Siamo grati per l&#39;eccellente assistenza tecnica di Janine Becker, Caroline Bickelmann e Ruth Nickels. Questo studio è stato finanziato da una sovvenzione della Deutsche Forschungsgemeinschaft (DFG - German Research Foundation) - LA 2682 / 7-1.

Tutte le procedure sono state eseguite secondo l&#39;Istituto Nazionale di Salute linee guida per l&#39;uso di animali da laboratorio e hanno seguito le linee guida istituzionali (Landesamt für Soziales, Gesundheit und Verbraucherschutz, Abt. Lebensmittel- und di veterinaria, Zentralstelle, Saarbrücken, Germania). 1. Preparazione di strumenti chirurgici <ol><li> Tenere pronte le forbici dissezione, pinze chirurgiche, forbici preparazione piccole pinza sottile e una piastra di Petri sterile con terreno Eagle modificato da 15 mL Dulbecco (DMEM, 10% siero di vitello fetale (FCS), 100 U / ml di penicillina, 0,1 mg / mL di streptomicina ) per la raccolta cuscinetti di grasso dell&#39;epididimo. </li><li> Esporre gli strumenti chirurgici per una soluzione disinfettante per 5 min. In alternativa, sterilizzarli (sterilizzazione a vapore; 121 ° C, 20 min). </li></ol> 2. Gli animali e l&#39;anestesia <ol><li> Scegli con attenzione il ceppo di topi come indicato per lo studio e la<html> soggetto sotto indagine. NOTA: In questo studio abbiamo utilizzato maschio wild-type C57BL 6 topi / come donatori per la raccolta dei grassi dell&#39;epididimo. Di interesse, ad-DMV può essere isolato anche da transgenici proteina fluorescente verde (GFP) animali donatori -positive (C57BL / 6-Tg (CAG-EGFP) 1Osb / J) 22. Questo porta il grande vantaggio che i frammenti sono facilmente individuabili dalla colorazione immunoistochimica di GFP dopo l&#39;impianto in topi wild-type GFP-negativi 14. </li><li> Anestetizzare gli animali con un&#39;iniezione intraperitoneale di xilazina (15 mg / kg) e ketamina (75 mg / kg). Assicurarsi che gli animali sono profondamente anestetizzati eseguendo un pizzico piedi con nessuna risposta. lubrificante occhio non è indicata come animali donatori vengono sacrificati dopo la raccolta grasso. NOTA: Assicurarsi che l&#39;analgesia e la sterilità chirurgica sono in accordo con le rispettive linee guida del paese e istituzione in cui sono previsti gli esperimenti. </li></ol>Le &quot;&gt; 3. La raccolta delle epididimali Fat Pads <ol><li> Trasferire l&#39;animale ad un tavolo operatorio. Posto l&#39;animale in posizione supina allo stereomicroscopio chirurgica e confermare anestesia profonda con punta pizzico. </li><li> Immobilizzare lo zampe da loro nastratura ad un telo chirurgico e disinfettare l&#39;addome con soluzione disinfettante. </li><li> Separare la pelle addominale libera dello strato muscolare sottostante con le forbici dissezione. </li><li> Eseguire una laparotomia mediana con le forbici dissezione e lateralmente spiegare i lembi della parete addominale. </li><li> Bilateralmente identificare testicolo, epididimo e il cuscinetto adiposo dell&#39;epididimo base alle pinza sottile (Figura 1). Non danneggiare le strutture intestinali per evitare la contaminazione fecale delle cuscinetti adiposi. </li><li> Raccogliere i cuscinetti adiposi dell&#39;epididimo con le forbici piccole di preparazione e le pinza sottile allo stereomicroscopio. Mantenere un margine di sicurezza di diversi mm tra l&#39;epididimo e il grasso perridurre il rischio di accidentali raccolta dell&#39;epididimo. NOTA: Questa procedura può essere eseguita anche senza uno stereomicroscopio. Tuttavia, questo può aumentare ulteriormente il rischio di lesioni accidentali dell&#39;epididimo e corde spermatiche. </li><li> Trasferire i cuscinetti adiposi dell&#39;epididimo in una capsula Petri contenente 15 ml di DMEM preriscaldato a 37 ° C per il trasporto al laboratorio cella. </li><li> Sacrificare l&#39;animale tramite incisione dell&#39;aorta addominale o dislocazione cervicale. </li></ol> 4. Isolamento di ad-MVF <ol><li> Preparare tre piastre sterili con 15 ml di soluzione salina tamponata con fosfato (PBS), provette sterili di polipropilene da 14 ml (PP), una beuta da 50 ml sterile, sterili provette coniche microcentrifuga