This work was funded by a New Faculty Award II from the California Institute of Regenerative Medicine (CIRM; RN2-00921-1), NIH-funded National Research Award (F32-HL104924), and CIRM Training Grant (TG21163). Materials were provided by: Glycosan Biosystems Inc / BioTime and Dr. Stephen Badylak (University of Pittsburgh)

1 Creazione di piatti pNIPAAM rivestite <ol><li> Sciogliere 2.6 g di pNIPAAM in 2 ml di una soluzione di esano al 60% di toluene / 40%. </li><li> Riscaldare la miscela a 60 ° C per 10 min agitati, finché il pNIPAAM è disciolto. </li><li> Tagliare carta da filtro in un cerchio di diametro di 60 mm e carta posto in imbuto Buchner. </li><li> Filtrare la soluzione attraverso imbuto Buchner nel bicchiere di vetro pre-pesato (non usare la plastica, come esano si scioglierà la plastica). </li><li> Porre il bicchiere e contenuto in un vuoto campana (24 psi) O / N (16 ore). Nota: Fino a quando il residuo viene fatto reagire con isopropilico esso si ossida in modo da assicurarsi che non venga a contatto con l&#39;ossigeno. </li><li> Pesare il becher per stabilire il peso del pNIPAAM. </li><li> Aggiungere alcool isopropilico al pNIPAAM, creando un 50/50 w / w soluzione. </li><li> Inserire 2 ml della soluzione sulla superficie della piastra di coltura tissutale, e cappotto per 5 min sotto luce UV. </li><li> Lavare la piastra con 2 ml di PBS caldo due volteprima di utilizzare per la coltura cellulare. </li></ol> 2 Creazione di fogli Cellulari Nota: fogli di cellule primarie per l&#39;organo bersaglio possono essere creati utilizzando una serie di metodi diversi, o da rivestimento di superfici di coltura di tessuti con polimero termo-sensibile come descritto qui. Lastre termo-sensibile pre-rivestiti sono offerti anche da un certo numero di fornitori. Nota: Questo protocollo è per la cultura con un piatto da 35 mm. Brevemente, le cellule vengono incubate a 37 ° C per un minimo di 24 ore a confluenza per stabilire connessioni laterali tra celle adiacenti. Per rilasciare fogli cellulari, piastre vengono sottoposti a temperature inferiori a 32 ° C. Il foglio cella viene poi trasferito al forte matrice fibrosa base contenente un idrogel permissiva neovascolare con cellule endoteliali vascolari. <ol><li> Isolare la popolazione cellulare. Nota: questo metodo dipende dai singoli procedimenti di derivazione e del tipo di cellule. Rat aortica mu lisciacellule SCLE (RASMC) sono utilizzati in questo esempio. Queste sono cellule muscolari lisce primarie isolate dall&#39;aorta addominale di un topo. </li><li> Lavare le cellule con 2 ml di PBS caldo. </li><li> Aggiungere 3 ml di tripsina (o altro / soluzione dissociare scissione) alle cellule per 5 min. </li><li> Inibire la tripsina con l&#39;aggiunta di 3 ml di terreno di coltura, o soluzione tampone fosfato (PBS) contenente 10% di siero fetale bovino (FBS). </li><li> Raccogliere le cellule in un tubo conico e contare una aliquota. </li><li> Spin le cellule a 1000 rpm (228 xg) per 5 min. </li><li> Aspirare il surnatante e risospendere le cellule in loro mezzi di crescita (SmGM2 più kit proiettile terreno di coltura viene utilizzato per RASMC). </li><li> Posizionare i supporti contenenti le cellule su un piatto di 35 millimetri termo-sensibili - piastra rivestita pNIPAAM ad una concentrazione che permetterà di conseguire il 100% di confluenza. Nota: Per RASMCs quel numero è stato determinato in 100.000 cellule / cm 2. Tuttavia, a causa della perdita di cellule durante il passaggio, 120% di detto va<html> viene utilizzato lue. </li><li> Mettere in un incubatore a 37 ° CO / N. Nota: È importante mantenere le cellule a 37 ° C per mantenere l&#39;adesione delle cellule alla piastra. </li></ol> 3 Preparazione di Matrix Foundational Nota: Vari matrici fibrosi 3D possono essere usati per sovrapporre forte matrice fibrosa tra i fogli di cellule delicate. Alcuni esempi includono: Gelfoam, Bioglass, materiali naturali acellularized 26 o nanospun materiali 27,28 Il suina matrice vescica urinaria (UBM) utilizzati in questi studi è stato generosamente fornito dal nostro collaboratore, il dott Badylak 29. <ol><li> Prima dell&#39;uso, determinare le caratteristiche della matrice, tra cui la mancanza di contenuto cellulare se si utilizza matrice decellularized, 27,28 cellulare vitalità specifico, e lo spazio vuoto. 