Queremos agradecer aos nossos muitos colegas que prestaram ajuda, conselhos e críticas de entrada em várias fases do projeto. Produção deste artigo vídeo foi apoiado com fundos do Ministério Alemão de Educação e Pesquisa (BMBF) para o Centro Alemão de Pesquisa do Diabetes (DZD, <a href="http://www.dzd-ev.de">http://www.dzd-ev.de</a> ), IMIDIA ( <a href="http://www.imidia.org">http://www . imidia.org</a> ) eo Hospital Universitário de Carl Gustav Carus, da Universidade de Tecnologia de Dresden. A pesquisa que levou a estes resultados beneficiaram de financiamento do Programa da Comunidade Europeia Quadro (FP7/2007-2013) para a Iniciativa de Medicamentos Inovadores no âmbito da convenção de subvenção n ° 115005.

1. Coleta de tecido pancreático na sala de cirurgia <ol><li> O cirurgião realiza uma ressecção parcial do pâncreas. </li><li> Depois de colocar a amostra de pâncreas em uma caixa de gelo, entregá-lo imediatamente para o patologista. </li></ol> 2. Seleção de tecidos para isolamento de ilhotas <ol><li> O patologista seleciona tecido que parece macia e saudável, mantendo o tecido danificado para fins de diagnóstico. Tecido fibrótico e espécimes oferecendo menos de 2 g de tecido utilizável são excluídos do isolamento de ilhotas. </li><li> Mergulhe o tecido pancreático em solução Euro Collins e entregar no gelo para o laboratório. </li></ol> 3. Isolamento de ilhotas humanas <ol><li> Pese o tecido pancreático, em seguida, coloque-o em um prato de 10 cm. </li><li> Coloque 150 ml RPMI mídia em um balão de 500 ml. </li><li> Em um frasco de 250 ml, preparar a solução de enzimas digestivas, combinando RPMI 130 ml de mídia com 100 mg / ml DNase e 20 ml de 5mg/ml RI Liberase. Draw 10 ml desta solução para uma seringa para injetar o tecido pancreático. </li><li> Injetar a solução de enzimas digestivas no tecido pancreático, com o objetivo de distender-lo de forma homogênea. Se este for impedido pela fibrose, a digestão, provavelmente, falhar. Em seguida, mediu o tecido em ~ 4 mm 3 peças no gelo. </li></ol> 4. Circuito digestão humana pâncreas <ol><li> Monte o circuito da digestão, como mostrado na Figura 1. O sentido do fluxo na câmara Ricordi é no fundo e sair no topo da câmara. </li><li> Adicionar a solução de enzima digestiva restantes para o balão de 500 ml até um volume total de 300 ml e coloque um termômetro. </li><li> Transferir o tecido pancreático para a câmara, insira o mesh (diâmetro dos poros: 600 mm) e três Marbles nitreto de silício (diâmetro: 15 mm), perto da câmara e começar a bomba com o fluxo fixado em 140 ml / min. </li><li> Quando a temperatura do circuito atinge 37 ° C, iniciar a cronometragem e, periodicamente, colher amostras para determinar quando parar a digestão. Coloração com ditizona (2 mg / ml) permite a visualização microscópica de ilhotas no tecido digerido 4. </li><li> Uma vez que os ilhéus são separados a partir do tecido acinar circundante, rapidamente parar a digestão, colocando a bobina de aquecimento no gelo e adicionando 200 ml de mídia lavagem a frio (900 ml RPMI 1640 com 5,5 mM de glicose e FBS 10%) para o circuito. </li><li> Recolher a solução de ilhotas em 250 ml tubos cônicos, uma vez que sai do tubo de amostra. Continue até que o circuito está vazio. </li></ol><img alt="Figura 1" src="/files/ftp_upload/2962/2962fig1.jpg" /> Figura 1. O circuito de digestão humana pâncreas O circuito digestão humana pâncreas inclui uma câmara de Ricordi contendo mesh (diâmetro dos poros: 600 mm) e três bolas de gude nitreto de silício (diâmetro: 15 mm). Saída da câmara é movida para o balão de coleta de tecido pela bomba peristáltica (140 ml / min). Setas indicam a direção do fluxo. Uma temperatura ideal para a digestão enzimática é mantida por imersão da bobina de aquecimento em banho-maria a 37 ° C. 5. Lavagem pós-digestão <ol><li> Centrifugar a 250 ml tubos cônicos contendo a solução de ilhotas a 1000 rpm, 4 ° C por 5 min. </li><li> Desprezar o sobrenadante, ressuspender o sedimento ilhota na lavagem de mídia (900 ml RPMI 1640 com 5,5 mM de glicose e FBS 10%) e distribuí-lo em frações iguais a 50 tubos de ml. (Opcional:. Distribuir em tubos cônicos adicionais para melhorar a lavagem) Centrifugar a 1000 rpm, 4 ° C por 5 min. </li><li> Elimine o sobrenadante e gentilmente afrouxar pellets por agitação manual. </li></ol> 6. Purificação em um gradiente de Ficoll <ol><li> Ressuspender o sedimento com Ficoll media a uma densidade de 1,125 g / ml, pH 7,4 e lentamente overlay alíquotas de 10 ml de Ficoll media com densidades de 1,080, 1,060 e 1,037 g / ml. </li><li> Centrifugar os gradientes de Ficoll a 2400 rpm, 4 ° C por 20 min. </li></ol> 7. Islet Recolha e Cultura <ol><li> Após a centrifugação, componentes do tecido diferentes podem ser distinguidos pela sua partição no gradiente. </li><li> Descartar a camada superior composta de gordura e tecido conjuntivo. </li><li> Cuidadosamente a colheita da primeira camada de agregados ilhota na interfase 1,037-1,060 g / ml. Esta camada contém as ilhotas mais puro isolado. Coletar fracções para isolamento eficiente. </li><li> Recolher a fracção de segundo, menos pura de agregados ilhota na interfase 1,060-1,080 g / ml. </li><li> Diluir o gradiente de Ficoll, adicionando media de lavagem (900 ml RPMI 1640 com 5,5 mM de glicose e FBS 10%) a cada tubo cônico até um volume total de 50 ml. Centrifugar a 1000 rpm, 4 ° C por 5 min. </li><li> Desprezar o sobrenadante por sucção. Tenha cuidado como o pellet pode estar solto devido ao gradiente de Ficoll. </li><li> Lave o wi pelletsª lavagem de mídia (900ml RPMI 1640 com 5,5 mM de glicose e FBS 10%) e centrifugar a 1000 rpm, 4 ° C por 5 min. Repita usando meios de cultura ilhéu </li><li> Volte a suspender as ilhotas em meios de cultura e colocá-los na incubadora com 5% de CO 2 a 37 ° C por 24-48 horas antes do processamento. </li></ol>

