Nós gostamos de reconhecer Marcelo Toro para a assistência com a citometria de fluxo. Este estudo foi financiado pelo sueco Coração e Pulmão Foundation, da Comissão Européia, FP7 &quot;CardioCell&quot;, Sueco de Pesquisa do Conselho, seguros AFA e ALF. OB foi apoiado pelo Deutsche Forschungsgemeinschaft.

1. Isolamento dos Núcleos Cardiac <ol><li> Revestimento tubos de ultracentrifugação (Tubos centrífuga Beckman # 363664) com 10 ml de solução de revestimento de 1% BSA / PBS. Tapar os tubos e deixá-los girar durante 30 min num rotor tubo. Remover a solução de revestimento e deixar que o tubos de centrifugação de ar seco (um tubo por coração do rato é necessária para a análise de corações de rato individuais, alternadamente até 5 corações de rato ou 1 g de tecido do coração a partir de uma espécie diferente (por exemplo, humanos) podem ser processados em um tubo). </li><li> Todos os passos seguintes devem ser realizados no gelo. Dissecar o ventrículo esquerdo do coração de rato fresco ou congelado snap-com um bisturi. Nota, este protocolo é optimizado para coração de rato, mas também pode ser adaptado para rato ou do coração humano. Alternativamente, usam-se a 1 g de tecido do coração a partir de uma espécie diferente. </li><li> Apare o espécime em cubículos pequenos, com um bisturi. </li><li> Transfira os pedaços de tecido em um tubo Falcon 50 ml cheia com 15 ml de tampão de lise. </li><li> Homogeneizar atecido do coração com uma T-25 Ultra-Turrax sonda homogeneizador (IKA) com 24.000 rpm durante 10 seg. </li><li> Diluir o homogeneizado com um volume igual de tampão de lise a 30 ml. </li><li> Utilizar um vidro douncer (40 ml) para continuar a homogeneizar o tecido e os núcleos livre. Executar oito traçados com um pilão de grande folga. </li><li> Passe o núcleo bruto isolar através de um 100 mm e 70 mM de nylon de malha de células do filtro (BD Biosciences), consecutivamente. </li><li> Girar para baixo os núcleos isolado bruto numa centrifugadora refrigerada (4 ° C) a 700 xg durante 10 min. </li><li> Remover o sobrenadante cuidadosamente por inversão dos tubos e limpar o interior do tubo com uma toalha de papel. Tenha cuidado para não perturbar o pellet núcleos. </li><li> Dissolve-se o isolado bruto núcleos em 5ml de tampão de sacarose por pipetagem a solução várias vezes para cima e para baixo. Adicionar mais 25 ml de tampão de sacarose para o sedimento dissolvido. </li><li> Adicionar 10 ml de tampão de sacarose recentemente preparada para o tubo de ultracentrifuga revestido (ver passo 1.1). &lt;/ Li&gt; <li> Cuidadosamente sobrepor a 10 ml adicional de tampão de sacarose, com a ressuspenso núcleos sedimento dissolvido em tampão de sacarose a partir do passo 1.9. </li><li> Equilibrar os tubos de centrifugação antes de os colocar num rotor JS13.1 livre oscilante e colocar o rotor em uma centrífuga de alta velocidade (Beckman Avanti-S 25). </li><li> Girar a amostra núcleos em 13.000 xg, a 4 ° C durante 60 min. </li><li> Quando a rotação foi concluída, remover os tubos cuidadosamente a partir do rotor e descartar o sobrenadante por inversão dos tubos e limpando os detritos remanescente a partir do interior dos tubos com toalha de papel. </li><li> Dissolve-se o pellet núcleos em 1 ml de tampão de núcleos de armazenamento (NSB mais tampão). Nota: NSB plus contém 1,5 mM espermina na forma de um estabilizador de DNA. </li><li> Prossiga com o passo 2.1, imunocoloração de núcleos de cardiomiócitos. </li></ol> 2. A imunocoloração para Citometria de Fluxo <ol><li> Prepare o controlo negativo para a imunocoloração. Tomar uma alíquota de 20 uL para fora da amostra núcleos e umadd ul 980 de NSB mais buffer. </li><li> Adicionar anti-pericentriolar material de um anticorpo (anticorpo de coelho anti-PCM-1, Antibodies Atlas) para a amostra núcleos em uma diluição de 1:500 a immunolabel núcleos de cardiomiócitos. Adicionar o anticorpo de isotipo para a diluição mesmo que o anticorpo anti-PCM-1 para o controlo negativo, preparado no passo 2.1. </li><li> Incubar controlo negativo e tubo de amostra a 4 ° C durante a noite. </li><li> Lavar controlo negativo e amostra, pelo menos, uma vez com NSB mais tampão (tubos de girar para baixo numa centrifugadora refrigerada (4 ° C) a 700 xg durante 10 min. Elimine o sobrenadante e ressuspender o pellet núcleos em 1 ml de tampão mais NSB). </li><li> Adicionar anti-coelho de anticorpo fluorescente secundário (FITC ou APC) para controlo negativo e tubo de amostra em uma