22 </li><li> Tagliare la matrice pre-sterilizzato in una dimensione e forma desiderata. Nota: Qui, un foro-perforazione è utilizzata per tagliare un cerchio del diametro di 4 mm. </li></ol>ve_title &quot;&gt; 4. Semina cellule endoteliali in un Neovascolare Permissivo Hydrogel Nota: Le cellule endoteliali possono essere ottenuti da una varietà di fonti, tra cui la differenziazione da cellule staminali o progenitrici. Qui, vengono utilizzati HUVECs. <ol><li> Utilizzare qualsiasi idrogel permissiva (fibrina, gel di collagene) fintanto che il tempo di reticolazione è abbastanza breve da consentire alle cellule rimangono vitali. Nota: Qui, un gel Hyaluronan (HA) basato reticolato con viene utilizzato un ponte disolfuro. </li><li> Preparare l&#39;idrogel HA in conformità con il protocollo aziendale. </li><li> Raccogliere le cellule endoteliali e disperdersi in una soluzione singola cella utilizzando tripsina 1x. Nota: Accutase o cellulare Dissociazione Buffer potrebbero essere utilizzati anche per la dispersione singola cellula. </li><li> Disattivare l&#39;enzima tripsina utilizzando una pari quantità di inibitore della tripsina di soia (se è importante che le cellule non vengano a contatto con il siero) o 10% FBS in PBS, raccogliendo la soluzione / cellule in un tubo da 15 ml. </li><li> Count le cellule, e calcolare il volume necessario per le dimensioni delle patch (precedentemente quantificato). Nota: Per una patch a 4 mm, sono usati qui 2 milioni di cellule endoteliali. </li><li> Estrarre 2 milioni di cellule, e inserire in un nuovo tubo da 15 ml. </li><li> Spin a (228 xg) per 5 min. </li><li> Aspirare il supernatante, lasciando le cellule come pellet in un tubo conico. </li><li> Mescolare i materiali liquidi HA e la gelatina in un 1: 1 razione. Aggiungere quindi 80% del volume totale nel tubo conico contenente il pellet. </li><li> Risospendere le cellule endoteliali nella miscela 1: 1 HA / gelatina </li><li> Posizionare le cellule in sospensione nella miscela HA / gelatina nella base matrice fibrosa dal punto 2. </li><li> Aggiungere 1/5 (20 percento) del volume totale desiderata del cross-linker </li><li> Incubare per 1 ora a 37 ° C. </li></ol> 5 Isolamento di fogli Cellulari <ol><li> Rimuovere le piastre 35 millimetri pNIPAAM trattati contenenti le cellule dal incubatore e posto in una cappa di coltura cellulare aRT. </li><li> Aspirare rapidamente il supporto dalle cellule, e aggiungere 2 ml di 6% di gelatina normale che è stato riscaldato a 37 ° C. </li><li> Mentre la gelatina è ancora caldo, posizionare il reticolo di metallo nella gelatina, immergendo sotto la superficie della gelatina normale (Film 1). </li><li> Inserire l&#39;intera piastra su ghiaccio per 5 a 7 minuti, consentendo la gelatina per indurire. </li><li> Dopo 7 minuti, utilizzare una spatola per separare accuratamente i bordi gelatina dal lato della piastra, e quindi utilizzare pinze per sollevare la grata metallica dalla piastra Nota: La gelatina 6%, e il foglio di cella deve sollevare con il reticolo. </li><li> Spostare il foglio cella al piatto e posto sulla sommità della combinazione matrice idrogel di base fibrosa, impostando attentamente il reticolo sopra il costrutto. Nota: La parte apicale dello strato di cellule sarà ancora in prima posizione. </li><li> Aggiungere 2 ml di supporti caldo (37 ° C). </li><li> Incubare O / N consentendo il foglio di cellule di aderire alla superficie idrogel. </li><li> Rimuovere tegli reticolo di metallo dopo la soluzione si riscalda (circa 1 ora), o il giorno successivo. </li></ol>

