Name: generation and grafting of tissue-engineered vessels in a mouse model
Uploaded: 2015-03-18T14:00:00
Description: Watch this Scientific Journal Video about Generation and Grafting of Tissue-engineered Vessels in a Mouse Model at JoVE.com

This work was supported by The British Heart Foundation and The Oak Foundation.

Voer alle dierproeven volgens de protocollen door de Institutional Comité voor gebruik en onderhoud van proefdieren goedgekeurd. 1. Voorbereiding van Cultuur Media <ol><li> Maak kweekmedia voor humane fibroblast cellijn CCL-153: F-12K Medium, 10% foetaal runderserum (FBS) en 100 U / ml penicilline en streptomycine. </li><li> Maak herprogrammeren Media voor PiPSC generatie Knockout Dulbecco&#39;s gemodificeerd Eagle&#39;s medium (DMEM) dat 20% Knockout Serum vervanging, 0,1 mM β-mercaptoethanol, 0,1 mM Minimu...

<ol>
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Het huidige protocol geeft een goed, snel, eenvoudig, efficiënt en reproduceerbaar strategie waarbij functionele tissue engineered schepen kunnen worden gegenereerd met behulp PiPSC uit humane fibroblasten. Deze techniek is een waardevol instrument voor de regeneratieve geneeskunde, tissue engineering en potentieel patiënt-specifieke celtherapie in de nabije toekomst. Kritische stappen om de effectiviteit van het protocol omvatten de bereiding van PiPSC, bereiding van steriele en volledig ontcelde aortische transplant...

