The authors would like to thank Joshua Kimani, clinical director of the research program at the University of Nairobi, for his assistance with mucosal immunology studies related to this protocol. The authors would like to acknowledge funding from CHVI grant MOP 86721.

Declaração de ética: a ética conselhos de administração da Universidade de Manitoba e Kenyatta National Hospital / Universidade de Nairobi de pesquisa aprovado este estudo e consentimento informado foi obtido de todos os participantes do estudo. 1. Preparação de Mídia e CMC Coleção Tubes <ol><li> Preparar a solução (PBS) (NaCl 137,93 mM, KCl 2,67 mM, 8,1 mM de Na 2 HPO 4, 1,47 mM, KH 2 PO 4) tampão fosfato salino. Autoclave para esterilidade. Este pode ser armazenado a 4 ° C durante vários meses. </li><li> Pré-alíquota de 5 ml PBS em 50 ml tubo falcon (uma por amostra a ser coletado) e armazenar a 4 º C até o tempo de coleta de amostras. </li><li> Rotular 2-4 criotubos ou 1,5 ml microtubos por amostra para coletar lavagem para armazenamento. </li><li> Preparar meios de cultura celular: RPMI 1640 + 1% de penicilina / estreptomicina / anfotericina B (concentração final de 100 unidades / ml, 100 ug / ml e 250 ng / ml, respectively, sem FCS / FBS acrescentou. </li></ol> 2. Coleta de Amostras cytobrush Coleta de amostras cytobrush é um procedimento não-invasivo, que deve ser realizada por um médico ou ginecologista treinado. <ol><li> Coletar células mononucleares do colo do útero (CMC) usando tanto um cytobrush e um raspador de madeira ou de plástico. Colete CMC do participante sob exame especular, inserindo o raspador e girando ao redor do orifício cervical (Figura 1). Insira o cytobrush no SO endocervical, gira 360 ° e coloque imediatamente tanto o cytobrush e raspador no tubo falcon 50 ml contendo 5 ml de PBS. </li><li> Manter as amostras em gelo / a 4 º C até o processamento. Para manter a viabilidade celular, amostras de processos dentro de 2 horas da coleta. </li><li> Se possível, coletar amostras de sangue compensadas em top vacutainers heparina verdes para células mononucleares do sangue periférico (PBMC) de isolamento para servir como um comparador / controle para análise CMC. </li&gt; </ol> 3. Isolamento de CMC Executar o processamento de amostras em um laboratório de biossegurança nível 2, em uma cabine de segurança biológica estéril com luvas duplas. <ol><li> Durante a coleta, as amostras de CMC podem ser contaminados com sangue. Excluir amostras com contaminação de sangue visível a partir do estudo, a fim de evitar a inclusão de confusão de PBMC na amostra final. Devido ao baixo número de linfócitos isolados a partir de amostras de CMC, contaminação sanguínea menor pode facilmente sobrecarregar o componente de linfócitos CMC. </li><li> Tubos Vortex falcon contendo tanto o cytobrush e raspador para 45 seg dissociar células das escovas. As amostras podem tornar espumoso durante este processo; isso é normal. </li><li> Use o cytobrush para raspar qualquer material restante fora do raspador, e descartar o raspador em uma solução de água sanitária. Para coletar as células continuem ligadas ao escova / raspador, use uma luva fresca para espremer o raspador entre o polegar eo indicador.Deslize a escova, permitindo que as células e PBS a ser recolhidos no mesmo tubo de 50 ml. Descarte o cytobrush em uma solução de água sanitária, e descarte a luva. IMPORTANTE: Use uma luva nova para cada amostra. </li><li> Instale um filtro de células 100 mm de nylon para um novo tubo de 50 ml falcão. Usando uma pipeta de transferência, recolher a suspensão PBS-célula do tubo de amostra e filtra-se através do filtro para dentro do tubo fresco. </li><li> Adicionar 5 ml de meio RPMI 1640 para o tubo de amostra original. Utilizando a pipeta de transferência, lavar os lados do tubo de ensaio com o meio RPMI, e transferir através do filtro para o novo tubo de recolha. Lava-se a parte inferior do filtro de nylon, com o meio RPMI bem. </li><li> Centrifugar as amostras durante 10 min a 514 x g. É crucial para deixar o freio de centrifugação, durante este passo, a fim de evitar desalojamento do sedimento CMC. </li><li> Remova cuidadosamente o sobrenadante do tubo, tomando cuidado para não perturbar o sedimento. Tamanho da pelota será altamente variável; no modome amostras, o sedimento é muito grande, devido à presença de grandes números de células epiteliais, enquanto em outros casos, a pelete é pouco visível e altamente transparente. </li><li> Ressuspender o sedimento por agitação suave do tubo falcon. Adicionar 5 ml de PBS para lavar a amostra. </li><li> Centrifugar novamente durante 10 min a 514 x g sem travão de centrifuga. </li><li> Cuidadosamente descarte do sobrenadante e re-suspensão da pelota como acima. As células estão prontas para qualquer criopreservação (usando um protocolo de criopreservação PBMC), a estimulação ou citometria de fluxo fenotipagem. </li></ol> 4. CMC manchas na superfície e Citometria de Fluxo <ol><li> Ressuspender o