Vorremmo ringraziare Jennifer Petrosino e David Disilvestro aiuto editoriale. Questa ricerca è stata sostenuta dal Premio numero 20020728 del consiglio Egg americano e Premio numero 10040042 da Novo Nordisk Farmaceutici, nonché dalla Food Innovation Center, Ufficio per gli Affari Internazionali, Center for Advanced Functional Foods ricerca, e l&#39;imprenditoria a OSU, nonché la National Science Foundation concessione CEE-0914790 (LJL). Il progetto descritto è stato sostenuto dal Premio Numero R21OD017244 (OZ) e UL1RR025755 (OSUCCC) dal Centro Nazionale per la Ricerca Risorse, finanziato dall&#39;Ufficio del Direttore, National Institutes of Health (OD) e sostenuto dal NIH tabella di marcia per la ricerca medica e NSC P30CA16058. Il contenuto è di esclusiva responsabilità degli autori e non rappresentano necessariamente il punto di vista ufficiale del Centro nazionale per le risorse di ricerca o il National Institutes of Health.

Il protocollo di studio è stato approvato da The Ohio State University comitati etici. Gli esperimenti sugli animali sono stati approvati dal protocollo IACUC. Tutte le procedure sono state eseguite sotto l&#39;armadio di livello 2 di biosicurezza con flusso laminare. Abbiamo seguito tutti i requisiti e le procedure di sicurezza standard. La tecnica di microincapsulazione per la preparazione di microcapsule è stata eseguita come descritto 17, 18. 1. Preparativi di materiali <ol><li> Preparare 10 ml di 2% di soluzione di alginato di sodio in soluzione salina fisiologica autoclavato (0,9% NaCl in acqua). Preparare 0,05% soluzione PLL in salina fisiologica. Preparare queste soluzioni il giorno prima e mescolare durante la notte. Filtrare le soluzioni con 0,22 micron filtro prima dell&#39;uso. </li><li> Preparare citrato di sodio 50 mM e 100 mM CaCl 2 0.9% soluzioni di NaCl e sterilizzare in autoclave. </li><li> Autoclavare tutti soluzioni, aghi, elettrodi, e bicchieri. Pulire accuratamente aghi puliti con fili per evitare intasamenti. </li><li> Determinare la quantità appropriata di cellule da utilizzare per le capsule (102 microcapsule sono necessari per ogni cm 2 di pozzo e ci sono circa 500 cellule per capsula 18). Usare 1 ml di alginato di sodio per due milioni di cellule. </li><li> Preparare una &#39;crescita dei fibroblasti Medium&#39; di siero di vitello contenente il 10%, e 100 U / ml di penicillina / streptomicina in un alto livello di glucosio (4.500 mg / l di glucosio) a medio Modified Eagle Dulbecco (DMEM). <ol><li> Preparare una &#39;Differenziazione medio I&#39; contenente 10% di siero fetale bovino, 10 ug / ml di insulina, 1 mM dexametasone, 0,5 mM xantina 3-isobutil-1-metil, e 100 U / ml di penicillina / streptomicina DMEM. </li><li> Preparare contenente siero un &#39;Differenziazione medio II&#39; 10% fetale bovino, 10 ug / ml di insulina, e 100 U / ml di penicillina / streptomicina DMEM. </li></ol></li><li> Preparare buffer di lisi contenente tablet inibitore della proteasi uno per 10 ml di Radio-Immunoprecipitazione tampone (RITampone PA). </li></ol> 2. alginato Microbeads Preparazione (Figura 1A) <ol><li> Rimuovere il vecchio mezzo da pallone di coltura cellulare. Lavare le cellule con 10 ml di PBS. Portare le cellule in sospensione con 0,25% tripsina-EDTA (2 ml per un pallone T175 confluenti). </li><li> Contare le cellule in sospensione di cellule utilizzando un emocitometro. Utilizzare 10 ml un&#39;aliquota dalla sospensione cellulare e contare le cellule secondo le istruzioni del produttore. Centrifugare le restanti cellule in terreno centrifugazione a 480 xg a temperatura ambiente per 5 min. </li><li> Sospendere il pellet cellulare in alginato di sodio, come descritto al punto 1.4. Trasferire la soluzione di sodio alginato-cella a una siringa da 5 ml. </li><li> Rimuovere le bolle d&#39;aria nella soluzione, aggiungere un ago 23-gauge e capovolgere la siringa per creare una tasca 1 ml di aria. </li><li> Mettere un piccolo bicchiere (180 ml) contenente 144 ml di CaCl mM soluzione 100 2 sotto il beccuccio ago della encapsulator. Collegare l&#39;elettrodo al encapsulator con la punta approximdiatamente 2,5 