Ringraziamo il dottor Kati Andreasson (dipartimento di neurologia e scienze neurologiche, Università di Stanford) per l'uso della macchina qRT-PCR. Il lavoro è stato supportato da nazionale istituti di salute sovvenzioni K08NS098876 (alla P.M.G) e Postdoctoral Fellowship di Stanford School of Medicine Dean (a B.O.).

Tutte le cellule staminali e procedure degli animali sono state approvate dal comitato di supervisione di ricerca sulle cellule staminali di Stanford e dal pannello di amministrazione dell'Università di Stanford su Laboratory Animal Care (SCRO-616 e APLAC-31909).
1. incisione del vetro di ITO
<ol><li>Preparare l'ossido della latta dell'indio (ITO)-vetrino coperto inserendo un agente mascherante sopra il lato conduttivo del vetro. Nota: Nastri trasparenti più commerciali possono essere utilizzati come un agente mascherante.<ol>
<li>Rimuovere la maschera in eccesso utilizzando una lama, lasciando solo la forma desiderata del progetto definitivo ITO coperto sul vetro.</li>
	</ol>
</li>
	<li>Combinare il 5% (v/v) di acido nitrico e 45% (v/v) di HCl in un becher di vetro all'interno di una cappa aspirante per preparare la soluzione di incisione.<ol>
<li>Utilizzare un piatto caldo e un termometro per garantire che la temperatura della soluzione di incisione è mantenuta a 70 ° C.</li>
	</ol>
</li>
	<li>Una volta che la soluzione di acquaforte raggiunge i 70 ° C, immergere il vetrino mascherato-ITO vetro nella soluzione per 2 minuti per rimuovere lo strato di ITO in eccesso. Nota: La mascheratura nastro protegge lo strato di ITO dall'esposizione a soluzione acida.</li>
	<li>Rimuovere il vetrino dalla soluzione e sciacquare con deionizzata (DI) H2O.</li>
	<li>Rimuovere con cautela la maschera e misura la resistenza utilizzando un multimetro per garantire che il restante ITO è intatto. Letture dell'area desiderata acidato dovrebbero essere circa 0 Ω.</li>
</ol>2. preparazione della soluzione di pirrolo
<ol><li>In una bottiglia di vetro da 1000 mL, sciogliere 70 g di sodio dodecylbenzenesulfonate (NaDBS) in 600 mL DI H2O.</li>
	<li>Una volta che il NaDBS è sciolto, aggiungere 14 mL di 0,2 M pirrolo e 386 mL DI H2O alla soluzione.</li>
	<li>Coprire la soluzione con foglio di alluminio e conservare a 4 ° C.</li>
</ol>3. placcatura elettrolitica di polipirrolo su vetro di ITO
<ol><li>Riscaldare la soluzione di pirrolo a temperatura ambiente fino a quando NaDBS è completamente ridisciolto. Nota: Questo richiede circa 15-20 minuti.</li>
	<li>Versare 25 mL della soluzione di pirrolo in un becher.</li>
	<li>Collegare il vetro acidato ITO di un multimetro e immergere il vetrino nella soluzione di pirrolo.<ol>
<li>Assicurarsi che il lato con 0 resistenza Ω è rivolto verso l'elettrodo di riferimento di mesh platino.</li>
	</ol>
</li>
	<li>Applicare una corrente elettrica per avviare Galvanotecnica di PPy sulla superficie ITO utilizzando un multimetro (corrente controllata a 3 mA/cm2 per 4 ore).</li>
	<li>Rimuovere il vetro ITO dal multimetro e lavare elettrolitico-PPy in DI H2O per rimuovere residui dell'agente tensioattivo.</li>
	<li>Staccare delicatamente la piastra elettrolitica-PPy i vetro di ITO con una lama di rasoio.<ol>
<li>Conservare la piastra PPy a temperatura ambiente.</li>
	</ol>
</li>
</ol>4. preparazione di polidimetilsilossano (PDMS)
<ol><li>Utilizzando una spatola e un piatto di pesatura, mescolare 18 g di agente base con 2 g di agente indurente e versare il composto in stampi di vetro due 10 x 8 cm.</li>
	<li>Rimuovere le bolle dagli stampi utilizzando una camera a vuoto per 15-20 minuti.</li>
	<li>Posizionare gli stampi in forno a 70 ° C per 3 h.</li>
	<li>Una volta raffreddato, è possibile utilizzare una spatola piatta per rimuovere il PDMS dagli stampi.</li>