sedimento do passo de 3,10 em 100 ul de solução de bloqueio e transferir quer numa placa de 96 poços ou de tubo de FACS. A solução de bloqueio (para bloquear os receptores Fcy) receita é como se segue: 1,8 ul de IgG (concentração final de 0,2 ug / ul), 5 ul de FBS e 93,2 ul de lavagem de FACS (PBS 2% de FBS).A coloração pode ser realizada em uma placa de 96 poços que os tamanhos de aglomerados são suficientemente pequenas, mas podem precisar de ser realizada em tubos de FACS se pelotas são rotineiramente grande. É importante para a titulação de anticorpos em conformidade. </li><li> Bloquear as amostras durante 10 min em gelo / a 4 ° C. </li><li> Lavar as células com 100 ul de FACS de lavagem (PBS 2% de FBS) em placas de 96 poços, ou 500 ul em tubos de FACS. Centrifugar a 600 xg por 10 minutos a 4 ° C com o freio baixo (ou fora, se um nível baixo de freio não estiver disponível). </li><li> Remover o sobrenadante e ressuspender o sedimento de células por agitação. Adicionar Stain viabilidade (corante de viabilidade solucionáveis ​​Morto Live). Prepare corante de viabilidade de acordo com as instruções do fabricante. Ela pode necessitar de ser ajustada para uso em cada configuração de laboratório / citómetro. Incubar durante 30 min no escuro a 4 ° C. </li><li> Lave as células 100 μl/500 ul de PBS (para tubos de placa / FACS de 96 poços). Centrifugar a 600 xg por 10 min com baixo / nenhum freio. Remover supernatant e ressuspender as células. </li><li> Incubar as células com o cocktail de anticorpos de marcadores de superfície, num volume total de 100 ul. IMPORTANTE: Otimizar e titular cada citometria de fluxo do painel antes de provar coloração. Incubar as células durante 30 min no escuro a 4 ° C. </li><li> Repita o passo 4.5. Se a coloração em uma placa de 96 poços, as células de transferência para um tubo de FACS e diluir para um volume final de 750 ul de FACS de lavagem contendo 1% de paraformaldeído (PFA) (ou Cytofix, diluídos 1 para 4). Continuar a aquisição de dados em um citómetro de fluxo. </li></ol> 5. Coleta e Preparação de colo do útero Vaginal Lavage (CVL) <ol><li> Antes da amostragem cytobrush, utilizar uma seringa, para lavar o endocérvix com 2 ml de PBS estéril e aspirar a lavagem do saco posterior. </li><li> Colete a amostra do lavado aspirado em um tubo falcon de 15 ml e manter em gelo / a 4 º C até o processamento. </li><li> Amostra Centrifugar a 400 g durante 7 min para remover os restos celulares. </li><li> Collect o sobrenadante, alíquota da amostra em 1 ml ou 500 mL alíquotas e armazenar a -70 ° C. As alíquotas podem ser descongelados para ELISA ou análise disposição talão de proteínas CVL, mas evite múltiplos ciclos de congelamento / descongelamento. </li><li> Se desejado, ressuspender o sedimento de restos celulares em ARN Posterior para medir a expressão de ARN seguindo o protocolo do fabricante. </li></ol> 6. Aquisição de Dados <ol><li> Preparar um conjunto de tubos de compensação que corresponde painel de anticorpos. Execute os tubos de compensação para ajustar sobreposição espectral. Execute as amostras de qualquer fluxo multicolor citômetro que está configurada para os anticorpos conjugados com fluorocromos utilizados no painel. </li><li> Adquirir uma amostra PBMC primeiro para ajustar Forward Scatter (FSC) e Side Scatter tensão (SSC), a fim de detectar a população de linfócitos. Tente mantê-los em 100 em cada eixo em uma escala linear. Executando um controle PBMC irá facilitar significativamente para encontrar a população de linfócitos CMC. </li><li> O limiar do FSC para amostras de CMC pode precisar de ser aumentada relativamente a amostras de PBMC devido a manchar-se o eixo de SSC a baixos valores de FSC. </li><li> Vortex cada tubo antes de adquirir. Adquirir todo o tubo para maximizar o número de eventos de porta de linfócitos recolhidos. Monitore a máquina com cuidado para evitar entupimento. </li><li> Exportar os dados em um programa de análise por citometria de fluxo, tais como FlowJo. </li></ol> 7. Estratégia de propagação <ol><li> Selecione Encaminhar dispersão lateral elevada (FSC-H) / área de dispersão lateral para a frente (FSC-A) para determinar a população singlet. </li><li> Selecione Tempo contra marcador fluorescente (como exemplo FITC) para controlar a qualidade da aquisição. Alteração da taxa de fluxo pode introduzir artefatos nos dados. Excluir qualquer área que mostra discrepância na taxa de fluxo. </li><li> Identificar a população de linfócitos em SSC-A/FSC-A trama. Use um controle PBMC para facilitar a identificação. Note-se que a população de linfócitos CMC pode não ser tão clara como o controle PBMC. &lt;/ Li&gt; <li> Portão em SSC-A/viability tingir para excluir as células mortas de células vivas. As células mortas pode não especificamente incorporar os anticorpos, que vai introduzir artefatos. </li><li> Porta na SSC-A/CD3 para identificar a população de células T. Deste portão, identificar as populações de células CD4 + e CD8 +. </li></ol>

