Nos gustaría agradecer Marcelo Toro para la asistencia con la citometría de flujo. Este estudio fue apoyado por el sueco de corazón y pulmón Fundación, la Comisión Europea el 7 º PM &quot;CardioCell&quot;, Consejo Sueco de Investigación, seguros AFA y ALF. OB fue apoyado por la Deutsche Forschungsgemeinschaft.

1. El aislamiento de los Núcleos cardiaca <ol><li> Tubos Coat ultracentrífuga Beckman (Tubos de centrífuga # 363664) con 10 ml de solución de revestimiento 1% de BSA / PBS. Tape los tubos y dejar que gire durante 30 minutos en un rotor de tubo. Eliminar la solución de revestimiento y dejar que la centrífuga tubos de aire seco (un tubo por el corazón del ratón se requiere para el análisis de los corazones de ratón individuales, alternativamente hasta 5 corazones de ratón o 1 g de tejido cardíaco de una especie diferente (por ejemplo, humano) puede ser procesado en un tubo). </li><li> Todos los pasos siguientes se debe realizar en hielo. Diseccionar el ventrículo izquierdo del corazón del ratón fresco o congelado complemento con un bisturí. Nota, este protocolo se ha optimizado para el corazón del ratón, pero también se puede adaptar a la rata o el corazón humano. Alternativamente, el uso de hasta 1 g de tejido cardíaco de una especie diferente. </li><li> Recorte la muestra en pequeños cubículos con un bisturí. </li><li> Transferir los trozos de tejido en un tubo Falcon de 50 ml lleno con 15 ml de tampón de lisis. </li><li> Homogeneizar elcorazón tejido con una T-25 homogeneizador Ultra-Turrax sonda (IKA) a 24.000 rpm durante 10 segundos. </li><li> Diluir el homogeneizado con un volumen igual de tampón de lisis a 30 ml. </li><li> Utilizar un vaso douncer (40 ml) para homogeneizar aún más el tejido y los núcleos libre el. Realizar ocho golpes con un mazo gran distancia. </li><li> Pasar de los núcleos a través de un crudo de aislar a 100 micras y 70 micras de malla de nylon de células colador (BD Biosciences), de forma consecutiva. </li><li> Girar hacia abajo los núcleos crudo aislado en una centrífuga refrigerada (4 ° C) a 700 xg durante 10 min. </li><li> Eliminar el sobrenadante cuidadosamente invirtiendo los tubos y limpie el interior del tubo con una toalla de papel. Tenga cuidado de no molestar a los núcleos de pellets. </li><li> Disolver los núcleos crudo aislado en 5 ml de tampón de sacarosa pipeteando la solución varias veces arriba y abajo. Añadir un nuevo 25 ml de tampón de sacarosa al disuelto precipitado. </li><li> Añadir 10 ml de tampón de sacarosa recién preparada en el tubo de ultracentrífuga recubierto (véase el paso 1.1). &lt;/ Li&gt; <li> Cuidadosamente superponer el añadido 10 ml de tampón de sacarosa con los núcleos precipitado resuspendido disolvió en tampón de sacarosa de 1,9 paso. </li><li> Balance de los tubos de centrifugación antes de colocarlos en un rotor de giro libre JS13.1 y colocar el rotor en una centrífuga de alta velocidad (Beckman Avanti-S 25). </li><li> Haga girar la muestra de los núcleos a 13.000 xg a 4 º C durante 60 min. </li><li> Cuando el giro se ha completado, retirar los tubos cuidadosamente del rotor y descartar el sobrenadante invirtiendo los tubos y limpiando la suciedad restante de la parte interior de los tubos con una toalla de papel. </li><li> Disolver el precipitado en los núcleos 1 ml de tampón de núcleos de almacenamiento (NSB más tampón). Nota: NSB además contiene 1,5 mM de espermina como un estabilizador de ADN. </li><li> Proceda con el paso 2.1, inmunotinción de los núcleos de cardiomiocitos. </li></ol> 2. La inmunotinción para citometría de flujo <ol><li> Preparar el control negativo para inmunotinción. Tomar una alícuota de 20 l fuera de la muestra núcleos y undd 980 l de tampón NSB más. </li><li> Añadir contra pericentriolar material de un anticuerpo (conejo anti-PCM-1, anticuerpos Atlas) a la muestra de los núcleos en una dilución de 1:500 a los núcleos immunolabel cardiomiocitos. Añadir el anticuerpo isotipo en la misma dilución como el anticuerpo anti-PCM-1 para el control negativo, preparada en la etapa 2.1. </li><li> Incubar control negativo y el tubo de muestra a 4 ° C durante la noche. </li><li> Lavar control negativo y la muestra al menos una vez con tampón NSB más (de girar los tubos en una centrífuga refrigerada (4 ° C) a 700 xg durante 10 min. Desechar el sobrenadante y se disuelve el precipitado en los núcleos 1 ml de tampón NSB más). </li><li> Añadir anticuerpo anti-conejo fluorescente secundario (FITC o APC) para el control negativo y el tubo de muestra en una dilución de 1:1000. </li><li> Incubar control negativo y el tubo de muestra