Os autores agradecem as famílias dos doadores e as Organizações de Procura de Órgãos envolvidos na pesquisa e Martino Maria e Irina Kusmartseva para a sua assistência especializada. Este trabalho foi financiado pela Juvenile Diabetes Research Foundation (MC-T., MA), em apoio da Rede de Doadores de Órgãos do pâncreas com Diabetes.

1. Set-up <ol><li> Cassetes de etiquetas para blocos de parafina manualmente com um lápis, ou automaticamente com uma impressora cassete. Incluir o identificador do doador (CaseID), tipo de amostra, o número de alíquota, e qualquer identificação adicional única (planilha Case, <a href="http://jove.com/files/ftp_upload/4039/4039appendix1.doc" target="_blank">Anexo 1</a> ). </li><li> Rotular moldes Oct como para cassetes com um marcador permanente. Corte retângulos de 5-10 cm de papel alumínio. </li><li> Imprima ou manualmente etiqueta com o marcador permanente todos os frascos e tubos. Incluir frascos suficientes para amostras de encaixe picados congelados e tubos com meios RPMI para remessas frescas. Para os frascos de encaixe congelados com RNAlater, adicionar 1 mL de cada RNAlater criotubo. </li><li> Placas de Set-up de dissecação em biossegurança capô e bancadas com instrumentos esterilizados dissecação (pinças, bisturis, lâminas, compressas de gaze (4x4)), bem como cassetes e moldes PTU dispostos em ordem de uso. </li><li> Preparar um banho de congelação, colocandogelo seco numa caixa de isopor de tamanho médio (10x10 cm) a uma profundidade de pelo menos 3 cm e adicionar isopentano até o gelo seco é completamente coberto. </li><li> Opcional: Encha um recipiente pequeno Dewar com nitrogênio líquido. </li><li> Se pâncreas é necessário para microscopia electrónica (ME), preparar fixador TAAB pela adição de 1,25 mL de paraformaldeído a 16%, 1,25 mL de glutaraldeído 8%, e 7,5 mL 0,1 M PBS. Preparar fixador Lowicryl por adição de 2,5 mL de 16% de paraformaldeído e 7,5 mL 0,1 M PBS. </li><li> Prepare soluções usadas para isolar as células. Para frasco de 500 ml de DMEF (com glutamina), siga técnicas assépticas, acrescentando o seguinte: <ol><li> Soro fetal de bovino (FBS): adicionar 50 mL, 10% final. </li><li> Penicilina / estreptomicina (Pen / Strep, 10.000 U / mL / 10.000 ug / mL de stock) - adicionar 5 ml de caldo de Pen / Strep para RPMI para a concentração final de 100 U / mL / 100ug/mL. Também adicionar 5 ml de uma garrafa de 500 ml de DPBS, quando utilizados para a realização de gânglios linfáticos ou de amostras de baço. </li></ol></li></ol> 2. Pessoal <ol&gt; <li> Procedimento é perfeitamente realizado com três funcionários no entanto, o procedimento pode ser realizado com um indivíduo bem treinado. Dois indivíduos podem geralmente completar o procedimento em 90 minutos. Duração real depende da quantidade de tecidos recebidos e facilidade de encontrar gânglios pancreáticos. As amostras para isolamentos de células são mantidas a 4 ° C até à conclusão da dissecção tecido, enquanto as amostras de soro são processadas em paralelo. </li><li> Patologista, assistente de patologia, ou equivalente, um membro do pessoal experiente leva a manipulação de tecidos e executa dissecações principais, incluindo divisão (s) de cabeça do pâncreas e colheita gânglios pancreáticos. Os esforços iniciais são direcionados para pâncreas, seguido de exame de peri-pancreático gordura para os gânglios linfáticos. </li><li> Técnico de laboratório ou equivalente pessoal-membro treinado em tecido de corte cuja principal responsabilidade é para pesar pâncreas e regiões e tecidos de acabamento para os blocos fixos ou congelada e pressão v congeladoIALS. Recebe duodeno e baço de assistente de patologia e processa esses órgãos, enquanto o assistente de patologia está processando o pâncreas. </li><li> Membro assistente de laboratório pessoal, que auxilia os outros, incluindo blocos e rotulagem do frasco, por exemplo picagem criotubos e processamento de soro. </li></ol> 3. Dissecação Pancreas <ol><li> Retire o duodeno eo baço e limpar o pâncreas de gordura estranhos, vasos e nervos com dissecção romba e aguda </li><li> Coloque o pâncreas limpos em um tabuleiro de dissecação coberta com toalhas de papel. Mergulhar um aplicador com ponta de algodão com tinta azul e propagação na superfície anterior do pâncreas. Seque o excesso. </li><li> Opcional: Mergulhar um novo aplicador de tinta preta e propagação na superfície posterior do pâncreas. Retornar pâncreas a orientação normal com a superfície anterior de frente para patologista. </li><li> Corte o pâncreas em 3 regiões: cabeça, corpo e cauda (Figura 1). Corte a junção entre head corpo e, em seguida, dividir o restante em partes iguais do ~ para o corpo e cauda. </li><li> Pesar cada região do pâncreas e registro pesos (folha Caso workup ( <a href="http://jove.com/files/ftp_upload/4039/4039appendix1.doc" target="_blank">Apêndice 1</a> )). </li><li> Seção (aprox. 0,5 cm) de cada região do pâncreas em um &quot;pão de forma&quot; transversal forma (Figura 1). </li><li> Use as seções de ambas as junções entre as regiões para as amostras moídas. Dependendo do tamanho do pâncreas, uma amostra a partir da região de junção do corpo da cauda-pode ser difícil de obter, caso em que o número de blocos de parafina pode ser reduzida para obter amostras de picadas ou apenas as amostras obtidas do pâncreas região cabeça-corpo pode ser obtida. Criotubos etiqueta de acordo com a cabeça ou o corpo para denotar região proximal na junção. <ol><li> Picar pedaços e dividir igualmente entre criotubos (≥ 1 grama por frasco). Rapidamente congelar os frascos sem RNALater em nitrogênio líquido ou gelo seco no - suspensão isopentano em seguida, transferir toa -80 ° C congelador. </li><li> Manter frascos com RNALater à temperatura ambiente durante 30 minutos para permitir o equilíbrio, remix e rapidamente congelar em azoto líquido ou na suspensão de gelo seco-isopentano, em seguida, transferidos para um congelador -80 ° C. </li></ol></li><li> Remover secções alternadas de parafina e blocos OCT começando com parafina (Figura 1). </li><li> Incline-se para todas as seções transversais para a direita. </li><li> Coloque ~ 1,5 x 1,5 x 0,5 cm em seções as cassetes. Se as secções transversais são muito grandes, cortado em meia (Figura 2b). Cassetes de etiquetas para metade anterior azul como &quot;A&quot; e metade posterior como &quot;B&quot;. </li><li> Se as peças são ainda muito grande, cortar cada secção perpendicular ao corte anterior (Figura 2c). Cassetes de etiquetas AD no sentido horário. Corte pedaços mais forem necessárias, para caber dentro de cassetes. </li><li> Blocos de números em seqüência dentro de região, começando com a parte mais medial. </li><li> Para blocos de parafina, a posição cassettes forma que o rótulo é para a esquerda e local do tecido na cassete, mantendo a sua orientação. Tampa da fita estreita e transferência para um recipiente com ~ 500 ml de 10% formalina tamponada neutra (NBF). Inicie a fixação de temporização, quando a cassete último é colocado em fixador. </li><li> Para blocos de PTU, despeje uma fina camada de outubro de mídia dentro do molde prelabeled em seguida, colocar o tecido no molde tendo a certeza de manter a orientação e cobrir o tecido com outubro Rapidamente mergulhe os moldes PTU em um gelo seco - suspensão isopentano de ~ 1 minuto. Remover a partir de pasta e local em gelo seco para o armazenamento temporário ou em -80 ° C congelador para armazenamento a longo prazo. </li><li> Obter ~ de 0,5 x 0,5 cm fatias a partir da junção cabeça-corpo para microscopia eletrônica (ME). Fatia único lugar do fixador TAAB e outra fatia no fixador Lowicryl. Armazenar os dois tipos de fixador com amostras a 4 ° C durante pelo menos 48 horas e / ou indefinidamente até processamento posterior para EM. </li></ol> 4. Baço, Linfonodo dissecçãoções e do mucosa duodenal dissecções <ol><li> Isolar nodos linfáticos pancreáticas (PLN) de gordura e aparar todo o tecido conjuntivo para a cápsula de nó. Coloque no prato estéril de cultura de células contendo DMEF. Dependendo números totais isoladas, dividir PLN em alíquotas para as transferências frescas, isolamentos de células criopreservadas, parafina e / ou blocos OCT e frascos. Picar em pedaços grandes nós não maiores que 0,5 cm. </li><li> Picar nós pancreáticas não-linfáticos e no baço em pequenos (aprox. 