centimetri sopra la superficie della soluzione di CaCl 2 100 mM. </li><li> Inserire saldamente la siringa contenente la soluzione di alginato di sodio-cellule nella pompa a siringa. Attaccare il tubo di gomma per l&#39;apertura della siringa. Spingere lo stantuffo fino a quando la soluzione di alginato di sodio a cellule entra a metà del tubo. Regolare la tensione di 5,4 kV. Impostare il diametro 12,06 millimetri sulla pompa a siringa. Regolare la velocità di 3 ml / h. Avviare la pompa. Accendere encapsulator e mantenere la tensione a 5,4 kV. </li><li> Chiudere la finestra della cappa ed evitare inutili vibrazioni fino alla fine della formazione delle microsfere di alginato. Dopo tutta la soluzione passa attraverso l&#39;ago, solidificare microsfere sferiche alginato nella soluzione di CaCl 2 100 mM per ulteriori 20 minuti prima del rivestimento con PLL. </li></ol> 3. Microbeads rivestimento con PLL (Figura 1B) <ol><li> Rimuovere il bicchiere contenente le microsfere a forma di palla di sodio alginato-cellula e trasferire questi microbeads in una provetta da centrifuga da 50 ml. Rimuovere CaCl soluzione 2 dal pellet microperla. </li><li> Lavare le microsfere a forma di palla alginato di cellule aggiungendo 30 ml di NaCl allo 0,9%. Agitare il tubo a mano delicatamente. Rimuovere 0,9% NaCl con 25 ml pipetta dopo le microsfere sono precipitati per gravità. Ripetere altre due volte per un totale di 3 lavaggi. </li><li> Utilizzare 10 ml di soluzione PLL 0,05% per ogni 1 ml di soluzione di alginato di sodio. Aggiungere il PLL 0,05% e agitare a 1000 giri al minuto per 10 min. Nota: Di solito, 10 min è sufficiente per il rivestimento PLL. </li><li> Dopo si forma PLL cappotto, rimuovere la soluzione PLL e lavare le capsule 3 volte come descritto al punto 3.2. </li></ol> 4. Rimozione di alginato core (Figura 1C) <ol><li> Aggiungere 30 ml di 50 mM soluzione di citrato di sodio. Attendere 5 minuti o fino a quando tutta l&#39;alginato di sodio è disciolto. Lavare capsule tre volte come descritto al punto 3.1.1. </li><li> Rimuovere il 0,9% NaCl, aggiungere 20 ml di mezzo di coltura al tubo 50 mle trasferire tutte le capsule contenenti le cellule in un pallone di coltura cellulare. Maneggiare cellule incapsulate in condizioni standard di coltura cellulare 18. </li></ol> 5. Le domande vitro per lo studio xenotrapianto e Host cellulari interazioni o Cinetica di Metabolita Afflusso / Efflux tra cellule (Figura 2) <ol><li> Cellule ospiti Cultura il 24 pozzetti fino confluenti per co-culture. Utilizzare il &#39;fibroblasti crescita medio&#39; per preadipocytes coltura. </li><li> Trasferire microcapsule in 24 piastra contenente cellule ospiti confluenti bene. Aggiungere microcapsule per ottenere un monostrato (102 microcapsule / cm 2 di pozzo). </li><li> Indurre la differenziazione pre-adipociti con Differenziazione Media I. Ogni 48 ore, cambio media differenziazione Media II per sei giorni. Lisare le cellule in tampone RIPA. Utilizzare 50 proteine ​​g per condizione di analizzare l&#39;espressione proteica con Western Blot. </li></ol> 6. In Vivo Domanda di Trattento di obesità (figura 3) <ol><li> Mescolare 4% isoflurano con l&#39;ossigeno per l&#39;induzione di anestesia e 2% isoflurano con ossigeno per il mantenimento dell&#39;anestesia. Confermare adeguata profondità dell&#39;anestesia per pizzico piedi. <ol><li> Applicare una pomata anti-prurito sugli occhi per proteggere le cornee si secchi. </li></ol></li><li> Utilizzare cellule incapsulate che sono etichettati in modo permanente con proteina fluorescente artificiale, come la proteina fluorescente verde (GFP). </li><li> Utilizzare una siringa da 3 ml e un ago calibro 20 per iniettare cellule incapsulate. <ol><li> Iniettare 0,5 x 10 6 cellule sospese in 0,2 ml di soluzione in ciascuno depot grasso catetere che si trova nella zona intraperitoneale tra un gonadi e reni come mostrato in figura 3. Utilizzare questo volume di PBS e il numero di cellule per topo con un peso medio di 40 g. Regolare numero di cellule e volume in topi che hanno peso diverso o per la determinazione degli effetti dose-dipendente. </li></ol></li></ol>