a 4 ° C durante 1 h. </li><li> Lavar control negativo y la muestra al menos una vez con tampón NSB más (de girar los tubos en una centrífuga refrigerada (4 ° C) a 700 xg durante 10 min. Desechar el sobrenadante y se disuelve el precipitado en los núcleos 1 ml de tampón NSB más). </li><li> Continuar con el análisis de citometría de flujo y separación. </li></ol> 3. Citometría de Flujo <ol><li> Cubra los tubos de recogida de los núcleos (Falcon de 15 ml) con BSA / PBS solución al 1% antes de empezar a ordenar la citometría de flujo como se describe en el paso 1.1. </li><li> Filtrar la muestra y el control negativo a través de un colador micras celda 30 y cargar primero el control negativo para el citómetro de flujo (afluencia BD). Definir la puerta primera y la segunda para definir los núcleos y los núcleos singletes (individual), sobre la base de dispersión frontal (FSC), la anchura del pulso de dispersión hacia adelante (FS ancho de pulso) y la dispersión lateral (SSC) (Fig. 2A y B). Adición de una mancha de ADN (DRAQ5 (1:500)) a la muestra puede ayudar a identificar la población de los núcleos inicialmente. </li><li> Cargue la muestra immunolabeled y definir la tercera puerta para aislar núcleos de cardiomiocitos (PCM-1-positve) de los no cardiomiocitos núcleos (PCM-1-negativo). Inicie la clasificación(Fig. 2C y D). </li></ol> Opcional: Con el fin de analizar el contenido de ADN nuclear (ploidía) y para realizar el análisis del ciclo celular añadir una mancha de ADN apropiada a los núcleos (por ejemplo, Hoechst 33342 o DRAQ5) (Fig. 2e). <ol start="4"><li> Después de la clasificación de citometría de flujo, coloque los núcleos de hielo y volver a analizar para determinar la pureza de clasificación (Figura 3a yb). </li><li> Girar hacia abajo los núcleos ordenados en los tubos de recogida a 1500 xg en una centrífuga refrigerada durante 15 min. </li><li> Disolver el precipitado núcleos en un tampón compatible con la aplicación de aguas abajo. </li></ol> 4. Los resultados representativos Núcleos morfología y la integridad puede ser evaluada por las manchas de ADN y se visualizaron por microscopía (fig. 1). El éxito de PCM-1 etiquetado puede ser evaluada por microscopía de epifluorescencia y por citometría de flujo (fig. 1 y fig. 2c y d). PCM-1-positiVe y las poblaciones negativas deben estar bien separados unos de otros (Fig. 2C y D). En ventrículo izquierdo murino aproximadamente el 30% de todos los núcleos deben ser núcleos de cardiomiocitos (Fig. 2d). Clasificación pureza puede ser evaluada por re-analizar los núcleos ordenados (fig. 3a yb). En ambos grupos, los núcleos deben tener una pureza superior al 95% de clasificación. <img alt="Figura 1" src="/files/ftp_upload/4205/4205fig1.jpg" /> Figura 1. PCM-1 identifica núcleos de cardiomiocitos. Núcleos cardíaca (a) se tiñen con anticuerpos frente a PCM-1 (b) y Nkx2.5 (c) en un corazón de ratón adulto. (D) PCM-1-etiquetados núcleos están rodeados por citoplasma de los cardiomiocitos (cadena pesada de miosina (MHC)) y expresar el factor de transcripción Nkx2.5, la documentación de la identificación precisa de los núcleos de cardiomiocitos por el PCM-1 tinción (barras de escala de 20 micras y 10 m (d, recuadro)). (E) de los aislamientos cardíacos núcleos visualizados con el ADN de manchas DRAQ5. (F y G) Cardiomyocnúcleos YTE están etiquetados con anticuerpos contra el PCM-1 (barra de escala de 10 micras). Nota, el patrón de tinción epinuclear de PCM-1 en núcleos de los miocitos en sección de tejido y en núcleos aislados (flechas). <img alt="Figura 2" src="/files/ftp_upload/4205/4205fig2.jpg" /> Figura 2. Citometría de flujo clasificación de los núcleos de cardiomiocitos. (A) los núcleos cardíaco se identifican por dispersión frontal (FSC) y dispersión lateral (SSC). (B) Una segunda puerta se identifica un solo núcleo por el FSC y FS ancho de pulso 5. (C, d) compuerta fluorescente permite la separación de núcleos de cardiomiocitos (PCM-1-positivo) y no cardiomiocitos-(PCM-1-negativa) núcleos de tejido del corazón. (E) de cardiomiocitos de ratón son en su mayoría (&gt; 80%) diploides (2n), sólo un pequeño subconjunto es tetraploide (4n) 6. Tenga en cuenta que los cardiomiocitos humanos contienen una mayor frecuencia de los núcleos de poliploidía (&gt; 2n) 7,8. <img alt="Figura 3" src="/files/ftp_upload/4205/4205fig3.jpg" /> <strong&gt; Figura 3. Pureza análisis de núcleos ordenados cardiomiocitos y no cardiomiocitos. Re-análisis de los núcleos ordenados no cardiomiocitos (a) y cardiomiocitos (b). Ambas poblaciones muestran una pureza superior al 99% de la clasificación.