0,5 cm) cubos. Coloque em fixador, mídia outubro, e ou DMEM para remessas de amostras frescas ou isolamentos de células criopreservadas de acordo com os valores das matérias-primas. </li><li> Abra o duodeno e limpe cuidadosamente a mucosa duodenal de muco limpo com uma gaze umedecida. Cortar secções da mucosa para parafina e / ou blocos congelados OCT e amostras moídas para frascos, com e sem RNAlater. Congelar como para outras amostras. </li></ol> 5. Quebrar blocos OCT no pré-rotulados papel alumínio e colocar blocos de PTU e<html> frascos em caixas para armazenamento a longo prazo a -80 ° C. 6. Enviar amostras fixas de Processamento de parafina <ol><li> Fixar tecidos durante 16 horas (intervalo de 20 ± 4 horas), utilizando um processador automático de parafina ou usando temporização manual. Processo para blocos de parafina, usando um processador automático ( <a href="http://jove.com/files/ftp_upload/4039/4039appendix2.doc" target="_blank">Apêndice 2</a> ). </li><li> Incorporar secções na orientação exacta tal como é colocado pelo patologista com o rótulo da cassete para a esquerda. Incorporar a mucosa duodenal com a superfície de corte para baixo para permitir o exame da perpendicular mucosa para a submucosa. </li></ol> 7. Limpe as áreas de dissecação e desinfectar. Lave instrumentos de dissecção e re-esterilizar. Coloque tecidos remanescentes humanos em fixador de formalina para o armazenamento como resíduos biomédica. Descartar lixo biomédico de acordo com os regulamentos locais dentro de um mês. 8. Atualizar o sistema de inventário Amostra ve_title &quot;&gt; 9. resultados representativos Este procedimento irá processar um pâncreas humano intacto com uma amostragem representativa em todo o órgão, incluindo demarcação das três grandes regiões, a saber, cabeça, corpo e cauda dentro de 2 horas. A região uncinado, encontrado na região posterior da cabeça, é incluído na dissecção cabeça. O peso médio de pâncreas de doadores de órgãos sem diabetes foi de 82,4 gramas (52,7 - 139,0 gramas). Pesos pâncreas regionais foram aproximadamente iguais (Figura 3). Este procedimento também pode permitir a reconstrução do pâncreas inteiro usando lâminas coradas de parafina seqüencial e blocos PTU. Vários formatos são viáveis ​​permitindo a máxima utilização de tecnologias atuais e futuras. A planilha caso atual nPOD é fornecido ( <a href="http://jove.com/files/ftp_upload/4039/4039appendix1.doc" target="_blank">Anexo 1</a> ) que é utilizado para documentar as amostras recuperadas e proc subseqüenteessing por tipos de alíquotas e quantidades. <img alt="A Figura 1" src="/files/ftp_upload/4039/4039fig1.jpg" /> Figura 1. Esquema geral do processo. O pâncreas inteiro é processado, mantendo orientações anatômicas. A região da cabeça é mais longo do eixo superior-inferior devido à presença do lobo pancreático ventral na região posterior, que inclui a região uncinado. Área hachurada nos cruzamentos denotar regiões usadas para amostras moídas em criotubos. P, parafina, O, outubro <img alt="A Figura 2" src="/files/ftp_upload/4039/4039fig2.jpg" /> Figura 2. Esquema de letras para as secções de pâncreas. Fatias transversais do pâncreas pode ser dividido em metades ou quartos e letras no sentido horário. <img alt="A Figura 3" src="/files/ftp_upload/4039/4039fig3.jpg" /> Figura 3. Pesos de pâncreas de doadores de órgãos sem diabetes. Ospâncreas intacta de doadores adultos (&gt; 17 anos) foi pesado, em seguida, dividido em regiões e pesos regionais obtidos. Os dados são médias ± SEM (n = 15).

