Name: measuring the confluence of ipscs using an automated imaging system
Uploaded: 2020-06-10T13:30:00
Description: Watch this Scientific Journal Video about Measuring the Confluence of iPSCs Using an Automated Imaging System at JoVE.com

Undersøgelsen blev støttet af tilskud fra Fondazione Bambino Gesù og Ricerca Corrente (det italienske sundhedsministerium) til C.C.  Vi vil gerne takke Dr. Enrico Bertini (Institut for Neurovidenskab, Enhed for Neuromuskulære og Neurodegenerative Sygdomme, Laboratorium for Molekylær Medicin, Bambino Gesù Children's Research Hospital), Dr. Stefania Petrini (Confocal Microscopy Core Facility, Research Laboratories, Bambino Gesù Children's Research Hospital), Giulia Pericoli (Institut for Onco-hæmatologi, Gen- og Celleterapi, Børneforskningshospital Bambino Gesù) og Roberta Ferretti (Institut for Onco-hæmatologi, Gen- og Celleterapi, Children's Research Hospital Bambino Gesù) for videnskabelige diskussioner og teknisk hjælp. Maria Vinci er modtager af et "Children with Cancer UK fellowship".

1. Belægning af 96 brøndplader
BEMÆRK: Forskellige belægninger blev testet i samme plade, men separate brønde (se supplerende fil).
<ol><li>Fortynd Matrigel 1:100 i DMEM. Der tilsættes 100 μL pr. brønd til de 96 brøndplader og inkuberes i 1 time ved stuetemperatur. Herefter fjernes opløsningen og brøndene vaskes med 100 μL DMEM to gange.</li>
	<li>Fortyndet laminin (20 μg/ml, LN-521) i PBS (med calcium og magnesium). Der tilsættes 100 μL til brønden, og der i...

<ol>
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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+defined+xeno-free+and+feeder-free+culture+system+for+the+derivation,+expansion+and+direct+differentiation+of+transgene-free+patient-specific+induced+pluripotent+stem+cells.">A defined xeno-free and feeder-free culture system for the derivation, expansion and direct differentiation of transgene-free patient-specific induced pluripotent stem cells.</a> Biomaterials. 35 (9), 2816-2826 (2015).</li><li>Miyazaki, T., Nakatsuji, N., Suemori, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+slow+cooling+cryopreservation+for+human+pluripotent+stem+cells.">Optimization of slow cooling cryopreservation for human pluripotent stem cells.</a> Genesis. 52 (1), 49-55 (2014).</li><li>Bergstr&#246;m, R., Str&#246;m, S., Holm, F., Feki, A., Hovatta, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xeno-free+culture+of+human+pluripotent+stem+cells.">Xeno-free culture of human pluripotent stem cells.</a> Methods in Molecular Biology. 767, 125-136 (2011).</li><li>Braam, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+vitronectin+is+a+functionally+defined+substrate+that+supports+human+embryonic+stem+cell+self-renewal+via+alphavbeta5+integrin.">Recombinant vitronectin is a functionally defined substrate that supports human embryonic stem cell self-renewal via alphavbeta5 integrin.</a> Stem Cells. 26 (9), 2257-2265 (2008).</li><li>Chen, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemically+defined+conditions+for+human+iPSC+derivation+and+culture.">Chemically defined conditions for human iPSC derivation and culture.</a> Nature Methods. 8 (5), 424-429 (2008).</li><li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+vitronectin+concentration+and+surface+properties+on+the+stable+propagation+of+human+embryonic+stem+cells.">Impact of vitronectin concentration and surface properties on the stable propagation of human embryonic stem cells.</a> Biointerphases. 5 (3), 132-142 (2010).</li><li>Prowse, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+culture+of+human+embryonic+stem+cells+on+recombinant+vitronectin+in+ascorbate+free+media.">Long-term culture of human embryonic stem cells on recombinant vitronectin in ascorbate free media.</a> Biomaterials. 31 (32), 8281-8288 (2010).</li><li>Rowland, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+integrins+in+human+induced+pluripotent+stem+cell+growth+on+Matrigel+and+vitronectin.">Roles of integrins in human induced pluripotent stem cell growth on Matrigel and vitronectin.</a> Stem Cells and Development. 19 (8), 1231-1240 (2010).</li><li>Ruoslahti, E., Engvall, E., Hayman, E. G., Spiro, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+studies+on+amniotic+fluid+and+plasma+fibronectins.">Comparative studies on amniotic fluid and plasma fibronectins.</a> Biochemistry. 193 (1), 295-299 (1981).</li><li>Ni, H., Li, A., Simonsen, N., Wilkins, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+activation+by+dithiothreitol+or+Mn2++induces+a+ligand-occupied+conformation+and+exposure+of+a+novel+NH2-terminal+regulatory+site+on+the+beta1+integrin+chain.">Integrin activation by dithiothreitol or Mn2+ induces a ligand-occupied conformation and exposure of a novel NH2-terminal regulatory site on the beta1 integrin chain.</a> Biological Chemistry. 273 (14), 7981-7987 (1998).</li><li>Seltana, A., Basora, N., Beaulieu, J. 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Brugen af iPSC'er til sygdomsmodellering og fremtidig lægemiddelscreening sammen med deres mulige anvendelse i præcisionsmedicin gør det til en teknologi af stor relevans, og derfor mener vi, at det er nødvendigt klart at forstå in vitro-dyrkningstilstanden, der bedre ligner den fysiologiske situation for embryonale stamceller. I denne sammenhæng testede vi forskellige ECM-belægninger ved hjælp af vildtype iPSC'er for at forstå de forhold, der gør det muligt for cellerne at forblive i en sund og udifferentieret...

