayen. Samla hela volymen av supernatant av en enda brunn, och ladda den i en 0,45 mikrometer centrifug filterrör, se till att analysera varje tidpunkt i tre exemplar.Centrifugera rören på 190 gånger G och 20 grader Celsius i fem minuter, och samla den filtrerade volymen. Sedan lagra proverna på minus 80 grader Celsius fram till bearbetningstiden. När du försöker detta protokoll, arbeta med fotoresist och utvecklare i rök huven.Har en IRB godkänt protokoll på plats innan du tar blod från mänskliga givare. Kom ihåg att material som härrör från humant blod är BSL2. När neutrofiler läggs till biopartikelmikroarrayen aktiveras neutrofiler som kontaktar biopartikelklusterna och initierar det svärmande svaret.Biopartikelmikrorrayen validerades med hjälp av tidsförloppslysrörsmikroskopi för att spåra neutrofilmigrationer mot biopartikelkluster. Stabila neutrofilsvärmar bildas runt varje kluster efter 30 till 60 minuters exponering. Däremot visar neutrofiler inte kollektiv migration i avsaknad av biopartikelkluster.Fluorescens intensiteten av färgade neutrofil kärnor användes för att fastställa att den genomsnittliga svärm storlek runt biopartikel kluster var cirka 1490 mikrometer kvadrat. I avsaknad av kluster förblev fluorescensen av en viss region av intresse konstant över tiden. Spår av neutrofil migration visar att neutrofiler konvergerade på en biopartikel kluster när en var närvarande, men ingen konvergens observerades i kontrollsystemet.Hastigheten på svärmande och nonactivated neutrophils mättes, och en statistiskt signifikant skillnad i hastigheten distributioner hittades. Den genomsnittliga hastigheten för svärmande neutrofiler var 20,6 mikrometer per minut, medan den genomsnittliga hastigheten för kontroll neutrofiler var två mikrometer per minut. Koncentrationerna av 16 proteiner som neutrofiler släpper under svärmning analyserades över tiden.10 proteiner ökade i koncentration. Två minskade. Och de återstående fyra ökade under den första timmen av svärmande men minskade därefter.Medan du utför detta förfarande, se till att allt är rent och dina stämplar torkas på rätt sätt, eftersom dessa är väsentliga för framgångsrika mönster av de bakteriella partiklarna. Utvecklingen av denna teknik kommer att göra det möjligt för forskare att utforska nya frågor inom området neutrofil svärmande, inklusive hur fria medlare och extracellulära vesiklar är involverade i neutrofil intercellulär kommunikation.

bioparticle-microarrays-for-chemotactic-molecular-analysis-human

Research

JoVE Journal

Bioengineering

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular machines centers on devices capable of directional motion, i.e. molecular motors, and on their scaled-down mechanical parts (wheels, axels, pendants etc) 7-9. This imitates the macro-machines, even though the physical properties essential for these devices, such as inertia and momentum conservation, are not usable in the nanoworld environments 10. Alternative designs, which do not follow the mechanical macromachines schemes and use mechanisms developed in the evolution of biological molecules, can take advantage of the specific conditions of the nanoworld. Besides, adapting actual biological molecules for the purposes of nano-design reduces potential dangers the nanotechnology products may pose. Here we demonstrate the assembly and application of one such bio-enabled construct, a semi-artificial molecular device which combines a naturally-occurring molecular machine with artificial components. From the enzymology point of view, our construct is a designer fluorescent enzyme-substrate complex put together to perform a specific useful function. This assembly is by definition a molecular machine, as it contains one 12. Yet, its integration with the engineered part - fluorescent dual hairpin - re-directs it to a new task of labeling DNA damage12.
Our construct assembles out of a 32-mer DNA and an enzyme vaccinia topoisomerase I (VACC TOPO). The machine then uses its own material to fabricate two fluorescently labeled detector units (Figure 1). One of the units (green fluorescence) carries VACC TOPO covalently attached to its 3'end and another unit (red fluorescence) is a free hairpin with a terminal 3'OH. The units are short-lived and quickly reassemble back into the original construct, which subsequently recleaves. In the absence of DNA breaks these two units continuously separate and religate in a cyclic manner. In tissue sections with DNA damage, the topoisomerase-carrying detector unit selectively attaches to blunt-ended DNA breaks with 5'OH (DNase II-type breaks)11,12, fluorescently labeling them. The second, enzyme-free hairpin formed after oligonucleotide cleavage, will ligate to a 5'PO4 blunt-ended break (DNase I-type breaks)11,12, if T4 DNA ligase is present in the solution 13,14 . When T4 DNA ligase is added to a tissue section or a solution containing DNA with 5'PO4 blunt-ended breaks, the ligase reacts with 5'PO4 DNA ends, forming semi-stable enzyme-DNA complexes. The blunt ended hairpins will interact with these complexes releasing ligase and covalently linking hairpins to DNA, thus labeling 5'PO4 blunt-ended DNA breaks.
This development exemplifies a new practical approach to the design of molecular machines and provides a useful sensor for detection of apoptosis and DNA damage in fixed cells and tissues.

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

We demonstrate the assembly and application of a molecular-scale device powered by a topoisomerase protein. The construct is a bio-molecular sensor which labels two major types of DNA breaks in tissue sections by attaching two different fluorophores to their ends.

In vitro-Montering av Semi-konstgjorda molekylär maskin och dess användning för detektion av DNA-skador

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular ...