<ol>
	<li>Ohashi, K., Okano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+tissue+engineering+of+the+liver+and+islets.">Functional tissue engineering of the liver and islets.</a> Anat Rec (Hoboken). 297, 73-82 (2014).</li><li>Chen, Q. Z., Harding, S. E., Ali, N. N., Lyon, A. R., Boccaccini, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterials+in+cardiac+tissue+engineering:+Ten+years+of+research+survey.">Biomaterials in cardiac tissue engineering: Ten years of research survey.</a> Mat Sci Eng R. 59, 1-37 (2008).</li><li>Jakob, P., Landmesser, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+of+cell-based+therapy+for+heart+failure.">Current status of cell-based therapy for heart failure.</a> Curr Heart Fail Rep. 10, 165-176 (2013).</li><li>Tongers, J., Losordo, D. W., Landmesser, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+and+progenitor+cell-based+therapy+in+ischaemic+heart+disease:+promise,+uncertainties,+and+challenges.">Stem and progenitor cell-based therapy in ischaemic heart disease: promise, uncertainties, and challenges.</a> Eur Heart J. 32, 1197-1206 (2011).</li><li>Etzion, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+embryonic+cardiomyocyte+transplantation+on+the+progression+of+heart+failure+in+a+rat+model+of+extensive+myocardial+infarction.">Influence of embryonic cardiomyocyte transplantation on the progression of heart failure in a rat model of extensive myocardial infarction.</a> J Mol Cell Cardiol. 33, 1321-1330 (2001).</li><li>Masuda, S., Shimizu, T., Yamato, M., Okano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+sheet+engineering+for+heart+tissue+repair.">Cell sheet engineering for heart tissue repair.</a> Adv Drug Deliv Rev. 60, 277-285 (2008).</li><li>Koh, G. Y., Soonpaa, M. H., Klug, M. G., Field, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+myocardial+repair.">Strategies for myocardial repair.</a> J Interv Cardiol. 8, 387-393 (1995).</li><li>Li, R. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+a+bioengineered+cardiac+graft.">Construction of a bioengineered cardiac graft.</a> J Thorac Cardiovasc Surg. 119, 368-375 (2000).</li><li>Muller-Ehmsen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rebuilding+a+damaged+heart:+long-term+survival+of+transplanted+neonatal+rat+cardiomyocytes+after+myocardial+infarction+and+effect+on+cardiac+function.">Rebuilding a damaged heart: long-term survival of transplanted neonatal rat cardiomyocytes after myocardial infarction and effect on cardiac function.</a> Circulation. , 105-1720 (2002).</li><li>Reinecke, H., Zhang, M., Bartosek, T., Murry, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival,+integration,+and+differentiation+of+cardiomyocyte+grafts:+a+study+in+normal+and+injured+rat+hearts.">Survival, integration, and differentiation of cardiomyocyte grafts: a study in normal and injured rat hearts.</a> Circulation. , 100-193 (1999).</li><li>Roell, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+cardiomyoplasty+improves+survival+after+myocardial+injury.">Cellular cardiomyoplasty improves survival after myocardial injury.</a> Circulation. 105, 2435-2441 (2002).</li><li>Soonpaa, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+approaches+for+myocardial+regeneration.">Potential approaches for myocardial regeneration.</a> Ann N Y Acad Sci. 752, 446-454 (1995).</li><li>Akins, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+tissue+engineering+mend+broken+hearts.">Can tissue engineering mend broken hearts.</a> Circ Res. 90, 120-122 (2002).</li><li>Goodell, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+plasticity+in+muscle+and+bone+marrow.">Stem cell plasticity in muscle and bone marrow.</a> Ann N Y Acad Sci. 938, 208-218 (2001).</li><li>Menasche, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myoblast+transplantation+for+heart+failure.">Myoblast transplantation for heart failure.</a> Lancet. 357, 279-280 (2001).</li><li>Murry, C. E., Wiseman, R. W., Schwartz, S. M., Hauschka, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+myoblast+transplantation+for+repair+of+myocardial+necrosis.">Skeletal myoblast transplantation for repair of myocardial necrosis.</a> J Clin Invest. 98, 2512-2523 (1172).