</ol>5. fabbricazione di vano di stimolazione elettrica In Vitro
<ol><li>Sterilizzare in autoclave il flusso e piastre di metallo (WxL, 2,5 cm x 12,5 cm) valvole a 120 ° C per 1 ora.</li>
	<li>Utilizzando una diapositiva di camera come un modello, tagliare due pezzi di PDMS. Nota: Un pezzo di PDMS serve come il perimetro della camera cellulare con ritagli di due piazze contigue all'interno della camera di cella. L'altro pezzo PDMS è un contorno del perimetro della camera.<ol>
<li>Tagliare l'interno camera di cella che è quadrato a forma di e corrisponde alla forma dell'alloggiamento delle cellule. Assicurarsi che la forma complessiva di entrambe le parti di PDMS è rettangolare e corrisponde a quello della diapositiva da camera.</li>
	</ol>
</li>
	<li>I materiali di strato come segue dal basso verso l'alto: (1) metallo piatto, (2) PDMS (senza aperture), (3) PPy piastra (perpendicolare alla PDMS), (4) PDMS (con ritagli) e camera (5) delle cellule.</li>
	<li>Bloccare l'apparato insieme una volta che completamente è allineato.</li>
	<li>Tagliare due pezzi di 60 cm di un filo metallico e utilizzare pasta d'argento per collegare un filo ad ogni estremità della piastra PPy che sporge dalla parte esterna della camera. Assicurarsi che i cavi siano abbastanza a lungo per estendere dall'apparecchio verso l'esterno di un incubatore.</li>
	<li>Una volta che la pasta d'argento è guarita, rafforzare e sigillare il collegamento del filo con resina epossidica.<ol>
<li>Registrare la resistenza utilizzando un multimetro per assicurarsi che il campo applicato è lo stesso in ogni camera.</li>
		<li>Per l'impianto, rimuovere i fili; sbloccare gli alloggiamenti di cella e PDMS e poi li separano dal patibolo conduttivo. La dimensione delle impalcature impiantate è circa 1 x 3 x 0,25 mm.</li>
	</ol>
</li>
</ol>6. cellule progenitrici neurali umane (hNPCs) su PPy di placcatura
<ol><li>Sterilizzare gli alloggiamenti assemblati sotto luce UV per 2 ore.<ol>
<li>Dopo 1 ora, ruotare l'assemblato chambers 90°.</li>
	</ol>
</li>
	<li>Dopo la sterilizzazione, rivestire la superficie di PPy nella parte inferiore della camera con 800 µ l di un substrato di rivestimento e consentire il substrato solidificare il PPy piastra in un incubatore a 37 ° C 5% CO2 per 1 h.</li>
	<li>Dopo 1 ora, rimuovere il surnatante dalle camere e risciacquare delicatamente con 1 mL di DPBS + Ca + Mg per bene. Nota: Evitare di lavare la superficie di PPy con forza come questo farà sì che il distacco del substrato rivestimento.</li>
	<li>Utilizzando supporti di cellule fresche, meccanicamente dissociare le cellule coltivate per uso con gli alloggiamenti da una piastra di coltura del tessuto con delicato pipettaggio. Nota: Le cellule dovrebbero essere 90-100% confluenti sulla dissociazione.</li>
	<li>Raccogliere hNPC pellet utilizzando una centrifuga a 8000 x g per 5 minuti.</li>
	<li>Utilizzando un emocitometro, contare e risospendere le cellule in un volume di 100.000 cellule/cm2.</li>
	<li>Piastra di 100.000 cellule in ogni camera ben (100.000 cellule/cm2 in 1 mL di media).</li>
	<li>Tornare chambers incubatore a 37 ° C 5% CO2.
</li>
</ol>7. elettrostimolazione del hNPCs
<ol><li>Un giorno dopo la semina le cellule su chambers, utilizzare un generatore di forme d'onda per applicare la stimolazione elettrica alle celle.</li>
	<li>Prima stimolazione, rimuovere i vecchi media da ogni camera ben e saziatevi con 800 µ l di media fresco.</li>
	<li>Dopo avere modificato i media, inserire nuovamente gli alloggiamenti dell'incubatrice, estendere i cavi dall'incubatrice e allegarli a un generatore di forme d'onda</li>
	<li>Applicare la stimolazione delle cellule con un segnale ad onda quadra a ± 400 mV con 100 Hz per 1 h.</li>