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The authors have nothing to disclose.

Dadas as grandes lacunas de conhecimento no que diz respeito à imunidade no trato genital feminino (FGT), análise fenotípica de CMC pode fornecer uma ampla gama de conhecimentos em várias populações linfocitárias no colo do útero. Juntamente com análise proteômica e medições da carga viral no lavado cervical, imunidade a infecções sexualmente transmissíveis (IST) s e outros patógenos pode ser dissecada em várias populações. Considerações técnicas - CMC:...

A maioria das novas infecções pelo HIV em todo o mundo surgem através de transmissão heterossexual, com as mulheres representando 47% das novas infecções em 2011 (UNAIDS 1). Compreender o trato genital feminino (FGT), um dos principais portais de entrada para o HIV e outros patógenos sexualmente transmissíveis, é de grande importância no caminho para encontrar estratégias eficazes para prevenir a infecção. As respostas imunitárias a mucosa genital são claramente único e diferem dos que foram medidos no sangue periférico 2. No entanto, o conhecimento atual sobre a dinâmica do sistema imunológico no FGT é limitada na melhor das hipóteses. Até o momento, os estudos sobre o ambiente imune da mucosa em grande parte focada nos tecidos linfóides associados ao intestino (GALT), onde se tornou claro que os primeiros eventos em tecidos da mucosa após a infecção ter um forte impacto na posterior progressão da doença 3,4. A recolha de amostras a partir da mucosa genital representa um grande desafio e é pelo menos parcialmente responsável pela lack de compreensão da imunologia da FGT. Resolvendo o quebra-cabeça da dinâmica imunológico entre o hospedeiro eo agente patogénico no contexto do ambiente distinto que é o FGT necessita de métodos eficientes para a coleta e análise de amostras a partir desta localidade. A FGT está dividido em duas partes: a parte superior do tracto reprodutivo, que inclui os tubos de Falópio, endométrio e endocervical, e do tracto inferior, o qual contém o ectocérvix e da vagina (revisto por Kaushic et al, 5). Não é ainda claro que a contribuição relativa de diferentes locais é a infecção pelo HIV, mas acredita-se que ambos os locais poderiam contribuir para a entrada do VIH 6. As células T representam 40-50% dos leucócitos do trato reprodutivo superiores e inferiores, enquanto que os macrófagos representam cerca de 10% (revisto em Rodriguez-Garcia et al 2). As células T podem ser detectadas na vagina, colo do útero, endométrio e. Os macrófagos são mais fortemente localized no endométrio e miométrio tecido conjuntivo do que o colo do útero, embora possam ser detectados em ambos os tecidos. Finalmente, as células dendríticas plasmacytoid (PDCs) e as células de Langerhans pode também ser detectada em tecidos FGT. O fenótipo e proporções de populações do sistema imunológico e a sua susceptibilidade à infecção por HIV pode variar de acordo com os ciclos importante hormonais, o uso de contraceptivos hormonais, a vaginose bacteriana ou actividades sexuais 5,7-9. Diversos métodos foram desenvolvidos para estudar as populações imunes e Meio Ambiente da FGT. Biópsia do colo do útero, colo do útero e cytobrushes lavagens cérvico-vaginais (CVL) 10-12 são os mais comumente usados ​​em literatura. Coleção CVL por PBS lavagem é o método mais simples e permite o estudo de proteínas moduladoras do sistema imunológico, mas os resultados no rendimento extremamente baixo da pilha, e não é, portanto, adequado