<ol>
	<li>Kahn, S. E., Zraika, S., Utzschneider, K. M., Hull, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+beta+cell+lesion+in+type+2+diabetes:+there+has+to+be+a+primary+functional+abnormality.">The beta cell lesion in type 2 diabetes: there has to be a primary functional abnormality.</a> Diabetologia. 52, 1003-1012 (2009).</li><li>Deng, S., Vatamaniuk, M., Huang, X., Doliba, N., Lian, M. M., Frank, A., Velidedeoglu, E., Desai, N. M., Koeberlein, B., Wolf, B., Barker, C. F., Naji, A., Matschinsky, F. M., Markmann, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+abnormalities+in+the+islets+isolated+from+type+2+diabetic+subjects.">Structural and functional abnormalities in the islets isolated from type 2 diabetic subjects.</a> Diabetes. 53, 624-632 (2004).</li><li>Ricordi, C., Lacy, P. E., Finke, E. H., Olack, B. J., Scharp, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+method+for+isolation+of+human+pancreatic+islets.">Automated method for isolation of human pancreatic islets.</a> Diabetes. 37, 413-420 (1988).</li><li>Latif, Z. A., Noel, J., Alejandro, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+staining+fresh+and+cultured+islets.">A simple method of staining fresh and cultured islets.</a> Transplantation. 45, 827-830 (1988).</li><li>Ehehalt, F., Knoch, K., Erdmann, K., Krautz, C., J&#228;ger, M., Steffen, A., Wegbrod, C., Meisterfeld, R., Kersting, S., Bergert, H., Kuhlisch, E., Bornstein, S., Bonifacio, E., Saeger, H. D., Solimena, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+insulin+turnover+in+islets+from+type+2+diabetic+patients.">Impaired insulin turnover in islets from type 2 diabetic patients.</a> Islets. 2, 30-36 (2010).</li><li>Hardt, P. D., Brendel, M. D., Kloer, H. U., Bretzel, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+pancreatic+diabetes+(type+3c+diabetes)+underdiagnosed+and+misdiagnosed?.">Is pancreatic diabetes (type 3c diabetes) underdiagnosed and misdiagnosed?.</a> Diabetes Care. 31, S165-S169 (2008).</li><li>Meisterfeld, R., Ehehalt, F., Saeger, H. D., Solimena, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+disorders+and+diabetes+mellitus.">Pancreatic disorders and diabetes mellitus.</a> Exp Clin Endocrinol Diabetes. 116, S7-S12 (2008).</li><li>Verge, C. F., Gianani, R., Kawasaki, E., Yu, L., Pietropaolo, M., Jackson, R. A., Chase, H. P., Eisenbarth, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+type+I+diabetes+in+first-degree+relatives+using+a+combination+of+insulin,+GAD,+and+ICA512bdc/IA-2+autoantibodies.">Prediction of type I diabetes in first-degree relatives using a combination of insulin, GAD, and ICA512bdc/IA-2 autoantibodies.</a> Diabetes. 45, 926-933 (1996).</li><li>Alberti, K. G., Zimmet, P. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Definition,+diagnosis+and+classification+of+diabetes+mellitus+and+its+complications.+Part+1:+diagnosis+and+classification+of+diabetes+mellitus+provisional+report+of+a+WHO+consultation.">Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus provisional report of a WHO consultation.</a> Diabet Med. 15, 539-553 (1998).</li><li>Marselli, L., Thorne, J., Ahn, Y. B., Omer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+Expression+of+Purified+{beta}-Cell+Tissue+Obtained+from+Human+Pancreas+with+Laser+Capture+Microdissection.">Gene Expression of Purified {beta}-Cell Tissue Obtained from Human Pancreas with Laser Capture Microdissection.</a> Journal of Clin. Endocrinol. Metab. 93, 1046-1053 (2008).</li></ol>