	<li>Dopo la stimolazione, rimuovere i fili e incubare gli alloggiamenti per un altro giorno in un incubatore a 37 ° C con 5% CO2.</li>
	<li>Eseguire successiva analisi biologiche tra cui la vitalità cellulare e reazione a catena della polimerasi in tempo reale quantitativa (qRT-PCR) seguendo il protocollo del produttore.</li>
</ol>8. in Vivo PPy impianto
<ol><li>Eseguire modelli di ictus di occlusione dell'arteria cerebrale centrale distale (dMCA) su T cellula-carenti ratti nudi maschio adulto (NIH-RNU 230 ± 30g) come descritto in precedenza2. Eseguire l'occlusione l'impianto una settimana post-dMCA.</li>
	<li>Un giorno prima dell'intervento, dare ampicillina (1 mg/mL) per i ratti in loro acqua di gabbia.</li>
	<li>Anestetizzare il ratto in una camera di induzione utilizzando 0,5 - 3% mg/kg di isoflurane somministrata tramite inalazione.</li>
	<li>Confermare amputate del ratto da una mancanza di risposta riflessa di punta pizzico.<ol>
<li>Mentre l'animale è sotto anestesia, continuare a monitorare la sua membrana colore, pattern respiratorio e temperatura rettale ogni 15 minuti.</li>
	</ol>
</li>
	<li>Una volta confermata la amputate, applicare le lacrime artificiali su occhi di topo per prevenire la secchezza.</li>
	<li>Assicurare che una tecnica asettica è mantenuta durante questa procedura utilizzando guanti sterili e un telo chirurgico sterile13. Mantenere gli strumenti chirurgici sterili all'interno di un campo sterile.</li>
	<li>Mentre il ratto è sotto anestesia, praticare un craniectomy sopra la corteccia sinistra. Aprire la dura madre.<ol>
<li>Rimuovere il mater di dura dal cervello utilizzando un ago chirurgico micro-sottile.</li>
	</ol>
</li>
	<li>Impiantare le impalcature conduttive da sistema in vitro sulla corteccia del ratto. Nota: Per i nostri esperimenti, abbiamo impiantato impalcature da in vitro giorno 3 dopo il placcaggio di hNPC.<ol>
<li>Inserire l'impianto principalmente sulla corteccia penombra mediale alla lesione di colpo.</li>
	</ol>
</li>
	<li>Dopo la messa il patibolo, è posto surgicel sopra l'impianto per impedire il movimento con la chiusura della pelle.</li>
	<li>Suturare la ferita chiusa e iniettare per via sottocutanea i ratti con buprenorfina SR ad un dosaggio di 1 mg/kg per gestire il dolore associato con la chirurgia stereotassica e occlusione dMCA.</li>
	<li>Posizionare il ratto in una gabbia di recupero come riprende conoscenza.<ol>
<li>Non lasciare mai l'animale incustodito mentre è incosciente.</li>
		<li>Non lasciare mai l'animale con gli altri fino a quando non si è pienamente guadagnato coscienza.</li>
	</ol>
</li>
	<li>Monitorare gli animali ogni giorno per segni di dolore, compreso perdita di peso corporeo, assunzione di cibo/acqua in diminuzione, ridotta cappotto governare/spettinati, diminuito/aumentato attività spontanea, postura anormale, disidratazione/pelle tenting, gli occhi infossati, nascondersi, automutilazioni, respirazione rapida, respirazione a bocca aperta, spasmi muscolari, tremore, tremito e vocalizzazione.<ol>
<li>Monitorare gli animali ogni giorno fino a quando sembra che essi sono mangiare, bere, toelettatura e aumento di peso; e poi settimanalmente dopo aumento di peso.</li>
	</ol>
</li>
	<li>Euthanization precoce tramite inalazione di CO2 e una conferma secondaria di euthanization (tale da garantire l'animale non farà rivivere a causa di inalazione di ossigeno normale alimentazione post CO2 ) si verificherà per qualsiasi animale che sembra essere non mangiare, bere e aumentare di peso dopo l'intervento chirurgico iniziale; appare nel dolore; o appare impossibile completare i test comportamentali a causa di un maggiore del previsto disavanzo corteccia motoria.</li>
</ol>

