Wir möchten Marcelo Toro für die Unterstützung bei der Durchflusszytometrie anerkennen. Diese Studie wurde vom schwedischen Herz-und Lungen-Stiftung, EU-Kommission RP7 &quot;CardioCell&quot;, Schwedischer Forschungsrat, AFA Versicherungen und ALF unterstützt. OB wurde von der Deutschen Forschungsgemeinschaft unterstützt.

1. Die Isolierung des Herz-Kerne <ol><li> Mantel Ultrazentrifuge Rohre (Beckman Zentrifugenröhrchen # 363.664) mit 10 ml 1% BSA / PBS Beschichtungslösung. Verschließen Sie die Röhrchen und lassen Sie sie für 30 min in einem Rohr Rotator drehen. Entfernen Sie die Beschichtungslösung und lassen Sie die Zentrifugenröhrchen Luft trocknen lassen (eine Tube pro Maus Herz für die Analyse der einzelnen Mäuseherzen, abwechselnd bis zu 5 Mäuseherzen oder 1 g Herzgewebe von einer anderen Spezies erforderlich ist (z. B. Mensch) können verarbeitet werden In einem Röhrchen). </li><li> Alle folgenden Schritte sollten auf Eis durchgeführt werden. Präparieren Sie die linke Herzkammer aus frischen oder gefrorenen Snap-Maus-Herz mit einem Skalpell. Man beachte, daß dieses Protokoll für Mäuseherz optimiert, sondern auch von Ratten oder menschlichen Herzens angepasst werden. Alternativ können bis zu 1 g des Herzgewebes von einer anderen Spezies. </li><li> Schneiden Sie die Probe in kleinen Kabinen mit einem Skalpell. </li><li> Übertragen der Gewebestücke in einen 50 ml-Falcon-Röhrchen mit 15 ml Lyse-Puffer gefüllt. </li><li> Homogenisieren derHerzgewebe mit einem T-25 Ultra-Turrax-Homogenisator Sonde (IKA) bei 24.000 UpM für 10 sek. </li><li> Verdünnen des Homogenats mit einem gleichen Volumen Lysepuffer bis 30 ml. </li><li> Verwenden Sie ein Glas Douncer (40 ml) zur weiteren Homogenisierung des Gewebes und die Kerne frei. Führen Sie acht Schläge mit einem großen Abstand Pistill. </li><li> Führen Sie das rohe Kerne durch ein 100 um und 70 um Nylon-Mesh Zellsieb (BD Biosciences), fortlaufend zu isolieren. </li><li> Anzentrifugieren das rohe Kerne in einer gekühlten Zentrifuge (4 ° C) bei 700 × g für 10 min isoliert. </li><li> Entfernen Sie den Überstand vorsichtig durch Umdrehen der Rohre und wischen Sie das Innere des Rohres mit einem Papiertuch. Achten Sie darauf, die Kerne Pellet stören. </li><li> Man löst das rohe Kerne in 5 ml Saccharose-Puffer zu isolieren durch Pipettieren der Lösung mehrmals auf und ab. Fügen Sie eine weitere 25 ml Saccharose-Puffer auf die gelöste Pellet. </li><li> 10 ml frisch zubereiteten Saccharose-Puffer auf die beschichtete Ultrazentrifuge Rohr (siehe Schritt 1.1). &lt;/ Li&gt; <li> Sorgfältig überlagern den zusätzlichen 10 ml Saccharose-Puffer mit dem resuspendierten Pellet Kerne in Saccharose-Puffer von 1,9 Schritt aufgelöst. </li><li> Gleichen Sie die Zentrifugenröhrchen, bevor Sie sie in eine JS13.1 frei schwingenden Rotor und platzieren Sie den Rotor in eine High-Speed-Zentrifuge (Beckman Avanti S-25). </li><li> Drehen der Kerne Probe bei 13.000 × g bei 4 ° C für 60 min. </li><li> Wenn die Spin abgeschlossen ist, entfernen die Röhrchen vorsichtig von dem Rotor und der Überstand verworfen durch Invertieren der Rohre und Abwischen der restlichen Ablagerungen aus dem Inneren der Rohre mit einem Papiertuch. </li><li> Lösen Sie die Kerne Pellet in 1 ml Kerne Pufferspeicher (NSB Plus-Puffer). Hinweis: NSB plus enthält 1,5 mM Spermin als DNA-Stabilisator. </li><li> Fahren Sie mit Schritt 2.1, Immunfärbung von Kardiomyozyten Kernen. </li></ol> 2. Immunfärbung für die Durchflusszytometrie <ol><li> Die negativen Kontrollen für Immunfärbung. Man entnimmt einen aliquoten von 20 ul aus den Kernen Probe und eineDD 980 ul von NSB und Puffer. </li><li> In Anti-pericentriolar Material 1-Antikörper (Kaninchen-Anti-PCM-1, Atlas Antikörper) zu den Kernen Probe in einer Verdünnung von 1:500 bis Immunmarkierung Kardiomyozyten Kernen. In den Isotyp-Antikörper in der gleichen Verdünnung des Anti-PCM-1-Antikörper mit der Negativkontrolle, in Schritt 2.1 hergestellt. </li><li> Inkubieren negativen Kontrolle und Probe-Röhrchen bei 4 ° C über Nacht. </li><li> Waschen Sie Negativ-Kontrolle und Probe mindestens einmal mit NSB Plus-Puffer (Spin-Down Rohre in einer gekühlten Zentrifuge (4 ° C) bei 700 xg für 10 min. Überstand verwerfen und lösen sich die Kerne Pellet in 1 ml NSB Plus-Puffer). </li><li> In Anti-Kaninchen-fluoreszierenden sekundären Antikörper (FITC oder APC) zu negativen Kontrolle und Probenröhrchen in