Não há conflitos de interesse declarados.

Usando nosso protocolo, as ilhotas humanas podem ser isoladas de tecido pancreático coletados a partir de uma pancreatectomia parcial. O sucesso deste protocolo baseia-se no cuidado durante alguns pontos críticos. Para preservar a viabilidade das células beta, é essencial que a amostra seja transportada rapidamente sobre o gelo para o laboratório. Além disso, a duração da digestão do tecido deve ser otimizado empiricamente de acordo com o grau de fibrose tecidual ea atividade enzimática da mídia. Isso também é verdadeiro para o grau de força mecânica aplicada manualmente à câmara Ricordi. Assim, para obter um rendimento bom ilhéu, o protocolo é melhor executada por um cientista dedicado ou assistente técnico. Fracasso inicial é a norma, a menos que a equipe já está experiente em isolamento de ilhotas. Existem diferenças fundamentais entre o nosso protocolo de isolamento de ilhotas eo método padrão de isolamento de ilhotas humanas: 1) Apesar de usarmos tecido pancreático submetido a várias horas de isquemia durante o procedimento cirúrgico, ele é imediatamente transformado em site. Isto está em contraste com pancreata explantado de doadores em morte encefálica para o pâncreas / transplante de ilhotas, que permanecem isquêmica por várias horas durante a alocação e entrega à instalação de isolamento de ilhotas. 2) Nós colagenase injetar diretamente no tecido pancreático, enquanto que o protocolo padrão é infundir-lo para o ducto pancreático. 3) Nós separar as ilhotas utilizando um gradiente de Ficoll descontínua em vez de recolher as ilhotas de um gradiente de Ficoll contínua usando um processador de células COBE. Nos casos em que a peça cirúrgica é muito fibrótica ou escassos para isolar um número adequado de ilhotas, o tecido ainda podem ser recuperados por laser microdissecção de captura (LCM) 10. Isso permite que dados de expressão gênica a ser recuperado a partir de praticamente todas as amostras, mesmo que o isolamento das ilhotas falhar ou o rendimento é muito baixo. Infelizmente, LCM não produz células vivas para estudos funcionais e sua quantidade é normalmente insuficiente para análise proteômica. Assim, usando LCM em paralelo com nosso protocolo de digestão da colagenase para recuperar as ilhotas podem ser a maneira mais eficaz de processo de peças cirúrgicas. Ao coletar tecidos para isolamento de ilhotas de pancreatectomies parcial, é importante examinar cuidadosamente a história clínica do paciente e estado metabólico. Um paciente parcialmente pancreatectomizado poderiam ser afetados por diabetes tipo 3c, ou seja, diabetes secundário ao distúrbio do pâncreas levando a cirurgia 6. Entre os 43 pacientes participantes que se submeteram a esta cirurgia em nosso serviço em 2010, 32 eram não-diabéticas, 5 foram afetadas pelo diabetes tipo 2 e 6 apresentavam diabetes tipo 3c. Estes dados concordam com estudos anteriores que apontam para o metabolismo da glicose prejudicada e diabetes em uma fração considerável de pacientes que sofrem de câncer de pâncreas ou pancreatite crônica 6. Consideramos diabetes ser de origem primária se foi diagnosticado pelo menos um ano antes do início dos sintomas principais da cirurgia pancreática 7. Os níveis de anticorpos contra ilhotas auto-antígenos também devem ser medidos para avaliar uma origem auto-imune potencial do diabetes 8. Porque um paciente submetido a pancreatectomia poderia sofrer de diabetes não diagnosticada ou ser intolerantes à glicose, todos os pacientes não-diabéticos deve ser dado um teste de tolerância oral à glicose antes da pancreatectomia 9.