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No hay conflictos de interés declarado.

La identificación precisa de los núcleos de cardiomiocitos es crucial para el análisis de los procesos regenerativos en el miocardio 2,3. Las técnicas convencionales para aislar cardiomiocitos a partir de tejido fresco se basan principalmente en la digestión enzimática de proteínas de matriz extracelular y la posterior purificación de las células intersticiales por centrifugación a baja velocidad. La purificación adicional de cardiomiocitos vivos a partir de células madre embrionarias (CES) puede ...

<table border="1"><tbody><tr><td> 1. Tampón de lisis </td></tr><tr><td> Nombre del reactivo </td></tr><tr><td> 0,32 M de sacarosa </td></tr><tr><td> 10 mM Tris-HCl (pH = 8) </td></tr><tr><td> 5 mM de CaCl 2 </td></tr><tr><td> 5 mM de acetato de magnesio </td></tr><tr><td> 2,0 mM de EDTA </td></tr><tr><td> 0,5 mM EGTA </td></tr><tr><td> 1 mM de DTT </td></tr></tbody></table><table border="1"><tbody><tr><td> 2. Sacarosa tampón </td></tr><tr><td> Nombre del reactivo </td></tr><tr><td> 2,1 M de sacarosa </td></tr><tr><td> 10 mM Tris-HCl (pH = 8) </td></tr><tr><td> 5 mM de acetato de magnesio </td></tr><tr><td> 1 mM de DTT </td></tr></tbody></table><table border="1"><tbody><tr><td> 3. Los núcleos buffer de almacenamiento (NSB más) </td></tr><tr&gt; <td> Nombre del reactivo </td></tr><tr><td> 0,44 M de sacarosa </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 MgCl 2 </td></tr><tr><td> 1,5 mM de espermina </td></tr></tbody></table><table border="1"><tbody><tr><td> Reactivos y Equipos </td><td> Empresa </td></tr><tr><td> Isotipo IgG de conejo-chip de grado, # ab37415 </td><td> Abcam </td></tr><tr><td> De conejo anti-PCM-1 anticuerpo, # HPA023374 </td><td> Atlas anticuerpos </td></tr><tr><td> Donkey seg. anticuerpos, anti-conejo Alexa 488 Fluor, # A-21206 o equivalente seg. anticuerpos fluorescentes </td><td> Tecnologías de la Vida </td></tr><tr><td> DRAQ5 </td><td> Biostatus </td></tr><tr><td> coladores de células de 30 m, micras y 70 micras 100 </td><td> BD Biosciences </td></tr><tr><td> Vidrio douncer (40 ml) y sela mano del mortero &quot;L&quot; </td><td> VWR (Wheaton Industries Inc.) </td></tr><tr><td> T-25 Ultra-Turrax </td><td> IKA Alemania </td></tr><tr><td> La dispersión de herramientas S25 N-18 G </td><td> IKA Alemania </td></tr><tr><td> Beckman Avanti Centrífuga </td><td> Beckman Coulter </td></tr><tr><td> Tubos Falcon de 15 ml y 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 de giro libre </td><td> Beckman Coulter </td></tr><tr><td> Afluencia citómetro </td><td> Beckman Coulter </td></tr><tr><td> Tubo de los rotadores </td><td> VWR </td></tr></tbody></table>