para estudar as populações de células imunes do FGT 13. CVL amostras são, por outro lado, vEry útil para avaliar o ambiente imunológico do FGT medindo a expressão de diversas citocinas, quimiocinas e factores anti-microbianos, utilizando métodos tais como ELISA, citocina talão de matriz 14, ou espectrometria de massa de 15,16. Caracterização das frequências de células imunitárias, fenótipos e funções pode ser alcançado através da recolha de células mononucleares do colo do útero (CMC) por cytobrush cervical ou por amostragem de biopsia cervical. Amostragem de biopsia cervical é um método invasivo, que aumenta o desconforto e risco de hemorragia e leva de 2 a 11 dias para curar seguindo o procedimento de acordo com o estado imunitário do mulher 12. Por outro lado, cytobrushes cervicais, apesar do rendimento mais baixo de células recolhidas, é um método menos invasivo e mais conveniente para recolher células do sistema imunológico do FGT. Ambos os métodos podem atingir o mesmo rendimento de leucócitos CD45 +, mas dois cytobrushes cervicais sequenciais são necessárias para se obter a mesma quantidade de células continha in uma biópsia 13. No entanto, a amostragem cytobrush ainda fornece um número aceitável de células (cerca de 5.000 células CD45 + / cytobrush) para posterior ex vivo fenotipagem por citometria de fluxo 14. Além disso, a caracterização funcional pode ser levada a cabo com as amostras, tal como a estimulação do fluxo intracelular e citometria de qPCR ou ter sido realizada utilizando derivados de CMC cytobrush para identificar as respostas imunitárias específicas para o HIV 17 ou polarização celular Th 18. Expansão da população de células T podem também facilitar estudos funcionais com CMC 19. É importante notar que as biópsias e cytobrushes amostra porções distintas do FGT. As biópsias são derivados a partir da porção superior do epitélio e estroma da ectocérvix 12,13, enquanto cytobrushes cervicais amostra do orifício cervical, recolhendo as células derivadas do epitélio endocervical e presumivelmente da zona de transformação. Portanto amostras cytobrush provar uma região composto por uma única camada de epitélio, enquanto que as biópsias, incluem uma região revestida por um epitélio escamoso estratificado 5. Como resultado, a natureza das populações de leucócitos recolhidos por biopsia cervical e cytobrush difere. Biópsias recolher uma proporção mais elevada de células T CD3 +, ao passo que cytobrushes resultam na recolha de uma maior proporção de monócitos CD14 + / macrófagos 13. O estudo da imunologia da FGT tem havido um interesse por muitos anos 20-22 e temos acumulado uma grande quantidade de conhecimentos com o estudo da CMC derivados de cytobrush. Nossos estudos se concentram principalmente no estudo do HIV-infectados, não infectados e expostas ao HIV soronegativos (HESN) profissionais do sexo de Nairobi, no Quênia. VIH replica preferencialmente em células T activadas 23 e inferior do número de células activadas que podem ser alvo de HIV no FGT poderiam contribuir para a protecção contra a infecção pelo VIH. Em linha com esta hipótese, vários studies descreveram ativação imune menor entre os trabalhadores do sexo HESN que estão altamente expostos ao HIV ainda permanecem não infectados 24,25, e este fenótipo quiescente também é observado no FGT 14. Aqui, descrevemos a metodologia para o processamento e avaliação de ativação de células T em amostras de CMC derivadas cytobrushes cervicais por ex vivo citometria de fluxo.

