Name: induction and testing of hypoxia in cell culture
Uploaded: 2011-08-12T13:00:00
Duration: 7 min 1 s
Description: Watch this Scientific Journal Video about Induction and Testing of Hypoxia in Cell Culture at JoVE.com

1. Hypoxie durch CoCl 2-Lösung induzierte Cobalt (II)-Chlorid Hexahydrat (CoCl 2 • 6 H 2 O, MG = 237,9) ist eine chemische Induktor von Hypoxie-induzierbaren Faktor-1.3 8. Dieses Produkt ist in Wasser löslich (100 mg / ml), wodurch eine klare, rote Lösung. <ol><li> Bereiten Sie eine 25mM Stammlösung in sterilem dd Wasser (unmittelbar vor Gebrauch vorbereitet) </li><li> Verwenden Sie CoCl 2 in der endgültigen Konzentration von 100 &amp;mgr; in Ihrem normalen Zellkulturmedien auf Hypoxie zu induzieren. </li><li> Fügen Sie die CoCl 2 mit Medien, um Ihre Zellen und inkubieren Sie die Kulturen für 24 Stunden in einem herkömmlichen Inkubator (37 ° C, 5% C0 2). </li></ol> Die oben genannten Konzentration arbeitet für die Zell-Linien die wir getestet haben aber jede Zelllinie sollte in verschiedenen Konzentrationen (um eine Dosis-abhängige Kurve zu etablieren) getestet sowie auch in unterschiedlichen Inkubationszeiten um Drogenkriminalität Toxizität zu begrenzen und Optimierung der Assays. 2. Hypoxie in Modular Incubator Chamber induzierten <ol><li> Bereiten Sie mindestens zwei identisch (twin Kultur) Zellkulturen. Zellkulturen können in Flaschen, Teller oder Kulturschalen werden. Zum Öffnen der Kammer, öffnen Sie zunächst die beiden weißen Kunststoff-Schellen an den Rohren befestigt, um die Kammer (Rohre für die Injektion / Spülen der hypoxischen Gas) und öffnen Sie vorsichtig den Stahlring Klemme befindet. </li><li> Lösen Sie die Klemme. Der Deckel und Schalen können nun entfernt werden. </li><li> Legen Sie die Zellkultur in der hypoxischen Kammer. Auch Platz eine Petrischale mit sterilem Wasser in die Kammer eine ausreichende Befeuchtung der Kulturen. </li><li> Platzieren Sie den &quot;Zwilling&quot; der Zellkultur in Normoxie als Kontrolle. </li><li> Stellen Sie sicher, Schalen befestigt sind und sich nicht bewegt, und schließen Sie dann die Kammer mit dem Deckel. Richtig zu positionieren den Stahlring Haken, um den hermetischen Verschluss der Kammer zu gewährleisten und zu schließen. </li><li> So erstellen Sie eine Hypoxie, bringen Sie den Schlauch mit einer &quot;Hypoxie-Tank&quot; mit einer 1% O 2-Gasgemisch. Wenn Sie ein Durchflussmesser angeschlossen, um den Tank Ihre Kammer wird direkt an ihn (Gas-Tank-Flow-Meter-Kammer) angeschlossen werden. Wir verwenden ein Durchflussmesser in unserem Regler eingebaut. </li><li> Es ist wichtig, die meisten zu entfernen, wenn nicht alle vorhandenen Sauerstoff in die Kammer und in Ihren Medien, dies zu tun bündig die Kammer durch Öffnen des Gas-Tank mit einer Flussrate von 20 Litern pro Minute für 7-10 Minuten, dann schnell schalten Sie die Gasfluss und vollständig schließen die Kammer durch Schließen sowohl weiße Klammern. </li><li> Return der Kammer zu einer herkömmlichen Inkubator für den gewünschten Zeitraum. Wenn Sie große Kulturen verwendet werden, können Medien in den Kulturen zu de-Gas für 1 bis 2 Stunden und wiederholen Sie dann die Spülung. Die oben genannten Schritte sind auf den Konstruktor Empfehlungen &quot;(Billups-Rothenberg, Inc) basiert. Stellen Sie sicher, Gas-Tank ordnungsgemäß zu jeder Zeit gesichert. </li></ol> Notes: <ul><li> Um die O 2 in den Medien enthalten sie wieder Gas geraten ist die Kammer wieder nach 1-3 Stunden zu beseitigen. </li><li> Für Inkubationszeit länger als 48 Stunden ist es auch wieder Gas in die Kammer beraten. </li><li> Oxygen Sensoren (Elektroden / Manometer), dass Messungen des O 2 in Zellen / Kammer enthaltene ermöglichen wird eine genauere Messung der intrazellulären / Kammer O 2 enthalten). </li><li> Kultivieren von Zellen in verschiedenen Zeitspannen zu einem Zeit-Kurve machen würde helfen, die optimale Zeit Expression von HIF-1α in Ihren