Gostaríamos de agradecer a Jennifer Petrosino e David DiSilvestro para a ajuda editorial. Esta pesquisa foi apoiada pelo Prêmio Número 20020728 da American Egg Board e Prêmio Número 10040042 da Novo Nordisk Farmacêutica, bem como pelo Centro de Inovação Alimentar, Instituto de Assuntos Internacionais, Centro de Pesquisa Avançada Functional Foods, e Empreendedorismo na OSU, bem como a National Science Foundation conceder CEE-0914790 (LJL). O projeto descrito foi apoiado pelo Prêmio Número R21OD017244 (OZ) e UL1RR025755 (OSUCCC) a partir do Centro Nacional de Pesquisa de Recursos, financiado pelo Escritório do Diretor, National Institutes of Health (OD) e apoiado pelo NIH Roteiro para a Investigação Médica e NCI P30CA16058. O conteúdo é da exclusiva responsabilidade dos autores e não representam necessariamente a posição oficial do Centro Nacional de Pesquisa de Recursos ou o National Institutes of Health.

O protocolo do estudo foi aprovado pelos Comitês de Ética da Universidade do Estado de Ohio. As experiências com animais foram aprovados pelo protocolo do IACUC. Todos os procedimentos foram realizados sob o nível 2 de biossegurança armário com fluxo laminar. Nós seguimos todos os requisitos e procedimentos de segurança padrão. A técnica de microencapsulação para a preparação de microcápsulas foi realizada tal como descrito 17, 18. 1. Preparações de Materiais <ol><li> Preparar 10 ml de solução de alginato de sódio a 2% em soro fisiológico esterilizado (0,9% NaCl em água). Preparar solução de 0,05% de PLL em solução salina fisiológica. Preparar estas soluções no dia anterior e agita-se durante a noite. Filtrar as soluções com filtro de 0,22 um antes da utilização. </li><li> Prepare citrato de sódio 50 mM e 100 mM CaCl 2 em 0,9% de solução de NaCl e autoclave eles. </li><li> Autoclave todas as soluções, agulhas, eletrodos e copos. Agulhas limpas cuidadosamente com fios para evitar entupimento. </li><li> Determinar a quantidade apropriada de células a ser usado para as cápsulas (102 microcápsulas são necessários para cada cm2 de poço e há aproximadamente 500 células por cápsula 18). Use 1 ml de alginato de sódio por dois milhões de células. </li><li> Prepara-se uma &quot;Meio de Crescimento de Fibroblastos &#39;contendo 10% de soro de vitelo, e 100 U / ml de penicilina / estreptomicina numa glucose elevada (4500 mg / l de glucose) meio de Eagle modificado por Dulbecco (DMEM). <ol><li> Prepara-se uma &#39;Diferenciação Meio I&#39; contendo 10% de soro fetal bovino, 10 ng / ml de insulina, 1 uM de dexametasona, 0,5 mM de xantina 3-isobutil-1-metilo, e 100 U / ml de penicilina / estreptomicina em DMEM. </li><li> Prepare contendo 10% de soro de um &#39;Diferenciação meio II&#39; fetal bovino, 10 ng / ml de insulina, e 100 U / ml de penicilina / estreptomicina em DMEM. </li></ol></li><li> Preparar tampão de lise contendo um inibidor de protease comprimido por 10 ml de tampão de Ensaio de Radio-imunoprecipitação (RITampão PA). </li></ol> 2. Preparação de alginato microesferas (Figura 1A) <ol><li> Remova o meio de idade de balão de cultura de células. Lavar as células com 10 ml de PBS. Traga células em suspensão com 0,25% de tripsina-EDTA (2 ml por um balão T175 confluente). </li><li> Contagem de células em suspensão de células utilizando um hemocitómetro. Utilizar 10 ml de alíquota da suspensão de células e contagem de células de acordo com as instruções do fabricante. Centrifugar as células restantes em meio de centrifugação a 480 xg à temperatura ambiente durante 5 min. </li><li> Suspende-se o sedimento celular em alginato de sódio como descrito no passo 1.4. Transferir a solução de células de alginato de sódio para uma seringa de 5 ml. </li><li> Retirar as bolhas de ar na solução, adicionar uma agulha de calibre 23 e inverter a seringa para criar uma bolsa de 1 ml de ar. </li><li> Coloque um pequeno copo de vidro (180 ml) contendo 144 ml de solução de CaCl 2 100 mM sob a saída da agulha da encapsulador. Fixe o eletrodo para o encapsulator com a ponta approximdamente 2,5 centímetros acima da superfície da solução de CaCl 2 100 