einer Verdünnung von 1:1000. </li><li> Inkubieren negativen Kontrolle und Probe-Röhrchen bei 4 ° C für 1 h. </li><li> Waschen Sie Negativ-Kontrolle und Probe mindestens einmal mit NSB Plus-Puffer (Spin-Down Rohre in einer gekühlten Zentrifuge (4 ° C) bei 700 xg für 10 min. Überstand verwerfen und lösen sich die Kerne Pellet in 1 ml NSB Plus-Puffer). </li><li> Fahren Sie mit der durchflusszytometrischen Analyse und Sortierung. </li></ol> 3. Durchflusszytometrie <ol><li> Coat Kerne Sammlung Röhrchen (Falcon 15 ml) mit 1% BSA / PBS-Lösung, bevor Sie die Durchflusszytometrie Sortierung wie in Schritt 1.1 beschrieben starten. </li><li> Filtern Sie die Probe und die negative Kontrolle durch einen 30 um Zellsieb und laden Sie zuerst die negative Kontrolle der Durchflusszytometer (BD Zufluss). Definieren Sie die erste und zweite Tor zu Kernen und Singuletts (Einzel-Kerne) zu definieren, basierend auf Vorwärtsstreuung (FSC), Vorwärtsstreuung Impulsbreite (FS Pulsbreite) und seitliche Streulicht (SSC) (Abb. 2a und b). Hinzufügen eines DNA-Färbung (DRAQ5 (1:500)) zu der Probe kann helfen, die Kerne Bevölkerung zunächst zu identifizieren. </li><li> Laden der Probe und immunmarkierten definieren das dritte Tor zu Kardiomyozyten Kerne (PCM-1-positve) aus nicht-Kardiomyozyten Kerne (PCM-1-negativen) zu isolieren. Starten Sie die Sortierung(Abb. 2c und d). </li></ol> Optional: Um die Kern-DNA-Gehalt (Ploidie) zu analysieren und Zellzyklus-Analyse durchzuführen fügen Sie eine entsprechende DNA-Färbung der Kerne (zB Hoechst 33342 oder DRAQ5) (Abb. 2e). <ol start="4"><li> Nach der Durchflusszytometrie Sortierung, legen Sie die Kerne auf Eis und neu analysieren, um die Sortierung Reinheit (Abb. 3a und b) zu bestimmen. </li><li> Drehen Sie den sortierten Kerne in den Sammelröhrchen bei 1500 xg in einer Kühlzentrifuge für 15 min. </li><li> Man löst die Kerne Pellets in einer Pufferlösung mit dem Downstream-Anwendungen. </li></ol> 4. Repräsentative Ergebnisse Kerne Morphologie und Integrität durch DNA Flecken beurteilt werden und visualisiert durch Mikroskopie (Abb. 1). Erfolgreiche PCM-1 Kennzeichnung kann durch Epifluoreszenzmikroskopie und mittels Durchflusszytometrie (Abb. 1 und Abb. 2c und d) beurteilt werden. PCM-1-Positive und negative Populationen sollten gut voneinander getrennt werden (Abb. 2c und d). In murinen linken Ventrikel ungefähr 30% aller Kerne Kerne sein sollte Kardiomyozyten (2d). Sortieren Reinheit kann durch neue Analyse der sortierten Kerne (Abb. 3a und b) beurteilt werden. Beide Kerne Bevölkerung sollte eine Sortierung Reinheitsgrad von mehr als 95%. <img alt="1" src="/files/ftp_upload/4205/4205fig1.jpg" /> Abbildung 1. PCM-1 identifiziert Kardiomyozyten Kerne. Cardiac Kerne (a) mit Antikörpern gegen PCM-1 (b) und Nkx2.5 (c) in einer erwachsenen Maus Herzen gefärbt werden. (D) PCM-1-markierte Kerne werden von Kardiomyozyten Zytoplasma (Myosin heavy chain (MHC)) umgeben ist und zum Ausdruck des Transkriptionsfaktors Nkx2.5 und dokumentiert die genaue Identifizierung von Kardiomyozyten Kerne von PCM-1-Färbung (Maßstab 20 pm und 10 um (d, Einschub)). (E) Cardiac Kerne Isolate visualisiert mit dem DNA-Farbstoff DRAQ5. (F und g) CardiomyocYTE Kerne sind mit Antikörpern gegen PCM-1 (Maßstab 10 um) beschriftet. Beachte, dass die epinuclear Färbungsmuster von PCM-1 in Myozyten Kerne im Gewebe aufweist und in isolierten Kernen (Pfeile). <img alt="2" src="/files/ftp_upload/4205/4205fig2.jpg" /> Abbildung 2. Durchflusszytometrische Sortierung von Kardiomyozyten Kernen. (A) Cardiac Kerne werden durch Vorwärtsstreuung (FSC) und Seitwärts-Streulicht (SSC) identifiziert. (B) ein zweites Tor identifiziert einzelne Kerne von FSC und FS Impulsbreite 5. (C, d) Fluoreszenz-Gating ermöglicht die Trennung von Kardiomyozyten Kerne (PCM-1-positiv) und nicht-Kardiomyozyten (PCM-1-negativen) Kerne von Herzgewebe. (E) Maus Kardiomyozyten meist (&gt; 80%) diploid (2n), ist nur eine kleine Teilmenge tetraploiden (4n) 6. Hinweis enthalten menschlichen Kardiomyozyten eine höhere Frequenz von Polyploidie Kerne (&gt; 2n) 7,8. <img alt="Abbildung 3" src="/files/ftp_upload/4205/4205fig3.jpg" /> <strong&gt; Abbildung 3. Reinheit Analyse der sortierten Kardiomyozyten und nicht-Kardiomyozyten Kerne. Re-Analyse der sortierten Nicht-Kardiomyozyten (a) und Kardiomyozyten Kerne (b). Beide Populationen zeigen eine Sortierung Reinheitsgrad von mehr als 99%.