<ol>
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Crescenti evidenze ha dimostrato la promessa delle cellule staminali come terapia romanzo tratto. Questa promessa ha provocato un grande sforzo per avanzare terapeutica delle cellule staminali al capezzale con almeno 40 studi clinici in corso o completati. Patologia di corsa offre un unico disturbo neurologico che si presta a terapia con cellule staminali, perché dopo l'insulto acuto, non c'è nessun processo neurodegenerativo, impedendo così il recupero. Il meccanismo esatto di cellule staminali-aumentata tempi di rec...

Il colpo è la seconda causa di morte nel mondo e la quinta causa di morte negli Stati Uniti. Nonostante questi alti tassi di mortalità, trattamenti per tempi di recupero attualmente rimangono una sfida con nessun valide opzioni mediche attualmente disponibili1. Ci sono attualmente circa 300 test clinici a che fare con colpi ischemici, di cui solo 40 utilizzare cellule staminali. Gli studi precedenti hanno dimostrato che le terapie con cellule staminali hanno un effetto benefico sulla corsa riparazione2,3. Fattori paracrini come fattore neurotrophic cervello-derivato (BDNF) e trombospondina-1 (THBS-1) rilasciati dalle cellule progenitrici neurali umane trapiantate (hNPCs) hanno mostrato una migliore recupero funzionale attraverso meccanismi associati con un aumento Synapse formazione, angiogenesi, ramificazioni dendritiche e nuove proiezioni assonali, come pure di modulazione del sistema immunitario4,5,6. Tuttavia, i metodi di consegna ottimale delle cellule staminali rimangono sfuggenti.
Consegna di successo sulle cellule staminali al cervello rimane una sfida. Attualmente, idrogel iniettabili e sistemi di ponteggio polimerici sono stati introdotti per fornire cellule staminali. Questi metodi di consegna proteggono le cellule staminali durante il trapianto, nonché offrono protezione da ambienti post-ictus tra cui risposta infiammatoria dell'ospite e condizioni ipossiche7,8,9 , 10. Tuttavia, i materiali più comunemente usati sono inerti, che limita l'uso della modulazione continua (cioè, stimolazione elettrica) delle cellule11. La stimolazione elettrica è uno spunto che influenza la differenziazione, densità di canali ionici e neuriti delle cellule staminali12. Rispetto ai polimeri inerti, polimeri conduttivi possono trasportare una corrente che consente per la stimolazione elettrica e la manipolazione di cellule staminali2. Tuttavia, il meccanismo preciso con cui stimolazione elettrica modula il rilascio di fattore neurotrophic (cioè, BDNF e THBS-1) è ancora non completamente esplorato.
In questo protocollo, descriviamo i passaggi per costruire un sistema di coltura cellulare che consiste di uno scaffold polimerici, polipirrolo (PPy), che permette per la stimolazione elettrica in vitro . A causa del modo in cui è realizzato il sistema di coltura cellulare, è possibile impianto successivo dell'impalcatura della cellula formativa-seminato sulla corteccia del peri-infarto. Per questo sistema, abbiamo presupposto elettricamente le cellule staminali sul patibolo per un breve periodo di tempo prima dell'impianto. A seguito di stimolazione elettrica, lo scaffold polimerici che trasportano le cellule è impiantato con successo intracranially utilizzando un metodo come minimo dilagante.