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

Polymer Microarrays för hög genomströmning Upptäckt av Biomaterial

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment 1,2 . These materials are desirable because they possess highly controllable dimensional and material characteristics, are biocompatible and promote cell adhesion, can be modified through topographic patterning or by chemically altering the surface, and can be used as a depot for biologically active molecules for drug delivery related applications 3-8 . In addition, silk films are relatively straightforward to custom design, can be designed to dissolve within minutes or degrade over years in vitro or in vivo, and are produce with the added benefit of being transparent in nature and therefore highly suitable for imaging applications 9-13. The culture system methodology presented here represents a scalable approach for rapid assessments of cell-silk film surface interactions. Of particular interest is the use of surface patterned silk films to study differences in cell proliferation and responses of cells for alignment 12,14 . The seeded cultures were cultured on both micro-patterned and flat silk film substrates, and then assessed through time-lapse phase-contrast imaging, scanning electron microscopy, and biochemical assessment of metabolic activity and nucleic acid content. In summary, the silk film in vitro culture system offers a customizable experimental setup suitable to the study of cell-surface interactions on a biomaterial substrate, which can then be optimized and then translated to in vivo models. Observations using the culture system presented here are currently being used to aid in applications ranging from basic cell interactions to medical device design, and thus are relevant to a broad range of biomedical fields.

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are a novel class of biomaterials readily customizable for an array of biomedical applications. The presented silk film culture system is highly adaptable to a variety of in vitro analyses. This system represents a biomaterial design platform offering in vitro optimization before direct translation to in vivo models.

Silk Film Kultur System för In vitro Analys och Biomaterial design

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Självrapporterings Ställningar för 3-dimensionell cellodling

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

Ögonirritationstest (EIT) för Hazard Identifiering av Eye irriterande kemikalier med hjälp av rekonstruerad Human Cornea liknande Epithelial (RHCE) Tissue Modell

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

Anpassning av Electro processen med att ge tre unika miljöer för en Tri-lager<em&gt; In Vitro</em&gt; Modell av Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

Beredning och analys av<em&gt; In Vitro</em&gt; Tredimensionell bröstkarcinom Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

En<em&gt; In Vitro</em&gt; Organ Kultur Modell av murina mellankotskivan

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical gradient, known as chemotaxis, plays an important role in development, the immune response, wound healing, and cancer metastasis. While chemotaxis modulates the migration of single cells as well as collections of cells in vivo, in vitro research focuses on single-cell chemotaxis, partly due to the lack of the proper experimental tools. To fill that gap, described here is a unique experimental system that combines microfluidics and micropatterning to demonstrate the effects of chemical gradients on collective cell migration. Furthermore, traction microscopy and monolayer stress microscopy are incorporated into the system to characterize changes in cellular force on the substrate as well as between neighboring cells. As proof-of-concept, the migration of micropatterned circular islands of Madin-Darby canine kidney (MDCK) cells is tested under a gradient of hepatocyte growth factor (HGF), a known scattering factor. It is found that cells located near the higher concentration of HGF migrate faster than those on the opposite side within a cell island. Within the same island, cellular traction is similar on both sides, but intercellular stress is much lower on the side of higher HGF concentration. This novel experimental system can provide new opportunities to studying the mechanics of chemotactic migration by cellular collectives.

Collective cell migration in development, wound healing, and cancer metastasis is often guided by the gradients of growth factors or signaling molecules. Described here is an experimental system combining traction microscopy with a microfluidic system and a demonstration of how to quantify the mechanics of collective migration under biochemical gradient.

Traktionsmikroskopi integrerat med mikrofluidik för Chemotactic kollektiv migration

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical ...

Production of Autologous Platelet-Rich Plasma for Boosting In Vitro Human Fibroblast Expansion

There is currently great clinical interest in the use of autologous fibroblasts for skin repair. In most cases, culture of skin cells in vitro is required. However, cell culture using xenogenic or allogenic culture media has some disadvantages (i.e., risk of infectious agent transmission or slow cell expansion). Here, an autologous culture system is developed for the expansion of human skin fibroblast cells in vitro using a patient&#8217;s own platelet-rich plasma (PRP). Human dermal fibroblasts are isolated from the patient while undergoing abdominoplasty. Cultures are followed for up to 7 days using a medium supplemented with either fetal bovine serum (FBS) or PRP. Blood cell content in PRP preparations, proliferation, and fibroblast differentiation are assessed. This protocol describes the method for obtaining a standardized, non-activated preparation of PRP using a dedicated medical device. The preparation requires only a medical device (CuteCell-PRP) and centrifuge. This device is suitable under sufficient medical practice conditions and is a one-step, apyrogenic, and sterile closed system that requires a single, soft spin centrifugation of 1,500 x g for 5 min. After centrifugation, the blood components are separated, and the platelet-rich plasma is easily collected. This device allows a quick, consistent, and standardized preparation of PRP that can be used as a cell culture supplement for in vitro expansion of human cells. The PRP obtained here contains a 1.5-fold platelet concentration compared to whole blood together, with a preferential removal of red and white blood cells. It is shown that PRP presents a boosting effect in cell proliferation compared to FBS (7.7x) and that fibroblasts are activated upon PRP treatment.

This protocol presents a device that produces PRP to boost the in vitro expansion of cells in a 100% autologous fibroblast culture system.