Inducerede pluripotente stamceller (iPSC'er) opnås fra somatiske celler og kan differentieres i forskellige celletyper. De bruges ofte som et system til at modellere sygdomspatogenese eller udføre lægemiddelscreening og tilbyder også potentialet til at blive brugt i forbindelse med personlig medicin. Da iPSC'er har stort potentiale, er det vigtigt at karakterisere dem fuldt ud til brug som et pålideligt modelsystem. Vi har tidligere vist vigtigheden af at dyrke iPSC'er i et hypoxisk miljø, da disse celler er afhængige af glykolyse, og et aerobt miljø kan forårsage redox ubalance1. iPSC'er er også sårbare over for andre kulturforhold, især det ekstracellulære miljø. Optimering af kulturforhold er et centralt spørgsmål for at holde dem sunde og udbredte. En sund iPSC-kultur vil føre til sunde differentierede celler, der generelt er endepunktet for den model, der bruges til at forstå molekylære, cellulære og funktionelle træk ved specifikke menneskelige lidelser eller cellulære processer.
I denne undersøgelse er en simpel protokol blevet brugt til at teste sammenløbet af iPSC'er ved hjælp af forskellige belægningsforhold i separate brønde. iPSC'er kræver et feederlag af murine embryonale fibroblaster (MEF) for at kunne fastgøres korrekt, men sameksistensen af iPSC'er og MEF gør det vanskeligt at udføre analyse som RNA- eller proteinekstraktion, da to populationer af celler er til stede. For at undgå feederlaget er forskellige proteiner, der tilhører den ekstracellulære matrix (ECM), blevet brugt til at genskabe den naturlige celleniche og til at have feederfri iPSC-kultur. Matrigel er især et opløseligt kældermembranpræparat ekstraheret fra Engelbreth-Holm-Swarm (EHS) musesarkom, som er beriget med ekstracellulære matrixproteiner (dvs. laminin, kollagen IV, heparansulfatproteoglycaner, entactin / nidogen og vækstfaktorer)2,3. De andre anvendte belægningsbetingelser er i stedet oprensede proteiner med kendt relevans i opbygningen af ECM'erne: laminin-521 vides at blive udskilt af humane pluripotente stamceller (hPSC'er) i embryoets indre cellemasse, og det er en af de mest almindelige lamininer i kroppen efter fødslen 4,5,6,7,8,9, 10,11; vitronectin er en xeno-fri cellekulturmatrix, der vides at understøtte vækst og differentiering af hPSC 12,13,14,15,16; fibronectin er et ECM-protein, der er vigtigt for hvirveldyrs udvikling og vedhæftning og vedligeholdelse af embryonale stamceller i en pluripotent tilstand 17,18,19,20,21,22,23,24,25. Da der findes forskellige belægningsbetingelser, sammenligner vi dem med hensyn til deres virkning på iPSC'ernes sammenløb.