De bouw van vasculaire leidingen is een fundamentele strategie voor chirurgische reparatie van beschadigde en gewonden schepen als gevolg van hart- en vaatziekten. Tot op heden, graft materialen die worden gebruikt in de chirurgie omvatten biocompatibele synthetische polymeren (polytetrafluoretheen [Teflon], geëxpandeerd polytetrafluorethyleen [ePTFE; Gore-Tex] of polyethyleentereftalaat [Dacron]), allogene, autoloog weefsel (pericardium of vene) en xenograften 1. Terwijl kunstmatige transplantaten (bv Gore-Tex en Dacron) worden het meest gebruikt, deze materialen waarschijnlijk leiden tot tal van korte en lange termijn complicaties die stenose, calcium afzetting, trombo-embolisatie en infecties omvatten. Hoewel patiënten met biologische enten aanwezig met verminderde trombo-embolische gebeurtenissen, ze nog steeds beperkingen tegenkomen zoals secundaire transplantaat falen en een kortere levensduur als gevolg van verkalking degradatie 2. Daarom, ondanks aanzienlijke verbeteringen in chirurgische techniques de jaren, onderzoekers en clinici nog belast met het nodig zijn de ideale geleider voor vaatziekten. Meer recent heeft het onderzoeksveld van de vasculaire tissue engineering een concept waarbij cellen worden opgenomen in biologisch afbreekbare steigers, met als doel het creëren van een biomimetische omgeving die een functioneel schip voor succesvolle enting 1 belichaamt gegenereerd. Fundamenteel, het succes van de vasculaire constructen afhankelijk drie essentiële componenten; cellen die de steiger, dwz een endotheliale binnenlaag en een gladde spiercel laag, een scaffold dat het geschikte extracellulaire matrix mechanische eigenschappen vergelijkbaar met de natieve vasculatuur verschaffen, en de moleculaire / cellulaire signalering die nodig is voor het initiëren / regulerend omvatten reparatie. Lange termijn patency en duurzame ontwikkeling van de neo-weefsels sterk afhankelijk effectieve zaaien van cellen van steigers, thereby waardoor de beslissing van celtype van cruciaal belang. Verscheidene rapporten tonen het gebruik van volwassen endotheliale en gladde spiercellen uit diverse bronnen kleine diameter leidingen 3-6 ontwikkelen. Hoewel veelbelovend, het ontbreken van voldoende autologe schepen te verkrijgen mature endotheelcellen en gladde spiercellen blijven een aanzienlijke last. Recenter zijn stamcellen uit diverse bronnen zijn gebruikt voor vaatweefsel technische toepassingen. Inderdaad, verschillende types stamcellen waaronder embryonale stamcellen 7, geïnduceerde pluripotente stamcellen (iPSCs) 8,9, PiPSC 10,11, beenmerg afgeleide mononucleaire cellen 12, mesenchymale stamcellen 13, endotheliale progenitor cellen en wand volwassen vat -afgeleide stamcel antigeen-1 (Sca-1) + stam / progenitorcellen 14,15 zijn allemaal aangetoond kan differentiatie worden in ofwel functioneel endotheel of gladde spiercellen in reactie op gedefinieerde media enkweekomstandigheden. Bovendien is de onbeperkte zelfvernieuwing capaciteit van de stamcellen ze betere kandidaten tegenstelling volwassen endotheliale en gladde spiercellen die slechts delen een eindig aantal malen ondergaan groeistop en afsterven. De keuze van dragermateriaal succesvol tissue engineered verblijf genereren voor enten afhankelijk van verschillende factoren zoals biocompatibiliteit, biomechanische eigenschappen en biologische afbraaksnelheden. Fundamenteel, materialen voor scaffolds voor het enten creëren biologisch afbreekbaar zijn en mogen geen onnodige ontvanger immuunreacties monteren. Bovendien moet een geschikte porositeit en microstructuur voor celhechting en daaropvolgende overleving omvatten. Tot op heden de meest voorkomende materialen voor steigers vaatweefsel techniek omvatten polymeren van polyglycolzuur, polymelkzuur, en poly ε-caprolacton 16. Recenter cellen ontdaan biologische materialenook toegepast met enig succes. Verschillende laboratoria hebben aangetoond dat het zaaien gedecellulariseerde mens, honden of varkens schepen met autologe cellen voorzien van een biologische graft die verzette stolling en intima hyperplasie 17-19. Andere strategieën in vasculaire tissue engineering omvatten extracellulaire matrix eiwitten gebaseerde vaattransplantaten bv, zaaien cellen in fibrine gel 13 en het genereren cel vellen zonder steiger steun 20, 21. Het huidige protocol toont de differentiatie van humane PiPSC in functionele endotheel- en gladde spiercellen, het genereren van een bioreactor die bestaat uit een gedecellulariseerde vaartuig scaffold functionele PiPSC afgeleide vasculaire cellen haven en enten van de tissue engineered vaartuigen in ernstige gecombineerde immunodeficiëntie (SCID ) muizen. PiPSC zijn een optimale celtype te gebruiken voor tissue engineering van het schip enten omdat deze cellen niet tumoren bij muizen te vormen of te verhogen ethische enallo- immuunresponsen. Verder hebben we aangetoond dat de strategie voor het genereren PiPS-endotheelcellen en PiPS gladde spiercellen efficiënt en reproduceerbaar 10,11. Daarna hebben we een gedecellulariseerde vaartuig voor het zaaien van PiPSC-afgeleide vasculaire cellen naar de matrix eiwitten die binnen een native vat bestaat na te bootsen, waardoor het verbeteren van enten en overleving werkzaamheid. Bovendien, de cellen ontdoen van de bloedvaten vóór PiPSC enten voorkomt het optreden van ontstekingsreacties gemonteerd immuunceltypen zoals macrofagen. Bovendien houdt dit protocol vormen niet alleen een methode voor het genereren veelbelovende vasculaire leidingen voor translatie in mensen, maar ook waardevol middel van het bestuderen en begrijpen van de moleculaire mechanismen die vasculair weefselregeneratie regeren door muismodellen.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Human Fibroblasts CCL-153</td>
			<td >ATCC</td>
			<td >CCL-153</td>