<ol>
	<li>Brissova, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+human+pancreatic+islet+architecture+and+composition+by+laser+scanning+confocal+microscopy.">Assessment of human pancreatic islet architecture and composition by laser scanning confocal microscopy.</a> J. Histochem. Cytochem. 53, 1087-1097 (2005).</li><li>Rahier, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pancreatic+polypeptide+cells+in+the+human+pancreas:+the+effects+of+age+and+diabetes.">The pancreatic polypeptide cells in the human pancreas: the effects of age and diabetes.</a> J. Clin. Endocrinol. Metab. 56, 441-444 (1983).</li><li>Uchida, T., Takada, T., Ammori, B. J., Suda, K., Takahashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+reconstruction+of+the+ventral+and+dorsal+pancreas:+a+new+insight+into+anatomy+and+embryonic+development.">Three-dimensional reconstruction of the ventral and dorsal pancreas: a new insight into anatomy and embryonic development.</a> J. Hepatobiliary Pancreat. Surg. 6, 176-180 (1999).</li><li>Wittingen, J., Frey, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+concentration+in+the+head,+body,+tail+and+uncinate+process+of+the+pancreas.">Islet concentration in the head, body, tail and uncinate process of the pancreas.</a> Ann. Surg. 179, 412-414 (1974).</li><li>Rahier, J., Goebbels, R., Henquin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+composition+of+the+human+diabetic+pancreas.">Cellular composition of the human diabetic pancreas.</a> Diabetologia. 24, 366-371 (1983).</li><li>Vaught, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+NCI+perspective+on+creating+sustainable+biospecimen+resources.">An NCI perspective on creating sustainable biospecimen resources.</a> J. Natl. Cancer Inst. Monogr. 42, 1-7 (2011).</li><li>Gianani, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+results+of+screening+of+nondiabetic+organ+donors+for+expression+of+islet+autoantibodies.">Initial results of screening of nondiabetic organ donors for expression of islet autoantibodies.</a> J. Clin. Endocrinol. Metab. 91, 1855-1861 (2006).</li><li>Tauriainen, S., Salmela, K., Rantala, I., Knip, M., Hy&#246;ty, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collecting+high-quality+pancreatic+tissue+for+experimental+study+from+organ+donors+with+signs+of+&#946;-cell+autoimmunity.">Collecting high-quality pancreatic tissue for experimental study from organ donors with signs of &#946;-cell autoimmunity.</a> Diabetes Metab. Res. Rev. 26, 585-592 (2010).</li><li>In't Veld, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+insulitis+in+adult+autoantibody-positive+organ+donors.">Screening for insulitis in adult autoantibody-positive organ donors.</a> Diabetes. 56, 2400-2404 (2007).</li><li>T&#246;ns, H. A., Terpstra, O. T., Bouwman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+human+pancreata+in+perspective+of+the+isolation+of+the+islets+of+langerhans.">Heterogeneity of human pancreata in perspective of the isolation of the islets of langerhans.</a> Transplant Proc. 40, 367-369 (2008).</li></ol>