mM. </li><li> Colocar firmemente a seringa contendo a solução de alginato de sódio na célula da bomba de seringa. Anexar o tubo de borracha para a abertura da seringa. Empurre o êmbolo até que a solução de células alginato de sódio entra no meio do tubo. Ajustar a tensão de 5,4 kV. Definir a 12,06 milímetros de diâmetro sobre a bomba de seringa. Ajuste a velocidade de 3 ml / h. Comece a bomba. Ligar o encapsulador e manter a voltagem de 5,4 kV. </li><li> Fechar a janela da cobertura e evitar qualquer vibração desnecessária até o fim da formação das micro-esferas de alginato. Depois de toda a solução passa através da agulha, solidificar as microesferas em forma de bola de alginato na solução de CaCl 2 100 mM para adicional de 20 min antes do revestimento com PLL. </li></ol> 3. microesferas de revestimento com PLL (Figura 1B) <ol><li> Retirar o copo contendo as micro-esferas em forma de bola de células alginato de sódio e de transferir estes Microbeads para um tubo de centrifugação de 50 ml. Remover solução de CaCl 2 a partir do sedimento microesferas. </li><li> Lavam-se as microesferas em forma de esferas de alginato de células por adição de 30 ml de NaCl a 0,9%. Agite o tubo com a mão suavemente. Retirar 0,9% de NaCl com 25 ml de pipeta depois de as microesferas foram precipitadas pela gravitação. Repita duas vezes mais para um total de 3 lavagens. </li><li> Utilizar 10 ml de solução de PLL 0,05% por cada 1 ml de solução de alginato de sódio. Adicionar o PLL 0,05% em vórtice e 1000 rotações por minuto durante 10 min. Nota: normalmente, 10 minutos é suficiente para revestimento de PLL. </li><li> Após revestimento de PLL é formado, remover a solução de PLL e lavar as cápsulas três vezes tal como descrito em 3.2. </li></ol> 4. Remoção de O alginato do núcleo (Figura 1C) <ol><li> Adicionar 30 ml de solução de citrato de sódio 50 mM. Esperar 5 min ou até que todo o alginato de sódio estar dissolvido. Lavar cápsulas três vezes tal como descrito no passo 3.1.1. </li><li> Remover a 0,9% de NaCl, adicionar 20 ml de meio de cultura para o tubo de 50 mle transferir todas as cápsulas contendo as células para um frasco de cultura de célula. Lidar com células encapsuladas em condições de cultura de células normais 18. </li></ol> 5. Em Aplicações vitro para estudar Xenoenxerto-célula hospedeira Interações ou Cinética de Metabolite Influx / Efluxo entre as células (Figura 2) <ol><li> Células hospedeiras Cultura na placa de 24 poços até à confluência de co-culturas. Use &quot;Meio de Crescimento de Fibroblastos &#39;para a cultura de pré-adipócitos. </li><li> Transferência de microcápsulas em 24 células hospedeiras confluentes placa contendo bem. Adicionar microcápsulas para alcançar uma monocamada microcápsulas (102 / cm 2 de bem). </li><li> Induzir a diferenciação de pré-adipócitos com Diferenciação Médio I. Cada 48 horas, media mudança para Diferenciação Médio II durante seis dias. Lisar as células em tampão RIPA. Utilizar 50 ug de proteína por condição para analisar a expressão de proteínas utilizando a técnica Western Blot. </li></ol> 6. In Vivo Pedido de Treatment da Obesidade (Figura 3) <ol><li> Mistura-se 4% de isoflurano com oxigénio para indução de anestesia e com isoflurano a 2% de oxigénio para a manutenção da anestesia. Confirme profundidade anestésica adequada por toe pitada. <ol><li> Aplicar pomada anti-coceira nos olhos para proteger as córneas de secar. </li></ol></li><li> Use células encapsuladas que estão permanentemente marcadas com a proteína de fluorescência artificial, como a proteína verde fluorescente (GFP). </li><li> Use uma seringa de 3 ml e uma agulha de calibre 20 para injetar células encapsuladas. <ol><li> Injectar 0,5 x 10 6 células em suspensão em 0,2 ml de PBS em cada depósito de gordura Iab que está localizado na área intraperitoneal gonadal entre um rim e, como mostrado na Figura 3. Use este volume de PBS e o número de células de ratos com um peso médio de 40 g. Ajustar o número de células e o volume em ratos que têm peso diferente ou para a determinação de efeitos dependentes da dose. </li></ol></li></ol>

