Name: evaporation-reducing culture condition increases the reproducibility of multicellular spheroid formation in microtiter plates
Uploaded: 2017-03-07T14:00:00
Description: Watch this Scientific Journal Video about Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates at JoVE.com

Dette arbejde blev støttet af tilskud fra det tjekkiske ministerium for uddannelse, ungdom og sport (LO1304) og Teknologisk Agentur for Den Tjekkiske Republik (TE01020028). Forfatterne vil gerne takke Dr. Lakshman Varanasi for at tage stillbilleder af miljømæssige låg.

1. Fremstilling af agarose-overtrukne plader <ol><li> Afvej 0,75 g af lav-smeltepunkt agarose og føje den til 100 ml McCoys 5A-medium (med eller uden phenolrødt) uden serum. Opvarm opløsningen i en mikrobølgeovn og swirl hver 1-2 min for at opløse agarosen. Autoklaver opløsningen at sterilisere den. </li><li> Afkøl autoklaveret agarose til ca. 70 ° C og filtrere det gennem en 500 ml, 0,22 um filter top ved vakuumfiltrering i en laminar strømning boks. Alikvot den 0,75% filtrerede agaroseopløsning (FAS) i min...

<ol>
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Coating 384-Well TC plader med Filtreret Agarose Den normale praksis i LOT er at anvende en 1-1,5% lavt smeltepunkt agarose at overtrække pladerne, hvilket kræver agarose og / eller dispenseringsindretning, der skal holdes opvarmet for at forhindre gelering af agarose 6. Den geldannende af agarose er potentielt problematiske samtidig forbereder flere plader under anvendelse af flydende udlevering kassetter med små slanger åbninger på mellem 0,2 og 0,4 mm i diameter....

Kræftceller i tumorer er fysiologisk anbragt i en kompleks, 3-dimensional (3D) struktur omgivet af ekstracellulær matrix og interagerende celler. Som næsten alle celler i væv opholde sig i en 3D-miljø, er behovet for mere fysiologisk relevant in vitro tumormodeller der efterligner tumor træk resulteret i udviklingen af en række 3D dyrkningsteknikker 1, 2, 3. Disse modeller er nu ved at blive grundforskning værktøjer til undersøgelser af betydningen af tumormikromiljøet på metastase og celle respons på terapeutiske midler i 3D 2. Endvidere sammenlignet med 2-dimensionale (2D) cellekulturer 4, 3D-modeller giver mulighed for en bedre forståelse af tumor-stroma-interaktioner, som påvirker celle signalveje. Flercellede tumor sfæroider (MCTSes) af cancer cellelinjer anvendes hyppigt i 3D celle cturprodukter modeller grund af deres relative nærhed til in vivo tumorer. Ud af de mange teknikker i brug, har den flydende-overlay teknik (LOT), i MCTS generation på agarose-belagte plader opnået betydelig interesse for bly optimering og målvalidering 5, 6, 7, 8, 9, 10, 11. Dette fremgår af de seneste undersøgelser, der var held i stand til at køre pilot skærme af sammensatte biblioteker i MCTS kulturer ved hjælp af LOT 6, 7. Men godt-til-brønd variabiliteten i MCTS morfologi og vækst som følge af fordampning-induceret ujævn tab af medium er almindelige forhindringer, der ledsager LOT hjælp mikrotiterplader (parlamentsmedlemmer). Følgelig er dannelsen af ​​uensartet MCTSes kompromitterer betydningen og relevansenaf data fra farmakologiske assays 8, 12, 13. Ud over de reproducerbarhed spørgsmål, en anden praktisk problem, der påvirker LOT-baserede high-throughput assays er coatingen af ​​MP&#39;er med agarose når bruger automatisk væskedispenserende enheder. Selv dispenseringsenheden kan holdes opvarmet for at forhindre gelering af agarose, tilstopning af dispenser kassette og slange er et potentielt problem for robotsystemer 6. For at overvinde nogle af disse udfordringer, har vi for nylig udviklet et par ændringer i partiet for MCTS kultur 8. Disse ændringer er hovedsagelig baseret på mulige måder at forhindre ujævn medium tab fra parlamentsmedlemmer bruger instrumenter, som er almindeligt forekommende i high-throughput screening laboratorier. En detaljeret procedure af den modificerede LOT til generering af ensartet størrelse og reproducerbare MCTSes tværs 384-brønds plader (WPS) præsenteres her. Manuskriptet præsenterer også en semi-automatiseret rutine for evaluering af MCTS størrelse, især i delvis nedbrudt, lægemiddelbehandlede MCTSes, der ikke har en klart defineret grænse for måling af tværsnitsarealet.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9414</td>
			<td>Low-melting</td>
		</tr>
		<tr>
			<td>McCOY&#39;s 5A Medium</td>
			<td>Sigma-Aldrich</td>
			<td>M8403</td>
			<td></td>
		</tr>
		<tr>
			<td>&#8220;rapid&#8221; Filtermax filter</td>
			<td>TPP</td>
			<td>99505</td>
			<td>0.22 &#956;m, 500 mL</td>
		</tr>
		<tr>
			<td>Multidrop&#8482; Combi Reagent Dispenser&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>5840300</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Tube Dispensing cassette&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>24073295</td>
			<td>Metal tip&#160;</td>
		</tr>
		<tr>
			<td>384-well TC plate&#160;</td>
			<td>PerkinElmer</td>
			<td>6057308</td>
			<td>Plate type- CellCarrier</td>
		</tr>
		<tr>
			<td>Standard Tube Dispensing Cassette</td>
			<td>Thermo Fisher Scientific</td>
			<td>24072670</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroClime Environmental Lid</td>
			<td>Labcyte</td>
			<td>LLS-0310</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D4540</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary Incubator (SteriStore )</td>
			<td>HighRes Biosolutions</td>
			<td>23641</td>
			<td>Serial No.: D00384</td>
		</tr>
		<tr>
			<td>Microplate Washer Dispenser&#160;</td>
			<td>BioTek</td>
			<td>Unspecified</td>
			<td>Model: EL406&#160;</td>
		</tr>
		<tr>
			<td>High-Content Imaging System (CellVoyager )</td>
			<td>Yokogawa Electric Corporation</td>
			<td>Unspecified</td>
			<td>Model: CV7000</td>
		</tr>
		<tr>
			<td>Orange G</td>
			<td>New England Biolabs</td>
			<td>B7022S</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE&#8482; Express recombinant cell dissociation reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>12604021</td>
			<td>Phenol red free</td>
		</tr>
	</tbody>
</table>