Não há conflitos de interesse declarados.

O objectivo deste procedimento é definir um processamento pâncreas padrão que pode ser harmonizadas entre os locais de recolha múltiplas e, assim, permitir a comparação dos resultados obtidos por diferentes grupos. O método de dissecção é baseada no padrão cirúrgicos práticas patologia com passos adicionais para anotação das regiões mais importantes do pâncreas seguido por intra-regionais subdivisões. Padronização de métodos de processamento biospecimen é necessário para facilitar a recolha de amo...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Instrumentos de dissecção incluindo bisturis com 22 lâminas </td><td> Vário </td><td> Vário </td><td></td></tr><tr><td> Tubos de centrífuga (15 e 50 ml) </td><td> Fisher Scientific </td><td> 05-539-5, 430828 </td><td> Estéril </td></tr><tr><td> Criotubos (1,8 ml) </td><td> VWR América do Norte </td><td> 89004-300 </td><td> Estéreis, códigos de barras opcionais </td></tr><tr><td> RNAlater </td><td> Ambion </td><td> AM7021 </td><td></td></tr><tr><td> Tinta Patologia Cirúrgica </td><td></td><td></td><td></td></tr><tr><td> Cassetes </td><td> Mercedes Medical </td><td> CAS ECO109 </td><td></td></tr><tr><td> 10% formalina tamponada neutra </td><td> Fisher Scientific </td><td> 23-245-685 </td><td></td></tr><tr><td> Congelamento de mídia PTU </td><td> TissueTek </td><td> 4583 </td><td></td></tr><tr><td> Moldes </td><td> ThermoScientific </td><td> 58952 </td><td> Vários tamanhos </td></tr><tr><td> Isopentano </td><td> Fisher Scientific </td><td> O3551-4 </td><td> Outubro banho frio </td></tr><tr><td> O gelo seco </td><td></td><td></td><td> Outubro banho frio </td></tr><tr><td> Nitrogênio líquido em frasco de Dewar </td><td></td><td></td><td> Encaixe criotubos de congelamento </td></tr><tr><td> Prato de cultura de células (100 mm) </td><td> Corning </td><td> 430167 </td><td> Estéril </td></tr><tr><td> Hyclone DMEF </td><td> ThermoScientific </td><td> SH30023.01 </td><td> Loja, estéril refrigerado, glutamina incluído </td></tr><tr> &lt;td&gt; Soro Fetal Bovino </td><td> Cellgro </td><td> 35-010-CV </td><td> Estéreis, loja de alíquotas congeladas </td></tr><tr><td> Penicilina / estreptomicina / Antifungal </td><td> GIBCO </td><td> 15240-062 </td><td> Estéreis, loja de alíquotas congeladas </td></tr><tr><td> Digite 9 Parafina </td><td> Richard Allan-Científico </td><td> 8337 </td><td></td></tr><tr><td> D-PBS 1X </td><td> GIBCO </td><td> 14190-144 </td><td> Estéril </td></tr><tr><td> Paraformaldeído 16% </td><td> EM Sciences </td><td> 15710 </td><td> Faça loja fresco, refrigerado </td></tr><tr><td> Glutaraldeído 8% </td><td> EM Sciences </td><td> 16019 </td><td> Faça loja fresco, refrigerado </td></tr><tr><td> Nível de Biossegurança 2 capa </td><td> Vário </td><td></td><td></td></tr><tr><td> Ultra-baixo congelador </td><td> Vário </td><td></td><td></td></tr><tr><td> Printer Cassete Sakura </td><td> Sakura </td><td></td><td></td></tr><tr><td> Sakura VIP300 </td><td> Sakura </td><td></td><td> Processador automático de parafina </td></tr><tr><td> Estação de Inclusão em Parafina </td><td> ThermoFisher </td><td></td><td></td></tr></tbody></table> Tabela 1. Reagentes específicos e equipamentos.

collection protocol for human pancreas

Esta dissecção e procedimento de amostragem foi desenvolvido para a Rede de Doadores de Órgãos do pâncreas com Diabetes programa (nPOD) para padronizar a preparação do pâncreas recuperado de doadores de órgãos cadáveres. O pâncreas é dividido em 3 regiões principais (cabeça, corpo, cauda), seguido por seriados transversais ao longo do eixo medial para lateral. Secções alternadas são utilizados para parafina fixo e blocos congelados frescos e amostras remanescentes são picados para encaixe preparações amostra congelada, com ou sem inibidores de RNAse, por DNA, RNA, ou o isolamento de proteínas. O objetivo geral do procedimento de dissecção pâncreas é para provar o pâncreas inteiro, mantendo a orientação anatômica. Heterogeneidade endócrino célula em termos de composição ilhéu, tamanho e número é relatado para ilhotas humanos em comparação com ilhotas de roedores 1. A maioria dos ilhéus humanos a partir da cabeça do pâncreas, regiões do corpo e da cauda são compostos de insulina containin-g beta-células seguido por proporções inferiores de glucagon contendo ct-células e somatostatina-ô-contendo células. Células pancreáticas polipeptídeo contendo PP e células que contêm grelina epsilon também estão presentes, mas em pequenas quantidades. Em contraste, a região uncinado contém ilhotas que são principalmente compostas por células pancreáticas polipéptido contendo 2 pp. Essas variações regionais ilhotas surgem diferenças de desenvolvimento. O pâncreas desenvolve a partir das gemas pancreáticas ventral e dorsal no estômago e depois da rotação do estômago e do duodeno, os movimentos do lóbulo ventral e os fusíveis com o 3 dorsal. O lóbulo ventral forma a porção posterior da cabeça, incluindo o processo uncinado enquanto o lobo dorsal dá origem ao resto do órgão. Variação de pâncreas Regional também é relatado com a região da cauda com maior densidade de ilhota em comparação com outras regiões e os dorsais lobo derivados componentes submetidos a atrofia seletiva na diabetes tipo 1 <saté&gt; 4,5. Órgãos, tecidos e muitas vezes são recuperadas a partir dos doadores de órgãos e incluir nós pancreáticas linfáticos, baço e gânglios linfáticos não-pancreáticas. Estas amostras são recuperados com os formatos similares às utilizadas para o pâncreas com a adição de isolamento de células criopreservadas. Quando o duodeno proximal está incluído com o pâncreas, mucosa duodenal pode ser recolhida para parafina e congeladas blocos e preparações instantâneas picados congelados.