<table border="0" cellpadding="0" cellspacing="0" style="width:1124px;" width="1123">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:589px;">10 mL Stripette Serological Pipets, Polystyrene, Individually Paper/Plastic Wrapped, Sterile</td>
			<td style="width:281px;">Corning</td>
			<td style="width:77px;">4488</td>
			<td style="width:176px;">Tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 mL high-clarity polypropylene (PP) conical centrifuge tubes</td>
			<td>Falcon</td>
			<td>352097</td>
			<td>Tool</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">1x PBS (With Ca2+; Mg2+)</td>
			<td>Thermofisher</td>
			<td>14040133</td>
			<td>Medium</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">1x PBS (without Ca2+; Mg2+)</td>
			<td>Euroclone</td>
			<td>ECB4004L</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5 mL Stripette Serological Pipets, Polystyrene, Individually Paper/Plastic Wrapped, Sterile</td>
			<td>Corning</td>
			<td>4487</td>
			<td>Tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture microplate, 96 WELL, PS, F-Bottom</td>
			<td>Greiner Bio One</td>
			<td>655090</td>
			<td>Support</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture plate, 6 well</td>
			<td>Costar</td>
			<td>3516</td>
			<td>Support</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM (Dulbecco&#39;s Modified Eagle&#39;s Medium- high glucose)</td>
			<td>Sigma</td>
			<td>D5671</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Sigma</td>
			<td>ED4SS-500g</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Epi Episomal iPSC Reprogramming Kit</td>
			<td>Invitrogen</td>
			<td>A15960</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FAST - READ 102</td>
			<td>Biosigma</td>
			<td>BVS100</td>
			<td>Tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>10270106</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fibronectin</td>
			<td>Merck</td>
			<td>FC010</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycerol</td>
			<td>Sigma</td>
			<td>G5516</td>
			<td>Reagent</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">H2O</td>
			<td></td>
			<td>MILLIQ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst</td>
			<td>Thermofisher</td>
			<td>33342</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laminin 521</td>
			<td>Stem Cell Technologies</td>
			<td>77003</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine (200 mM)</td>
			<td>Gibco</td>
			<td>LS25030081</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrigel</td>
			<td>Corning Matrigel hESC-Qualified Matrix</td>
			<td>354277</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:589px;">Mouse embryonic fibroblasts (MEF)</td>
			<td>Life Technologies</td>
			<td>A24903</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MTESR1 Medium</td>
			<td>Stem Cell Technologies</td>
			<td>85851</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MTESR1 Supplement</td>
			<td>Stem Cell Technologies</td>
			<td>85852</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phalloidin</td>
			<td>Sigma</td>
			<td>P1951</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vitronectin</td>
			<td>Stem Cell Technologies</td>
			<td>7180</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Y-27632</td>
			<td>Sigma</td>
			<td>Y0503</td>
			<td>Reagent</td>
		</tr>
	</tbody>
</table>

measuring the confluence of ipscs using an automated imaging system

Denne undersøgelse fokuserer på at forstå, hvordan voksende iPSC'er på forskellige ECM-belægningssubstrater kan påvirke cellesammenløbet. En protokol til vurdering af iPSC-sammenløb i realtid er blevet etableret uden behov for at tælle celler i enkeltcellesuspension for at undgå vækstforstyrrelser. Et billedanalysesystem med højt indhold blev brugt til at vurdere iPCS-sammenløb på 4 forskellige ECM'er over tid på en automatiseret måde. Forskellige analyseindstillinger blev brugt til at vurdere cellesammenløbet af vedhæftede iPSC'er, og kun en lille forskel (ved 24 og 48 timer med laminin) er blevet observeret, om en 60, 80 eller 100% maske blev påført. Vi viser også, at laminin fører til den bedste sammenløb sammenlignet med Matrigel, vitronectin og fibronectin.

I denne undersøgelse undersøgte vi sammenløbet af iPSC'er, når de dyrkes under forskellige belægningsforhold. Ved hjælp af et cytometer var vi i stand til at opnå let informative resultater i tredoblinger på 5 dage. Da iPSC'er næppe fastgøres til plastbeholdere, og en belægning er nødvendig for at understøtte deres spredning, besluttede vi at overvåge sammenløbet af humane iPSC'er, da det er tegn på cellekulturens sundhed, og det kan afspejle deres differentieringspotentiale. Efter in vitro-ekspansion så...

Watch this Scientific Journal Video about Measuring the Confluence of iPSCs Using an Automated Imaging System at JoVE.com

Measuring the Confluence of iPSCs Using an Automated Imaging System

Målet med protokollen er at sammenligne forskellige ekstracellulære matrix (ECM) belægningsbetingelser for at vurdere, hvordan differentiel belægning påvirker væksthastigheden for inducerede pluripotente stamceller (iPSC'er). Vi sigter især mod at skabe betingelser for at opnå optimal vækst af iPSC-kulturer.

Måling af sammenløbet af iPSC'er ved hjælp af et automatiseret billedbehandlingssystem.

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence ...

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JoVE Journal

Biology

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