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No conflicts of interest declared.

Vari metodi sono stati utilizzati per incapsulare cellule, comprese l&#39;essiccazione, estrusione, ed emulsione 19. In questo metodo, le perline alginato vengono estrusi attraverso un ago, poi rivestito con PLL e il nucleo alginato sarà sciolto per completare l&#39;incapsulamento. Anche se questo metodo è stato usato per anni, formazione delle perline con la dimensione desiderata e la forma sferica è ancora difficile. La dimensione delle capsule è fortemente dipendente dalla viscosità della soluzione di...

L&#39;aumento dell&#39;incidenza delle malattie croniche 1 ha stimolato studi sul trapianto di popolazioni di cellule terapeutiche 2. Le cellule staminali singenici o allogeniche sono i tipi di cellule più comunemente usati per queste applicazioni 2. Tuttavia, questi trattamenti non consentono il controllo della differenziazione e migrazione delle cellule staminali dopo l&#39;impianto e non sono convenienti. Il trapianto di cellule geneticamente modificate con funzioni benefiche anticipa migliorare il trattamento di molte malattie. Tuttavia, le modifiche genetiche di cellule vengono riconosciuti dal sistema immunitario dell&#39;ospite, quindi, questi trattamenti richiedono immunosoppressione 3. L&#39;incapsulamento di cellule che producono l&#39;insulina è stato sviluppato dal Dr. Chang 4. La tecnica si basa sulla incapsulamento delle cellule in goccioline alginato che vengono immerse in una soluzione di cloruro di calcio. Molecole alginato consistono di acido mannuronico (M) e guluronico (G) e possono essere collegati da Ca 2+. Dopo gelificazione, le perle sono sospese una soluzione di poli-L-lisina (PLL). Durante questa fase, PLL lega al G e M nelle molecole alginato che stabilisce membrana della capsula. La porosità della membrana della capsula può essere modulata variando le concentrazioni M e PLL, il tempo di incubazione, e la temperatura. Il legame del PLL dipende anche dal tipo e dalla concentrazione di alginato. Matrici alginato reticolato con ioni Ca 2+, sono instabili in ambiente fisiologico o in soluzioni tampone comuni con alta concentrazione di fosfato e ioni citrato. Questi buffer possono estrarre Ca 2+ dal alginato e liquefare il nucleo. Liquefazione del nucleo alginato fornisce spazio all&#39;interno delle capsule per il movimento e la crescita cellulare. Cellule incapsulate in alginato polyanionic con policationico poli-L-lisina (APL) sono impermeabili per immunoglobuline ma hanno afflusso di nutrienti e l&#39;efflusso di tossine. Queste proprietà consentono APL lungo termine survival di cellule incapsulate dopo il trapianto in host geneticamente diverse. Elliott et al. Riportato la sopravvivenza di funzionamento delle cellule pancreatiche suina incapsulati in un paziente umano nove anni dopo l&#39;impianto 5. Tecniche di incapsulamento possono essere classificati in microincapsulazione (3-800 micron) e