da 1,5 ml e forbici sottili sterilizzati. </li><li> Lavare i cuscinetti adiposi tre volte in piastre Petri con 15 ml di PBS sotto una cappa a flusso laminare. </li><li> Trasferire il grasso in un tubo da 14 ml in PP. Determinare il volume del tessuto adiposo raccolto (in mL) per mezzo di tHe scala tubo. Tritare il tessuto grasso meccanicamente con fini forbici fino ad ottenere una sospensione omogenea tessuto. </li><li> Trasferire il tessuto tritato con due volumi di collagenasi NB4G (0,5 U / mL PBS) in un pallone da 50 mL beuta con 10 mL di misurazione pipetta. Il volume totale diventa tre volte il volume del tessuto grasso, determinato nel passo 4.3. Eseguire digestione tissutale in un incubatore per 10 min sotto vigorosa agitazione mediante un agitatore automatico (dimensione di ancoretta magnetica: 25 mm) a 37 ° C e le condizioni atmosferiche umidificati con 5% di CO 2. </li><li> Osservare una piccola frazione (10 ml) del tessuto digerito sotto un microscopio per giudicare se la digestione può essere fermato. Accertarsi che il digestato contiene principalmente &quot;libero&quot; ad-DMV accanto a singole cellule, che indica il punto appropriato per arrestare il processo di digestione enzimatica (Figura 2). NOTA: Questo passaggio richiede esperienza con la procedura ed è fondamentale per la qualitàdi isolato ad-MVF. Prolungato risultati digestione dei grassi in una cella singola sospensione senza ad-DMV. </li><li> Neutralizzare dell&#39;enzima con due volumi di PBS / FCS 20%. Il volume totale diventa tre volte il volume della sospensione cellulare vasi nel passaggio 4.4. Trasferire la sospensione cellulare-nave di nuovo in nuovi tubi PP. </li><li> Incubare la sospensione per 5 min a 37 ° C per separare ad-DMV rimanga grasso per gravità. Quindi rimuovere con attenzione il surnatante grasso principale con una precisione pipetta da 1 ml. Ripetere questo ciclo più volte con una pipetta di precisione da 100 ml fino a quando la sospensione sembra essere priva di grassi. </li><li> Mettere un filtro da 500 micron in cima ad una provetta da centrifuga conica da 50 ml. Trasferire la sospensione cellulare vasi con una pipetta di misurazione 10 mL dai tubi 14-mL PP sulla membrana filtrante per rimuovere i coaguli residuo grasso. Trasferire la sospensione filtrata in nuove provette da 14 mL PP secondo il numero dei singoli ad-DMV isolati per gli esperimenti pianificati. NOTA: Per in Vitro saggi incentrati sulla formazione rete microvascolare, può essere utile per purificare ulteriormente la sospensione cellulare vasi per migliorare la qualità delle immagini durante analisi al microscopio. A tale scopo, la sospensione può essere ulteriormente filtrata una volta con un filtro da 20 micron per rimuovere le cellule singole dal ad-DMV raccolti sul filtro. </li><li> Centrifugare la sospensione cellulare cardiovascolare (600 xg, 5 min, temperatura ambiente) per ottenere un pellet contenente ad-DMV. </li><li> Dopo la centrifugazione, rimuovere il surnatante fino 1 mL viene lasciata. Risospendere il pellet con ad-DMV in 1 ml e trasferire la sospensione in una provetta conica da 1,5 mL. </li><li> Centrifugare la sospensione ad-DMV nella provetta (600 xg, 5 min) per ottenere un pellet. </li><li> Rimuovere il surnatante per risospendere il pellet nel volume finale richiesto di PBS / FCS 20%. NOTA: Il numero di ad-DMV per isolare può essere valutata mediante conteggio al microscopio. A tale scopo, 1/10 della cellula-vesse finalel sospensione viene diluita 1:10 in PBS e 100 pl di questa diluizione vengono trasferiti in un pozzetto di una piastra a 96 pozzetti. L&#39;intero numero di ad-DMV presentante una morfologia a recipiente viene contato e estrapolato a tutta isolare. La concentrazione ad-MVF richiesto e la purificazione possono essere adattati individualmente al rispettivo in vitro o in vivo applicazione dell&#39;annuncio-DMV. Ciò può includere l&#39;incorporamento di annuncio-DMV in gel di collagene per l&#39;analisi in vitro di formazione rete microvascolare o la semina di ad-DMV su scaffold per l&#39;impianto in vivo in difetti del tessuto. </li></ol>