diluição de 1:1000. </li><li> Incubar controlo negativo e tubo de amostra a 4 ° C durante 1 h. </li><li> Lavar controlo negativo e amostra, pelo menos, uma vez com NSB mais tampão (tubos de girar para baixo numa centrifugadora refrigerada (4 ° C) a 700 xg durante 10 min. Descartar o sobrenadante e dissolver os núcleos sedimento em 1 ml de tampão mais NSB). </li><li> Continuar com a análise por citometria de fluxo e triagem. </li></ol> 3. Citometria de Fluxo <ol><li> Revestimento tubos núcleos de recolha (Falcon de 15 ml) com 1% de solução de BSA / PBS antes de começar a citometria de fluxo de triagem, tal como descrito no passo 1,1. </li><li> Filtrar a amostra eo controlo negativo através de um filtro 30 uM célula e carregar primeiro o controlo negativo para o citómetro de fluxo (afluxo BD). Definir o portão primeiro e segundo para definir núcleos e singuletos (núcleos único), com base na dispersão para a frente (FSC), largura de pulso para a frente de dispersão (FS largura de pulso) e dispersão lateral (SSC) (Fig. 2a e b). Adicionando um DNA mancha (DRAQ5 (1:500)) para a amostra pode ajudar a identificar a população núcleos inicialmente. </li><li> Carregar a amostra immunolabeled e definir a terceira porta para isolar os núcleos dos cardiomiócitos (PCM-1-positve) de não-cardiomiócitos núcleos (PCM-1-negativo). Inicie a triagem(Fig. 2c, d). </li></ol> Opcional: A fim de analisar o conteúdo de DNA nuclear (ploidia) e para executar a análise do ciclo celular adicionar uma mancha de DNA adequado para os núcleos (por exemplo, Hoechst 33342 ou DRAQ5) (fig. 2e). <ol start="4"><li> Após a triagem de citometria de fluxo, colocar os núcleos em gelo e re-analisar para determinar a pureza de classificação (Fig. 3a e b). </li><li> Girar para baixo os núcleos classificados nos tubos de recolha a 1500 xg numa centrifugadora refrigerada durante 15 min. </li><li> Dissolve-se o pellet núcleos num tampão compatível com a aplicação a jusante. </li></ol> 4. Os resultados representativos Morfologia núcleos e integridade pode ser avaliada por manchas de DNA e visualizada por microscopia (Fig. 1). Bem sucedida PCM-1 rotulagem pode ser avaliado por microscopia de epifluorescência e por citometria de fluxo (Fig. 1 e Fig.. 2c e d). PCM-1-positive e populações negativas devem ser bem separadas umas das outras (Fig. 2c, d). Em ventrículo esquerdo murino cerca de 30% de todos os núcleos deve ser os núcleos dos cardiomiócitos (Fig. 2d). Triagem pureza pode ser avaliada por re-analisando os núcleos ordenadas (Fig. 3a e b). Ambas as populações núcleos deve ter uma pureza superior a 95% de triagem. <img alt="A Figura 1" src="/files/ftp_upload/4205/4205fig1.jpg" /> Figura 1. PCM-1 identifica os núcleos dos cardiomiócitos. Núcleos Cardíaca (a) são coradas com anticorpos para PCM-1 (b), e para Nkx2.5 (c) em um coração de rato adulto. (D) PCM-1-rotulado núcleos são cercados por citoplasma dos cardiomiócitos (miosina de cadeia pesada (MHC)) e expressar o fator de transcrição Nkx2.5, documentando a identificação precisa de núcleos de cardiomiócitos pelo PCM-1 coloração (barras de escala 20 microns e 10 iM (d, inserção)). (E) isolados núcleos cardíacas visualizado com o DNA mancha DRAQ5. (Feg) Cardiomyocnúcleos YTE são rotulados com anticorpos contra PCM-1 (barra de escala 10 mm). Note, o padrão de coloração epinuclear de PCM-1 em núcleos dos miócitos em secção de tecido e em núcleos isolados (setas). <img alt="A Figura 2" src="/files/ftp_upload/4205/4205fig2.jpg" /> Figura 2. Classificação por citometria de fluxo de núcleos de cardiomiócitos. (A) núcleos cardíaca são identificados por dispersão frontal (FSC) e dispersor lateral (SSC). (B) Um segundo portão identifica núcleos único pelo FSC e largura de pulso FS 5. (C, d) gating fluorescente permite a separação de núcleos de cardiomiócitos (PCM-1-positivo) e não-cardiomiócitos (PCM-1-negativa) núcleos a partir de tecido do coração. (E) cardiomiócito do rato são na maior parte (&gt; 80%) diplóide (2n), apenas um pequeno subconjunto é tetraplóide (4n) 6. Note, cardiomiócitos humanos contêm uma maior frequência de núcleos poliploidia (&gt; 2n) 7,8. <img alt="A Figura 3" src="/files/ftp_upload/4205/4205fig3.jpg" /> <strong&gt; Figura 3. Análise de pureza de classificados núcleos dos cardiomiócitos e não-cardiomiócitos. Re-análise de núcleos não-ordenados dos cardiomiócitos (a) e cardiomiócitos (b). Ambas as populações apresentam um grau de pureza superior a 99% de classificação.