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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.

Citometria de fluxo Multi é uma poderosa ferramenta para dissecar os fenótipos e funções de subpopulações de células em tecidos previamente descaracterizados. Análise de amostras de CMC pode render informações sobre ambos linfócitos e monócitos populações com as estratégias de propagação apropriados. Uma estratégia de propagação CMC representativo, em comparação com um perfil combinado de PBMC, é mostrada na Figura 2. O FSC-A versus FSC-H trama permite...

Watch this Scientific Journal Video about Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples at JoVE.com

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

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 ...

collection-isolation-flow-cytometric-analysis-human-endocervical

Research

JoVE Journal

Medicine

19.0K visualizações.  University of Manitoba. 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. 

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

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

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 ...

Medicina

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

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 ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

Isolamento, cultura, e imagem de Fetal Humano pancreáticas grupos de células

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

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 ...

Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both with and without preexisting conditions. Rapid processing of these samples is essential to avoid degradation of key cellular components. Included here are methods for simultaneous peripheral blood mononuclear cell (PBMC), DNA, RNA and serum isolation from a single blood draw performed in the homes of consenting participants across a metropolitan area, with processing initiated within 2 hr of collection. We have used these techniques to process over 1,600 blood specimens yielding consistent, high quality material, which has subsequently been used in successful DNA methylation, genotyping, gene expression and flow cytometry analyses. Some of the methods employed are standard; however, when combined in the described manner, they enable efficient processing of samples from participants of population- and/or community-based studies who would not normally be evaluated in a clinical setting. Therefore, this protocol has the potential to obtain samples (and subsequently data) that are more representative of the general population.

We outline a methodology for the processing of whole blood to obtain a variety of components for further analysis. We have optimized a streamlined protocol that enables rapid, high-throughput simultaneous processing of whole blood samples in a non-clinical setting.

Fracionamento rápido e isolamento de componentes de sangue total em amostras obtidas a partir de uma configuração de base comunitária

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both ...

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 ...

Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central nervous system (CNS). However, further optimization of these technologies would benefit from methods allowing rapid and streamline determination of the extent of CNS and cell-specific targeting upon administration of viral vectors or nanoparticles in the body. Here, we present a protocol that takes advantage of the high throughput and multiplexing capabilities of flow cytometry to allow a straightforward quantification of different cell subtypes isolated from mouse brain or spinal cord, namely microglia/macrophages, lymphocytes, astrocytes, oligodendrocytes, neurons and endothelial cells. We apply this approach to highlight critical differences between two tissue homogenization methods in terms of cell yield, viability and composition. This could instruct the user to choose the best method depending on the cell type(s) of interest and the specific application. This method is not suited for analysis of anatomical distribution, since the tissue is homogenized to generate a single-cell suspension. However, it allows to work with viable cells and it can be combined with cell-sorting, opening the way for several applications that could expand the repertoire of tools in the hands of the neuroscientist, ranging from establishment of primary cultures derived from pure cell populations, to gene-expression analyses and biochemical or functional assays on well-defined cell subtypes in the context of neurodegenerative diseases, upon pharmacological treatment or gene therapy.

We present a flow cytometry method to identify simultaneously different cell types retrieved from mouse brain or spinal cord. This method could be exploited to isolate or characterize pure cell populations in neurodegenerative diseases or to quantify the extent of cell targeting upon in vivo administration of viral vectors or nanoparticles.

Caracterização citométrica de fluxo simultâneo de múltiplos tipos de células recuperadas do cérebro do camundongo/medula espinhal através de diferentes métodos de homogeneização

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central ...

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body&#39;s circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 &#176;C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (&#946;-thromboglobulin [&#946;-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

This protocol describes a comprehensive hemocompatibility evaluation of blood-contacting devices using laser-cut neurovascular implants. A flow loop model with fresh, heparinized human blood is applied to mimic blood flow. After perfusion, various hematologic markers are analyzed and compared to the values gained directly after blood collection for hemocompatibility evaluation of the tested devices.

Teste de hemocompatibilidade de implantes de contato com o sangue em um modelo de loop de fluxo imitando o fluxo sanguíneo humano

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the ...

Validated LC-MS/MS Panel for Quantifying 11 Drug-Resistant TB Medications in Small Hair Samples

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. Plasma drug levels are the current gold standard assessment, but require phlebotomy and a cold chain, and capture only very recent adherence. Our method uses hair, a matrix that is easily collected and reflective of long-term adherence, to test for 11 anti-TB medications. Previous work by our group shows that antiretroviral drug levels in hair are associated with HIV outcomes. Our method for DR-TB drugs uses 2 mg of hair (3 cm proximal to the root), which is pulverized and extracted in methanol. Samples are analyzed with a single LC-MS/MS method, quantifying 11 drugs in a 16 min run. Lower limits of quantification (LLOQs) for the 11 drugs range from 0.01 ng/mg to 1 ng/mg. Drug presence is confirmed by comparing ratios of two mass spectrometry transitions. Samples are quantified using the area ratio of the drug to the deuterated, 15N-, or 13C-labeled drug isotopologue. We used a calibration curve ranging from 0.001-100 ng/mg. Application of the method to a convenience sample of hair samples collected from DR-TB patients on directly observed therapy (DOT) indicated drug levels in hair within the linear dynamic range of nine of the eleven drugs (isoniazid, pyrazinamide, ethambutol, linezolid, levofloxacin, moxifloxacin, clofazimine, bedaquiline, pretomanid). No patient was on prothionamide, and the measured levels for ethionamide were close to its LLOQ (with further work instead examining the suitability of ethionamide&#8217;s metabolite for monitoring exposure). In summary, we describe the development of a multi-analyte panel for DR-TB drugs in hair as a technique for therapeutic drug monitoring during drug-resistant TB treatment.

Current methods of analyzing patients&#8217; adherence to complex drug resistant-tuberculosis (DR-TB) regimens can be inaccurate and resource-intensive. Our method analyzes hair, an easily collected and stored matrix, for concentrations of 11 DR-TB medications. Using LC-MS/MS, we can determine sub-nanogram drug levels that can be utilized to better understand drug adherence.

Painel LC-MS/MS validado para quantificar 11 medicamentos resistentes a medicamentos para tuberculose em amostras de cabelos pequenos

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. ...