macroincapsulazione (maggiore di 1000 micron). Le microcapsule sono più durevoli macrocapsules 6. Fin dalla sua scoperta dal Dr. Chang e colleghi nel 1964, microincapsulazione è stato ampiamente utilizzato per l&#39;incapsulamento delle cellule anabolizzanti che producono insulina, altri ormoni e molecole bioattive 7. Questi trattamenti hanno affrontato diverse sfide nel tessuto ospite tra cui la fibrosi e la risposta immunitaria 8. Inizialmente, gli effetti collaterali legati alla qualità dei biopolimeri sono stati risolti. Tuttavia, il trapianto di cellule anabolizzanti avvia ancora effetti collaterali, quali la fibrosi, come risultato di ormone overproduzione di fuori di una ghiandola specializzata. Negli ultimi decenni, l&#39;obesità e il diabete di tipo 2 ha raggiunto proporzioni epidemiche 9. Più del 30% delle persone adulte in tutto il mondo sono in sovrappeso e obesi 10. Aumento intra-addominale (IAB) la formazione di grasso aumenta l&#39;incidenza di infiammazione cronica e promuove diabete di tipo 2, le malattie cardiovascolari, alcuni tipi di cancro, e di altre patologie 11-13. Molte evidenze suggeriscono che la patogenesi associato con il grasso IAB può essere evitato dagli adipociti specifici. Recenti studi hanno dimostrato che il trapianto di adipociti sottocutanei in regione IAB può migliorare il metabolismo e ridurre l&#39;obesità e insulino-resistenza nei roditori in vivo 14. Efficace riduzione di obesità e insulino resistenza è stata associata con adipociti termogenica grado di dissipare l&#39;energia sotto forma di calore 15,16. Modifica termogenica adipociti può essere ottenuto mediante trasfezione stabiledi geni che partecipano al disaccoppiamento protonico mitocondriale, come la proteina di disaccoppiamento 1 (Ucp1) o di geni che regolano l&#39;espressione di altri geni Ucp1 e termogenica 15,16. Nostri studi recenti hanno dimostrato che la deficienza in aldeide deidrogenasi 1 a1 (Aldh1a1) porta al rimodellamento termogenico di grasso IAB che riduce l&#39;obesità e insulino-resistenza in questi topi 17,18. In particolare, l&#39;incapsulamento di termogenico Aldh1a1 carente (Aldh1a1 - / -) preadipocytes media stesso effetto terapeutico di grassi IAB in obesi topi wild-type, suggerendo nuove opportunità terapeutiche per il trattamento di IAB grasso 18. Nelle impostazioni sperimentali, le cellule incapsulate permettono ai ricercatori di studiare gli effetti di specifiche popolazioni cellulari in modo economicamente vantaggioso 19. Qui si discute il metodo di incapsulamento di una linea cellulare catabolico termogenica e il suo laboratorio e applicazione terapeutica in un modello murino di obesità. Il protocollo descrive three fasi per la produzione di microcapsule (Figura 1): la formazione delle microsfere di alginato (Figura 1A), la formazione del policationico poli-L-lisina (PLL) membrane sulla superficie di microsfere (Figura 1B), e la rimozione del nuclei di alginato (Figura 1C).