<table><tbody><tr><td>FGF-Basic</td><td>Invitrogen</td><td>CTP0261</td><td>20 ng/mL for working media</td></tr><tr><td>Matrigel</td><td>Corning</td><td>cb40234a</td><td>1:200 dilution</td></tr><tr><td>LIF Protein, Recom. Hum. (10 &amp;micro;g/mL)</td><td>EMD Millipore</td><td>LIF1010</td><td>10 ng/mL for working media</td></tr><tr><td>Sylgard 184 silicone 3.9 kg</td><td>Fisherbrand</td><td>NC0162601</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Fisherbrand</td><td>SA56-4</td><td></td></tr><tr><td>Nitric Acid Concentrate (Certified) ACS, Fisher Chemical</td><td>Fisherbrand</td><td>SA95</td><td></td></tr><tr><td>ITO Glass</td><td>Delta Technologies</td><td>CG-40IN-0115</td><td></td></tr><tr><td>Sodium dodecylbenzenesulfonate</td><td>Sigma</td><td>289957-1KG</td><td></td></tr><tr><td>Pyrrole</td><td>Sigma</td><td>131709-500ML</td><td>Protect pyrrole solution from light and room temperature</td></tr><tr><td>8 well glass slide chambers</td><td>Thermo Sci Nuc&amp;nbsp;</td><td>125658</td><td>Detach the cell chamber and keep it under sterilized conditions</td></tr><tr><td>Flat-Surface Bracket, 3&quot;x1&quot;</td><td>McMaster-Carr&amp;nbsp;</td><td>1030A4</td><td></td></tr><tr><td>TWO PART SILVER PAINT 14G</td><td>Electron Microscopy Sciences&amp;nbsp;</td><td>1264214</td><td>Mix two parts (1:1) in plastic plate</td></tr><tr><td>DPBS, 1x, with Ca and Mg, No Phenol Red</td><td>Genesse&amp;nbsp;</td><td>25-508C</td><td></td></tr><tr><td>AB2 ArunA Neural Cell Culture Media Kit</td><td>Aruna Biomedical&amp;nbsp;</td><td>ABNS7013.2</td><td></td></tr><tr><td>hNP1 Human Neural Progenitor Expansion Kit</td><td>Aruna Biomedical&amp;nbsp;</td><td>&amp;nbsp;hNP7013.1</td><td></td></tr><tr><td>Noncontact Flow-Adjustment Valve, Nickel-Plated Brass, for 3/32&quot; to 5/8&quot; Tube OD</td><td>McMaster-Carr&amp;nbsp;</td><td>5330K22</td><td></td></tr><tr><td>Multimeter</td><td>Keysight&amp;nbsp;</td><td>E3641A</td><td></td></tr><tr><td>Wavefoam generator</td><td>Keysight</td><td>33210A-10MHz</td><td></td></tr><tr><td>Pt meshes</td><td>Sigma-Aldrich&amp;nbsp;</td><td>298107-425MG</td><td>Reference electrode with dimensions, 1x1 cm</td></tr><tr><td>LIVE/DEAD Viability/Cytotoxicity Kit, for mammalian cells</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>L3224</td><td></td></tr><tr><td>BDNF</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>Hs02718934_s1</td><td></td></tr><tr><td>THBS1</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>Hs00962908_m1</td><td></td></tr><tr><td>GAPDH</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>Hs02758991_g1</td><td></td></tr><tr><td>RNeasy Mini Kit (250)</td><td>Qiagen</td><td>74106</td><td></td></tr><tr><td>QIAshredder (250)</td><td>Qiagen</td><td>79656</td><td></td></tr><tr><td>RNase-Free DNase Set (50)</td><td>QIAGEN</td><td>79254</td><td></td></tr><tr><td>iScript cDNA Synthesis Kit, 100 x 20 &amp;micro;L rxns</td><td>BIORAD</td><td>1708891</td><td></td></tr><tr><td>TaqMan Gene Expression Master Mix</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>4369510</td><td></td></tr><tr><td>7-8 Week Old, male, RNU Rats</td><td>NCI-Frederick</td><td></td><td></td></tr><tr><td>4-0 Ethicon Silk Suture</td><td>eSutures.com</td><td>683G</td><td></td></tr><tr><td>Isoflurane</td><td>Henry Schein</td><td>29405</td><td></td></tr><tr><td>V-1 Tabletop Laboratory Animal Anesthesia System</td><td>VetEquip</td><td>901806</td><td></td></tr><tr><td>Surgicel Original Absorbable Hemostat</td><td>Ethicon</td><td>1952</td><td></td></tr><tr><td>Lab Standard Stereotaxic Instrument, Rat</td><td>Stoelting</td><td>51600</td><td></td></tr><tr><td>Kimberly-Clark Professional&amp;nbsp;Safeskin Purple Nitrile Sterile Exam Gloves</td><td>Fisherbrand</td><td>19-063-130</td><td></td></tr><tr><td>Sterile Drape</td><td>Medline</td><td>DYNJSD1092</td><td></td></tr><tr><td>Thermo Scientific&amp;nbsp;Shandon Stainless-Steel Scalpel Blade Handle, Holds No. 20-25 Blades</td><td>Fisherbrand</td><td>53-34</td><td></td></tr><tr><td>Walter Stern Scalpel Blade Series 300</td><td>Fisherbrand</td><td>17-654-456</td><td></td></tr><tr><td>QuantStudio 6 Flex Real-Time PCR System</td><td>Thermo Fisher Scientific</td><td>4484642</td><td></td></tr><tr><td>Frazier Micro Dissecting Hook</td><td>Harvard Apparatus</td><td>52-2706</td><td></td></tr></tbody></table>

