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><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&amp;nbsp;</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&#039;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

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

9.3K 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

A Microphysiologic Platform for Human Fat: Sandwiched White Adipose Tissue

White adipose tissue (WAT) plays a crucial role in regulating weight and everyday health. Still, there are significant limitations to available primary culture models, all of which have failed to faithfully recapitulate the adipose microenvironment or extend WAT viability beyond two weeks. The lack of a reliable primary culture model severely impedes research in WAT metabolism and drug development. To this end we have utilized NIH's standards of a microphysiologic system to develop a novel platform for WAT primary culture called 'SWAT' (sandwiched white adipose tissue). We overcome the natural buoyancy of adipocytes by sandwiching minced WAT clusters between sheets of adipose-derived stromal cells. In this construct, WAT samples are viable over eight weeks in culture. SWAT maintains the intact ECM, cell-to-cell contacts, and physical pressures of in vivo WAT conditions; additionally, SWAT maintains a robust transcriptional profile, sensitivity to exogenous chemical signaling, and whole tissue function. SWAT represents a simple, reproducible, and effective method of primary adipose culture. Potentially, it is a broadly applicable platform for research in WAT physiology, pathophysiology, metabolism, and pharmaceutical development.

White adipose tissue (WAT) has critical deficiencies in its current primary culture models, hindering pharmacological development and metabolic studies. Here, we present a protocol to produce an adipose microphysiological system by sandwiching WAT between sheets of stromal cells. This construct provides a stable and adaptable platform for primary WAT culture.

Una piattaforma di Microphysiologic per grasso umano: Sandwich tessuto adiposo bianco

White adipose tissue (WAT) plays a crucial role in regulating weight and everyday health.  Still, there are significant limitations to available primary ...

Ricerca

Comportamento

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease.
This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) &#62;35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2&#8211;3 g of fat. These cells can be expanded in culture over 10&#8211;14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5&#8211;6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPAR&#947; agonist-rosiglitazone.
This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

Isolamento, espansione e adipogenico induzione di CD34 + CD31 + cellule endoteliali dal tessuto adiposo umano omentale e sottocutaneo

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...

Immunologia e infezioni

Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

Brown adipocytes have the ability to uncouple the respiratory chain in mitochondria and dissipate chemical energy as heat. Development of UCP1-positive brown adipocytes in white adipose tissues (so called beige or brite cells) is highly induced by a variety of environmental cues such as chronic cold exposure or by PPAR&gamma; agonists, therefore, this cell type has potential as a therapeutic target for obesity treatment. Although most immortalized adipocyte lines cannot recapitulate the process of "browning" of white fat in culture, primary adipocytes isolated from stromal vascular fraction in subcutaneous white adipose tissue (WAT) provide a reliable cellular system to study the molecular control of beige/brite cell development. Here we describe a protocol for effective isolation of primary preadipocytes and for inducing differentiation to beige/brite cells in culture. The browning effect can be assessed by the expression of brown fat-selective markers such as UCP1.

Primary white preadipocytes isolated from white adipose tissues in mice can be differentiated into beige/brite cells. Presented here is a reliable cellular model system to study the molecular regulation of "browning" of white fat.

Isolamento e la differenziazione di cellule stromali vascolari alle celle Beige / Brite

Brown adipocytes have the ability to uncouple the  respiratory chain in mitochondria and dissipate chemical energy as heat.  Development of UCP1-positive ...

Biologia

Three-Dimensional Culture of Vascularized Thermogenic Adipose Tissue from Microvascular Fragments

Engineering thermogenic adipose tissue (e.g., beige or brown adipose tissues) has been investigated as a potential therapy for metabolic diseases or for the design of personalized microtissues for health screening and drug testing. Current strategies are often quite complex and fail to accurately fully depict the multicellular and functional properties of thermogenic adipose tissue. Microvascular fragments, small intact microvessels comprised of arteriole, venules, and capillaries isolated from adipose tissue, serve as a single autologous source of cells that enable vascularization and adipose tissue formation. This article describes methods for optimizing culture conditions to enable the generation of three-dimensional, vascularized, and functional thermogenic adipose tissues from microvascular fragments, including protocols for isolating microvascular fragments from adipose tissue and culture conditions. Additionally, best practices are discussed, as are techniques for characterizing the engineered tissues, and sample results from both rodent and human microvascular fragments are provided. This approach has the potential to be utilized for the understanding and development of treatments for obesity and metabolic disease.

