Name: improving 2d and 3d skin in vitro models using macromolecular crowding
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Description: Watch this Scientific Journal Video about Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding at JoVE.com

This work was supported by the Biomedical Research Council, Singapore through core support to the Institute of Medical Biology and grant SPF2013/005. M.R. was supported by a NUS Faculty Research Committee Grant (Engineering in Medicine) (M.R.) R-397-000-081-112, and the NUS Tissue Engineering Program (NUSTEP). The authors would like to thank Professor Irene Leigh for providing the collagen VII antibody. Electron microscopy work was carried out at the National University of Singapore Electron Microscopy Unit.

1. makromolekylær gruppering i 2D Skin cellekulturer <ol><li> Seed 50.000 celler (fibroblaster primære eller primære keratinocyter eller ko-kultur av primære fibroblaster og keratinocytter) per brønn i en 24-brønns plate. Seed celler i 1 ml av den tilsvarende celletype vekstmedier. </li><li> La cellene få feste seg over natten i en 37 ° C inkubator med 5% CO2. </li><li> Kast gamle media og erstatte med 1 ml ferske medier som inneholder makromolekylCrowDers og 100 mikrometer askorbinsyre. Bruke en Cr...

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Enhanced extracellular matrix is obtained upon introduction of macromolecular crowders to cell culture, owing to the excluded volume effect which increases the propeptide cleavage by proteinases. This results in extracellular matrix, in particular collagen, to be processed faster and deposited on the culture surface. While other groups have obtained thick fibroblast cell layers, it involved a culture time of several weeks20. In contrast, MMC described in this Protocol, dramatically shortens the culture time wh...