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Encapsulation device (VAR V1)</td>
			<td>Nisco</td>
			<td>LIN-0042</td>
			<td>None</td>
		</tr>
		<tr>
			<td>KD scientific syringe pump</td>
			<td>KD scientific</td>
			<td>780100Y</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Olympus microscope&#160;</td>
			<td>Olympus Optical</td>
			<td>IX70-S8F2</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Sodium alginate</td>
			<td>Sigma</td>
			<td>MKBP8122V</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Poly-l-lysine hydrobromide (PLL)</td>
			<td>Sigma</td>
			<td>020M5006V</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma</td>
			<td>SLBJ2662V</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Sodium citrate tribasic dihydrate</td>
			<td>Sigma</td>
			<td>030M0200</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma</td>
			<td>SLBD2595V</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Mini-PROTEAN TGX Gels</td>
			<td>Bio-Rad</td>
			<td>456-1093</td>
			<td>None</td>
		</tr>
		<tr>
			<td>ATGL primary antibody (from rabbit)</td>
			<td>Cell Signaling</td>
			<td>2138S</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Secondary anti body (anti rabbit)</td>
			<td>LI-COR</td>
			<td>926-68071</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Radio-Immunoprecipitation Assay (RIPA) buffer</td>
			<td>Boston BioProducts</td>
			<td>D25Y6Z</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma</td>
			<td>RNBD2893</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Cortizone 10 anti-itch ointment</td>
			<td>Cortizone 10</td>
			<td>C4029138</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Newborn calf serum (CS)</td>
			<td>Sigma</td>
			<td>N4762</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Sigma</td>
			<td>F4135</td>
			<td>None</td>
		</tr>
		<tr>
			<td>3-Isobutyl-1-methylxanthine (IBMX)</td>
			<td>Sigma</td>
			<td>I0516</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma</td>
			<td>D4902</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Insulin (bovine)</td>
			<td>Sigma</td>
			<td>I5879</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail tablets</td>
			<td>Roche</td>
			<td>4693159001</td>
			<td>None</td>
		</tr>
	</tbody>
</table>

encapsulation thermogenic preadipocytes for transplantation into adipose tissue depots

Incapsulamento delle cellule è stato sviluppato per intrappolare cellule vitali all&#39;interno delle membrane semi-permeabili. Le cellule incapsulate trapiantate possono scambiare metaboliti a basso peso molecolare in tessuti dell&#39;ospite trattato per conseguire sopravvivenza a lungo termine. La membrana semipermeabile permette innestato cellule incapsulate per evitare il rigetto da parte del sistema immunitario. La procedura di incapsulamento è stato progettato per consentire un rilascio controllato di composti bioattivi, come l&#39;insulina, altri ormoni e citochine. Qui si descrive un metodo per l&#39;incapsulamento di cellule catabolici, che consumano lipidi per la produzione di calore e la dissipazione di energia (termogenesi) nel tessuto adiposo intra-addominale di topi obesi. L&#39;incapsulamento di cellule catabolici termogenica potrebbe essere potenzialmente applicabili alla prevenzione e il trattamento dell&#39;obesità e il diabete di tipo 2. Un&#39;altra potenziale applicazione di cellule catabolici può includere la disintossicazione da alcool o altri metaboliti tossici e inquinanti ambientali.

La Figura 1 mostra che ogni fase della produzione microsfere può essere controllato al microscopio. La Figura 2A mostra come co-coltura adipociti con un monostrato di cellule incapsulate. Figura 2B è un esempio rappresentativo di uno studio quantitativa utilizzando adipociti / microcapsule co-culture sono stati descritti nella sezione 5. Lisati degli adipociti sono stati analizzati usando Western Blot. Cellule incapsulate non sono state analizzate in questo esperiment...

Watch this Scientific Journal Video about Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots at JoVE.com

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

Incapsulamento termogenico preadipocytes al trapianto nel tessuto adiposo Depositi tessuti

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

encapsulation-thermogenic-preadipocytes-for-transplantation-into

Research

JoVE Journal

Medicine

9.2K Views.  The Ohio State University. Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