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Keine Interessenskonflikte erklärt.

Genaue Identifizierung der Kardiomyozyten Kerne ist entscheidend für die Analyse der regenerativen Vorgänge im Myokard 2,3. Herkömmliche Techniken zu Kardiomyozyten aus frischem Gewebe zu isolieren sind hauptsächlich auf enzymatischen Abbau von Proteinen der extrazellulären Matrix und die anschließende Reinigung in der interstitiellen Zellen durch Zentrifugation bei niedriger Geschwindigkeit basiert. Die weitere Reinigung des lebenden Kardiomyozyten aus embryonalen Stammzellen (ESC) Immunmarkierung mit ...

<table border="1"><tbody><tr><td> 1. Lysispuffer </td></tr><tr><td> Name des Reagenzes </td></tr><tr><td> 0,32 M Saccharose </td></tr><tr><td> 10 mM Tris-HCl (pH = 8) </td></tr><tr><td> 5 mM CaCl 2 </td></tr><tr><td> 5 mM Magnesiumacetat </td></tr><tr><td> 2,0 mM EDTA </td></tr><tr><td> 0,5 mM EGTA </td></tr><tr><td> 1 mM DTT </td></tr></tbody></table><table border="1"><tbody><tr><td> 2. Saccharose-Puffer </td></tr><tr><td> Name des Reagenzes </td></tr><tr><td> 2,1 M Saccharose </td></tr><tr><td> 10 mM Tris-HCl (pH = 8) </td></tr><tr><td> 5 mM Magnesiumacetat </td></tr><tr><td> 1 mM DTT </td></tr></tbody></table><table border="1"><tbody><tr><td> 3. Kerne Speicherpuffer (NSB plus) </td></tr><tr&gt; <td> Name des Reagenzes </td></tr><tr><td> 0,44 M Saccharose </td></tr><tr><td> 10 mM Tris-HCl (pH = 7,2) </td></tr><tr><td> 70 mM KCl </td></tr><tr><td> 10 mM MgCl 2 </td></tr><tr><td> 1,5 mM Spermin </td></tr></tbody></table><table border="1"><tbody><tr><td> Chemikalien und Geräte </td><td> Firma </td></tr><tr><td> Kaninchen-IgG-Isotyp-ChIP Grade, # ab37415 </td><td> Abcam </td></tr><tr><td> Kaninchen anti-PCM-1-Antikörper, # HPA023374 </td><td> Atlas Antibodies </td></tr><tr><td> Donkey sek. Antikörper, Anti-Kaninchen Alexa 488 Fluor, # A-21.206 oder gleichwertige sek. Fluoreszenz-Antikörper </td><td> Life Technologies </td></tr><tr><td> DRAQ5 </td><td> Biostatus </td></tr><tr><td> Zelle Siebe 30 um, 70 um und 100 um </td><td> BD Biosciences </td></tr><tr><td> Glass Douncer (40 ml) undPistill &quot;L&quot; </td><td> VWR (Wheaton Industries Inc.) </td></tr><tr><td> T-25 Ultra-Turrax-Homogenisator </td><td> IKA Deutschland </td></tr><tr><td> Dispergierwerkzeug S25 N-18 G </td><td> IKA Deutschland </td></tr><tr><td> Beckman Avanti Zentrifuge </td><td> Beckman Coulter </td></tr><tr><td> Falcon-Tubes 15 ml und 50 ml </td><td> VWR </td></tr><tr><td> Beckman Zentrifugenröhrchen # 363664 </td><td> Beckman Coulter </td></tr><tr><td> JS13.1 frei schwingenden Rotor </td><td> Beckman Coulter </td></tr><tr><td> Influx Zytometer </td><td> Beckman Coulter </td></tr><tr><td> Tube Rotator </td><td> VWR </td></tr></tbody></table>