<ol>
	<li>Vogeli, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+chronic+conditions:+prevalence,+health+consequences,+and+implications+for+quality,+care+management,+and+costs.">Multiple chronic conditions: prevalence, health consequences, and implications for quality, care management, and costs.</a> Journal of general internal medicine. 22, 391-395 (2007).</li><li>Vija, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells:+Stem+cell+therapy+perspectives+for+type+1+diabetes.">Mesenchymal stem cells: Stem cell therapy perspectives for type 1 diabetes.</a> Diabetes &amp; metabolism. 35, 85-93 (2009).</li><li>Acarregui, A., Orive, G., Pedraz, J. L., Hernandez, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+applications+of+encapsulated+cells.">Therapeutic applications of encapsulated cells.</a> Methods in molecular biology. 1051, 349-364 (2013).</li><li>Chang, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semipermeable+Microcapsules.">Semipermeable Microcapsules.</a> Science. 146, 524-525 (1964).</li><li>Elliott, R. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+encapsulated+porcine+islets+from+a+type+1+diabetic+patient+9.5+yr+after+xenotransplantation.">Live encapsulated porcine islets from a type 1 diabetic patient 9.5 yr after xenotransplantation.</a> Xenotransplantation. 14, 157-161 (2007).</li><li>Lim, F., Sun, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microencapsulated+islets+as+bioartificial+endocrine+pancreas.">Microencapsulated islets as bioartificial endocrine pancreas.</a> Science. 210, 908-910 (1980).</li><li>Vos, P., Spasojevic, M., Faas, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+diabetes+with+encapsulated+islets.">Treatment of diabetes with encapsulated islets.</a> Advances in experimental medicine and biology. 670, 38-53 (2010).</li><li>Cotton, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+challenges+in+cell-encapsulation+technology.">Engineering challenges in cell-encapsulation technology.</a> Trends in biotechnology. 14, 158-162 (1996).</li><li>Yach, D., Stuckler, D., Brownell, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiologic+and+economic+consequences+of+the+global+epidemics+of+obesity+and+diabetes.">Epidemiologic and economic consequences of the global epidemics of obesity and diabetes.</a> Nature medicine. 12, 62-66 (2006).</li><li>Ng, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global,+regional,+and+national+prevalence+of+overweight+and+obesity+in+children+and+adults+during+1980-2013:+a+systematic+analysis+for+the+Global+Burden+of+Disease+Study+2013.">Global, regional, and national prevalence of overweight and obesity in children and adults during 1980-2013: a systematic analysis for the Global Burden of Disease Study 2013.</a> Lancet. 384, 766-781 (2014).</li><li>Kissebah, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relation+of+body+fat+distribution+to+metabolic+complications+of+obesity.">Relation of body fat distribution to metabolic complications of obesity.</a> The Journal of clinical endocrinology and metabolism. 54, 254-260 (1982).</li><li>Bray, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relation+of+central+adiposity+and+body+mass+index+to+the+development+of+diabetes+in+the+Diabetes+Prevention+Program.">Relation of central adiposity and body mass index to the development of diabetes in the Diabetes Prevention Program.</a> The American journal of clinical nutrition. 87, 1212-1218 (2008).</li><li>Klein, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+are+subcutaneous+adipocytes+really+good+for.">What are subcutaneous adipocytes really good for.</a> Experimental dermatology. 16, 45-70 (2007).</li><li>Tran, T. T., Yamamoto, Y., Gesta, S., Kahn, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beneficial+effects+of+subcutaneous+fat+transplantation+on+metabolism.">Beneficial effects of subcutaneous fat transplantation on metabolism.</a> Cell metabolism. 7, 410-420 (2008).</li><li>Seale, P., Kajimura, S., Spiegelman, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+control+of+brown+adipocyte+development+and+physiological+function--of+mice+and+men.">Transcriptional control of brown adipocyte development and physiological function--of mice and men.</a> Genes &amp; development. 23, 788-797 (2009).</li><li>Kozak, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+variation+in+brown+fat+activity+and+body+weight+regulation+in+mice:+lessons+for+human+studies.">Genetic variation in brown fat activity and body weight regulation in mice: lessons for human studies.</a> Biochimica et biophysica acta. 1842, 370-376 (2014).</li><li>Zhang, X., He, H., Yen, C., Ho, W., Lee, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+biodegradable,+immunoprotective,+dual+nanoporous+capsule+for+cell-based+therapies.">A biodegradable, immunoprotective, dual nanoporous capsule for cell-based therapies.</a> Biomaterials. 