Video: Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability in industrialized countries. Replacement of degenerated photoreceptors by cell transplantation represents a possible treatment option in future clinical applications. Indeed, recent preclinical studies demonstrated that immature photoreceptors, isolated from the neonatal mouse retina at postnatal day 4, have the potential to integrate into the adult mouse retina following subretinal transplantation. Donor cells generated a mature photoreceptor morphology including inner and outer segments, a round cell body located at the outer nuclear layer, and synaptic terminals in close proximity to endogenous bipolar cells. Indeed, recent reports demonstrated that donor photoreceptors functionally integrate into the neural circuitry of host mice. For a future clinical application of such cell replacement approach, purified suspensions of the cells of choice have to be generated and placed at the correct position for proper integration into the eye. For the enrichment of photoreceptor precursors, sorting should be based on specific cell surface antigens to avoid genetic reporter modification of donor cells. Here we show magnetic-associated cell sorting (MACS) - enrichment of transplantable rod photoreceptor precursors isolated from the neonatal retina of photoreceptor-specific reporter mice based on the cell surface marker CD73. Incubation with anti-CD73 antibodies followed by micro-bead conjugated secondary antibodies allowed the enrichment of rod photoreceptor precursors by MACS to approximately 90%. In comparison to flow cytometry, MACS has the advantage that it can be easier applied to GMP standards and that high amounts of cells can be sorted in relative short time periods. Injection of enriched cell suspensions into the subretinal space of adult wild-type mice resulted in a 3-fold higher integration rate compared to unsorted cell suspensions.

Cell transplantation represents a strategy for the treatment of retinal degeneration characterized by photoreceptor loss. Here we describe a method for enrichment of transplantable photoreceptors and their subretinal grafting into adult mice.

Trapianto subretinale di cellule precursori del fotorecettore purificato MACS nella retina del topo adulto

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability ...

Ricerca

Medicina

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

Il trapianto di valvola polmonare Utilizzando un modello murino di trapianto cardiaco eterotopico

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely related to metabolism and energy expenditure, and its dysfunction is one important contributor for obesity and diabetes. Contrary to previous belief, recent PET/CT imaging studies indicated the BAT depots are still present in human adults. PET imaging clearly shows that BAT has considerably high uptake of 18F-FDG under certain conditions. In this video report, we demonstrate that Cerenkov luminescence imaging (CLI) with 18F-FDG can be used to optically image BAT in small animals. BAT activation is observed after intraperitoneal injection of norepinephrine (NE) and cold treatment, and depression of BAT is induced by long anesthesia. Using multiple-filter Cerenkov luminescence imaging, spectral unmixing and 3D imaging reconstruction are demonstrated. Our results suggest that CLI with 18F-FDG is a practical technique for imaging BAT in small animals, and this technique can be used as a cheap, fast, and alternative imaging tool for BAT research.

In this video report, we show the application of Cerenkov Luminescence Imaging (CLI) for interscapular brown adipose tissue in mice under activated and depressed conditions.

Cerenkov Luminescenza Imaging di interscapolare Brown Tessuto adiposo

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely ...

Localization, Identification, and Excision of Murine Adipose Depots

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 reached a staggering sum of $190.2 billion in direct medical costs alone. Obesity is a major risk factor for a wide host of diseases. Historically, little was known regarding adipose and its major and essential functions in the body. Brown and white adipose are the two main types of adipose but current literature has identified a new type of fat called brite or beige adipose. Research has shown that adipose depots have specific metabolic profiles and certain depots allow for a propensity for obesity and other related disorders. The goal of this protocol is to provide researchers the capacity to identify and excise adipose depots that will allow for the analysis of different factorial effects on adipose; as well as the beneficial or detrimental role adipose plays in disease and overall health. Isolation and excision of adipose depots allows investigators to look at gross morphological changes as well as histological changes. The adipose isolated can also be used for molecular studies to evaluate transcriptional and translational change or for in vitro experimentation to discover targets of interest and mechanisms of action. This technique is superior to other published techniques due to the design allowing for isolation of multiple depots with simplicity and minimal contamination.

Due to the drastic and negative connection between obesity and other comorbidities, research on the role adipose plays in disease and overall health is warranted. We present a protocol for the isolation and excision of adipose depots allowing for the study of adipose using in situ and in vitro methods.

Localizzazione, identificazione, e l&#39;escissione dei Depositi Murine adipose

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 ...