</li><li>Orlic, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplanted+adult+bone+marrow+cells+repair+myocardial+infarcts+in+mice.">Transplanted adult bone marrow cells repair myocardial infarcts in mice.</a> Ann N Y Acad Sci. 938, 221-229 (2001).</li><li>Elia, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silk-hyaluronan-based+composite+hydrogels:+a+novel,+securable+vehicle+for+drug+delivery.">Silk-hyaluronan-based composite hydrogels: a novel, securable vehicle for drug delivery.</a> J Biomater Appl. 27, 749-762 (2013).</li><li>Kai, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell-loaded+nanofibrous+patch+promotes+the+regeneration+of+infarcted+myocardium+with+functional+improvement+in+rat+model.">Stem cell-loaded nanofibrous patch promotes the regeneration of infarcted myocardium with functional improvement in rat model.</a> Acta Biomater. , (2014).</li><li>Hong, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracheal+reconstruction+using+chondrocytes+seeded+on+a+poly(l-lactic-co-glycolic+acid)-fibrin/hyaluronan.">Tracheal reconstruction using chondrocytes seeded on a poly(l-lactic-co-glycolic acid)-fibrin/hyaluronan.</a> J Biomed Mater Res A. , (2014).</li><li>Serpooshan, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+bioengineered+acellular+collagen+patch+on+cardiac+remodeling+and+ventricular+function+post+myocardial+infarction.">The effect of bioengineered acellular collagen patch on cardiac remodeling and ventricular function post myocardial infarction.</a> Biomaterials. 34, 9048-9055 (2013).</li><li>Turner, W. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+tissue+development+for+delivery+of+embryonic+stem+cell-derived+endothelial+and+cardiac+cells+in+natural+matrices.">Cardiac tissue development for delivery of embryonic stem cell-derived endothelial and cardiac cells in natural matrices.</a> J Biomed Mater Res B Appl Biomater. 100, 2060-2072 (2012).</li><li>Sato, M., Yamato, M., Hamahashi, K., Okano, T., Mochida, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Articular+cartilage+regeneration+using+cell+sheet+technology.">Articular cartilage regeneration using cell sheet technology.</a> Anat Rec (Hoboken). 297, 36-43 (2014).</li><li>Sawa, Y., Miyagawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Present+and+future+perspectives+on+cell+sheet-based+myocardial+regeneration+therapy.">Present and future perspectives on cell sheet-based myocardial regeneration therapy.</a> Biomed Res Int. 2013, 583912 (2013).</li><li>Demirbag, B., Huri, P. Y., Kose, G. T., Buyuksungur, A., Hasirci, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+cell+therapies+with+and+without+scaffolds.">Advanced cell therapies with and without scaffolds.</a> Biotechnol J. 6, 1437-1453 (2011).</li><li>Song, J. J., Ott, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+engineering+based+on+decellularized+matrix+scaffolds.">Organ engineering based on decellularized matrix scaffolds.</a> Trends Mol Med. 17, 424-432 (2011).</li><li>Badylak, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+extracellular+matrix+as+an+inductive+scaffold+for+the+partial+replacement+of+functional+myocardium.">The use of extracellular matrix as an inductive scaffold for the partial replacement of functional myocardium.</a> Cell Transplant. 15, S29-S40 (2006).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+restriction+of+human+hepatic+stem+cells+to+mature+fates+is+made+efficient+by+tissue-specific+biomatrix+scaffolds.">Lineage restriction of human hepatic stem cells to mature fates is made efficient by tissue-specific biomatrix scaffolds.</a> Hepatology. 53, 293-305 (2011).</li><li>Gilbert, T. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+fiber+alignment+and+biaxial+mechanical+behavior+of+porcine+urinary+bladder+derived+extracellular+matrix.">Collagen fiber alignment and biaxial mechanical behavior of porcine urinary bladder derived extracellular matrix.</a> Biomaterials. 29, 4775-4782 (2008).</li><li>Luna, J. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiscale+biomimetic+topography+for+the+alignment+of+neonatal+and+embryonic+stem+cell-derived+heart+cells.">Multiscale biomimetic topography for the alignment of neonatal and embryonic stem cell-derived heart cells.</a> Tissue Eng Part C Methods. 17, 579-588 (2011).</li></ol>