Watch this Scientific Journal Video about Collection Protocol for Human Pancreas at JoVE.com

Collection Protocol for Human Pancreas

Este vídeo demonstra um processo para o processamento de dissecção pâncreas humano em formatos de armazenamento. Orientação anatómica é mantida ao longo das regiões pancreáticas para permitir a definição da composição de ilhotas regional e densidade.

Protocolo de Coleta para Pâncreas Humano

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

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14.7K visualizações.  University of Florida. Este vídeo demonstra um processo para o processamento de dissecção pâncreas humano em formatos de armazenamento. Orientação anatómica é mantida ao longo das regiões pancreáticas para permitir a definição da composição de ilhotas regional e densidade.

Vídeo: Collection Protocol for Human Pancreas

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 ...

Medicina

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

Coleção, isolamento e Citometria de Fluxo de Análise de Amostras endocervix Humano

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female ...

A Detailed Protocol for Perspiration Monitoring Using a Novel, Small, Wireless Device

Perspiration monitoring can be utilized for the detection of certain diseases, such as thermoregulation and mental disorders, particularly when the patients are unaware of such disorders or are having difficulty expressing their symptoms. Until now, several devices for perspiration monitoring have been developed; however, such devices tend to have a relatively large exterior, considerable power consumption, and/or less sensitivity.
Recently, we developed a small, wireless device for perspiration monitoring. The device consists of a temperature/relative humidity (T/RH) sensor, battery-driven small data logger, and silica gel as a desiccant in a small cylindrical exterior. The T/RH sensor is placed between the detection windows (through which the water vapor from the skin enters) and the silica gel. The underlying principle of the perspiration monitoring device is based on Fick&#39;s law of diffusion, which means that water vapor flux from the skin to the silica gel (i.e.&#160;transepidermal water loss and perspiration) can be captured by change in humidity at the T/RH sensor. In addition, a baseline subtraction method was adopted to distinguish perspiration and transepidermal water loss.
As shown in the previous report, the developed device can monitor the perspiration at any sites of the body in an easy, wireless manner. However, detailed methods of how to use the device have not been disclosed yet. In this article, therefore, we would like to show the point-by-point tutorials of how to use the device for perspiration monitoring, by showing the sympathetic activity test with the sympathetic skin response monitoring as an example.

Recently, we developed a small wireless device for perspiration monitoring. In this article, we present detailed protocols on how to use the device for perspiration monitoring with an example of the sympathetic activity test.

Um protocolo detalhado para Monitoramento transpiração Usando um Novel, Pequeno, dispositivo sem fios

Perspiration monitoring can be utilized for the detection of certain diseases, such as thermoregulation and mental disorders, particularly when the ...

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

Modelo do rato para transplante de pâncreas, usando uma técnica modificada do manguito

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the ...

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at the 12 codon (Ptf1acre/+;LSL-KrasG12D/+) mouse strain as a model of human pancreatic cancer. The goal of our current studies is to identify novel metabolic biomarkers of pancreatic cancer progression. We have performed metabolic profiling of urine, feces, blood, and pancreas tissue extracts, as well as histological analyses of the pancreas to stage the cancer progression. The mouse pancreas is not a well-defined solid organ like in humans, but rather is a diffusely distributed soft tissue that is not easily identified by individuals unfamiliar with mouse internal anatomy or by individuals that have little or no experience performing mouse organ dissections. The purpose of this article is to provide a detailed step-wise visual demonstration to guide novices in the removal of the mouse pancreas by dissection. This article should be especially valuable to students and investigators new to research that requires harvesting of the mouse pancreas by dissection for metabolic profiling or histological analyses.

This video article provides a detailed demonstration of the procedures required to successfully remove the pancreas from a mouse by dissection for histological analysis and metabolic profiling.

Dissecação do pâncreas Mouse para análise histológica e perfis metabólicos

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at ...