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A identificação precisa de núcleos de cardiomiócitos é crucial para a análise dos processos regenerativos no miocárdio 2,3. As técnicas convencionais para isolar cardiomiócitos de tecido fresco baseiam-se principalmente a digestão enzimática de proteínas da matriz extracelular e da subsequente purificação a partir de células intersticiais por centrifugação a baixa velocidade. A purificação adicional de cardiomiócitos de vida a partir de células estaminais embrionárias (ESC) pode ser real...

<table border="1"><tbody><tr><td> 1. Tampão de Lise </td></tr><tr><td> Nome do reagente </td></tr><tr><td> 0,32 M de sacarose </td></tr><tr><td> 10 mM Tris-HCl (pH = 8) </td></tr><tr><td> 5 mM de CaCl2 </td></tr><tr><td> 5 mM de acetato de magnésio </td></tr><tr><td> 2,0 mM de EDTA </td></tr><tr><td> 0,5 mM de EGTA </td></tr><tr><td> 1 mM de DTT </td></tr></tbody></table><table border="1"><tbody><tr><td> 2. Tampão de sacarose </td></tr><tr><td> Nome do reagente </td></tr><tr><td> 2,1 M de sacarose </td></tr><tr><td> 10 mM Tris-HCl (pH = 8) </td></tr><tr><td> 5 mM de acetato de magnésio </td></tr><tr><td> 1 mM de DTT </td></tr></tbody></table><table border="1"><tbody><tr><td> 3. Tampão de núcleos de armazenamento (NSB plus) </td></tr><tr&gt; <td> Nome do reagente </td></tr><tr><td> 0,44 M de sacarose </td></tr><tr><td> 10 mM Tris-HCl (pH = 7,2) </td></tr><tr><td> 70 mM de KCl </td></tr><tr><td> 10 mM MgCl2 </td></tr><tr><td> 1,5 mM de espermina </td></tr></tbody></table><table border="1"><tbody><tr><td> Reagentes e Equipamentos </td><td> Companhia </td></tr><tr><td> Isotipo IgG de coelho-CHIP Grau, # ab37415 </td><td> Abcam </td></tr><tr><td> De coelho anti-PCM-1 anticorpo, # HPA023374 </td><td> Anticorpos Atlas </td></tr><tr><td> Burro seg. anticorpo, anti-coelho Alexa 488 Fluor, # A-21.206 ou sec equivalente. anticorpo fluorescente </td><td> Life Technologies </td></tr><tr><td> DRAQ5 </td><td> Biostatus </td></tr><tr><td> Os filtros de células de 30 uM, 70 uM e 100 uM </td><td> BD Biosciences </td></tr><tr><td> Vidro douncer (40 ml) epilão &quot;L&quot; </td><td> VWR (Wheaton Industries, Inc.) </td></tr><tr><td> T-25 homogeneizador Ultra-Turrax </td><td> IKA Alemanha </td></tr><tr><td> Dispersão ferramenta S25 N-18 G </td><td> IKA Alemanha </td></tr><tr><td> Centrífuga Beckman Avanti </td><td> Beckman Coulter </td></tr><tr><td> Tubos Falcon de 15 ml e 50 ml </td><td> VWR </td></tr><tr><td> Tubos de centrífuga Beckman # 363664 </td><td> Beckman Coulter </td></tr><tr><td> JS13.1 rotor livre balançando </td><td> Beckman Coulter </td></tr><tr><td> Citómetro Influx </td><td> Beckman Coulter </td></tr><tr><td> Rotator tubo </td><td> VWR </td></tr></tbody></table>