We have nothing to disclose.

I passaggi critici nel protocollo comprendono: rivestimento delle superfici delle piastre con il polimero thermoresponsive e manipolare i fogli di cellule dopo il raffreddamento delle piastre. Poiché le cellule differenti presentano differenti proprietà fisiche, come adesività, il tempo di sollevamento deve essere ottimizzata per ogni tipo di cellula diversa. Il secondo, e più significativamente impegnativo componente di questo protocollo, incentrata sulla manipolazione dello strato di cellule, un aspetto critico di...

Injection of cells and/or single materials alone has shown variable success in other organ systems and limited success in cardiac regeneration.5,7-12 Currently, stem cell-derived cells are delivered to damaged tissue using a variety of delivery methods including: direct cell injection into tissue and perfusion into the blood supply.13-17 Others have implanted cells alone, materials alone and/or in combination with material carriers to help regenerate damaged organs.18-21 This design combines multiple strategies that provide material strength, patterning in multiple materials and multiple cell types. 
Specifically, the base acellularized fibrous matrix provides the foundational physical strength to the construct, making it suitable for suturing in into the patient, if necessary. The void spaces in the base matrix are filled with endothelial cells in a neovascular permissive hydrogel22 for rapidly establishing vascularization of the implanted construct. This composite is then integrated with pre-patterned cell sheets that allow enhanced cell-to-cell communication, more closely mimic the native tissue.1,23-25 The overall production process for the layered cellular patch is outlined by the flowchart in Figure 1.

<table border="0" cellpadding="0" cellspacing="0" width="695">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:207px;">Table of Reagents:</td>
			<td style="width:128px;"></td>
			<td style="width:140px;"></td>
			<td style="width:221px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Reagent</td>
			<td style="width:128px;">Company</td>
			<td style="width:140px;">Catalogue number</td>
			<td style="width:221px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Calcein-AM</td>
			<td style="width:128px;">Invitrogen</td>
			<td style="width:140px;">C3099</td>
			<td style="width:221px;">Cell tracker / live dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Lysotracker Red</td>
			<td style="width:128px;">Invitrogen</td>
			<td style="width:140px;">L7528</td>
			<td style="width:221px;">Cell tracker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Neutral Red</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:140px;">N7005</td>
			<td style="width:221px;">Visible Cell dye</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:207px;">pNIPAAM</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:140px;">412780250</td>
			<td style="width:221px;">Poly(N-isopropylacrylamide)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Toluene</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:140px;">244511-1L</td>
			<td style="width:221px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Hexane</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:140px;">296090-1L</td>
			<td style="width:221px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">RAOSMC</td>
			<td style="width:128px;">Lonza</td>
			<td style="width:140px;">R-ASM-580</td>
			<td style="width:221px;">Rat Aortic Smooth Muscle Cells</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:207px;">SmGM2</td>
			<td style="width:128px;">Lonza</td>
			<td style="width:140px;">CC-4149</td>
			<td style="width:221px;">Smooth Muscle Media</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:207px;">HUVEC</td>
			<td style="width:128px;">Invitrogen</td>
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tissue engineering: construction of a multicellular 3d scaffold for the delivery of layered cell sheets

Molti tessuti, come i cuori umani adulti, non sono in grado di rigenerarsi in modo adeguato dopo un danno. 2,3 Strategie di ingegneria dei tessuti propongono innovazioni per aiutare il corpo nel recupero e riparazione. Ad esempio, gli approcci TE possono essere in grado di attenuare il rimodellamento cardiaco dopo infarto miocardico (MI) e possibilmente aumentare la funzione cardiaca totale a un livello normale di MI-pre vicino. 4 Come con qualsiasi tessuto funzionale, la rigenerazione del tessuto cardiaco di successo comporta la corretta consegna dei più tipi di cellule con stimoli ambientali che favoriscono l&#39;integrazione e la sopravvivenza del trapianto di cellule / tessuti impiantati. Tessuti ingegnerizzati dovrebbero affrontare più parametri tra cui: segnali solubili, cell-to-cell interazioni e materiali a matrice, calcolati come veicoli per le consegne, i loro effetti sulla sopravvivenza delle cellule, la forza materiale, e la facilitazione di organizzazione cellula-tessuto. Gli studi che utilizzano l&#39;iniezione diretta di cellule madri per marze ignorare solo questi elementi essenziali. 2,5,6Un design di tessuto che unisce questi ingredienti deve ancora essere sviluppato. Qui, presentiamo un esempio di design integrate con stratificazione di fogli di cellule fantasia con due tipi distinti di materiali biologici di derivazione contenenti il ​​tipo di cellule dell&#39;organo bersaglio e le cellule endoteliali per migliorare la formazione di nuovi vasi nel &quot;tessuto&quot;. Sebbene questi studi si concentrano sulla generazione di tessuto cardiaco-like, questo disegno tessuto può essere applicato a molti altri organi oltre cuore con minimi cambiamenti di design e materiali, ed è pensato per essere un prodotto off-the-shelf per terapie rigenerative. Il protocollo contiene cinque passaggi dettagliati. Una temperatura sensibile Poli (N -isopropylacrylamide) (pNIPAAM) viene utilizzato per tessuti cappotto coltura piatti. Poi, le cellule del tessuto specifico sono coltivate sulla superficie delle piastre rivestite / superfici micropattern per formare fogli di cellule con forti aderenze laterali. In terzo luogo, una matrice di supporto è per il tessuto combinando matrice porosa con Permissi neovascolareve idrogel e cellule endoteliali. Infine, i fogli di cellule vengono sollevati dai piatti pNIPAAM rivestiti e trasferiti all&#39;elemento di base, rendendo il costrutto completo.