evaporation-reducing culture condition increases the reproducibility of multicellular spheroid formation in microtiter plates

Tumor modeller, der nøje efterligner in vivo forhold bliver stadig mere populære i lægemiddelforskning og udvikling til screening af potentielle anti-cancer medicin. Multicellulære tumor sfæroider (MCTSes) effektivt efterligne de fysiologiske betingelser i faste tumorer, hvilket gør dem fremragende in vitro modeller for bly optimering og målvalidering. Ud af de forskellige teknikker til rådighed for MCTS kultur, væske-overlay metode på agarose er en af ​​de mest billige metoder til MCTS generation. Imidlertid kan den pålidelige overdragelse af MCTS kulturer under anvendelse væske-overlay for high-throughput screening blive kompromitteret af en række begrænsninger, herunder belægning af mikrotiterplader (MP) med agarose og irreproducibility af ensartet MCTS dannelse tværs brønde. MP&#39;er er betydeligt tilbøjelige til randeffekter, der skyldes for kraftig fordampning af medium fra det ydre af pladen, hvilket forhindrer brugen af ​​hele pladen til lægemiddeltests. Dette håndskrift indeholder detaljerede tekniske forbedringer af væske-overlay teknik til at øge skalerbarheden og reproducerbarhed af ensartet MCTS dannelse. Derudover detaljer om en simpel, semi-automatisk, og universelt anvendelig softwareværktøj til evaluering af MCTS funktioner efter lægemiddelbehandling præsenteres.

Den ujævne tab af medium, især fra perifere brønde, er en ofte stødt problem i parlamentsmedlemmer med små mængder kultur. Væsentligt forbedrede dyrkningsbetingelser, såsom væksthuse med velkontrollerede temperatur / befugtningsanlæg og fordampning-reducerende parlamentsmedlemmer og plade låg, reducere betydeligt tab af medium tværs brønde 8. For at måle den relative fordampning, blev lige store volumener Orange G (OG) til hver brønd, og ændringen ...

Watch this Scientific Journal Video about Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates at JoVE.com

Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates

Den ujævne tab af medium fra mikrotiterplader påvirker reproducerbarheden af ​​ensartet flercellede tumor klumpformet dannelse. Forbedring dyrkningsbetingelser til at reducere væsentlig medium tab vil forbedre reproducerbarheden af ​​sfæroid dannelse og resultaterne af sfæroid assays under anvendelse af flydende-overlay teknik.

Fordampning-reducerende Kultur Betingelse Øger Reproducerbarhed af flercellet Sfæroide Formation i mikrotiterplader

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