Here, we present a detailed protocol outlining the use of microvascular fragments isolated from rodent or human fat tissue as a straightforward approach to engineer functional, vascularized beige adipose tissue.

Coltura tridimensionale di tessuto adiposo termogenico vascolarizzato da frammenti microvascolari

Engineering thermogenic adipose tissue (e.g., beige or brown adipose tissues) has been investigated as a potential therapy for metabolic diseases or for ...

Bioingegneria

Fat-Covered Islet Transplantation Using Epididymal White Adipose Tissue

Islet transplantation is a cellular replacement therapy for severe diabetes mellitus. The intraperitoneal cavity is typically the transplant site for this procedure. However, intraperitoneal islet transplantation has some limitations, including poor transplant efficacy, difficult graft detection ability, and a lack of graftectomy capability for post-transplant analysis. In this paper, "fat-covered islet transplantation", an intraperitoneal islet transplantation method that utilizes epididymal white adipose tissue, is used to assess the therapeutic effects of bioengineered islets. The simplicity of the method lies in the seeding of islets onto epididymal white adipose tissue and using the tissue to cover the islets. While this method can be categorized as an intraperitoneal islet transplantation technique, it shares characteristics with intra-adipose tissue islet transplantation. The fat-covered islet transplantation method demonstrates more robust therapeutic effects than intra-adipose tissue islet transplantation, however, including the improvement of blood glucose and plasma insulin levels and the potential for graft removal. We recommend the adoption of this method for assessing the mechanisms of islet engraftment into white adipose tissue and the therapeutic effects of bioengineered islets.

This fat-covered islet transplantation method is suitable for the detection of engrafted islets in the intraperitoneal cavity. Notably, it does not require the use of biobinding agents or suturing.

Trapianto di isole ricoperte di grasso utilizzando tessuto adiposo bianco epididimo

Islet transplantation is a cellular replacement therapy for severe diabetes mellitus. The intraperitoneal cavity is typically the transplant site for this ...

Medicina

Human Adipose Tissue Micro-fragmentation for Cell Phenotyping and Secretome Characterization

In the past decade, adipose tissue transplants have been widely used in plastic surgery and orthopaedics to enhance tissue repletion and/or regeneration. Accordingly, techniques for harvesting and processing human adipose tissue have evolved in order to quickly and efficiently obtain large amounts of tissue. Among these, the closed system technology represents an innovative and easy-to-use system to harvest, process, and re-inject refined fat tissue in a short time and in the same intervention (intra-operatively). Adipose tissue is collected by liposuction, washed, emulsified, rinsed and minced mechanically into cell clusters of 0.3 to 0.8 mm. Autologous transplantation of mechanically fragmented adipose tissue has shown remarkable efficacy in different therapeutic indications such as aesthetic medicine and surgery, orthopedic and general surgery. Characterization of micro-fragmented adipose tissue revealed the presence of intact small vessels within the adipocyte clusters; hence, the perivascular niche is left unperturbed. These clusters are enriched in perivascular cells (i.e., mesenchymal stem cell (MSC) ancestors) and in vitro analysis showed an increased release of growth factors and cytokines involved in tissue repair and regeneration, compared to enzymatically derived MSCs. This suggests that the superior therapeutic potential of microfragmented adipose tissue is explained by a higher frequency of presumptive MSCs and enhanced secretory activity. Whether these added pericytes directly contribute to higher growth factor and cytokine production is not known. This clinically approved procedure allows the transplantation of presumptive MSCs without the need for expansion and/or enzymatic treatment, thus bypassing the requirements of GMP guidelines, and reducing the costs for cell-based therapies.

Here, we present human adipose tissue enzyme-free micro-fragmentation using a closed system device. This new method allows the obtainment of sub-millimeter clusters of adipose tissue suitable for in vivo transplantation, in vitro culture, and further cell isolation and characterization.

Tessuto Adiposo Umano Microframmentazione per la Fenotipizzazione Cellulare e la Caratterizzazione del Secretome

In the past decade, adipose tissue transplants have been widely used in plastic surgery and orthopaedics to enhance tissue repletion and/or regeneration. ...