Il diagramma di flusso (figura 1) illustra il metodo generale di rendere la patch multistrato. Fogli cellulari sono staccati dalla piastra trattata pNIPAAM facendo cadere la temperatura sotto i 32 ° C. Poi il foglio cella è posto in cima al idrogel reticolato contenente le cellule endoteliali seminate nella matrice fibrosa sottostante (Figura 1). Le piastre termosensibili pretrattati possono essere utilizzati anche per creare i fogli di cellule. Superfici speciali topologiche sono usa...

Watch this Scientific Journal Video about Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets at JoVE.com

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

Ingegneria dei tessuti: Costruzione di un multicellulare 3D Ponteggio per la consegna di Layered fogli Cellulari

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

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Bioengineering

14.3K Views.  University of California, Merced. For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

Video: Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

Progettazione di un biassiale Carico meccanico bioreattore per l&#39;ingegneria tissutale

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

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Bioingegneria

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Ingegneria dei tessuti di un Umano 3D<em&gt; In vitro</em&gt; Sistema Tumore test

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Ponteggi self-reporting per coltura cellulare 3-Dimensional

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

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. ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

Adattamento del processo Electrospinning per fornire tre ambienti unici per un Tri-livello<em&gt; In Vitro</em&gt; Modello della parete delle vie aeree

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue geometry. The resulting 3D hydrogel-based cellular constructs have been introduced as an alternative to animal experiments for advanced biological studies, pharmacological assays and organ transplant applications. Although hydrogel-based particles and fibers can be easily fabricated, it is difficult to manipulate them for tissue reconstruction. In this video, we describe a fabrication method for micropatterned alginate hydrogel sheets, together with their assembly to form a macro-scale 3D cell culture system with a controlled cellular microenvironment. Using a mist form of the calcium gelling agent, thin hydrogel sheets are easily generated with a thickness in the range of 100 - 200 &#181;m, and with precise micropatterns. Cells can then be cultured with the geometric guidance of the hydrogel sheets in freestanding conditions. Furthermore, the hydrogel sheets can be readily manipulated using a micropipette with an end-cut tip, and can be assembled into multi-layered structures by stacking them using a patterned polydimethylsiloxane (PDMS) frame. These modular hydrogel sheets, which can be fabricated using a facile process, have potential applications of in vitro drug assays and biological studies, including functional studies of micro- and macrostructure and tissue reconstruction.

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

Costruzione di fogli modulare idrogel per micropatterned macro-scala 3D Cellular Architettura

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

Fabbricazione di invertito colloidale cristallo poli (glicole etilenico) Scaffold: una piattaforma Cell Culture tridimensionale per il fegato Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

Fabbricazione di Schiume matrice extracellulare-derivati ​​e microcarriers come tessuto-specifica delle cellule, Cultura e piattaforme di distribuzione

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

Fabbricazione di pozzi di agarosio personalizzato per la semina delle cellule e tessuto anello auto-assemblaggio utilizzando stampi 3D-stampato

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes&#39; functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups&#39; brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% &#177; 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% &#177; 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This&#160;reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells&#39; native microenvironment, useful to researchers that aim to understand astrocytes&#39; functionality and their relation to the mechanisms involved in neurological diseases.

Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.

Bioprinting 3D di astrociti corticali murini per l'ingegneria del tessuto neurale

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also ...