Human Brown Adipose Tissue Depots Automatically Segmented by Positron Emission Tomography/Computed Tomography and Registered Magnetic Resonance Images

Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in humans. Detecting BAT is typically confirmed by the uptake of the injected radioactive tracer 18F-Fluorodeoxyglucose (18F-FDG) into adipose tissue depots, as measured by positron emission tomography/computed tomography (PET-CT) scans after exposing the subject to cold stimulus. Fat-water separated magnetic resonance imaging (MRI) has the ability to distinguish BAT without the use of a radioactive tracer. To date, MRI of BAT in adult humans has not been co-registered with cold-activated PET-CT. Therefore, this protocol uses 18F-FDG PET-CT scans to automatically generate a BAT mask, which is then applied to co-registered MRI scans of the same subject. This approach enables measurement of quantitative MRI properties of BAT without manual segmentation. BAT masks are created from two PET-CT scans: after exposure for 2 hr to either thermoneutral (TN) (24 &#176;C) or cold-activated (CA) (17 &#176;C) conditions. The TN and CA PET-CT scans are registered, and the PET standardized uptake and CT Hounsfield values are used to create a mask containing only BAT. CA and TN MRI scans are also acquired on the same subject and registered to the PET-CT scans in order to establish quantitative MRI properties within the automatically defined BAT mask. An advantage of this approach is that the segmentation is completely automated and is based on widely accepted methods for identification of activated BAT (PET-CT). The quantitative MRI properties of BAT established using this protocol can serve as the basis for an MRI-only BAT examination that avoids the radiation associated with PET-CT.

The method presented here uses 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET-CT) and fat-water separated magnetic resonance imaging (MRI), each scanned following 2 hr exposure to thermoneutral (24 &#176;C) and cold conditions (17 &#176;C) in order to map brown adipose tissue (BAT) in adult human subjects.

Umano Brown adipose Depositi tessuti segmentato automaticamente da Positron Emission Tomography / tomografia computerizzata e registrati Risonanza Magnetica Images

Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in ...

Transplantation Into the Mouse Ovarian Fat Pad

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic transplantation of ovarian surface and tubal epithelia include intraperitoneal and intrabursal administration of cells. The respective limitations of these methods include poorly defined location of injected cells and limited space volume. Furthermore, they are poorly suited for long-term structural preservation of transplanted organs. To address these challenges, we have developed an alternative approach, which is based on the introduction of cells and tissue fragments into the mouse fat pad. The mouse ovarian fat pad is located in the immediate vicinity of the ovary and uterine tube (aka oviduct, fallopian tube), and provides a familiar microenvironment for cells and tissues of these organs. In our approach fluorescence-labeled mouse and human cells, and fragments of the uterine tube are engrafted by using minimally traumatic dorsal incision surgery. Transplanted cells and their outgrowths are easily located in the ovarian fat pad for over 40 days. Long-term transplantation of the entire uterine tube allows correct preservation of all principle tissue components, and does not result in adverse side effects, such as fibrosis and inflammation. Our approach should be uniquely applicable for answering important biological questions such as differentiation, regenerative and neoplastic potential of specific cell populations. Furthermore, it should be suitable for studies of microenvironmental factors in normal development and cancer.

We describe an ovarian fad pad transplantation assay suitable for studies of normal and transformed epithelia of the female reproductive tract. The mouse fat pad allows transplantation of large tissue fragments, is easily accessible for surgery and imaging, and provides the most favorable native environment for tissues of M&#252;llerian origin.

Trapianto nel Mouse Pad ovarico Fat

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic ...

A Technique for Subcutaneous Abdominal Adipose Tissue Biopsy via a Non-diathermy Method

Adipose tissue biopsies offer tissue samples that, upon analysis, may provide insightful overviews of mechanisms relating to metabolism and disease. To obtain subcutaneous adipose tissue biopsies in the abdominal area, researchers and physicians use either a surgical or a needle-based technique. However, surgical subcutaneous fat biopsies can offer tissue samples that may provide a more comprehensive overview of the complexities of biological indices in white adipose tissue. Usually, a surgical adipose tissue biopsy includes a diathermy treatment for cauterizing blood vessels to prevent excessive bleeding. Nevertheless, side effects, such as flash fires and skin lesions in the tissue, have been reported after diathermy. Therefore, we aimed to standardize a surgical abdominal adipose tissue biopsy performed under local anesthesia using a non-diathermy method. We conducted 115 subcutaneous adipose tissue biopsies in healthy men using a non-diathermy abdominal surgical biopsy method. Our results showed three cases of excessive post-operation bleeding out of 115 operations (2.61%).In conclusion, our standardized subcutaneous abdominal adipose tissue surgical biopsy using a non-diathermy method can be safely applied to healthy men at the bedside, with minimal side effects.