isolation of cardiomyocyte nuclei from post-mortem tissue

Identificação de núcleos de cardiomiócitos tem sido um desafio em cortes de tecido como a maioria das estratégias de contar apenas com proteínas marcadoras citoplasmáticos 1. Eventos raros em miócitos cardíacos, tais como a proliferação e apoptose necessitam de uma identificação precisa dos núcleos dos miócitos cardíacos para analisar a renovação celular e na homeostasia em condições patológicas 2. Aqui, nós proporcionar um método para isolar a partir de núcleos de cardiomiócitos pós tecido mortem por sedimentação densidade e imunomarcação com anticorpos contra o material pericentriolar 1 (PCM-1) e triagem de citometria de fluxo posterior. Esta estratégia permite uma análise com rendimento elevado e de isolamento, com a vantagem de se trabalhar igualmente bem em tecido fresco e material de arquivo congelado. Isto torna possível estudar o material já recolhidos na biobancos. Esta técnica é aplicável e testado em uma vasta gama de espécies e adequados para várias aplicações tais como a jusante de carbono-14 namoro 3, a célula-cycle análise 4, visualização de análogos da timidina (por exemplo, BrdU e UDI) 4, a análise de transcriptoma e epigenética.

Watch this Scientific Journal Video about Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue at JoVE.com

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Núcleos cardíaca são isolados através de sedimentação densidade e immunolabeled com anticorpos contra o material pericentriolar 1 (PCM-1) para identificar e classificar os núcleos dos cardiomiócitos por citometria de fluxo.

Isolamento dos núcleos dos cardiomiócitos a partir de Post-mortem de tecidos

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

isolation-of-cardiomyocyte-nuclei-from-post-mortem-tissue

Research

JoVE Journal

Medicine

16.9K visualizações.  University of Lund. Núcleos cardíaca são isolados através de sedimentação densidade e immunolabeled com anticorpos contra o material pericentriolar 1 (PCM-1) para identificar e classificar os núcleos dos cardiomiócitos por citometria de fluxo.

Vídeo: Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

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

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 ...

Isolation of Atrial Myocytes from Adult Mice

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents responsible for the action potential can cause pro-arrhythmic substrates that underlie arrhythmias, such as atrial fibrillation, which are highly prevalent in many conditions and disease states. Isolating adult mouse atrial cardiomyocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding of the cellular electrophysiology in the healthy atrial myocardium and in the setting of atrial pathophysiology. In addition, studies using genetic mouse models have elucidated the role of a vast array of proteins in regulating atrial electrophysiology. Here we provide a detailed protocol for the isolation of cardiomyocytes from the atrial appendages of adult mice using a combination of enzymatic digestion and mechanical dissociation of these tissues. This approach consistently and reliably yields isolated atrial cardiomyocytes that can then be used to characterize cellular electrophysiology by measuring action potentials and ionic currents in patch-clamp experiments under a number of experimental conditions.

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.

Isolamento de miócitos atriais de camundongos adultos

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

Electromechanical Assessment of Optogenetically Modulated Cardiomyocyte Activity

Over the past two decades, optogenetic tools have been established as potent means to modulate cell-type specific activity in excitable tissues, including the heart. While Channelrhodopsin-2 (ChR2) is a common tool to depolarize the membrane potential in cardiomyocytes (CM), potentially eliciting action potentials (AP), an effective tool for reliable silencing of CM activity has been missing. It has been suggested to use anion channelrhodopsins (ACR) for optogenetic inhibition. Here, we describe a protocol to assess the effects of activating the natural ACR GtACR1 from Guillardia theta in cultured rabbit CM. Primary readouts are electrophysiological patch-clamp recordings and optical tracking of CM contractions, both performed while applying different patterns of light stimulation. The protocol includes CM isolation from rabbit heart, seeding and culturing of the cells for up to 4 days, transduction via adenovirus coding for the light-gated chloride channel, preparation of patch-clamp and carbon fiber setups, data collection and analysis. Using the patch-clamp technique in whole-cell configuration allows one to record light-activated currents (in voltage-clamp mode, V-clamp) and AP (current-clamp mode, I-clamp) in real time. In addition to patch-clamp experiments, we conduct contractility measurements for functional assessment of CM activity without disturbing the intracellular milieu. To do so, cells are mechanically preloaded using carbon fibers and contractions are recorded by tracking changes in sarcomere length and carbon fiber distance. Data analysis includes assessment of AP duration from I-clamp recordings, peak currents from V-clamp recordings and force calculation from carbon fiber measurements. The described protocol can be applied to the testing of biophysical effects of different optogenetic actuators on CM activity, a prerequisite for the development of a mechanistic understanding of optogenetic experiments in cardiac tissue and whole hearts.