Huden danner en beskyttende barriere ved å hindre vanntap og patogen oppføring. Den består av tre hovedkomponenter; de stromale rike dermis 1, et stratifisert epidermis 2,3,4 på toppen av det, og det Dermo-epidermal junction i mellom 5,6. Dermis består hovedsakelig av kollagen og elastiske fibre, og er tynt befolket med fibroblaster 7. I motsetning til dette er cellerike epidermis sammensatt av flere lag av keratinocytter. Keratinocytter fra den innerste lag er proliferative og gi nye basale celler som fornye og erstatte terminalt differensierende keratinocytter som hele tiden beveger seg til den ytterste lag av huden og har mistet sine kjerner og cytoplasmatisk materiale, noe som resulterer i en forhomede sjikt som undergår avskalling. Den dermale-epidermale knutepunkt, en bestemt type grunnmembranen, er en sammensatt struktur bestående av sammenhengende matriks-molekyler, som TetheRS epidermis til dermis. Collagen I fibre av dermis sammenflettes med kollagen VII forankrings fibriller som er forankret til kollagen IV rik lamina densa. Forankring filamenter (laminin 5, kollagen XVII og inte) i sin tur kobler lamina densa med hemidesmosomes av basale keratinocytter. Basale keratinocytter (basal) har kapasitet til å proliferere og fornye, samt differensiere og stratify å danne suprabasal lagene; stratum spinosum, stratum granulosum, og endelig stratum corneum, som representerer kontakt overflaten av huden med omgivelsene. Underveis fra basallaget til forhomede lag, keratinocytter bytte uttrykk mønstre av cytokeratins og til slutt gjennomgår apoptose og omslutter seg i forhomede konvolutter, skrog av spesifikke proteiner som er kovalent kryssbundet av transglutaminase aktivitet. Gjenskape hud og dens lagene in vitro, inkludert de komplekse strukturene i<html> dermal-epidermal junction og dermal ekstracellulære matrise, og å etterligne horndannelse prosessen, har lenge fascinert forskere og bioingeniører som en utfordrende oppgave. Det har vært betydelige fremskritt innen hud tissue engineering, for eksempel, en vellykket utvinning av hudceller fra pasient biopsier og generering av huden organotypiske kulturer ved hjelp av pasient avledet hudceller 8. Men uløste problemer forbli knyttet til dårlig sekresjon av ekstracellulære matrix proteiner ved hudcellene seg og resulterer i sub-optimale hud modeller. Videre den tiden som kreves for å generere 3D organotypiske hud co-kultur med dagens protokoller varierer mellom fire til åtte uker, en tidsramme som potensielt kan bli forkortet med inkorporering av makromolekylære Crowders. Redusere kultur tide sparer reagens kostnader, reduserer forekomsten av cellen senescence og reduserer ventetiden for pasienten bør preparatet brukes i klinikken. t &quot;&gt; Macromolecular crowding (MMC) involverer å introdusere spesifikke makromolekyler til kulturmediet for å generere utelukkede volumeffekter. Disse påvirker enzymatiske reaksjonshastigheter, inkludert den proteolytiske spaltning av prokollagen som er treg under standard vandige kulturbetingelser 9-13. Under MMC, enzymatiske reaksjoner er ilte uten å øke mengden av reagenser 14,15 resulterer i, i tilfelle av prokollagen spalting, en økt mengde av kollagen i-molekyler i overfylte kulturer sammenlignet med kontroller urørte 10. som omdannelsen av prokollagen til kollagen tillater dannelse av kollagen forsamlinger, fibroblaster dyrket med MMC i 48 timer ga betydelig mer kollagen i forhold til urørte fibroblast kulturer overvåkes i opptil fire uker 11,16,17. Foruten effekter på enzymatiske aktiviteter som påvirker dannelsen, stabilisering og ombygging av ECM, MMC også har vist seg å direkte øke og modulere kollagenfiberdannelse 18,19. Vi presenterer her en metode for å øke den ekstracellulære matriks (ECM) produksjon av hudceller, særlig, dermale fibroblaster og epidermale keratinocytter. I tillegg, viser vi at det anrikede ECM fremstilt under MMC i monolagskulturer kan decellularized og brukes som ren celle-avledet matrise (CDM). Vi bruker en ikke-konvensjonell tilnærming til å visualisere og fullt utbytte av den ECM avsatt av hudceller dyrket med MMC. Interferens refleksjon mikroskopi brukes typisk for å studere celle-matriks-interaksjoner eller celle-til-glass kontaktpunkter. Denne teknikk ble benyttet i systemet for å vise den totale mengden av matrise avsatt på glassoverflaten. Interferens refleksjon mikroskopi ble kombinert med fluorescerende farging for å oppnå den mest mengden av informasjon i form av ekstracellulære matrise sammensetning og mønster, i nærvær og fravær av MMC. Organotypic hud ko-kulturer er en klassisk metode for å modellere hud in vitro i en tre-dimensjonal kontekst. Selv om todimensjonale ko-kulturer kan gi vesentlig informasjon, er det begrenset til å oversette disse data, og å anvende det tilbake til et in vivo miljø, som i seg selv er en tre-dimensjonal struktur. Skin keratinocytter, i særdeleshet, er polarisert og inneholder apikale og basale segmenter som er avgjørende for homeostase og celle etterlevelse. I tillegg er ekspresjonen av typiske suprabasal proteiner i keratinocytter over basallaget, så som keratin 1, keratin 10 og filaggrin bare til stede i løpet av lagdelingen og terminal differensiering av keratinocytter. Som terminal differensiering er nesten ikke til stede i typiske monolagskulturer, er suprabasal protein uttrykk vanligvis ikke oppnås i dette kultursystem. Derfor organotypiske kulturer start neddykket i kulturmedium, men blir deretter løftet til en luft-væske-grenseflate for å drive keratinocyte differensiering. Dette resulterer i ekspresjonen av lagdeling markører, og med horndannelse og en generelt bedre refleksjon av epidermal fysiologi. Mens andre grupper har tidligere generert organotypiske hud co-kulturer med hell, har etableringen av en funksjonell Dermo-epidermal junction sonen vært et problem. Her presenterer vi en ny metode for dyrking organotypiske hud co-kulturer med en forbedret basalmembran, i en kondensert tidsramme og uten at løpetiden på disse konstruksjonene. Dette vil gi huden etterligninger for in vitro modellering, studiet av huden biologi og et utvalg av utvelgelsesassays.