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident adipose progenitor cells (APCs) play a key role in adipose tissue homeostasis and can contribute to the tissue pathology. The growing use of single cell analysis technologies &#8211; including single-cell RNA-sequencing and single-cell proteomics &#8211; is transforming the stem/progenitor cell field by permitting unprecedented resolution of individual cell expression changes within the context of population- or tissue-wide changes. In this article, we provide detailed protocols to dissect mouse epididymal adipose tissue, isolate single adipose tissue-derived cells, and perform fluorescence activated cell sorting (FACS) to enrich for viable Sca1+/CD31-/CD45-/Ter119- APCs. These protocols will allow investigators to prepare high quality APCs suitable for downstream analyses such as single cell RNA sequencing.

We present a simple method to isolate highly viable adipose progenitor cells from mouse epididymal fat pads using fluorescence activated cell sorting.

Preparazione di cellule progenitrici adipose dai tessuti adiposi epididimali del topo

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident ...

<meta charset="utf8"></head><body>SATMVEC umane: isolamento e studi cross-talk con gli adipociti

Studying Adipose Endothelial Cell/Adipocyte Cross-Talk in Human Subcutaneous Adipose Tissue

Microvascular endothelial cells (MVECs) have many critical roles, including control of vascular tone, regulation of thrombosis, and angiogenesis. Significant heterogeneity in endothelial cell (EC) genotype and phenotype depends on their vascular bed and host disease state. The ability to isolate MVECs from tissue-specific vascular beds and individual patient groups offers the opportunity to directly compare MVEC function in different disease states. Here, using subcutaneous adipose tissue (SAT) taken at the time of insertion of cardiac implantable electronic devices (CIED), we describe a method for the isolation of a pure population of functional human subcutaneous adipose tissue MVEC (hSATMVEC) and an experimental model of hSATMVEC-adipocyte cross-talk.
hSATMVEC were isolated following enzymatic digestion of SAT by incubation with anti-CD31 antibody-coated magnetic beads and passage through magnetic columns. hSATMVEC were grown and passaged on gelatin-coated plates. Experiments used cells at passages 2-4. Cells maintained classic features of EC morphology until at least passage 5. Flow cytometric assessment showed 99.5% purity of isolated hSATMVEC, defined as CD31+/CD144+/CD45-. Isolated hSATMVEC from controls had a population doubling time of approximately 57 h, and active proliferation was confirmed using a cell proliferation imaging kit. Isolated hSATMVEC function was assessed using their response to insulin stimulation and angiogenic tube-forming potential. We then established an hSATMVEC-subcutaneous adipocyte co-culture model to study cellular cross-talk and demonstrated a downstream effect of hSATMVEC on adipocyte function.
hSATMVEC can be isolated from SAT taken at the time of CIED insertion and are of sufficient purity to both experimentally phenotype and study hSATMVEC-adipocyte cross-talk.

<meta charset="utf8"></head><body>Autore in primo piano: Approfondimenti sulle malattie cardiometaboliche con studi di microvascolarizzazione del tessuto adiposo sottocutaneo

Here, we describe a protocol for isolating, culturing, and phenotyping microvascular endothelial cells from human subcutaneous adipose tissue (hSATMVECs). Additionally, we describe an experimental model of hSATMVEC-adipocyte cross-talk.

Incapsulamento termogenico preadipocytes al trapianto nel tessuto adiposo Depositi tessuti

Riepilogo

Esplora Altri Video

Capitoli in questo video

Una piattaforma di Microphysiologic per grasso umano: Sandwich tessuto adiposo bianco

Isolamento, espansione e adipogenico induzione di CD34 + CD31 + cellule endoteliali dal tessuto adiposo umano omentale e sottocutaneo

Isolamento e la differenziazione di cellule stromali vascolari alle celle Beige / Brite

Coltura tridimensionale di tessuto adiposo termogenico vascolarizzato da frammenti microvascolari

Trapianto di isole ricoperte di grasso utilizzando tessuto adiposo bianco epididimo

Tessuto Adiposo Umano Microframmentazione per la Fenotipizzazione Cellulare e la Caratterizzazione del Secretome

Preparazione di cellule progenitrici adipose dai tessuti adiposi epididimali del topo

Studio del cross-talk tra cellule endoteliali adipose e adipociti nel tessuto adiposo sottocutaneo umano

Studio del cross-talk tra cellule endoteliali adipose e adipociti nel tessuto adiposo sottocutaneo umano

Studio del cross-talk tra cellule endoteliali adipose e adipociti nel tessuto adiposo sottocutaneo umano