We standardized an abdominal adipose tissue biopsy using a non-diathermy method performed under local anesthesia. Three cases of excessive post-operation bleeding out of 115 operations (2.61%) occurred.We conclude that an abdominal adipose tissue surgical biopsy using a non-diathermy method can be safely applied to healthy men.

Una tecnica per la biopsia del tessuto adiposo sottocutaneo addominale tramite un metodo Non-diatermia

Adipose tissue biopsies offer tissue samples that, upon analysis, may provide insightful overviews of mechanisms relating to metabolism and disease. To ...

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 &#181;m internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

Valutazione della funzione microvascolare di tessuto adiposo umano mediante videomicroscopia

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. ...

Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of clinical efficacy, they are until now imperfect solutions. Stem cell therapy using ASC sheets could provide a solution to this problem. ASCs are considered as promising candidates for promoting tissue healing because of their trophic and immunomodulatory properties. Here, we provide methods to produce high-density ASC sheets, that are transplanted onto a colorectal anastomosis in a rat model to reduce the leakage. ASCs formed cell sheets in thermo-responsive culture dishes that could be easily detached. On the day of the transplantation, a partial colectomy with a 5-suture colorectal anastomosis was performed. Animals were immediately transplanted with 1 ASC sheet per rat. ASC sheets adhered spontaneously to the anastomosis without any glue, suture, or any biomaterial. Animal groups were sacrificed 3 and 7 days postoperatively. Compared to transplanted animals, the incidence of anastomotic abscesses and leakage was higher in control animals. In our model, the transplantation of ASC sheets after colorectal anastomosis was successful and associated with a lower leakage rate.

The preparation and transplantation of adipose tissue derived stem cell (ASC) sheets onto insufficiently sutured colorectal anastomoses in a rat model is presented. This novel application shows that ASC sheets can reduce colorectal anastomosis leakage.

Trapianto di cellule staminali di tessuto adiposo-derivato foglio per ridurre le perdite dopo la colectomia parziale in un modello del ratto

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of ...

Clinical Protocol of Producing Adipose Tissue-Derived Stromal Vascular Fraction for Potential Cartilage Regeneration

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the knees by using different forms of mesenchymal stem cells (MSCs). In Korea, bone marrow concentrates and cord blood-derived stem cells have been approved by the Korean Food and Drug Administration (KFDA) for cartilage regeneration. In addition, an adipose tissue-derived stromal vascular fraction (SVF) has been allowed by the KFDA for joint injections in human patients. Autologous adipose tissue-derived SVF contains extracellular matrix (ECM) in addition to mesenchymal stem cells. ECM excretes various cytokines that, along with hyaluronic acid (HA) and platelet-rich plasma (PRP) activated by calcium chloride, may help MSCs to regenerate cartilage and improve knee functions. In this article, we presented a protocol to improve knee functions by regenerating cartilage-like tissue in human patients with OA. The result of the protocol was first reported in 2011 followed by a few additional publications. The protocol involves liposuction to obtain autologous lipoaspirates that are mixed with collagenase. This lipoaspirates-collagenase mixture is then cut and homogenized to remove large fibrous tissue that may clog up the needle during the injection. Afterwards, the mixture is incubated to obtain adipose tissue-derived SVF. The resulting adipose tissue-derived SVF, containing both adipose tissue-derived MSCs and remnants of ECM, is injected into knees of patients, combined with HA and calcium chloride activated PRP. Included are three cases of patients who were treated with our protocol resulting in improvement of knee pain, swelling, and range of motion along with MRI evidence of hyaline cartilage-like tissue.

Here, we present a protocol to produce an adipose tissue-derived stromal vascular fraction and its application to improve knee functions by regenerating cartilage-like tissue in human patients with osteoarthritis.

Protocollo clinico di produrre tessuto adiposo-derivato Stromal frazione vascolare per potenziale rigenerazione della cartilagine

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the ...