Robotic Lateral Pancreaticojejunostomy for Chronic Pancreatitis

Lateral pancreaticojejunostomy (LPJ) has shown good postoperative outcomes in patients with painful, morphine dependent, chronic pancreatitis (CP). The recent rise of robotic and laparoscopic pancreatic surgery has found benefits such as reduced time to functional recovery. Few studies have reported on the feasibility, technique and outcome of robotic LPJ, especially including transection of the gastroduodenal artery. The present study describes the main steps for robotic LPJ in a patient with painful chronic pancreatitis with a dilated main pancreatic duct. The patient underwent robotic LPJ. The LPJ anastomosis is performed using a running suture technique in a longitudinal side-to-side manner. Routinely, the gastroduodenal artery is transected to drain the entire length of the main pancreatic duct. The patient is in French position; 7 trocars are placed (4 robotic, 2 laparoscopic assistants, 1 liver-retractor). After docking of the robot system, the omental bursa is opened, and the right gastroepiploic artery and vein are ligated at their base at the lower border of the pancreas. Intraoperative ultrasonography is performed in order to determine trajectory of the dilated main pancreatic duct which is opened for its entire length after the gastroduodenal artery has been suture ligated both cranially and caudally from the main pancreatic duct. A Roux limb is created, and a latero-lateral PJ is fashioned using several 3-0 barbed sutures. A stapled jejuno-jejunostomy is created at sufficient distance from the pancreatic anastomosis, aided by a 50 cm suture. The described technique for robotic LPJ is a complex but feasible operation for patients with treatment refractory CP and a dilated main pancreatic duct. Due to its complexity, implementation in high volume centers with extensive experience with CP surgery may improve outcomes.

Robotic lateral pancreaticojejunostomy (RLPJ) may be used in patients with painful, morphine dependent, chronic pancreatitis and a dilated main pancreatic duct. We describe a standardized and reproducible technique for RLPJ, which includes transection of the gastroduodenal artery.

Pancreaticojejuntomia lateral robótica para pancreatite crônica

Lateral pancreaticojejunostomy (LPJ) has shown good postoperative outcomes in patients with painful, morphine dependent, chronic pancreatitis (CP). The ...

Robot-Assisted Kidney Transplantation

This paper describes robot-assisted kidney transplantation (RAKT) from a living donor. The robot is docked between the parted legs of the patient, placed in the supine Trendelenburg position. Kidney allografts are provided by a living donor. Before vascular anastomosis, the kidney allograft is prepared by inserting a double-J stent in the ureter, and the temperature for the anastomosis is lowered by wrapping it in an ice-packed gauze. A 12 mm or 8 mm port for the robotic camera and three 8 mm ports for robotic arms are placed. A peritoneal pouch is created for the kidney allograft by raising the peritoneal flaps on both sides over the psoas muscle before dissecting the iliac vessels and bladder. A 6 cm Pfannenstiel incision is made to insert the kidney into the peritoneal pouch, lateral to the right iliac vessels. 
After clamping the external iliac vein with Bulldogs clamps, a venotomy is performed, and the graft renal vein is anastomosed to the external iliac vein in an end-to-side continuous manner with a 6/0 polytetrafluoroethylene suture. After clamping the graft renal vein, the iliac vein is declamped. This is followed by clamping of the external iliac artery, arteriotomy, arterial anastomosis with a 6/0 polytetrafluoroethylene suture, clamping of the graft renal artery, and declamping of the external iliac artery. Reperfusion is then carried out, and ureteroneocystostomy is performed using the Lich-Gregoir technique. The peritoneum is closed at a few locations with polymer locking clips, and a closed-suction drain is placed through one of the working ports. After deflating the pneumoperitoneum, all incisions are closed.

This paper provides technical details for robot-assisted kidney transplantation from a living donor.

Transplante Renal Assistido por Robô

This paper describes robot-assisted kidney transplantation (RAKT) from a living donor. The robot is docked between the parted legs of the patient, placed ...