Zellen. </li></ul> 3. Evaluation von Hypoxie <ol><li> HIF-1 α-Erkennung mittels Western Blot. <ol><li> Re-open deine Kammer, wie in Abschnitt 2.2 beschrieben und sofort platzieren Sie Ihre Kulturen auf Eis. </li><li> Lyse Hypoxie behandelten und nicht behandelten Zellen mit 5% SDS-Lösung in die Platten übertragen Sie dann die Zelllysat zu Rohren, beschallen, spindown die Zelltrümmer und den Überstand. </li><li> Messen Proteinkonzentration mit BCA-Kit, bereiten Probe durch Zugabe von Ladepuffer und Erhitzen auf 95 ° C für 5 min. </li><li> Elektrophorese Proteine ​​auf einem 8% SDS-Polyacrylamidgel und Transfer auf Nitrozellulose-Membranen. </li><li> Blockieren Sie die Membran in PBS mit 5% fettfreier Trockenmilch und 0,1% Tween 20 bei Raumtemperatur für 1 Stunde oder bei 4 ° C über Nacht auf Schüttler. </li><li> Immunoblot Übernachtung mit anti-HIF-1α primären monoklonalen Antikörper (1 / 600) im gleichen Puffer bei 4 ° C. </li><li> Wash 3 mal in PBS mit 0,1% Tween 20 bei Raumtemperatur 5min jeder Zeit und inkubieren mit HRP-Sekundärantikörper (1 / 2000) in PBS + 0,1% Tween 20 für 45 min. </li><li> Wash 3 mal in PBS mit 0,1% Tween 20 bei Raumtemperatur 5min / Zeit. </li><li> Verwenden Sie Pierce ECL-Kit und Röntgenfilm auf die Protein-Bande zeigen </li></ol></li><li> Hypoxie Responsive Element (HRE). HIF-1α reguliert zahlreiche Gene (dh VEGF, EPO) durch die Bindung an eine Sequenz namens Hypoxie regulierendes Element (HRE). Mit HRE verbunden mit einem Marker-Gen it ist möglich, HIF Aktivität 9 zu erkennen. Um diese Methode verwenden, müssen Sie zuerst Ihre Zellen mit einem HRE-Luciferase-Plasmid mit einem Transfektionsmethode angepasst an Ihre Zellen zu transfizieren. Zum Beispiel haben wir genutzt Lipofectamine 2000. Zellen benötigen, um transfizierte mindestens 4 Stunden vor dem in Hypoxie und Normoxie inkubiert werden. Dieses Mal kann die DNA in die Zellen eindringen, so dass dieser erste Schritt ist nicht durch Hypoxie beeinflusst. Die Luciferase-Signal erkannt wird mit Hilfe eines Luciferase Assay Kit. <ol><li> Inkubieren Sie HRE-transfizierten Zellen in Hypoxie in modularen Kammer oder benutzen HRE-transfizierten Zellen mit CoCl 2 inkubiert, eine Normoxie Kontrolle. </li><li> Nachdem die gewünschte Inkubationszeit entfernen Wachstumsmedium von den Zellen. </li><li> Rinse-Zellen in PBS, entfernen Sie die PBS. </li><li> Dispense 400μl 1X Lyse-Reagenz in Kulturschale und kratzen die anhaftenden Zellen, übertragen sie dann auf eine Mikro-Schlauch. </li><li> Pellet Schmutz durch kurze Zentrifugation. </li><li> Mix 20 &amp;mgr; l der Zelllysate Überstand mit 100 &amp;mgr; l des Luciferase Assay Reagent und messen Sie die Lumineszenz mit einem Luminometer. </li></ol></li></ol> 4. Repräsentative Ergebnisse: In K562-Zellen (humane Leukämie-Zelllinie) kultiviert 2 Tage in geringem Sauerstoffgehalt, im Vergleich zu Normoxie, Hypoxie induziert eine Erhöhung der HIF-1α Protein, das durch Western-Blot (Abbildung 1) erkannt wurde. Mit HRE-Luciferase modifizierten 293-Zellen (embryonale Nieren-Zelllinie) kultiviert in Hypoxie eine signifikante erhöht wurde von HIF-1α Aktivität in hypoxischen Zellen nachgewiesen werden (Abbildung 2). <img src="/files/ftp_upload/2899/2899fig1.jpg" alt="Abbildung 1"/> Abbildung 1: Zunahme HIF-1α in hypoxischen Zellen. K562-Zellen wurden in Kultur Normoxie und Hypoxie (Hypoxie-Kammer) für 48 Stunden und analysiert durch Western-Blot mit einem Antikörper, der spezifisch für die HIF-1α. Ein Antikörper, der spezifisch für Aktin wurde für die Be-Kontrolle eingesetzt. <img src="/files/ftp_upload/2899/2899fig2.jpg" alt="Abbildung 2"/> Abbildung 2: HIF-1α Aktivität. HRE-Luciferase modifizierten 293-Zellen in Hypoxie und Normoxie für 48 Stunden kultiviert und anschließend lysiert, um Luciferase-Signal mit Hilfe eines Luciferase-Assay-Kit und einem Luminometer zu erkennen. Die Ergebnisse werden als relative Lumineszenz-Einheit (RLU) angegeben.