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Gli autori dichiarano di non avere interessi finanziari in competizione.

In questo studio vi presentiamo un protocollo consolidata per l&#39;isolamento di ad-MVF. Ottenere ad-MVF dal tessuto adiposo murino è una procedura semplice con pochi passaggi critici. I topi mostrano sottocutaneo diverso e depositi di grasso intra-addominale. Come precedentemente descritto per ratti, la fonte di grassi più adatto per l&#39;isolamento di annuncio-DMV sono i cuscinetti adiposi dell&#39;epididimo causa delle loro dimensioni, struttura omogenea e poco contaminate con vasi sanguigni più grandi <sup clas...

Ingegneria tissutale si concentra sulla fabbricazione di tessuti e organi succedanei mantenere, ripristinare o aumentare la funzione di inutilizzabile in vivo controparti 1, 2. Il destino di costrutti di tessuto ingegnerizzati dipende essenzialmente una vascolarizzazione adeguata 3. Reti microvascolari all&#39;interno di questi costrutti dovrebbero essere organizzati gerarchicamente con arteriole, capillari e venule per consentire efficiente perfusione sanguigna dopo inosculation alla vascolarizzazione del destinatario 4. La generazione di queste reti è tra le principali sfide nel campo dell&#39;ingegneria dei tessuti. A tale scopo, un ampio spettro di strategie di vascolarizzazione sperimentali è stata introdotta nel corso degli ultimi due decenni 5, 6. approcci angiogenici stimolano la crescita interna dei microvasi dei destinatari nel tiss ingegnerizzatiues mediante modificazione ponteggio strutturale o fisico-chimiche, come ad esempio l&#39;incorporazione di fattori di crescita 7. Tuttavia, per la vascolarizzazione di grandi costrutti tridimensionali, le strategie di angiogenesi-dipendenti sono notevolmente limitate da tassi di crescita lenta di sviluppo di microvasi 8. Al contrario, il concetto di prevascularization mira per la generazione di reti microvascolari funzionali all&#39;interno del tessuto costruisce prima del loro impianto 9. Prevascularization convenzionale comporta la co-coltura delle cellule del vaso che formano, come le cellule endoteliali, cellule murali o cellule staminali 10, all&#39;interno ponteggi. Dopo la formazione della rete microvascolare, i costrutti prevascularized possono essere impiantati in difetti del tessuto. Degno di nota, questo approccio prevascularization è di difficile applicazione in un ambiente clinico, perché si basa sulla complessa e richiede molto tempo in vitro &lt;/ em&gt; le procedure, che sono limitati dai maggiori ostacoli normativi 9. Di conseguenza, v&#39;è ancora una necessità per lo sviluppo di strategie prevascularization nuovi che sono più adatti per un&#39;ampia applicazione clinica. Tale strategia prevascularization può essere l&#39;applicazione di frammenti di tessuto adiposo derivate microvascolari (ad-MVF). ad-MVF rappresentano le unità di vascolarizzazione potenti che possono essere raccolti in grandi quantità dal tessuto adiposo dei ratti 11, 12 e 13 topi. Sono costituiti da arteriolare, capillare e segmenti di flotta venulari, che presentano una morfologia microrecipiente fisiologico con un lume e stabilizzanti cellule perivascolari 14, 15. Questa caratteristica unica permette l&#39;inserimento immediato di ponteggi ad-MVF-seminato in difetti del tessuto, senza precoltura. C&#39;è, l&#39;annuncio-DMV rapidamente rimontare inalle reti microvascolari funzionali. Inoltre, ad-DMV rappresentano una ricca fonte di cellule staminali mesenchimali 16, che possono inoltre contribuire alla loro capacità rigenerativa sorprendente. Di conseguenza, ad-DMV sono sempre più utilizzati in diversi campi dell&#39;ingegneria dei tessuti 14, 15, 17, 18, 19, 20, 21. L&#39;isolamento di ad-DMV è stato originariamente istituito nel ratti 11, 12. Qui, descriviamo un protocollo, che permette l&#39;isolamento standardizzato di murino ad-DMV da cuscinetti di grasso dell&#39;epididimo. Ciò può fornire ulteriori informazioni sui meccanismi molecolari alla base della funzione ad-DMV utilizzando modelli di topi transgenici.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5-mL conical microcentrifuge tube</td>
			<td>VWR, Kelsterbach, Germany</td>
			<td>700-5239</td>
			<td></td>
		</tr>
		<tr>
			<td>100-&#181;L precision pipette</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>4920000059</td>
			<td></td>
		</tr>
		<tr>
			<td>10-mL measuring pipette</td>
			<td>Costar, Corning Inc., New York, USA</td>
			<td>4488</td>
			<td></td>
		</tr>
		<tr>
			<td>14-mL PP tubes</td>