			<td>Prenatal human embryonic fibroblasts</td>
		</tr>
		<tr >
			<td >ATCC F-12K Medium (Kaighn&#39;s Modification of Ham&#39;s F-12 Medium)</td>
			<td >ATCC</td>
			<td >30-2004</td>
			<td></td>
		</tr>
		<tr >
			<td >Fetal Bovine Serum</td>
			<td >ATCC</td>
			<td >30-2020</td>
			<td></td>
		</tr>
		<tr >
			<td >Knockout DMEM medium optimized for embryonic stem cells</td>
			<td >Life technologies (Gibco)</td>
			<td >12660-012</td>
			<td></td>
		</tr>
		<tr >
			<td >Knockout Serum Replacement</td>
			<td >Life technologies (Invitrogen)</td>
			<td >10828-028</td>
			<td></td>
		</tr>
		<tr >
			<td >Human Basic FGF-2&#160;</td>
			<td >Miltenyi Biotech</td>
			<td >130-093-837</td>
			<td></td>
		</tr>
		<tr >
			<td >alpha-MEM medium</td>
			<td >Life technologies (Invitrogen)</td>
			<td >32571093</td>
			<td></td>
		</tr>
		<tr >
			<td >Human PDGF</td>
			<td >R&#38;D System</td>
			<td >120-HD-001</td>
			<td></td>
		</tr>
		<tr >
			<td >Gelatin Solution 2%</td>
			<td >Sigma</td>
			<td >G1393</td>
			<td></td>
		</tr>
		<tr >
			<td >Plasmid 20866: pCAG2LMKOSimO (SOX2, OCT4, KLF4, C-MYC)</td>
			<td >Addgene</td>
			<td >20866</td>
			<td></td>
		</tr>
		<tr >
			<td >&#160;PvuI Restriction Enzyme</td>
			<td >New England Biolabs</td>
			<td >RO150S</td>
			<td></td>
		</tr>
		<tr >
			<td >SureClean Plus</td>
			<td >Bioline</td>
			<td >BIO-37047</td>
			<td></td>
		</tr>
		<tr >
			<td >Nucelofection Kit (NHDF Kit)</td>
			<td >LONZA</td>
			<td >VPD-1001</td>
			<td></td>
		</tr>
		<tr >
			<td >Neomycin</td>
			<td >SIGMA</td>
			<td >G418</td>
			<td>Selection of&#160;</td>
		</tr>
		<tr >
			<td >KL 1500 LCD, Illumination for Stereo Microscopy</td>
			<td >SCHOTT</td>
			<td >KL 1500 LCD</td>
			<td>Cold light illumination for stereo microscopy</td>
		</tr>
		<tr >
			<td >Nikon Zoom Steromicroscope SMZ800</td>
			<td >Nikon</td>
			<td >SMZ800</td>
			<td></td>
		</tr>
		<tr >
			<td >Heparin sodium salt</td>
			<td >Sigma</td>
			<td >H3393</td>
			<td></td>
		</tr>
		<tr >
			<td >10% SDS Stock Solution Molecular Biology Reagent</td>
			<td >Severn Biotech</td>
			<td >CAS 151-21-3</td>
			<td></td>
		</tr>
		<tr >
			<td >Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td >Sigma</td>
			<td >D8537</td>
			<td></td>
		</tr>
		<tr >
			<td >Matrigel (10mg/ml)</td>
			<td >BD</td>
			<td >A6661</td>
			<td></td>
		</tr>
		<tr >
			<td >Shaker IKA Vibrax with Shaking platform VX 7&#160;</td>
			<td >Jepson Bolton&#39;s, Janke&#38;Kunkel</td>
			<td >S32-102</td>
			<td></td>
		</tr>
		<tr >
			<td >Masterflex L/S Digital Pump Drive</td>
			<td >Cole-Parmer</td>
			<td >WZ-07523-80</td>
			<td></td>
		</tr>
		<tr >
			<td >Masterflex L/S 6-channel, 6-roller cartridge pump head</td>
			<td >Cole-Parmer</td>
			<td >EW-07519-15</td>
			<td></td>
		</tr>
		<tr >
			<td >Masterflex L/S large cartridges for pump head</td>
			<td >Cole-Parmer</td>
			<td >EW-07519-75</td>
			<td></td>
		</tr>
		<tr >
			<td >Masterflex platinum-cured silicone pump tubing, L/S 14, 25 ft</td>
			<td >Cole-Parmer&#160;</td>
			<td >WZ-96410-14</td>
			<td >Tubing goes through the peristaltic pump</td>
		</tr>
		<tr >
			<td >0.5mm ID, 0.8 mm OD Silicone Tubing</td>
			<td >SILEX</td>
			<td >N/A</td>
			<td>Tubings connect incubation chamber, media reservoir and compliance chamber&#160;</td>
		</tr>
		<tr >
			<td >Fitting Reducer 0.5 to 1.6, natural Polypropyline</td>
			<td >Ibidi</td>
			<td >10829</td>
			<td>Adapter connect above two types of tubings</td>
		</tr>
		<tr >
			<td >1/32&#34; Tubing, ID 0.01&#34; (250&#181;m) Material: PEEK</td>
			<td >LabSmith</td>
			<td >T-132-010P</td>
			<td>Tubing through the incubation chamber wall which connects the graft with outside tubing</td>
		</tr>
		<tr >
			<td >One-Piece Fittings&#160;</td>
			<td >LabSmith</td>
			<td >T-132-100</td>
			<td>Fix the above tubings through the incubation chamber wall</td>
		</tr>
		<tr >
			<td >Nylon tubes (OD 0.9mm, ID 0.75mm)&#160;</td>
			<td >Smiths Medical</td>
			<td >N/A</td>
			<td>Tubings insert into two ends of the aorta graft</td>
		</tr>
		<tr >
			<td >NOD.CB17-Prkdcscid/NcrCrl mouse</td>
			<td >Charles River</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Surgical sutures, 8-0&#160; silk</td>
			<td >ETHICON</td>
			<td >W819</td>
			<td ></td>
		</tr>
		<tr >
			<td >Hypnorm</td>
			<td >Vetapharm</td>
			<td >Vm21757/4000</td>
			<td >Neuroleptanalgesic for use in mice</td>
		</tr>
		<tr >
			<td >Hypnovel (Midazolam)</td>
			<td >Roche</td>
			<td >59467-70-8</td>
			<td >Induction of anaesthesia</td>
		</tr>
		<tr >
			<td >Dissecting microscope</td>
			<td >Carl Zeiss</td>
			<td >Stemi 2000</td>
			<td ></td>
		</tr>
		<tr >
			<td >Nylon Tubing</td>
			<td >Portex LTD</td>
			<td >800/200/100/200</td>
			<td >0.65 mm in diameter and 1 mm in length; to make artery cuff</td>
		</tr>
		<tr >
			<td >Electrocoagulator</td>
			<td >Martin&#160;</td>
			<td >SN 54.131</td>
			<td >Ligation of artery branches on aorta</td>
		</tr>
		<tr >
			<td >Bipolar micro hemostat forceps</td>
			<td >Martin</td>
			<td >80-91-12-04</td>
			<td >Fixation of vessel ends</td>
		</tr>
		<tr >
			<td >Vessel Dilator</td>
			<td >S&#38;T</td>
			<td >JFX-7</td>
			<td ></td>
		</tr>
		<tr >
			<td >Vessel Dilator</td>
			<td >S&#38;T</td>
			<td >JFL-3dZ</td>
			<td ></td>
		</tr>
		<tr >
			<td >Vessel Dilator</td>
			<td >S&#38;T</td>
			<td >D-5aZ</td>
			<td ></td>
		</tr>
		<tr >
			<td >Mini applier&#160;</td>
			<td >AESCULAP</td>
			<td >FE572K</td>
			<td ></td>
		</tr>
		<tr >
			<td >Micro hemostats clips</td>
			<td >AESCULAP</td>
			<td >FE720K</td>
			<td ></td>
		</tr>
		<tr >
			<td >Surgical sutures, 6-0 VICRYL</td>
			<td >ETHICON</td>
			<td >V489</td>
			<td ></td>
		</tr>
	</tbody>
</table>