electrically conductive scaffold to modulate and deliver stem cells

Terapia con cellule staminali è emerso come un'eccitante corsa terapeutica, ma il metodo di consegna ottimale rimane poco chiaro. Mentre la tecnica di microiniezione è stata utilizzata per decenni per fornire cellule staminali in modelli di ictus, questa tecnica è limitata dalla mancanza di capacità di manipolare le cellule staminali prima dell'iniezione. Questa carta descrive in dettaglio un metodo di utilizzo di un'impalcatura polimero conduttivo per la consegna di cellule staminali. La stimolazione elettrica delle cellule staminali utilizzando un'impalcatura polimero conduttivo altera geni di cellule staminali coinvolti nella sopravvivenza delle cellule, la risposta infiammatoria e rimodellamento sinaptico. Dopo precondizionamento elettrici, le cellule staminali sul patibolo sono trapiantate intracranially in un modello del ratto di occlusione dell'arteria cerebrale centrale distale. Questo protocollo descrive una tecnica potente per manipolare le cellule staminali tramite un'impalcatura polimero conduttivo e crea un nuovo strumento per sviluppare ulteriormente la terapia a base di cellule staminali.

Lo schema illustrato nella Figura 1 rappresenta il flusso di lavoro complessivo della stimolazione elettrica dei hNPCs e potenziali applicazioni a valle. Una limitazione corrente nella terapia con cellule staminali è che le cellule staminali sono esposti a un ambiente di post-trapianto duro tra cui l'infiammazione e condizioni ischemiche. Queste difficili condizioni probabilmente limitano la loro efficacia terapeutica14,<su...

Watch this Scientific Journal Video about Electrically Conductive Scaffold to Modulate and Deliver Stem Cells at JoVE.com

Electrically Conductive Scaffold to Modulate and Deliver Stem Cells

Questo protocollo descrive la realizzazione di un sistema di coltura cellulare per consentire la semina delle cellule staminali su un'impalcatura polimero conduttivo per elettrostimolazione in vitro e successive in vivo l'impianto della cellula formativa-seminato impalcatura utilizzando un tecnica mininvasiva.

Elettricamente conduttivo impalcatura di modulare e fornire cellule staminali

Stem cell therapy has emerged as an exciting stroke therapeutic, but the optimal delivery method remains unclear. While the technique of microinjection ...

electrically-conductive-scaffold-to-modulate-and-deliver-stem-cells

Research

JoVE Journal

Bioengineering

12.4K Views.  Stanford University School of Medicine. Questo protocollo descrive la realizzazione di un sistema di coltura cellulare per consentire la semina delle cellule staminali su un'impalcatura polimero conduttivo per elettrostimolazione in vitro e successive in vivo l'impianto della cellula formativa-seminato impalcatura utilizzando un tecnica mininvasiva.