29, 4253-4259 (2008).</li><li>Yang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prolonged+survival+of+fibroblasts+with+forced+lipid+catabolism+in+visceral+fat+following+encapsulation+in+alginate-poly-L-lysine.">The prolonged survival of fibroblasts with forced lipid catabolism in visceral fat following encapsulation in alginate-poly-L-lysine.</a> Biomaterials. 33, 5638-5649 (2012).</li><li>Chang, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+cells+with+emphasis+on+bioencapsulation+in+biotechnology.">Artificial cells with emphasis on bioencapsulation in biotechnology.</a> Biotechnology annual review. 1, 267-295 (1995).</li><li>Chang, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+artificial+cells:+microencapsulation+of+living+cells.">Hybrid artificial cells: microencapsulation of living cells.</a> ASAIO journal. 38, 128-130 (1992).</li><li>Koo, J., Chang, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secretion+of+erythropoietin+from+microencapsulated+rat+kidney+cells:+preliminary+results.">Secretion of erythropoietin from microencapsulated rat kidney cells: preliminary results.</a> The International journal of artificial organs. 16, 557-560 (1993).</li><li>Weidenauer, U., Bodmer, D., Kissel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microencapsulation+of+hydrophilic+drug+substances+using+biodegradable+polyesters.+Part+I:+evaluation+of+different+techniques+for+the+encapsulation+of+pamidronate+di-sodium+salt.">Microencapsulation of hydrophilic drug substances using biodegradable polyesters. Part I: evaluation of different techniques for the encapsulation of pamidronate di-sodium salt.</a> Journal of microencapsulation. 20, 509-524 (2003).</li><li>Smidsrod, O., Skjak-Braek, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alginate+as+immobilization+matrix+for+cells.">Alginate as immobilization matrix for cells.</a> Trends in biotechnology. 8, 71-78 (1990).</li><li>Lewinska, D., Rosinski, S., Werynski, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+process+conditions+during+impulsed+electrostatic+droplet+formation+on+size+distribution+of+hydrogel+beads.">Influence of process conditions during impulsed electrostatic droplet formation on size distribution of hydrogel beads.</a> Artificial cells, blood substitutes, and immobilization biotechnology. 32, 41-53 (2004).</li><li>Chan, E. S., Lee, B. B., Ravindra, P., Poncelet, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+models+for+shape+and+size+of+ca-alginate+macrobeads+produced+through+extrusion-dripping+method.">Prediction models for shape and size of ca-alginate macrobeads produced through extrusion-dripping method.</a> Journal of colloid and interface science. 338, 62-72 (2009).</li><li>Bhujbal, S. V., Paredes-Juarez, G. A., Niclou, S. P., de Vos, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+influencing+the+mechanical+stability+of+alginate+beads+applicable+for+immunoisolation+of+mammalian+cells.">Factors influencing the mechanical stability of alginate beads applicable for immunoisolation of mammalian cells.</a> Journal of the behavior of biomedical materials. 37, 196-208 (2014).</li><li>Gushchina, L. V., Yasmeen, R., Ziouzenkova, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moderate+vitamin+A+supplementation+in+obese+mice+regulates+tissue+factor+and+cytokine+production+in+a+sex-specific+manner.">Moderate vitamin A supplementation in obese mice regulates tissue factor and cytokine production in a sex-specific manner.</a> Archives of biochemistry and biophysics. 539, 239-247 (2013).</li><li>Ziouzenkova, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinaldehyde+represses+adipogenesis+and+diet-induced+obesity.">Retinaldehyde represses adipogenesis and diet-induced obesity.</a> Nature. 13, 695-702 (2007).</li><li>Yasmeen, R., Jeyakumar, S. M., Reichert, B., Yang, F., Ziouzenkova, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+contribution+of+vitamin+A+to+autocrine+regulation+of+fat+depots.">The contribution of vitamin A to autocrine regulation of fat depots.</a> Biochimica et biophysica acta. 1821, 190-197 (2012).</li><li>Liu, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-133a+regulates+adipocyte+browning+in+vivo.">miR-133a regulates adipocyte browning in vivo.</a> PLoS genetics. 9, e1003626 (2013).</li><li>Rao, R. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meteorin-like+is+a+hormone+that+regulates+immune-adipose+interactions+to+increase+beige+fat+thermogenesis.">Meteorin-like is a hormone that regulates immune-adipose interactions to increase beige fat thermogenesis.</a> Cell. 157, 1279-1291 (2014).</li><li>Kir, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-derived+PTH-related+protein+triggers+adipose+tissue+browning+and+cancer+cachexia.">Tumour-derived PTH-related protein triggers adipose tissue browning and cancer cachexia.</a> Nature. 513, 100-104 (2014).</li></ol>