			<td>Greiner bio-one, Frickenhausen, Germany</td>
			<td>187261</td>
			<td></td>
		</tr>
		<tr>
			<td>1-mL precision pipette</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>4920000083</td>
			<td></td>
		</tr>
		<tr>
			<td>500-&#181;m filter (pluriStrainer 500 &#181;m)</td>
			<td>HISS Diagnostics, Freiburg, Germany</td>
			<td>43-50500-03</td>
			<td></td>
		</tr>
		<tr>
			<td>50-mL conical centrifuge tube</td>
			<td>Greiner bio-one, Frickenhausen, Germany</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr>
			<td>50-mL Erlenmeyer flask</td>
			<td>VWR, Kelsterbach, Germany</td>
			<td>214-0211</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Greiner bio-one, Frickenhausen, Germany</td>
			<td>65518</td>
			<td></td>
		</tr>
		<tr>
			<td>cell detachment solution (Accutase)</td>
			<td>eBioscience, San Diego, CA USA</td>
			<td>00-4555-56</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 mice</td>
			<td>Charles River, Cologne, Germany</td>
			<td>027</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6-Tg(CAG-EGFP)1Osb/J mice</td>
			<td>The Jackson Laboratory, Bar Harbor, USA</td>
			<td>003291</td>
			<td></td>
		</tr>
		<tr>
			<td>CD117-FITC</td>
			<td>BD Biosciences, Heidelberg, Germany</td>
			<td>553373</td>
			<td></td>
		</tr>
		<tr>
			<td>CD31-PE</td>
			<td>BD Biosciences, Heidelberg, Germany</td>
			<td>553354</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase NB4G&#160;</td>
			<td>Serva Electrophoresis GmbH, Heidelberg, Germany</td>
			<td>17465.02</td>
			<td>Lot tested by manufacturer for enzymatic activity</td>
		</tr>
		<tr>
			<td>Dissection scissors</td>
			<td>Braun Aesculap AG &#38;CoKG, Melsungen, Germany</td>
			<td>BC 601</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA-binding dye (Bisbenzimide H33342)</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany</td>
			<td>B2261</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle medium (DMEM)&#160;</td>
			<td>PAN Biotech, Rickenbach, Germany</td>
			<td>P04-03600</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal calf serum (FCS)</td>
			<td>Biochrom GmbH, Berlin, Germany</td>
			<td>S0615</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>S&#38;T AG, Neuhausen, Switzerland</td>
			<td>FRS-15 RM-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine scissors</td>
			<td>World Precision Instrumets, Sarasota, FL, USA</td>
			<td>503261</td>
			<td></td>
		</tr>
		<tr>
			<td>Dermal skin substitute (Integra)</td>
			<td>Integra Life Sciences, Sain Priest, France</td>
			<td>62021</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine&#160;</td>
			<td>Serumwerk Bernburg AG, Bernburg, Germany</td>
			<td>7005294</td>
			<td></td>
		</tr>
		<tr>
			<td>M-IgG2akAL488&#160;&#160;</td>
			<td>eBioscience, San Diego, CA USA</td>
			<td>53-4724-80</td>
			<td></td>
		</tr>
		<tr>
			<td>Octeniderm (disinfecting solution)</td>
			<td>Sch&#252;lke &#38; Mayer, Norderstedt, Germany</td>
			<td>118211</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Biochrom, Berlin, Germany</td>
			<td>A2213</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Greiner bio-one, Frickenhausen, Germany</td>
			<td>664160</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Lonza Group, Basel, Switzerland</td>
			<td>17-516F</td>
			<td></td>
		</tr>
		<tr>
			<td>pluriStrainer 20-&#181;m (20 &#181;m filter)</td>
			<td>HISS Diagnostics, Freiburg, Germany</td>
			<td>43-50020-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat-IgG2akFITC</td>
			<td>BD Biosciences, Heidelberg, Germany</td>
			<td>553988</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat-IgG2akPE</td>
			<td>BD Biosciences, Heidelberg, Germany</td>
			<td>553930</td>
			<td></td>
		</tr>
		<tr>
			<td>Small preparation scissors</td>
			<td>S&#38;T AG, Neuhausen, Switzerland</td>
			<td>SDC-15 R-8S</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical forceps</td>
			<td>Braun Aesculap AG &#38;CoKG, Melsungen, Germany</td>
			<td>BD510R</td>
			<td></td>
		</tr>
		<tr>
			<td>Tape (Heftpflaster Seide) 1.25 cm</td>
			<td>Fink &#38; Walter GmbH, Mechweiler, Germany</td>
			<td>1671801</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine&#160;</td>
			<td>Bayer Vital GmbH, Leverkusen, Germany</td>
			<td>1320422</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-SMA-AL488</td>
			<td>eBioscience, San Diego, CA USA</td>
			<td>53-9760-82</td>
			<td>Intracellular labeling additionally requires Cytofix/Cytoperm (BD Biosciences, Heidelberg, Germany; #554722)</td>
		</tr>
	</tbody>
</table>