<ol>
	<li>Swinson, D. E., O'Byrne, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+hypoxia+and+epidermal+growth+factor+receptor+in+non-small-cell+lung+cancer.">Interactions between hypoxia and epidermal growth factor receptor in non-small-cell lung cancer.</a> Clin Lung Cancer. 7, 250-256 (2006).</li><li>van Laarhoven, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+in+relation+to+vasculature+and+proliferation+in+liver+metastases+in+patients+with+colorectal+cancer.">Hypoxia in relation to vasculature and proliferation in liver metastases in patients with colorectal cancer.</a> Int J Radiat Oncol Biol Phys. 64, 473-482 (2006).</li><li>Hockel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratumoral+pO2+predicts+survival+in+advanced+cancer+of+the+uterine+cervix.">Intratumoral pO2 predicts survival in advanced cancer of the uterine cervix.</a> Radiother Oncol. 26, 45-50 (1993).</li><li>Semenza, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIF-1+and+human+disease:+one+highly+involved+factor.">HIF-1 and human disease: one highly involved factor.</a> Genes Dev. 14, 1983-1991 (2000).</li><li>Semenza, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-inducible+factor+1:+master+regulator+of+O2+homeostasis.">Hypoxia-inducible factor 1: master regulator of O2 homeostasis.</a> Curr Opin Genet Dev. 8, 588-594 (1998).</li><li>Tatum, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia:+importance+in+tumor+biology,+noninvasive+measurement+by+imaging,+and+value+of+its+measurement+in+the+management+of+cancer+therapy.">Hypoxia: importance in tumor biology, noninvasive measurement by imaging, and value of its measurement in the management of cancer therapy.</a> Int J Radiat Biol. 82, 699-757 (2006).</li><li>Brahimi-Horn, M. C., Pouyssegur, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIF+at+a+glance.">HIF at a glance.</a> J Cell Sci. 122, 1055-1057 (2009).</li><li>Piret, J. P., Mottet, D., Raes, M., Michiels, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CoCl2,+a+chemical+inducer+of+hypoxia-inducible+factor-1,+and+hypoxia+reduce+apoptotic+cell+death+in+hepatoma+cell+line+HepG2.">CoCl2, a chemical inducer of hypoxia-inducible factor-1, and hypoxia reduce apoptotic cell death in hepatoma cell line HepG2.</a> Ann N Y Acad Sci. 973, 443-447 (2002).</li><li>Emerling, B. M., Weinberg, F., Liu, J. L., Mak, T. W., Chandel, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PTEN+regulates+p300-dependent+hypoxia-inducible+factor+1+transcriptional+activity+through+Forkhead+transcription+factor+3a+(FOXO3a).">PTEN regulates p300-dependent hypoxia-inducible factor 1 transcriptional activity through Forkhead transcription factor 3a (FOXO3a).</a> Proc Natl Acad Sci U S A. 105, 2622-2627 (2008).</li></ol>