Video: Electrically Conductive Scaffold to Modulate and Deliver Stem Cells

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

L&#39;uso di cellule staminali per la rigenerazione ossea perivascolari

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

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Bioingegneria

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

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

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 ...

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(&#949;-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37&#160;oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged &#8211; the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

Ottimizzare Allegato di cellule staminali mesenchimali umane su Poli (ε-caprolattone) elettrofilate Filati

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

Elettrofilate nanofibre Ponteggi con gradazioni Organizzazione Fiber

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

3D Stem magnetica cellulare Aggregazione e bioreattore maturazione per la cartilagine Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated researchers in the stem cell and bioengineering fields to develop in vitro high-fidelity human myocardium for both basic research and clinical applications. However, the immature phenotype of cardiac cells is a limitation on obtaining tissues that functionally mimic the adult myocardium, which is mainly characterized by mechanical and electrical signals. Thus, the purpose of this protocol is to prepare and mature the target cell population through electromechanical stimulation, recapitulating physiological parameters. Cardiac tissue engineering is evolving toward more biological approaches, and strategies based on biophysical stimuli, thus, are gaining momentum. The device developed for this purpose is unique and allows individual or simultaneous electrical and mechanical stimulation, carefully characterized and validated. In addition, although the methodology has been optimized for this stimulator and a specific cell population, it can easily be adapted to other devices and cell lines. The results here offer evidence of the increased cardiac commitment of the cell population after electromechanical stimulation. Electromechanically stimulated cells show an increased expression of main cardiac markers, including early, structural, and calcium-regulating genes. This cell conditioning could be useful for further regenerative cell therapy, disease modeling, and high-throughput drug screening.

Here we present a protocol for training a cell population using electrical and mechanical stimuli emulating cardiac physiology. This electromechanical stimulation enhances the cardiomyogenic potential of the treated cells and is a promising strategy for further cell therapy, disease modeling, and drug screening.

Simultanea stimolazione elettrica e meccanica per migliorare il potenziale di cardiomiogenici delle cellule

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated ...

Construction and Use of an Electrical Stimulation Chamber for Enhancing Osteogenic Differentiation in Mesenchymal Stem/Stromal Cells In Vitro

Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) has been demonstrated to increase MSC osteogenic differentiation in vitro and promote bone healing in clinical settings. Here we describe the construction of an EStim cell culture chamber and its use in treating rat bone-marrow-derived MSC to enhance osteogenic differentiation. We found that treating MSCs with EStim for 7 days results in a significant increase in the osteogenic differentiation, and importantly, this pro-osteogenic effect persists long after (7 days) EStim is discontinued. This approach of pretreating MSCs with EStim to enhance osteogenic differentiation could be used to optimize bone tissue engineering treatment outcomes and, thus, help them to achieve their full therapeutic potential. In addition to this application, this EStim cell culture chamber and protocol can also be used to investigate other EStim-sensitive cell behaviors, such as migration, proliferation, apoptosis, and scaffold attachment.

Here we present a protocol for the construction of a cell culture chamber designed to expose cells to various types of electrical stimulation, and its use in treating mesenchymal stem cells to enhance osteogenic differentiation.

Costruzione e l'uso di una camera di stimolazione elettrica per migliorare la differenziazione osteogenica in cellule mesenchimali staminali/Stromal In Vitro

Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) ...

Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the major glial cells of the PNS, play a vital role in promoting PNS regeneration by dedifferentiating into a regenerative cell phenotype following injury. However, the dedifferentiated state of SCs is challenging to maintain through the time-period needed for regeneration and is impacted by changes in the surrounding extracellular matrix (ECM). Therefore, determining the complex interplay between SCs and differing ECM to provide cues of regenerative potential of SCs is essential. To address this, a strategy was created where different ECM proteins were adsorbed onto a tunable polydimethylsiloxane (PDMS) substrate which provided a platform where stiffness and protein composition can be modulated. SCs were seeded onto the tunable substrates and critical cellular functions representing the dynamics of SC phenotype were measured. To illustrate the interplay between SC protein expression and cellular morphology, differing seeding densities of SCs in addition to individual microcontact printed cellular patterns were utilized and characterized by immunofluorescence staining and western blot. Results showed that cells with a smaller spreading area and higher extent of cellular elongation promoted higher levels of SC regenerative phenotypic markers. This methodology not only begins to unravel the significant relationship between the ECM and cellular function of SCs, but also provides guidelines for the future optimization of biomaterials in peripheral nerve repair.

