Wir bedanken uns bei U. Ganapati für die Errichtung und Prüfung der mStrawberry OP9 Zelllinie. Darüber hinaus danken wir H. Shafifor ihre Unterstützung bei der Co-Kultur. HP wird durch ein Stipendium des National Heart, Lung, and Blood Institute (F31HL087714) (HP) unterstützt. Der Inhalt dieser Arbeit ist ausschließlich in der Verantwortung des Autors und nicht notwendigerweise die offizielle Meinung des National Heart, Lung, and Blood Institute oder die National Institutes of Health (NIH). Die UCLA Durchflusszytometrie Core Facility wird durch die NIH (CA-16042 und AI-28697), der Jonsson Cancer Center der UCLA AIDS-Institut und der UCLA School of Medicine unterstützt.

1. Vorbereitung der ES-Zellen <ol><li> Kultur ES-Zellen nach Standardprotokoll für Ihre speziellen Zell-Linie. Achten Sie darauf, genug ES-Zellen auf den Teller vorzubereiten auf der mStrawberry OP9 Zellen für Tag 0 (ES-Zellen werden auf 1x10 ^ 3 Zellen pro cm 2 am Tag 0 beschichtet werden) </li></ol> 2. Vorbereitung der OP9 Zellen <ol><li> Bevor wachsenden mStrawberry OP9 Zellen, bereiten die OP9 Nährmedien OP-9 Zellkulturmedium <table border="1"><tbody><tr><td></td><td> Lager </td><td> Endkonzentration. </td><td> Volumen (ml) </td></tr><tr><td> aMEM </td><td></td><td></td><td> 39,5 </td></tr><tr><td> FBS (OP-9 getestet) </td><td> 100% </td><td> 20% </td><td> 10 </td></tr><tr><td> L-Glutamin </td><td> 100X (200mm) </td><td> 2mM </td><td> 0,5 </td></tr><tr><td></td><td></td><td></td><td> 50 mL </td></tr></tbody></table> Lagerung bei 4 ° C lagern und innerhalb von einer Woche Vorbereitung </li><li> Um mStrawberry OP9 Zellen zu erhalten, müssen Medien alle 2 Tage und Zellen sollten vor der Zellen zu erreichen Konfluenz (Subkultur bei 70-90% Konfluenz) subkultiviert werden. mStrawberry OP9 Zellkulturen sind nie über 90% Konfluenz gezüchtet, wie diese in ihrer Differenzierung in Adipozyten führt. Siehe Abbildung 3. <ol class="lalpha"><li> Subculture Zellen durch Waschen der Zellen 2X mit PBS (oder 1x mit PBS + 1x 0,1% mit Trypsin-EDTA) </li><li> Add vorgewärmten 0,1% Trypsin-EDTA bei 37 ° C für 5 Minuten (oder bis die Zellen zu lösen) </li><li> Wenn die Zellen abgelöst haben, fügen OP-9 Wachstumsmedium. Transfer-Zellen auf eine 50 ml konischen Rohr. </li><li> Pellet-Zellen bei 300 xg für 5 Minuten bei Raumtemperatur. Überstand. </li><li> Die Zellen in frischem mStrawberry OP9 Nährmedien </li><li> Platte Zellen bei einer Dichte von 10 4 Zellen / cm 2 </li><li> Wachsen mStrawberry OP9 Zellen bis zur Konfluenz ab 3 Tage vor Tag für Co-Kultivierung ES-Zellen (Tag 0, Tag 5 oder Tag8 / 9 des Experiments) benötigt </li></ol></li></ol> 3. ES / mStrawberry OP9 Co-Kultur <ol><li> Bereiten Sie Co-Kultur Differenzierung Medien <table border="1"><tbody><tr><td></td><td> Lager </td><td> Finale </td><td> Volumen (ml) </td></tr><tr><td> aMEM </td><td></td><td></td><td> 39,5 </td></tr><tr><td> FBS </td><td> 100% </td><td> 20% </td><td> 10 </td></tr><tr><td> L-Glutamin </td><td> 200 mM </td><td> 2mM </td><td> 0,5 </td></tr><tr><td></td><td></td><td></td><td> 50 mL </td></tr></tbody></table></li><li> mStrawberry OP9 Zellen (Tag -3) <ol class="lalpha"><li> IMMER Subculture OP9 Zellen am Tag -3, so dass Sie konfluenten Schichten mStrawberry OP9 Zellen für Tag 0 der Co-Kultur haben. Siehe Abbildung 4b. Platte mStrawberry OP9 Zellen bei 1x10 4 / cm 2 auf Konfluenz am Tag 0. Achten Sie darauf, Subkultur zusätzliche Zellen für Tag 5 und Tag 8 / 9 für die Fortsetzung der Co-Kultur. </li></ol></li><li class="jove_content"> ES / mStrawberry OP9 Co-Kultur Set-Up (Tag 0) <ol class="lalpha"><li class="jove_content"> Wash ES-Zellen 2x mit PBS </li><li> Trypsinize ES-Zellen mit vorgewärmten 0,25% Trypsin-EDTA, Inkubation 5min bei 37 ° C </li><li> Add Co-Kultur Differenzierung Medien Zellen </li><li> Pellet ES-Zellen bei 400xg, 5-7min, RT. Überstand </li><li> Die Zellen in Co-Kultur Differenzierung Medien </li><li> Entfernen Sie das Medium aus mStrawberry OP9 Zellen </li><li> Add Co-Kultur Differenzierung Medien mStrawberry OP9 Flaschen </li><li> Platte 1x10 3 ES-Zellen / cm 2 bis Flasche mStrawberry OP9 Zellen. Inkubieren Zellen bei 37 ° C, 5% CO 2 mit Feuchtigkeit. </li></ol></li><li> mStrawberry OP9 Zellen (Tag 2) <ol class="lalpha"><li> Subculture mStrawberry OP9 so dass Sie konfluenten Schichten mStrawberry OP9 Zellen für Tag 5 der Co-Kultur. Platte mStrawberry OP9 Zellen bei 1x10 4 / cm 2 auf Konfluenz am Tag 5 (wie in Schritt 3,2). Achten Sie darauf, Subkultur zusätzliche Zellen für Day 8 / 9, falls erforderlich (wie in Schritt 2,2). </li></ol></li><li> ES / mStrawberry OP9 Co-Kultur (Tag 3) <ol class="lalpha"><li> Entfernen Sie vorsichtig Medium von Co-Kultur und ersetzen mit frischen Co-Kultur Differenzierungsmedium </li></ol></li><li> ES / mStrawberry OP9 Co-Kultur (Tag 5) <ol class="lalpha"><li> Sorgfältig waschen Co-Kulturen 2x mit PBS </li><li> Trypsinize Zellen mit vorgewärmten 0,25% Trypsin-EDTA </li><li> Wash-Zellen aus Kolben mit Co-Kultur Differenzierung Medien </li><li> Pellet-Zellen bei 400xg, Discard 7min, RT. Überstand </li><li>Add Co-Kultur Differenzierung Medien Zellen. Verwenden Sie eine 21-Gauge bluntended Nadel Zellen resuspendieren und für einzelne Zellsuspension gewährleisten </li><li> Zählen von Zellen mit einer Zählkammer. Sollte 100-150 fachen Anstieg der ES-Zell-Zahl von Tag 0 zu haben. </li><li> Re-Seed 6.48x10 4 / cm 2 - 7.76x10 4 / cm 2 Co-Kultur-Zellen auf eine neue Ebene der konfluent, mStrawberry OP9 Zellen </li><li> Tag 5 ASSAYS-Remaining Co-Kultur-Zellen können für die Durchflusszytometrie-Analyse, cytopreps (30K-Zellen), Chip-Assays, etc. verwendet werden </li></ol></li><li> ES / mStrawberry OP9 Co-Kultur (Tag 8 / 9 und Tag 12) <ol class="lalpha"><li> Entfernen Differenzierung Medien von Kolben und