<table border="1"><tbody><tr><th> Nome </th><th> Companhia </th><th> Número de catálogo </th></tr><tr><th> Equipamento </th><th></th><th></th></tr><tr><td> 1 ml sorológicos pipeta (CELLSTAR) </td><td> Greiner Bio-One </td><td> 604181 </td></tr><tr><td> 10 ml sorológicos pipeta (CELLSTAR) </td><td> Greiner Bio-One </td><td> 607180 </td></tr><tr><td> 25 ml sorológicos pipeta (CELLSTAR) </td><td> Greiner Bio-One </td><td> 760180 </td></tr><tr><td> Seringa de 10 ml </td><td> BD (Becton, Dickinson and Company) </td><td> 300912 </td></tr><tr><td> Seringa 50 ml </td><td> BD (Becton, Dickinson and Company) </td><td> 300865 </td></tr><tr><td> Seringa 5 ml </td><td> BD (Becton, Dickinson and Company) </td><td> 309050 </td></tr><tr><td> 18 Agulha 1,1 G / 2 Microlance 3 </td><td> BD (Becton, Dickinson and Company) </td><td> 304622 </td></tr><tr><td> 27 GX1 / 2 Agulha </td><td> Braun </td><td> 4658300 </td></tr><tr><td> 27 GX3 / 4 Microlance Agulha 3 </td><td> BD (Becton, Dickinson and Company) </td><td> 302200 </td></tr><tr><td> 3 centímetros de cultura de células de grade prato milímetros 2x2 </td><td> Nunc </td><td> 174926 </td></tr><tr><td> 20 centímetros prato de cultura de tecidos </td><td> BD (Becton, Dickinson and Company) </td><td> 353003 </td></tr><tr><td> 6 centímetros Fácil aderência placa de Petri </td><td> BD (Becton, Dickinson and Company) </td><td> 351016 </td></tr><tr><td> 150 ml garrafa de armazenamento </td><td> Corning Incorporated </td><td> 431175 </td></tr><tr><td> 250 ml Erlenmeyers </td><td> Schott Duran </td><td> 21 217 36 </td></tr><tr><td> 500 ml Erlenmeyers </td><td> Schott Duran </td><td> 21 217 44 </td></tr><tr><td> 250 ml Screw Cap tubo de centrífuga cônico inferior </td><td> Corning Incorporated </td><td> 430776 </td></tr><tr><td> Falcon 50 ml tubo cônico </td><td> BD (Becton, Dickinson and Company) </td><td> 352070 </td></tr><tr><td> 260 ml Easyflask não-tratados </td><td> Nunc </td><td> 156800 </td></tr><tr><td> Millex GV Filtro Unidade Duropore (0,22 mm) </td><td> Millipore </td><td> SLGV033RB </td></tr><tr><td> Vacuum Bottle Top Filter (PES 70 milímetros de diâmetro Membran, 0,22 mM tamanho dos poros, tamanho 45 pescoço mm, volume de 500 mL) </td><td> Corning Incorporated </td><td> 431118 </td></tr><tr><td> Densitômetro (Densito 30px) LWE37463 </td><td> Mettler Toledo </td><td> 51324450 </td></tr><tr><td> Unidade PES rápido Filtro 0,2 mM </td><td> Nalgene </td><td> 569-0020 </td></tr><tr><td> Bobina de aquecimento </td><td> BioRep Technologies Inc </td><td> HC-02 </td></tr><tr><td> Multifuge 4 KS-R </td><td> Heraeus </td><td> 75015680 </td></tr><tr><td> Bomba Typ PD5206 </td><td> Heidolph </td><td> 523-52060-00-2 </td></tr><tr><td> Ricordi câmara (malha de metal com 600 mM de diâmetro dos poros) </td><td> BioRep Technologies Inc. </td><td> Modelo 50-U-01, 0503-001 Serial </td></tr><tr><td> Fique preparação </td><td> BioRep Technologies Inc. </td><td> 50-PS-01 </td></tr><tr><td> O-Rings </td><td> BioRep Technologies Inc. </td><td> OR-50-U-01 </td></tr><tr><td> Mármores de nitreto de silício (15 set mm de 9) </td><td> BioRep Technologies Inc. </td><td> SN-01 </td></tr><tr><td> Tubulação de silicone </td><td> Tygon </td><td> R 3603 8,0 X4, 0 mm </td></tr><tr><td> Pinça cirúrgica </td><td> Braun </td><td> BD168R </td></tr><tr><td> Tesoura cirúrgica (Aesculap) </td><td> Braun </td><td> BC273R </td></tr><tr><td> Banho de água OLS 200 </td><td> Conceder </td><td> OLS 200 </td></tr><tr><th> Reagentes </th><th></th><th></th></tr><tr><td> Colagenase - RI Liberase&gt; (100 mg) </td><td> Roche </td><td> 11815032001 </td></tr><tr><td> D (+)-Glicose </td><td> Merck </td><td> 1.08337.1000 </td></tr><tr><td> Dimetilsulfóxido (DMSO) </td><td> Merck </td><td> 1.16743.1000 </td></tr><tr><td> Diphenylthiocarbazone (DTZ) </td><td> Sigma Aldrich </td><td> D5130 </td></tr><tr><td> DNase I (100 mg) </td><td> Roche </td><td> 10104159001 </td></tr><tr><td> Dulbeccos 1xPBS </td><td> PAA Laboratories </td><td> H15-002 </td></tr><tr><td> Electrolytsolution E154 </td><td> Serumwerk </td><td> 00509 </td></tr><tr><td> De soro fetal bovino (FBS) </td><td> PAA Laboratories </td><td> A15-101 </td></tr><tr><td> Ficoll 400 </td><td> Sigma Aldrich </td><td> F9378 </td></tr><tr><td> Fetal bovino Serum (FBS) </td><td> Gibco </td><td> 10270-106 </td></tr><tr><td> 2 - (4 - (2-hidroxietil) - 1-piperazinil)-ethansulfons ‰ ure (HEPES) </td><td> Roth </td><td> 9105,3 </td></tr><tr><td> NaOH, 5 M </td><td> farmácia clínica, hospital </td><td> - </td></tr><tr><td> Penicilina estreptomicina / (100x) </td><td> PAA Laboratories </td><td> P11-010 </td></tr><tr><td> Pipettaid (EXPRESS) </td><td> BD (Becton, Dickinson and Company) </td><td> 357591 </td></tr><tr><td> Potassiumchlorid </td><td> Fluka </td><td> 60132 </td></tr><tr><td> Potassiumdihydrogenphospate anidro </td><td> JT Baker </td><td> 0241 </td></tr><tr><td> Potassiummonohydrogenphosphate </td><td> JT Baker </td><td> 3246-01 </td></tr><tr><td> Meio RPMI 1640 [-] D-Glicose [+] L-Glutamina </td><td> Gibco </td><td> 11879-020 </td></tr><tr><td> Meio RPMI 1640 [+] L-Glutamina </td><td> PAA Laboratories </td><td> E15-840 </td></tr><tr><td> Sodiumhydrogencarbonat </td><td> Merck </td><td> 1.06329.0500 </td></tr></tbody></table> Mídia e Soluções Solução ditizona <ul><li> Dissolver 100 mg DTZ em 10 ml DMSO e diluir em 40 ml de PBS </li><li> Filtro estéril a solução (0,22 mm) </li></ul> Solução da enzima <ul><li> Dissolver 100 mg Liberase RI em 20 ml de mídia RPMI 1640 (sem suplementos) </li><li> Dissolver 100 mg em 1 ml DNase RPMI 1640 mídia (sem suplementos) </li><li> Filtro estéril a solução (0,22 mm) e manter a enzima no gelo </li></ul> Lavar media (1000ml/5.5mM Glicose) <ul><li> Dissolver 100 ml FBS (inactivado pelo calor) </li><li> Adicione 450 ml RPMI 1640 (sem glicose) </li><li> Adicione 450 ml RPMI 1640 (11 mM de glicose) </li><li> Filtro estéril a solução (0,22 mm) </li></ul> Islet cultura da mídia (500ml / 5,5 mM de glicose) <ul><li> Dissolver 10 ml 1M HEPES (pH 7,4) </li><li> Adicionar 50 ml FBS </li><li> Adicionar 5 ml de penicilina / estreptomicina (100x) </li><li> Adicionar 2,75 ml uma solução de glicose M </li><li> Adicionar 432,25 ml RPMI 1640 (sem glicose) </li><li> Filtro estéril a solução (0,22 mm) </li></ul> Solução Euro Collins <ul><li> 4.083 g de potássio hydrogenphosphate </li><li> 70,0 g de glicose </li><li> 2.237 g de cloreto de potássio </li><li> 14,80 g de potássio monohydrogenphosphate </li><li> 1.680 g de hidrogenocarbonato de sódio </li><li> 40 ml solução eletrolítica (E 154) </li><li> pH 7,4 </li><li> Adicionar água destilada até 2000 ml </li><li> Filtro estéril a solução (0,22 mm) </li></ul> Ficoll Solução stock <ul><li> Adicionar 1000 ml de solução Euro Collins a 500 g Ficoll </li><li> Adicionar 8,94 g HEPES </li><li> Mexa durante a noite até Ficoll é resolvido </li><li> pH 7,4 </li><li> Medir a densidade </li></ul> Ficoll Gradients <ul><li> Calcular a quantidade de FBS, Euro Collins e solução Ficoll Solução stock de acordo com a densidade </li><li> Combinar as três soluções e medir a densidade com um densitômetro </li><li> Adicionar Euro Collins Solução ou Ficoll Solução stock até que a densidade é ajustada </li><li> Estéril solução do filtro (0,22 mm) e armazenar a 4 ° C </li></ul>