isolation of cardiomyocyte nuclei from post-mortem tissue

Identificación de los núcleos de cardiomiocitos ha sido un desafío en secciones de tejido, como la mayoría de las estrategias se basan sólo en proteínas marcadoras frente al citoplasma de 1. Los eventos raros en los miocitos cardíacos, como la proliferación y la apoptosis requiere una identificación precisa de los núcleos de los miocitos cardíacos para analizar la renovación celular en la homeostasis y en condiciones patológicas 2. En este caso, se proporciona un método para aislar núcleos de cardiomiocitos de post mortem del tejido por la sedimentación y la densidad de inmunomarcación con anticuerpos contra el material pericentriolar 1 (PCM-1) y la clasificación posterior de citometría de flujo. Esta estrategia permite un análisis de alto rendimiento y el aislamiento con la ventaja de trabajar igualmente bien en tejido fresco y material de archivo congelado. Esto hace posible el estudio de material ya reunido en biobancos. Esta técnica es aplicable y probado en una amplia gama de especies y adecuado para múltiples aplicaciones posteriores tales como el carbono-14 contactos 3, célula-CYculo 4 de análisis, la visualización de los análogos de timidina (por ejemplo, BrdU y UDI) 4, el análisis de transcriptoma y epigenéticos.

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 están aislados cardíaca a través de la sedimentación y la densidad de immunolabeled con anticuerpos contra el material pericentriolar 1 (PCM-1) para identificar y clasificar los núcleos de cardiomiocitos mediante citometría de flujo.

El aislamiento de núcleos de cardiomiocitos a partir de tejidos post-mortem

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 vistas.  University of Lund. Núcleos están aislados cardíaca a través de la sedimentación y la densidad de immunolabeled con anticuerpos contra el material pericentriolar 1 (PCM-1) para identificar y clasificar los núcleos de cardiomiocitos mediante citometría de flujo.

Video: 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.

El aislamiento de islotes humanos de pacientes parcialmente Pancreatectomized

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

Investigación

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.

El aislamiento de las células madre del cáncer de muestras de cáncer 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.

Aislamiento de miocitos auriculares de ratones 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.

Evaluación electromecánica de la actividad de cardiomiocitos moduladas optogenéticamente

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 cardiopatía isquémica y comparación basada en vídeo de la contracción de cardiomiocitos utilizando cardiomiocitos derivados de 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.

Aislamiento de adipocitos viables y fracción vascular estromal a partir de tejido adiposo visceral humano adecuado para análisis de ARN y fenotipado 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.

Implantación de cardiomiocitos derivados de células madre pluripotentes inducida por ultrasonido en ratones con infartado de miocardio

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.

Preparación de una suspensión de células no cardiomiocitos para la secuenciación de ARN unicelular de un corazón de ratón adulto postinfarto de miocardio

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.

Aislamiento y cultivo de células madre derivadas de tejido adiposo humano con un innovador método libre de xenogénicos 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.

Aislamiento, cultivo y caracterización de fibroblastos dérmicos primarios a partir de tejido 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 ...