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Zellproliferation und Lebensfähigkeit in hypoxischen Bedingungen variieren stark in Abhängigkeit von der Zelltypen. Daher sollten Sie die Zelle oder die Nummer der Kulturen Platten starten Sie mit, damit Sie genügend Zellen / Proteine ​​für Ihre Experimente haben. Kobaltchlorid-Methode hat den Vorteil, kostengünstig und schnell. Dieses Produkt imitiert Hypoxie durch Induktion HIF-1/3α können aber auch andere Gene regulieren, stellen Sie sicher, dass dieses Produkt für Ihr Projekt wird durch die Überprüfung, was andere Effekte könnte auf die Funktion und Phänotyp der bestimmte Zellen unabhängig sind von der &quot;Mimik-adaptierten hypoxischen &quot;-Effekt. Ein weiteres Medikament auch zur Hypoxie nachahmen ist Deferoxamin Mesylat (DFO, verwendet bei 100 uM Endkonzentration). Der Einsatz dieser Medikamente kann der Experimentator die Kultur Platte / Schale / Kolben mehrmals ohne Beeinträchtigung der &quot;hypoxischen Zustand&quot; zu öffnen. Der Gehalt an Sauerstoff in dem Gasgemisch enthalten ist, kann abhängig von Ihren Experimenten und Zelltypen als Hypoxie-Werte in Abhängigkeit von der Gewebe-und Zelltypen variieren. Tatsächlich sind einige Zellen in 5% hypoxische O 2 andere brauchen weniger als 1% O 2 bis hypoxisch sein. 5% CO 2, das Gasgemisch wird hinzugefügt, um den pH-Wert der Kulturen zu stabilisieren, ist der Rest des Gases in der Regel Stickstoff. Hypoxische Kammern haben den Vorteil, nicht mit Medikamenten, die das Verhalten der Zelle wechseln kann unabhängig von der Sauerstoff-Partialdruck. Allerdings können nicht alle Arten von Experimenten durchgeführt, wie Sauerstoff wieder in die Kammer bei jeder Öffnung und damit verringert Hypoxie werden. Sie sollten diesen Faktor in Ihrer Experimente betrachten, Hypoxie / re-Oxygenierung ist eine bestimmte Bedingung, dass einige Zelltypen beeinflussen können. Eine Alternative ist die Hypoxie-Workstations (verbunden mit vorgemischten Gas-Tanks oder Gas Mischsysteme) oder größer Hypoxie Inkubator (Hypoxie Verarbeitung Kammer mit Handschuhfach), dass die Experimente zu den Medien zu ändern und zu manipulieren Zellen in kontinuierlichen hypoxische Umgebung ermöglicht den Einsatz. Verschiedene hypoxischen Kammern haben in den vergangenen zehn Jahren kommerzialisiert worden und sollte nach Ihrem Labor eingesetzt, Projekte, Raum, Größe und Budget gewählt werden. Achten Sie immer darauf den guten Zweck und Bedingung für die Kammer zu korrigieren Kultur zu garantieren. Im Allgemeinen, wenn Sie eine Kammer, kann der Grad der Hypoxie moduliert werden, indem Sie die verschiedenen Gasgemischen (dh 1%, 5% 10% Sauerstoff). In Bezug auf den Nachweis von HIF-1α, ist es wichtig zu wissen, dass einige Krebszellen line express HIF-1α in Normoxie. Es ist daher entscheidend für eine Normoxie-Steuerelement verwenden, um das Grundniveau von HIF in diesen Zelllinien zu bestimmen. HIF-1α kann auch in Normoxie bei nicht-malignen Zellen nach Zell-Stimulation oder Stress. Dies könnte auch auftreten, wenn diese Zellen verhungert sind sicher, dass Ihr Zellkulturen richtig ernähren und unterhalten.