generation and grafting of tissue-engineered vessels in a mouse model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

De succesvolle generatie PiPSC werd 4 dagen bevestigd na nucleofecting humane fibroblasten met een gelineariseerde pCAG2LMKOSimO plasmide dat 4 transcriptiefactoren, OCT4, SOX2, KLF4 en c-MYC (OSKM). PiPSC vertoonde een duidelijk onderscheiden fenotype in vergelijking met fibroblasten (Figuur 2A) en uitgedrukt 4 herprogrammering factoren mRNA (Figuur 2B) en eiwit (Figuur 2C) niveau 10. De werkzaamheid van een PiPSC gebaseerde vasculaire transplantaat is sterk...

Watch this Scientific Journal Video about Generation and Grafting of Tissue-engineered Vessels in a Mouse Model at JoVE.com

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

Hier presenteren we een protocol om tissue engineered vaartuig enten die functioneel zijn voor het enten in muizen door dubbel zaaien gedeeltelijk geïnduceerde pluripotente stamcellen genereren (PiPSC) - afgeleide gladde spiercellen en PiPSC - afgeleide endotheelcellen op een gedecellulariseerde schip steiger bioreactor.

Generatie en enten van-Weefselmanipulatieproducten Schepen in een muismodel

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Research

JoVE Journal

Bioengineering

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Protocol voor Relatieve Hydrodynamische Beoordeling van Tri-folder Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

Voorbereiding van de Light-responsieve membranen door een gecombineerde oppervlakte-enting en Postmodification Proces

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is ...

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

Chirurgische techniek voor de inplanting van Tissue Engineered vaattransplantaten en de daaropvolgende<em&gt; In Vivo</em&gt; Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

Genereren van een drie-dimensionale volledige dikte huidequivalent en Automated Verwonden

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

Productie, karakterisering en toepassingsmogelijkheden van een 3D Tissue-engineered Human Slokdarmkanker Mucosale Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

Een gecombineerde 3D Weefselmanipulatieproducten<em&gt; In Vitro</em&gt; /<em&gt; In Silico</em&gt; Lung Tumor Model voor het voorspellen van de Drug Effectiveness in Specifieke Mutatie Achtergronden

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

toepassingen van<em&gt; In Vivo</em&gt; Functioneel testen van de Rat tibialis anterior voor de evaluatie van Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Drie-dimensionale weefsel ontworpen uitgelijnd Astrocyt netwerken vergemakkelijken zenuwstelsel regeneratie te recapituleren Developmental mechanismen

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Een nieuwe tenorrfaphy hechttechniek met weefselmanipulatie collageentransplantaat om grote peesdefecten te repareren

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Zaaien en implantatie van een Biosynthetic weefsel-engineered tracheale Graft in een muismodel

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...