No conflicts of interest declared.

Vários métodos têm sido usados ​​para encapsular células, incluindo a secagem, extrusão, e emulsão 19. Neste método, as drageias de alginato são extrudidas através de uma agulha, em seguida, revestidas com PLL e o núcleo de alginato será dissolvido para completar o encapsulamento. Embora este método tenha sido utilizado durante anos, a formação dos grânulos com o tamanho desejado e a forma esférica é ainda difícil. O tamanho das cápsulas é altamente dependente da viscosidade da soluç?...

Aumento da incidência de doenças crônicas um tem estimulado estudos sobre o transplante de populações de células terapêuticas 2. Ou células-tronco alogénicas, isogénicas são os tipos de células mais comumente utilizados para estas aplicações 2. No entanto, estes tratamentos não permitem o controlo de diferenciação e migração de células estaminais, após o implante e não são economicamente eficiente. Transplante de células geneticamente modificadas com funções benéficas antecipa melhorar o tratamento de muitas doenças. No entanto, as modificações genéticas de células são reconhecidas pelo sistema imunológico do hospedeiro, por isso, estes tratamentos requerem imunossupressão 3. A encapsulação de células produtoras de insulina tem sido desenvolvido pelo Dr. Chang 4. A técnica baseia-se na encapsulação de células em gotículas de alginato que são imersos numa solução de cloreto de cálcio. Moléculas de alginato composto por ácido manurónico (M) e ácido gulurónico (G) e pode ser ligado por Ca 2+. Após a gelificação, as esferas são suspensas numa solução de poli-L-lisina (PLL). Durante este passo, PLL se liga a G e H nas moléculas de alginato que estabelece membrana da cápsula. A porosidade da membrana da cápsula pode ser modulada por variação das concentrações de H e de PLL, o tempo de incubação e temperatura. A ligação de PLL também depende do tipo e concentração do alginato. Matrizes de alginato reticulados com iões Ca2 +, são instáveis ​​no ambiente fisiológico ou em soluções tampão comuns com elevada concentração de fosfato e iões citrato. Estes tampões podem extrair de Ca2 + a partir do alginato e liquefazer o núcleo. A liquefacção do núcleo alginato fornece espaço no interior das cápsulas para movimento celular e crescimento. As células encapsuladas em alginato polianiónico com policatiónico poli-L-lisina (APL) são impermeáveis ​​para as imunoglobulinas mas têm influxo de nutrientes e de efluxo de toxinas. Estas propriedades da APL permitir a longo prazo survival de células encapsuladas após o transplante em geneticamente diferentes hosts. Elliott et ai. Relataram a sobrevivência de funcionamento das células pancreáticas porcinas encapsulados num paciente humano nove anos após o implante 5. Técnicas de encapsulação podem ser classificados em microencapsulação (3-800 pM) e macroencapsulação (maiores do que 1000 mm). As microcápsulas são mais duráveis ​​que macrocápsulas 6. Desde a sua descoberta pelo Dr. Chang e colegas em 1964, a microencapsulação tem sido amplamente utilizada para a encapsulação de células produtoras de insulina anabolizantes, hormonas, e outras moléculas bioactivas 7. Estes tratamentos enfrentou vários desafios no tecido hospedeiro, incluindo fibrose e resposta imune 8. Inicialmente, os efeitos secundários relacionados com a qualidade de biopolímeros foram resolvidos. No entanto, o transplante de células anabólicos ainda inicia efeitos colaterais, tais como fibrose, como um resultado da hormona overprodução fora de uma glândula especializado. Nas últimas décadas, a obesidade e diabetes tipo 2 tem alcançado proporções epidêmicas 9. Mais de 30% das pessoas adultas em todo o mundo estão acima do peso e obesos 10. (IAB) a formação de gordura aumento da pressão intra-abdominal aumenta a incidência de inflamação crônica e promove diabetes tipo 2, doenças cardiovasculares, certos tipos de câncer, e outras morbidades 11-13. Várias linhas de evidência sugeriram que a patogénese associada com gordura Iab pode ser evitada por adipócitos específicos. Estudos recentes demonstraram que o transplante de adipócitos subcutâneos na região Iab pode melhorar o metabolismo e diminuir a obesidade e resistência à insulina em roedores in vivo 14. Redução efectiva da obesidade e resistência à insulina está associada com adipócitos termogénicos, capazes de dissipar a energia na forma de calor 15,16. Termogênico modificação dos adipócitos pode ser conseguida por transfecção estávelde genes que participam no desacoplamento mitocondrial de protões, tais como proteína desacopladora 1 (UCP1) ou de genes que regulam a expressão de UCP1 e outros genes termogênicos 15,16. Os nossos estudos recentes mostraram que a deficiência em aldeído desidrogenase 1 a1 (Aldh1a1) leva à remodelação de gordura termogénico Iab que reduz a obesidade e resistência à insulina nestes ratos 17,18. Notavelmente, o encapsulamento de termogénico Aldh1a1 deficiente (Aldh1a1 - / - pré-adipócitos) medeia mesmo efeito terapêutico em gordura Iab em ratos obesos do tipo selvagem, sugerindo novas oportunidades terapêuticas para o tratamento de gordura Iab 18. Em situações experimentais, células encapsuladas permitir aos investigadores estudar efeitos das populações de células específicas em uma forma rentável 19. Aqui nós discutimos o método de encapsulação de uma linha celular catabólico termogénico e seu laboratório e aplicação terapêutica num modelo de rato de obesidade. O protocolo descreve três fases para a produção de microcápsula (Figura 1): a formação das microesferas de alginato (Figura 1a), a formação do policatiónico poli-L-lisina (PLL) membranas na superfície de microesferas (Figura 1B), e a remoção do núcleos de 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