<table border="1"><tbody><tr><th> Name des Reagenzes </th><th> Firma </th><th> Katalog-Nummer </th></tr><tr><td> Anti-HIF-1α </td><td> Invitrogen </td><td> 458400 </td></tr><tr><td> Cobalt (II)-Chlorid </td><td> Sigma </td><td> C8661-25G </td></tr><tr><td> Incubator Kammer </td><td> Billups-Rothenberg </td><td> MIC-101 </td></tr><tr><td> HRE-Luciferase-Plasmid </td><td> Adgene </td><td> Plasmid 26731 </td></tr></tbody></table>

induction and testing of hypoxia in cell culture

Hypoxie ist die Reduzierung oder das Fehlen von Sauerstoff in Organe, Gewebe oder Zellen definiert. Dieser Rückgang der Sauerstoffspannung kann durch eine reduzierte Versorgung mit Sauerstoff (Ursachen sind unzureichende Blutgefäß-Netzwerk, defekte Blutgefäß, und Anämie) oder zu einem erhöhten Verbrauch von Sauerstoff im Verhältnis zum Angebot (verursacht durch eine plötzliche höhere Zellteilungsrate) . Hypoxie kann physiologische oder pathologische wie in soliden Tumoren 1-3, rheumatoide Arthritis, Arteriosklerose usw. ... Jedes Gewebe und Zellen haben eine andere Fähigkeit, sich an diesem neuen Zustand anzupassen. Während Hypoxie, ist Hypoxie-induzierbaren Faktors alpha (HIF) stabilisiert und reguliert verschiedene Gene, wie sie in der Angiogenese oder den Transport von Sauerstoff 4 beteiligt. Die Stabilisierung dieses Proteins ist ein Markenzeichen von Hypoxie, also Erkennen HIF routinemäßig zum Screening auf Hypoxie 5-7 verwendet. In diesem Artikel schlagen wir zwei einfache Methoden, um eine Hypoxie in Säugetier-Zellkulturen und einfache Tests veranlassen, die hypoxischen Status dieser Zellen zu evaluieren.

Watch this Scientific Journal Video about Induction and Testing of Hypoxia in Cell Culture at JoVE.com

Induction and Testing of Hypoxia in Cell Culture

Hier schlagen wir einfache Methoden, um eine Hypoxie in Zellkulturen und einfache Tests veranlassen, die hypoxischen Zustand der Kulturen zu bewerten.

Induktion und Prüfung von Hypoxie in der Zellkultur

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

induction-and-testing-of-hypoxia-in-cell-culture

Research

JoVE Journal

Biology

Culture of Mouse Neural Stem Cell Precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types.  This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

Kultur der Maus Neural Stem Cell Precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase ...