isolation of murine adipose tissue-derived microvascular fragments as vascularization units for tissue engineering

Una rete microvascolare funzionale è di importanza fondamentale per la sopravvivenza e l&#39;integrazione dei costrutti ingegneria tessutale. A questo scopo, sono state stabilite diverse strategie angiogenici e prevascularization. Tuttavia, la maggior parte degli approcci basati su celle includono passi in termini di tempo in vitro per la formazione di una rete microvascolare. Quindi, essi non sono adatti per intraoperatori procedure one-step. Adiposo derivate dal tessuto frammenti microvascolari (ad-MVF) rappresentano le unità vascolarizzazione promettenti. Essi possono essere facilmente isolati dal tessuto grasso e presentano una morfologia microvasi funzionale. Inoltre, essi rapidamente rimontare in nuove reti microvascolari dopo l&#39;impianto in vivo. Inoltre, ad-DMV hanno dimostrato di indurre linfangiogenesi. Infine, sono una ricca fonte di cellule staminali mesenchimali, che possono ulteriormente contribuire alla loro elevato potenziale di vascolarizzazione. In studi precedenti abbiamo dimostrato la notevole vascularizatisulla capacità di ad-MVF in sostituti ossei e cutanei ingegnerizzati. Nel presente studio, riportiamo un protocollo standardizzato per l&#39;isolamento enzimatica di ad-DMV da tessuto adiposo murino.