isolation of human islets from partially pancreatectomized patients

Investigações sobre a patogênese da diabetes tipo 2 e ilhotas de Langerhans um mau funcionamento tem sido prejudicada pela disponibilidade limitada de ilhotas diabéticos tipo 2 de 2 doadores de órgãos. Aqui nós compartilhamos nosso protocolo para isolar ilhotas pancreáticas humanas a partir de tecido obtido a partir de diabéticos tipo 2 e não diabéticos que foram submetidos a pancreatectomia parcial devido a doenças pancreáticas diferentes (tumores pancreáticos benignos ou malignos, pancreatite crônica, e ducto biliar comum ou tumores duodenal) . Todos os pacientes envolvidos deram o seu consentimento a este estudo, que também havia sido aprovado pelo Comitê de Ética local. Os espécimes cirúrgicos foram imediatamente entregues ao patologista que selecionou tecido pancreático macia e saudável aparecendo para o isolamento de ilhotas, mantendo o tecido danificado para fins de diagnóstico. Descobrimos que para isolar mais de 1.000 ilhotas, tivemos que começar com pelo menos 2 g de tecido pancreático. Também essencial para o nosso protocolo foi visivelmente a distender o tecido ao injetar a mídia contendo enzima e, posteriormente, mediu-lo para ajudar a digestão, aumentando a área de superfície. Alargar a aplicabilidade do nosso protocolo para incluir o caso ocasional em que uma grande quantidade (&gt; 15g) de tecido pancreático humano está disponível, foi utilizado uma câmara de Ricordi (50 ml) para digerir o tecido. Durante a digestão, que balançou a câmara manualmente Ricordi 3 em uma intensidade que variou de amostra de acordo com seu nível de fibrose tecidual. A descontínua gradiente Ficoll foi então usada para separar as ilhotas do tecido acinar. Notamos que o tecido pellet deve ser pequeno o suficiente para ser homogeneamente ressuspenso em meio Ficoll com uma densidade de 1,125 g / ml. Após o isolamento, as ilhotas cultivadas que sob condições de estresse livre (sem agitação ou rotação) com 5% de CO 2 a 37 ° C durante pelo menos 48 h, a fim de facilitar a sua recuperação funcional. Aplicação generalizada do nosso protocolo e seu aperfeiçoamento futuro poderia permitir a colheita oportuna de grandes quantidades de ilhotas humanas de diabéticos e clinicamente combinado não-diabéticos, com grande avanço da diabetes tipo de pesquisa 2.