We present a protocol for evaluating the electromechanical effects of GtACR1 activation in rabbit cardiomyocytes. We provide detailed information on cell isolation, culturing and adenoviral transduction, and on functional experiments with the patch-clamp and carbon-fiber techniques.

Avaliação eletromecânica da atividade cardiomiocito optogeneticamente modulada

Over the past two decades, optogenetic tools have been established as potent means to modulate cell-type specific activity in excitable tissues, including ...

Model of Ischemic Heart Disease and Video-Based Comparison of Cardiomyocyte Contraction Using hiPSC-Derived Cardiomyocytes

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with small-animal models such as rodents. However, the physiology of the human heart differs significantly from that of the rodent heart, underscoring the need for clinically relevant models to study heart disease. Here, we present a protocol to model ischemic heart disease using cardiomyocytes differentiated from human induced pluripotent stem cells (hiPS-CMs) and to quantify the damage and functional impairment of the ischemic cardiomyocytes. Exposure to 2% oxygen without glucose and serum increases the percentage of injured cells, which is indicated by staining of the nucleus with propidium iodide, and decreases cellular viability. These conditions also decrease the contractility of hiPS-CMs as confirmed by displacement vector field analysis of microscopic video images. This protocol may furthermore provide a convenient method for personalized drug screening by facilitating the use of hiPS cells from individual patients. Therefore, this model of ischemic heart disease, based on iPS-CMs of human origin, can provide a useful platform for drug screening and further research on ischemic heart disease.

We present a model of ischemic heart disease using cardiomyocytes derived from human induced pluripotent stem cells, together with a method for quantitative evaluation of tissue damage caused by ischemia. This model can provide a useful platform for drug screening and further research on ischemic heart disease.

Modelo de Doença Cardíaca Isquêmica e Comparação Baseada em Vídeo de Contração de Cardiomiócitos usando cardiomiócitos derivados do hiPSC

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with ...

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow cytometry.

Isolamento de Adipócitos Viáveis e Fração Vascular Estromal do Tecido Adiposo Visceral Humano Adequado para Análise de RNA e Fenotipagem de Macrófagos

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...

Ultrasound-Guided Induced Pluripotent Stem Cell-Derived Cardiomyocyte Implantation in Myocardial Infarcted Mice

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a promising cell source for cardiac repair after ischemic damage. However, a low grafted rate is a significant obstacle for effective cardiac tissue regeneration after transplantation. This protocol shows that multiple hiPSC-CM ultrasound-guided percutaneous injections into an MI area effectively increase cell transplantation rates. The study also describes the entire hiPSC-CM culture process, pretreatment, and ultrasound-guided percutaneous delivery methods. In addition, the use of human mitochondrial DNA help detect the absence of hiPSC-CMs in other mouse organs. Lastly, this paper describes the changes in cardiac function, angiogenesis, cell size, and apoptosis at the infarcted border zone in mice 4 weeks after cell delivery. It can be concluded that echocardiography-guided percutaneous injection of the left ventricular myocardium is a feasible, relatively invasive, satisfactory, repeatable, and effective cellular therapy.

Ultrasound-guided cell delivery around the site of myocardial infarction in mice is a safe, effective, and convenient way of cell transplantation.

Implante de Cardiomiócitos Derivados de Células-Tronco Pluripotentes Induzidos por Ultrassom em Camundongos Infartados do Miocárdio

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem ...