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		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Ascorbic acid</td>
			<td style="width:181px;">Wako</td>
			<td style="width:175px;">013-12061</td>
			<td style="width:499px;">Cell culture media additive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell culture inserts&#160; (6-well format)</td>
			<td style="width:181px;">Greiner Bio-One</td>
			<td style="width:175px;">657610</td>
			<td>Organotypic cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Citrate solution</td>
			<td style="width:181px;">Dako</td>
			<td style="width:175px;">S2369</td>
			<td style="width:499px;">DakoCytomation Target Retrieval Solution Citrate, pH 6 (x10)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Collagen (rat tail)</td>
			<td style="width:181px;">Corning</td>
			<td style="width:175px;">354236</td>
			<td>Organotypic cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">CnT-57&#160;</td>
			<td style="width:181px;">CELLnTEC</td>
			<td style="width:175px;">CnT-57</td>
			<td>Cell culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">DAB Substrate + chromogen kit</td>
			<td style="width:181px;">Dako</td>
			<td style="width:175px;">K3468</td>
			<td>Histology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Deep-well plate (6-well format)</td>
			<td style="width:181px;">Corning</td>
			<td style="width:175px;">355467</td>
			<td>Organotypic cultures</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">ECL detection reagent</td>
			<td style="width:181px;">GE Healthcare Life Sciences</td>
			<td style="width:175px;">RPN2106</td>
			<td>Amersham ECL Western Blotting Detection Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Electron microscope (TEM)</td>
			<td>&#160;JEOL&#160;</td>
			<td>&#160;JEM-1010 (100kV)</td>
			<td>Transmission electron microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Fibroblast media (FM)</td>
			<td></td>
			<td></td>
			<td>High Glucose-DMEM + 10% Fetal Bovine Serum + 1% Penicillin Streptomycin</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Ficoll PM70</td>
			<td style="width:181px;">GE Healthcare Life Sciences</td>
			<td style="width:175px;">17-0310-05</td>
			<td>Macromolecular crowder</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Ficoll PM400</td>
			<td style="width:181px;">GE Healthcare Life Sciences</td>
			<td style="width:175px;">17-0300-05</td>
			<td>Macromolecular crowder</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Keratinocyte serum-free media (KSFM)</td>
			<td style="width:181px;">Life Technologies</td>
			<td style="width:175px;">17005-042</td>
			<td>Cell culture media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Lysis buffer</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">89900</td>
			<td style="width:499px;">Lysis buffer for protein extraction. A protease inhibitor (Roche, #11836170001) was added to this lysis buffer.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Microscope</td>
			<td style="width:181px;">Zeiss</td>
			<td style="width:175px;">LSM510</td>
			<td style="width:499px;">Red, green and blue channels to visualize AF594, AF488 and DAPI fluorescent immunostainings (40X mag).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Mounting media (Hydromount)</td>
			<td style="width:181px;">National Diagnostics</td>
			<td style="width:175px;">HS-106</td>
			<td>Fluorescent staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Mounting media (Cytoseal)</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">8310-16</td>
			<td>Histology (HRP)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">OCT compound (Tissue Tek)</td>
			<td style="width:181px;">Sakura</td>
			<td style="width:175px;">4583</td>
			<td>Embedding for cryotomy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Penicillin-streptomycin antibiotics</td>
			<td style="width:181px;">Sigma Aldrich</td>
			<td style="width:175px;">A5955</td>
			<td>Cell culture media additive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Primary antibodies</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Anti-Collagen I antibody</td>
			<td style="width:181px;">Abcam</td>
			<td style="width:175px;">#ab6308</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Anti-Collagen Type IV</td>
			<td style="width:181px;">Novocastra</td>
			<td style="width:175px;">#NCL-COLL-IV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Anti-collagen VII</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>LH7.2 (in house)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Anti-Fibronectin antibody</td>
			<td style="width:181px;">Abcam</td>
			<td style="width:175px;">#ab2413</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Reducing agent&#160;</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">NP0009</td>
			<td>Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Sample buffer</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">NP0008</td>
			<td>Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Secondary antibodies (Immunostaining)</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">4&#8242;,6-Diamidino-2-phenylindole dihydrochloride (DAPI)</td>
			<td style="width:181px;">Sigma Aldrich</td>
			<td style="width:175px;">#D9542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">AlexaFluor 594 goat-anti-rabbit</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">#A-11037&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">AlexaFluor 594 goat-anti-mouse</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">&#160;#A-11005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">AlexaFluor 488 chicken-anti-rabbit</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">#A-21441</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">AlexaFluor 488 goat-anti-mouse</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">#A-11001</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:299px;">Secondary antibodies (HRP)</td>
			<td style="width:181px;">Dako</td>
			<td style="width:175px;">Envision + System - HRP Labelled Polymer Anti-Rabbit and Anti-Mouse</td>
			<td>undiluted</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Sodium deoxycholate</td>
			<td style="width:181px;">Prodotti Chimicie Alimentari</td>
			<td style="width:175px;"></td>
			<td>Decellularization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Stratification media</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Dulbecco&#39;s Modified Eagle&#39;s Medium&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Ham&#8217;s F12&#160;&#160;&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">10% fetal bovine serum&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">100U/mL penicillin and 100U/mL streptomycin&#160;&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">0.4mg/mL hydrocortisone&#160;&#160;&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">5mg/mL insulin</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">1.8&#183;10-4 M adenine&#160;&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">5mg/mL transferrin&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">2&#183;10- 11 M triiodothyronine&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Trypsin</td>
			<td style="width:181px;">Biopolis Shared Facilities</td>
			<td style="width:175px;">0.125% Trypsin/Versene&#160;&#160; pH 7.0 + 0.3&#160;</td>
			<td>Used to trypsinize cells</td>
		</tr>
	</tbody>
</table>