Nel presente studio abbiamo eseguito sei procedure di isolamento ad-MVF con il tessuto grasso da 7 a 12 mesi maschio wild-type C57BL 6 topi / (peso corporeo medio: 35 ± 1 g). La Figura 1 illustra la raccolta dei cuscinetti adiposi dell&#39;epididimo murini con conseguente isolamento meccanico ed enzimatica ad-DMV. Il tempo necessario per la raccolta di grasso era 30 minuti e per l&#39;isolamento di ad-DMV era di 120 min. In totale, la procedura ha avuto 150 min. <p ...

Watch this Scientific Journal Video about Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering at JoVE.com

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

Vi presentiamo un protocollo per isolare frammenti adiposo microvascolari derivate dal tessuto che rappresentano le unità di vascolarizzazione promettenti. Possono essere isolati rapidamente, non richiedono l&#39;elaborazione in vitro e, pertanto, può essere utilizzato per un passo prevascularization in diversi campi dell&#39;ingegneria dei tessuti.

L&#39;isolamento di Murine adiposo derivate dal tessuto microvascolare Frammenti come vascolarizzazione Unità per Tissue Engineering

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...

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Research

JoVE Journal

Bioengineering

12.3K Views.  Saarland University.  Vi presentiamo un protocollo per isolare frammenti adiposo microvascolari derivate dal tessuto che rappresentano le unità di vascolarizzazione promettenti. Possono essere isolati rapidamente, non richiedono l'elaborazione in vitro e, pertanto, può essere utilizzato per un passo prevascularization in diversi campi dell'ingegneria dei tessuti. 

Video: Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

Fabbricazione di derivazione biologica Materiali iniettabili per l&#39;ingegneria tissutale miocardica

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

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Bioingegneria

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Elaborazione Postproduzione di fibre elettrofilate per l&#39;ingegneria tissutale

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

Ingegneria tessuti muscolo scheletrico di cellule murine progenitrici mioblasti e di applicazione di stimolazione elettrica

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Tissue Engineering of the Intestine in a Murine Model

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to preoperative weights within 40 days.1 In humans, massive small bowel resection can result in short bowel syndrome, a functional malabsorptive state that confers significant morbidity, mortality, and healthcare costs including parenteral nutrition dependence, liver failure and cirrhosis, and the need for multivisceral organ transplantation.2 In this paper, we describe and document our protocol for creating tissue-engineered intestine in a mouse model with a multicellular organoid units-on-scaffold approach. Organoid units are multicellular aggregates derived from the intestine that contain both mucosal and mesenchymal elements,3 the relationship between which preserves the intestinal stem cell niche.4 In ongoing and future research, the transition of our technique into the mouse will allow for investigation of the processes involved during TESI formation by utilizing the transgenic tools available in this species.5The availability of immunocompromised mouse strains will also permit us to apply the technique to human intestinal tissue and optimize the formation of human TESI as a mouse xenograft before its transition into humans. Our method employs good manufacturing practice (GMP) reagents and materials that have already been approved for use in human patients, and therefore offers a significant advantage over approaches that rely upon decellularized animal tissues. The ultimate goal of this method is its translation to humans as a regenerative medicine therapeutic strategy for short bowel syndrome.

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach.

L&#39;ingegneria dei tessuti del colon in un modello murino

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Capillare Forza litografia per Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Ingegneria 3D Cellularized collagene Gel per Vascolare Rigenerazione Tissutale

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

Preparazione di Thermoresponsive Nanostrutturati Superfici Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

Ingegneria dei tessuti da parte intrinseca Vascolarizzazione in un<em&gt; In Vivo</em&gt; Camera ingegneria dei tessuti

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Una piattaforma Microfluidic per l&#39;isolamento Single-cell high-throughput e cultura

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

Due metodi per decellularizzazione di tessuti vegetali per applicazioni di ingegneria del tessuto

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

L'isolamento di Murine adiposo derivate dal tessuto microvascolare Frammenti come vascolarizzazione Unità per Tissue Engineering

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