Preparation of a Non-Cardiomyocyte Cell Suspension for Single-Cell RNA Sequencing from a Post-Myocardial Infarction Adult Mouse Heart

Myocardial infarction (MI) is one of the most common cardiovascular diseases, with increasing mortality worldwide. Non-cardiomyocytes account for more than half of the total cardiac cell population, and they contribute to adaptive compensations upon myocardial injury, including inflammatory responses, tissue repair, and scar formation. To study the post-MI cardiac microenvironment, single-cell RNA sequencing (scRNA-seq) is widely used to identify different cardiac cell types and intercellular communications. Among the procedures of scRNA-seq sample preparation, preparing the cell suspension is one of the most critical steps, because the cell viability can affect the quality of the scRNA-seq results. Therefore, we designed an experimental protocol for preparing a non-cardiomyocyte cell suspension from post-MI mouse hearts with an extra focus on improving the cell viability by choosing mild digestive enzymes, controlling the digestion time, and applying fluorescence-activated cell sorting (FACS). Finally, we isolated CD45+ cells from the non-cardiomyocyte cell suspension obtained through this protocol, and then we performed scRNA-seq.

Here, we describe a protocol to isolate a sufficient amount of single non-cardiomyocytes with high viability from a post-myocardial infarction (MI) mouse heart. This can be used for subsequent single-cell sequencing, flow cytometry analysis, and primary cell culture.

Preparação de uma Suspensão de Células Não-Cardiomiócitos para Sequenciamento de RNA de Célula Única de um Coração de Camundongo Adulto Pós-Infarto do Miocárdio

Myocardial infarction (MI) is one of the most common cardiovascular diseases, with increasing mortality worldwide. Non-cardiomyocytes account for more ...

Isolation and Culture of Human Adipose-Derived Stem Cells With an Innovative Xenogeneic-Free Method for Human Therapy

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission due to the introduction of animal-derived products during in vitro manipulation. The xenogeneic components, such as collagenase or fetal bovine serum, commonly used during the cell isolation and expansion steps could be associated with the potential risks of immune reactivity or viral, bacterial, and prion infection in the receiving patients. Following good manufacturing practice guidelines, chemical tissue dissociation should be avoided, while fetal bovine serum (FBS) can be substituted with xenogeneic-free supplements. Moreover, to ensure the safety of cell products, the definition of more reliable and reproducible methods is important. We have developed an innovative, completely xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells without altering their properties compared to collagenase FBS-cultured standard protocols. Here, human adipose-derived stem cells (hASCs) were isolated from abdominal adipose tissue. The sample was mechanically minced with scissors/a scalpel, micro-dissected and mechanically dispersed in a 10 cm Petri dish, and prepared with scalpel incisions to facilitate the attachment of the tissue fragments and the migration of hASCs. Following the washing steps, hASCs were selected due to their plastic adherence without enzymatic digestion. The isolated hASCs were cultured with medium supplemented with 5% heparin-free human platelet lysate and detached with an animal-free trypsin substitute. Following good manufacturing practice (GMP) directions on the production of cell products intended for human therapy, no antibiotics were used in any culture media.

Xenogeneic (chemical or animal-derived) products introduced in the cell therapy preparation/manipulation steps are associated with an increased risk of immune reactivity and pathogenic transmission in host patients. Here, a complete xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells is described.

Isolamento e cultura de células-tronco derivadas de tecido adiposo humano com um método inovador livre de xenogênicos para terapia humana

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission ...

Isolation, Culture, and Characterization of Primary Dermal Fibroblasts from Human Keloid Tissue

Fibroblasts, the major cell type in keloid tissue, play an essential role in the formation and development&#160;of keloids. The isolation and culture of primary fibroblasts derived from keloid tissue are the basis for further studies of the biological function and molecular mechanisms of keloids, as well as new therapeutic strategies for treating them. The traditional method of obtaining primary fibroblasts has limitations, such as poor cellular state, mixing with other types of cells, and susceptibility to contamination. This paper describes an optimized and easily reproducible protocol that could reduce the occurrence of possible issues when obtaining fibroblasts. In this protocol, fibroblasts can be observed 5 days after isolation and reach nearly 80% confluency after 10 days of culture. Then, the fibroblasts are passaged and verified using PDGFR&#945; and vimentin antibodies for immunofluorescence assays and CD90 antibodies for flow cytometry. In conclusion, fibroblasts from keloid tissue can be easily acquired through this protocol, which can provide an abundant and stable source of cells in the laboratory for keloid research.

This study describes an optimized protocol for establishing primary fibroblasts from keloid tissues that can effectively and steadily provide pure and viable fibroblasts.

Isolamento, Cultura e Caracterização de Fibroblastos Dérmicos Primários de Tecido Queloide Humano

Fibroblasts, the major cell type in keloid tissue, play an essential role in the formation and development&#160;of keloids. The isolation and culture of ...