improving 2d and 3d skin in vitro models using macromolecular crowding

Den glykoprotein familien av kollagener representerer de viktigste strukturelle proteiner i menneskekroppen, og er viktige komponenter i biomaterialer som anvendes i moderne prosjektering vev. En teknisk flaskehals er avsetningen av kollagen in vitro, som det er svært langsom, noe som resulterer i sub-optimal dannelse av bindevev og kohesjon påfølgende vev, særlig i huden modeller. Her beskriver vi en fremgangsmåte som innebærer tilsetning av forskjellig størrelse sukrose kopolymerer til huden kulturer for å generere makromolekylære trenging (MMC), noe som resulterer i en dramatisk forbedring av kollagen avsetning. Spesielt, dermale fibroblaster avsatt en vesentlig mengde av kollagen I / IV / VII og fibronektin i henhold til MMC i forhold til kontrollene. Protokollen beskriver også en fremgangsmåte for å decellularize fylt cellelag, utsette betydelige mengder av ekstracellulær matriks (ECM) som ble holdt tilbake på kulturoverflaten som vist ved immunocytochemistry. Total matrise masse og distribusjon mønsteret ble undersøkt ved hjelp av forstyrrelser refleksjon mikroskopi. Interessant, fibroblaster, keratinocytter og ko-kulturer produserte celle-stammer matriser (CDM) med varierende sammensetning og morfologi. CDM kan brukes som &quot;Bio-stillas&quot; for sekundær cellen såing, hvor den nåværende bruk av belegg eller stillaser, typisk fra xenogene animalske kilder, kan unngås, og dermed beveger seg i retning av mer klinisk relevante anvendelser. I tillegg beskriver denne protokollen anvendelsen av MMC under den neddykkede fase av en 3D-organotypiske hud ko-kulturmodell som var tilstrekkelig til å forbedre ECM deponering i den dermo-epidermale knutepunkt (DEJ), særlig kollagen VII, hovedkomponenten av forankring fibriller. Elektronmikroskopi bekreftet tilstedeværelsen av forankrings fibriller i kulturer utviklet med MMC, sammenlignet med kontroller. Dette er viktig som forankring fibriller tjore dermis til epidermis, dermedå ha en pre-formet moden DEJ kan ha nytte hud pode mottakere i form av pode stabilitet og generell sårtilheling. Videre ble kulturen tid kondensert fra 5 uker til 3 uker for å oppnå en moden konstruksjon, ved bruk av MMC, redusere kostnader.

Makromolekylært trenging var i stand til å forbedre ECM avsetning, særlig fibroblaster avsatt mer kollagen I, IV og fibronektin sammenlignet med kontrollkulturer (fig 1, Cell lag, 1A, collagen I, 1B, kollagen IV; 1C, fibronektin). Ved decellularization, var det tydelig at fibroblaster var hoved innskytere av kollagen I, IV og fibronektin sammenlignet med keratinocytter (figur 1, Matrix). <p class=...

Watch this Scientific Journal Video about Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding at JoVE.com

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

Vi presenterer en protokoll for å oppnå celle-stammer matriser rik på ekstracellulære matrix proteiner, ved hjelp av makromolekylære Crowders (MMC). I tillegg presenterer vi en protokoll som inkorporerer MMC i 3D organotypiske hud co-kultur generasjon, noe som reduserer kultur tid samtidig modenhet konstruksjon.

Forbedre 2D- og 3D-Skin<em&gt; In Vitro</em&gt; Modeller Bruke makromolekylær gruppering

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

Research

JoVE Journal

Bioengineering

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

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