Nosso protocolo renderam uma média de ~ 500 ilhotas por grama de tecido pancreático, embora este muito variadas entre as preparações em grande parte devido a diferenças na fibrose e atividade da colagenase. Conseguimos&gt; pureza das ilhotas de 90% através da coloração pela primeira vez com Ditizona durante o processamento de tecidos, então handpicking-los 24 horas depois do isolamento. Para determinar a qualidade das ilhotas purificadas, testamos para 25 mM de glicose estimulada a secreção de insulina em condições estáticas, por 2 horas. Encontramos a secreção de insulina comparável à de ilhotas obtidas de ECIT isolamento de ilhotas e centros de transplantação. Como ilhotas isoladas de pancreata parcialmente pancreatectomizado não são destinadas para transplante de ilhotas, a avaliação da IEQ total foi de que não foi considerada um fator crítico. Importante, o nosso método nos permitiu isolar ilhotas para estudos funcionais de 25 pacientes parcial pancreatectomizado entre 2005 e 2008 5. Estas ilhas foram analisadas para glicose estimulada a secreção de insulina e expressão de proteínas específicas borrando ocidental e imuno-histoquímica. Ilhotas isoladas de pancreata parcialmente pancreatecomized também poderia ser usado para comparar genes e de perfis de proteínas de expressão, aparência ultra-estruturais e respostas funcionais de ilhotas não-diabéticos e diabéticos. Porque há mais pacientes submetidos à pancreatectomia parcial do que há doadores de órgãos, o nosso protocolo poderia permitir que amostras suficientes e dados a serem coletados para análise estatística robusta.

Watch this Scientific Journal Video about Isolation of Human Islets from Partially Pancreatectomized Patients at JoVE.com

Isolation of Human Islets from Partially Pancreatectomized Patients

O fornecimento de diabéticos tipo 2 ilhotas para pesquisa é insuficiente. Aqui nós compartilhamos nosso protocolo para isolar ilhotas de pacientes submetidos à pancreatectomia parcial. Esta abordagem representa um local único para a obtenção de ilhotas de diabéticos tipo 2 e clinicamente combinado não-diabéticos em número adequado para estudos básicos e clínicos.

Isolamento de Ilhotas Humanos de pacientes parcialmente pancreatectomizado

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

isolation-of-human-islets-from-partially-pancreatectomized-patients

Research

JoVE Journal

Medicine

15.9K visualizações.  University Hospital Carl Gustav Carus, University of Technology Dresden. O fornecimento de diabéticos tipo 2 ilhotas para pesquisa é insuficiente. Aqui nós compartilhamos nosso protocolo para isolar ilhotas de pacientes submetidos à pancreatectomia parcial. Esta abordagem representa um local único para a obtenção de ilhotas de diabéticos tipo 2 e clinicamente combinado não-diabéticos em número adequado para estudos básicos e clínicos.

Vídeo: Isolation of Human Islets from Partially Pancreatectomized Patients

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

Protocolos de coloração de ilhotas pancreáticas humanas

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

Medicina

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

Protocolo melhorado para microdissecção a laser de ilhotas pancreáticas humanas a partir de espécimes cirúrgicos

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Dynamic Visual Tests to Identify and Quantify Visual Damage and Repair Following Demyelination in Optic Neuritis Patients