Forschung

Biologie

Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of an antigen against which the immune system has been primed. The inflammatory reaction is characterized by redness and swelling of the site of antigenic challenge. It is a convenient model to determine the in vivo efficacy of immunosuppressants. Cutaneous DTH can be induced either by adoptive transfer of antigen-specific T lymphocytes or by active immunization with an antigen, and subsequent intradermal challenge with the antigen to induce the inflammatory reaction in a given skin area. DTH responses can be induced to various antigens, for example ovalbumin, tuberculin, tetanus toxoid, or keyhole limpet hemocyanin. Such reactions can also be induced against autoantigen, for example to myelin basic protein (MBP) in rats with experimental autoimmune encephalomyelitis induced with MBP, an animal model for multiple sclerosis (1).
Here we demonstrate how to induce an adoptive DTH reaction in Lewis rats. We will first stimulate ovalbumin-specific T cells in vitro and inject these activated cells intraperitoneally to naive rats. After allowing the cells to equilibrate in vivo for 2 days, we will challenge the rats with ovalbumin in the pinna of one ear, while the other ear wil receive saline. The inflammatory reaction will be visible 3-72 hours later and ear thickness will be measured as an indication of DTH severity.

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T (TEM) lymphocytes. Here we demonstrate how to activate antigen-specific TEM cells, induce adoptive DTH in Lewis rats and monitor the inflammatory response.

Induktion und Überwachung von Adoptive Allergien vom verzögerten Typ in Ratten

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Effiziente Ableitung von Human Cardiac Vorstufen und Kardiomyozyten aus pluripotenten humanen embryonalen Stammzellen mit Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Isolierung und Primärkultur von Ratten Leberzellen

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

Die Slice-Kultur-Verfahren zur Nach Entwicklung der Zahnkeime In Explantation Kultur

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

Isolierung, Kultur und Transplantation von Muskel-Satelliten-Zellen

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Analyse der globalen RNA-Synthese auf der Einzelzellebene folgende Hypoxie

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

Die c-fos-Protein Immunohistologische Nachweis: Ein nützliches Werkzeug als Marker der Mittelwegen beteiligt sind in spezifischen physiologischen Antworten<em&gt; In Vivo</em&gt; und<em&gt; Ex Vivo</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs&#39; as well as long non-coding RNAs&#39; (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor&#39;s (AR) 3&#39; untranslated region (3&#39;UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

Nachweis von RNA-bindenden Proteinen durch<em&gt; In Vitro</em&gt; RNA Pull-down in Adipocyte Kultur

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for developing methods to study the migration. The scratch assay has long been the most common in vitro method to test compounds with anti- and pro-migration properties because of its low cost and simple procedure. However, an often-reported problem of the assay is the accumulation of cells across the edge of the scratch. Furthermore, to obtain data from the assay, images of different exposures must be taken over a period of time at the exact same spot to compare the movements of the migration. Different analysis programs can be used to describe the scratch closure, but they are labor intensive, inaccurate, and forces cycles of temperature changes. In this study, we demonstrate an optimized method for testing the migration effect, e.g. with the naturally occurring proteins Human- and Bovine-Lactoferrin and their N-terminal peptide Lactoferricin on the epithelial cell line HaCaT. A crucial optimization is to wash and scratch in PBS, which eliminates the aforementioned accumulation of cells along the edge. This could be explained by the removal of cations, which have been shown to have an effect on keratinocyte cell-cell connection. To ensure true detection of migration, pre-treating with mitomycin C, a DNA synthesis inhibitor, was added to the protocol. Finally, we demonstrate the automated optical camera, which eliminates excessive temperature cycles, manual labor with scratch closure analysis, while improving on reproducibility and ensuring analysis of identical sections of the scratch over time.

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Here we describe a procedure to test the cell migration in vitro with an automated optical camera microscope. The scratch assay has been widely used where the closure of the scratch is followed for a set period. The optical microscope enables dependable and cheap detection of cell migration.