Collection, Expansion, and Differentiation of Primary Human Nasal Epithelial Cell Models for Quantification of Cilia Beat Frequency

Measurements of cilia function (beat frequency, pattern) have been established as diagnostic tools for respiratory diseases such as primary ciliary dyskinesia. However, the wider application of these techniques is limited by the extreme susceptibility of ciliary function to changes in environmental factors e.g., temperature, humidity, and pH. In the airway of patients with Cystic Fibrosis (CF), mucus accumulation impedes cilia beating. Cilia function has been investigated in primary airway cell models as an indicator of CF Transmembrane conductance Regulator (CFTR) channel activity. However, considerable patient-to-patient variability in cilia beating frequency has been found in response to CFTR-modulating drugs, even for patients with the same CFTR mutations. Furthermore, the impact of dysfunctional CFTR-regulated chloride secretion on ciliary function is poorly understood. There is currently no comprehensive protocol demonstrating sample preparation of in vitro airway models, image acquisition, and analysis of Cilia Beat Frequency (CBF). Standardized culture conditions and image acquisition performed in an environmentally controlled condition would enable consistent, reproducible quantification of CBF between individuals and in response to CFTR-modulating drugs. This protocol describes the quantification of CBF in three different airway epithelial cell model systems: 1) native epithelial sheets, 2) air-liquid interface models imaged on permeable support inserts, and 3) extracellular matrix-embedded three-dimensional organoids. The latter two replicate in vivo lung physiology, with beating cilia and production of mucus. The ciliary function is captured using a high-speed video camera in an environment-controlled chamber. Custom-built scripts are used for the analysis of CBF. Translation of CBF measurements to the clinic is envisioned to be an important clinical tool for predicting response to CFTR-modulating drugs on a per-patient basis.

This protocol describes nasal epithelial cell collection, expansion, and differentiation to organotypic airway epithelial cell models and quantification of cilia beat frequency via live-cell imaging and custom-built scripts.

Coleta, Expansão e Diferenciação de Modelos Primários de Células Epiteliais Nasais Humanas para Quantificação da Frequência de Batimento de Cílios

Measurements of cilia function (beat frequency, pattern) have been established as diagnostic tools for respiratory diseases such as primary ciliary ...

Normothermic Ex Vivo Pancreas Perfusion for the Preservation of Pancreas Allografts before Transplantation

Pancreas transplantation (PTx) is a curative treatment for people who live with the burden of a diagnosis of diabetes mellitus (DM). However, due to organ shortages and increasing numbers of patients being listed for PTx, new strategies are needed to increase the number of available grafts for transplantation. 
Static cold storage (SCS) is considered the gold standard for standard criteria organs. However, standard criteria donors (SCD) are becoming scarce and new strategies that can increase the rate of organ acceptance from extended criteria donors (ECD) are urgently needed. 
Normothermic ex vivo perfusion (NEVP) is one of the strategies that has become increasingly popular over the past couple of decades. This preservation method has already been used successfully in other organs (liver, kidneys, and lungs) but has been minimally explored in pancreas transplantation. The few papers that describe the method for pancreas show little success, edema being one of the major issues. The following manuscript describes the successful NEVP method and setup developed by our group to perfuse swine pancreas.

Normothermic Ex Vivo Pancreas Perfusion for the Preservation of Pancreas Allografts before Transplantation

Normothermic ex vivo machine perfusion (NEVP) has scarcely been explored for the preservation of pancreas allografts. We present an innovative preservation technique for pancreas allografts before transplantation.

Perfusão Normotérmica Ex Vivo do Pâncreas para Preservação de Aloenxertos de Pâncreas antes do Transplante

Pancreas transplantation (PTx) is a curative treatment for people who live with the burden of a diagnosis of diabetes mellitus (DM). However, due to organ ...

Surgical Tips and Tricks for Performing Porcine Pancreas Transplantation

Despite the promising results of pancreas transplantation in type 1 diabetes mellitus and metabolic syndrome, the biggest concern around this state-of-the-art technique remains the paucity of organs deemed fit for transplantation. High intravascular resistance, delicate intraparenchymal capillary framework, and complex lobular anatomy around the mesenteric vasculature are what make this organ more susceptible to injury and less tolerant to trivial trauma compared to organs such as the liver and kidney. Meticulous surgical dissection and judicious tissue handling form the cornerstone of the entire exercise of pancreas transplantation. Owing to morphological similarity between the anatomy of the porcine pancreas to the surrounding mesenteric vessels and the organs when compared to the human anatomy, demonstration of the technique in the porcine model could help to most accurately extrapolate this to a human setting. The present article aims to outline the essential surgical tips and tricks that need to be followed, in order to ensure a higher success rate of transplantation of this highly susceptible organ in a porcine 3-day survival model.

The video article summarizes the technique of pancreatectomy and pancreas allotransplantation in a porcine 3-day survival model with a step-by-step description of the method and emphasis on the surgical tips and tricks to deal with the precarious and delicate porcine visceral anatomy.