In order to follow optic neuritis patients and evaluate the effectiveness of their treatment, a handy, accurate and quantifiable tool is required to assess changes in myelination at the central nervous system (CNS). However, standard measurements, including routine visual tests and MRI scans, are not sensitive enough for this purpose. We present two visual tests addressing dynamic monocular and binocular functions which may closely associate with the extent of myelination along visual pathways. These include Object From Motion (OFM) extraction and Time-constrained stereo protocols. In the OFM test, an array of dots compose an object, by moving the dots within the image rightward while moving the dots outside the image leftward or vice versa. The dot pattern generates a camouflaged object that cannot be detected when the dots are stationary or moving as a whole. Importantly, object recognition is critically dependent on motion perception. In the Time-constrained Stereo protocol, spatially disparate images are presented for a limited length of time, challenging binocular 3-dimensional integration in time. Both tests are appropriate for clinical usage and provide a simple, yet powerful, way to identify and quantify processes of demyelination and remyelination along visual pathways. These protocols may be efficient to diagnose and follow optic neuritis and multiple sclerosis patients.
In the diagnostic process, these protocols may reveal visual deficits that cannot be identified via current standard visual measurements. Moreover, these protocols sensitively identify the basis of the currently unexplained continued visual complaints of patients following recovery of visual acuity. In the longitudinal follow up course, the protocols can be used as a sensitive marker of demyelinating and remyelinating processes along time. These protocols may therefore be used to evaluate the efficacy of current and evolving therapeutic strategies, targeting myelination of the CNS.

Object From Motion (OFM) and Time-constrained stereo protocols are sensitive tools to identify monocular and binocular dynamic visual function deficits, which are uniquely affected in optic neuritis patients. Furthermore, these tests may be used as quantitative noninvasive tools to assess the extent of myelination along visual pathways.

Testes dinâmicos visuais para identificar e quantificar danos Visual e Reparação Após desmielinização na neurite óptica Pacientes

In order to follow optic neuritis patients and evaluate the effectiveness of their treatment, a handy, accurate and quantifiable tool is required to ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

Isolamento e caracterização funcional de Ventricular Humano cardiomiócitos a partir de amostras cirúrgicas frescos

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

Isolamento de células-tronco cancerosas em amostras de câncer de próstata humano

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

Lepr<sup&gt; Db</sup&gt; Mouse Modelo de Diabetes Tipo 2: Isolamento de ilhotas pancreáticas e células vivas 2-Photon Imagens de ilhotas intactas

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

Isolamento e Caracterização do perfil de exossomas de bílis humana contendo MicroRNA-

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

Full-root Aortic Valve Replacement by Stentless Aortic Xenografts in Patients with Small Aortic Roots

In patients with small aortic roots who need an aortic valve replacement with biological valve substitutes, the implantation of the stented pericardial valve might not meet the functional needs. The implantation of a too-small stented pericardial valve, leading to an effective orifice area indexed to a body surface area less than 0.85 cm2/m2, is regarded as prosthesis-patient mismatch (PPM). A PPM negatively affects the regression of left ventricular hypertrophy and thus the normalization of left ventricular function and the alleviation of symptoms. Persistent left ventricular hypertrophy is associated with an increased risk of arrhythmias and sudden cardiac death. In the case of predictable PPM, there are three options: 1) accept the PPM resulting from the implantation of a stented pericardial valve when comorbidities of the patient forbid the more technically demanding operative technique of implanting a larger prosthesis, 2) enlarge the aortic root to accommodate a larger stented valve substitute, or 3) implant a stentless biological valve or a homograft. Compared to classical aortic valve replacement with stented pericardial valves, the full-root implantation of stentless aortic xenografts offers the possibility of implanting a 3-4 mm larger valve in a given patient, thus allowing significant reduction in transvalvular gradients. However, a number of cardiac surgeons are reluctant to transform a classical aortic valve replacement with stented pericardial valves into the more technically challenging full-root implantation of stentless aortic xenografts. Given the potential hemodynamic advantages of stentless aortic xenografts, we have adopted full-root implantation to avoid PPM in patients with small aortic roots necessitating an aortic valve replacement. Here, we describe in detail a technique for the full-root implantation of stentless aortic xenografts, with emphasis on the management of the proximal suture line and coronary anastomoses. Limitations of this technique and alternative options are discussed.

Full-root aortic valve replacement by stentless aortic xenograft is a viable option in patients with small aortic roots. We describe, a technique for the full-root implantation of stentless aortic xenografts, with emphasis on the management of the proximal suture line and coronary anastomoses, and discuss its limitations and alternative options.

Substituição da valva aórtica de raiz completa por Xenotransplantes aórticos sem stent em pacientes com pequenas raízes aórticas

In patients with small aortic roots who need an aortic valve replacement with biological valve substitutes, the implantation of the stented pericardial ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

Isolamento de células humanas primárias do Decidual de membranas fetais de termo placentas

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation.
This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 &#176;C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

Intraportal transplante de ilhotas pancreáticas no modelo de Mouse

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...