This methodology aims to illustrate the mechanisms by which extracellular matrix cues such as substrate stiffness, protein composition and cell morphology regulate Schwann cell (SC) phenotype.

Preparazione di microambienti a matrice extracellulare tunable per valutare schwann Cell Phenotype Specification

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the ...

Finite Element Modelling of a Cellular Electric Microenvironment

Clinical studies show electrical stimulation (ES) to be a potential therapy for the healing and regeneration of various tissues. Understanding the mechanisms of cell response when exposed to electrical fields can therefore guide the optimization of clinical applications. In vitro experiments aim to help uncover those, offering the advantage of wider input and output ranges that can be ethically and effectively assessed. However, the advancements in in vitro experiments are difficult to reproduce directly in clinical settings. Mainly, that is because the ES devices used in vitro differ significantly from the ones suitable for patient use, and the path from the electrodes to the targeted cells is different. Translating the in vitro results into in vivo procedures is therefore not straightforward. We emphasize that the cellular microenvironment's structure and physical properties play a determining role in the actual experimental testing conditions and suggest that measures of charge distribution can be used to bridge the gap between in vitro and in vivo. Considering this, we show how in silico finite element modelling (FEM) can be used to describe the cellular microenvironment and the changes generated by electric field (EF) exposure. We highlight how the EF couples with geometric structure to determine charge distribution. We then show the impact of time dependent inputs on charge movement. Finally, we demonstrate the relevance of our new in silico model methodology using two case studies: (i) in vitro fibrous Poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT-PSS) scaffolds and (ii) in vivo collagen in extracellular matrix (ECM).

This paper presents a strategy for building finite element models of fibrous conductive materials exposed to an electric field (EF). The models can be used to estimate the electrical input that cells seeded in such materials receive and assess the impact of changing the scaffold's constituent material properties, structure or orientation.

Modellazione agli elementi finiti di un microambiente elettrico cellulare

Clinical studies show electrical stimulation (ES) to be a potential therapy for the healing and regeneration of various tissues. Understanding the ...

Optimizing Extracellular Vesicle Delivery Using a Core-Sheath 3D-Bioprinted Scaffold for Chronic Wound Management

This study outlines a detailed protocol for the fabrication of core-sheath 3D-bioprinted scaffolds designed to enhance chronic wound healing. The protocol involves isolating extracellular vesicles (EVs) from mesenchymal stem cells (MSCs), known for their regenerative and immunomodulatory properties. These EVs are then incorporated into a unique scaffold structure. The scaffold features a core composed of alginate loaded with EVs, surrounded by a sheath made of carboxymethyl cellulose and alginate lyase. This innovative design ensures controlled scaffold degradation while promoting efficient and controlled release of EVs at the wound site. The protocol covers key steps, including the preparation and characterization of the EVs, the formulation of bio-inks for 3D bioprinting, and the optimization of printing parameters to achieve the desired core-sheath architecture. By combining structural integrity and bioactivity, the scaffold aims to address the limitations of conventional wound dressings, offering a targeted approach to accelerate tissue regeneration and reduce inflammation in chronic wounds. This method provides a reproducible and scalable strategy for developing advanced biomaterials with potential clinical applications in chronic wound management. The protocol also highlights critical considerations for achieving consistent results, ensuring adaptability for future therapeutic applications.

This study presents a protocol for fabricating core-sheath 3D-bio-printed scaffolds for chronic wound healing. Extracellular vesicles are isolated from mesenchymal stem cells, and loaded into the core (alginate) with the sheath made from carboxymethyl cellulose and alginate lyase. This design allows controlled scaffold degradation and efficient EVs release.

Ottimizzazione dell'erogazione di vescicole extracellulari utilizzando un'impalcatura biostampata in 3D con guaina centrale per la gestione delle ferite croniche

This study outlines a detailed protocol for the fabrication of core-sheath 3D-bioprinted scaffolds designed to enhance chronic wound healing. The protocol ...