isolation of cardiomyocyte nuclei from post-mortem tissue

Identifizierung von Kardiomyozyten Kerne hat in Gewebeschnitten Herausforderung gewesen, da die meisten Strategien nur auf zytoplasmatische Markerproteine ​​1 verlassen. Seltene Ereignisse in Herzmuskelzellen wie Proliferation und Apoptose erfordern eine genaue Identifizierung von Kardiomyozyten-Kerne auf die Zellerneuerung in der Homöostase und bei pathologischen Zuständen 2 zu analysieren. Hier stellen wir eine Methode, um Kardiomyozyten Kerne aus post mortem Gewebe zu isolieren durch Sedimentation und Dichte Immunomarkierung mit Antikörpern gegen pericentriolar Material 1 (PCM-1) und anschließende Durchflusszytometrie-Sortierung. Diese Strategie ermöglicht einen hohen Durchsatz-Analyse und Isolation mit dem Vorteil bei der Arbeit ebenso gut auf frischen und gefrorenen Gewebe Archivalien. Dies macht es möglich, das Material bereits in Biobanken gesammelten studieren. Diese Technik ist und die Messung in einer Vielzahl von Arten und mehrfach Downstream-Anwendungen, wie Kohlenstoff-14-3, Zell-cycle-Analyse 4, Visualisierung von Thymidin-Analoga (zB BrdU und IdU) 4, Transkriptom-und epigenetische Analyse.

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

Cardiac Kerne über Dichte Sedimentation isoliert und mit Antikörpern gegen immunmarkiert pericentriolar Material 1 (PCM-1) zu identifizieren und zu sortieren Kardiomyozyten Kerne durch Durchflusszytometrie.

Isolierung von Kardiomyozyten Kerne von Post-mortem Gewebe

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 Aufrufe.  University of Lund. Cardiac Kerne über Dichte Sedimentation isoliert und mit Antikörpern gegen immunmarkiert pericentriolar Material 1 (PCM-1) zu identifizieren und zu sortieren Kardiomyozyten Kerne durch Durchflusszytometrie.

Serie: 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.

Isolierung humaner Inselzellen aus teilweise Pankreatektomierten

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

Forschung

Medizin

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.

Isolierung von Krebs Stammzellen aus menschlichen Prostatakrebs-Proben

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.

Isolierung von Atrial Myozyten von erwachsenen Mäusen

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.

Elektromechanische Bewertung der optogenetisch modulierten Kardiomyozytenaktivität

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.

Modell der ischämischen Herzkrankheit und videobasierter Vergleich der Kardiomyozytenkontraktion mit hiPSC-abgeleiteten Kardiomyozyten

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.

Isolierung lebensfähiger Adipozyten und stromaler vaskulärer Fraktion aus menschlichem viszeralem Fettgewebe, geeignet für RNA-Analyse und Makrophagen-Phänotypisierung

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.

Ultraschallgesteuerte induzierte pluripotente Stammzell-abgeleitete Kardiomyozyten-Implantation bei myokardinfarktierten Mäusen

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.

Herstellung einer Nicht-Kardiomyozyten-Zellsuspension für die Einzelzell-RNA-Sequenzierung aus einem erwachsenen Mausherzen nach Myokardinfarkt

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.

Isolierung und Kultivierung von humanen Fettstammzellen mit einer innovativen xenogenfreien Methode für die Humantherapie

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.

Isolierung, Kultivierung und Charakterisierung von primären dermalen Fibroblasten aus humanem Keloidgewebe

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 ...