Encapsulamento celular foi desenvolvido para reter células viáveis ​​dentro das membranas semi-permeáveis. As células encapsuladas podem trocar enxertados metabolitos de baixo peso molecular em tecidos do hospedeiro a ser tratado para conseguir a sobrevivência a longo prazo. A membrana semipermeável permite enxertados células encapsuladas para evitar a rejeição pelo sistema imunológico. O processo de encapsulação foi concebido para permitir uma libertação controlada de compostos bioactivos, tais como insulina, hormonas, e outras citoquinas. Descrevemos aqui um método para a encapsulação de células catabólicos, que consomem lípidos para a produção de calor e dissipação de energia (termogénese) no tecido adiposo intra-abdominal de ratos obesos. A encapsulação de células catabólicos termogénicos podem ser potencialmente aplicáveis ​​para a prevenção e tratamento da obesidade e diabetes do tipo 2. Uma outra aplicação potencial das células catabólicos pode incluir a desintoxicação de álcoois ou outros metabolitos tóxicos e poluentes ambientais.

A Figura 1 mostra que todos os passos da produção de microesferas pode ser controlada ao microscópio. A Figura 2A mostra como a co-cultura de adipócitos com uma monocamada de células encapsuladas. A Figura 2B é um exemplo representativo de um estudo quantitativo utilizando adipócitos / microcápsulas co-culturas que foram descritas na secção 5. Os lisados ​​de adipócitos foram analisadas utilizando a técnica Western Blot. Células encapsuladas não foram ...

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.

Encapsulation termogênico pré-adipócitos para transplante em adiposo Depots Tissue

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 visualizações.  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.

Vídeo: 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.

Transplante subretinal de células precursoras do fotoreceptor purificado MACS na retina do rato 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 ...

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.

O transplante de válvula pulmonar Usando um modelo do rato de Heterotópica Transplante de Coração

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 luminescência imagem de Interscapular Brown tecido 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.

Localização, identificação e excisão de Depots Murino adiposo

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.

Brown Human Tissue adiposo Depots automaticamente segmentada por Positron Emission Tomography / Tomografia Computadorizada e registrado ressonância magnética

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.

Transplantação no Pad Mouse ovário 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.

Uma técnica para biopsia de tecido adiposo Abdominal subcutâneo através de um método não-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.

Avaliação da função Microvascular de tecido adiposo humano usando tele-microscopia

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.

Transplante de células-tronco derivadas de tecido adiposo folha para reduzir o vazamento após colectomia parcial em um modelo do rato

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.

Protocolo clínico de produzir tecido adiposo-derivado do estroma Vascular fração para a regeneração da cartilagem potenciais

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