speichern. Wash Monoschicht 1x mit PBS, um hämatopoetische Zellen in Suspension zu entfernen. Nicht wegwerfen hämatopoetischen Zellen. </li><li> Trypsinize adhärente Zellen mit vorgewärmten 0,25% Trypsin-EDTA </li><li> Wash-Zellen aus Kolben mit ES Differenzierung Medien und in den blutbildenden Zellen in Suspension = gefunden </li><li> Pellet-Zellen bei 400xg, Discard 7min, RT. Überstand </li><li> Add Co-Kultur Differenzierung Medien oder PBS zu den Zellen. Verwenden Sie eine 21-Gauge bluntended Nadel Zellen resuspendieren und für einzelne Zellsuspension Zählen von Zellen mit einer Zählkammer zu gewährleisten. </li><li> Co-Kultur kann für 14 Tage fortgesetzt werden, um reifer hämatopoetischer Vorläuferzellen durch erneutes Seeding Co-Kultur-Zellen zu bewerten auf einer neuen Ebene der konfluent, mStrawberry OP9 Zellen an Tag 8 oder 9 und dann wieder am 12. Tag. Re-Seed optimierte Zellzahl auf gewünschte Zelle Erholung für den Tag des Tests basiert. </li></ol></li><li> Evaluation von ES-derived Nachkommen (Tag 1-14). ES-derived Nachkommen können an jedem Tag der Co-Kultur von flow Aussortieren mStrawberry negativen Zellen (ES-derived Nachkommen) ausgewertet werden. <ol class="lalpha"><li> Entfernen Differenzierung Medien von Kolben und speichern. Wash Monoschicht 1x mit PBS, um hämatopoetische Zellen in Suspension zu entfernen. Nicht wegwerfen hämatopoetischen Zellen. </li><li> Trypsinize adhärente Zellen mit vorgewärmten 0,25% Trypsin-EDTA </li><li> Wash-Zellen aus Kolben mit Co-Kultur Differenzierung Medien und in den blutbildenden Zellen in Suspension gefunden </li><li> Pellet-Zellen bei 400xg, 7min, RT. Überstand </li><li> Die Zellen mit 21-Gauge bluntended Nadel und zählen </li><li> ES-derived Nachkommen (mStrawberry negativen Zellen) kann Flow aus mStrawberry positive OP9 Zellen sortiert werden. ES-derived Nachkommen können dann mittels Durchflusszytometrie-Analyse, cytopreps, ChIP-Assays, etc. charakterisiert werden </li></ol></li></ol> 4. Repräsentative Ergebnisse: <img src="/files/ftp_upload/2559/2559fig1.jpg" alt="Abbildung 1" /> Abbildung 1. ES / mStrawberry OP9 Co-Kultur-System. Unser Labor verwendet eine in vitro ES-Zell-Co-Kultur Systems zu modellieren hämatopoetischen Entwicklung. In dieser Co-Kultur-System, zu differenzieren ES-Zellen in hemangioblasts am Tag 5, wenn sie auf eine konfluente Schicht von mStrawberry OP9 Zellen gelegt. Als Co-Kultur geht, sind hämatopoetischen Vorläufer an Tag 8 vorhanden, während terminal differenzierten hämatopoetischen Linien von Tag 14 erscheinen. Die Pfeile zeigen die Tage der Differenzierung bei Passagieren der ES-derived Nachkommen auf neue konfluenten Schichten mStrawberry OP9 Zellen in notwendig. <img src="/files/ftp_upload/2559/2559fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Proper ES-Zell-Differenzierung. ES-Zellen werden auf eine konfluente Schicht von mStrawberry OP9 am Tag 0 ausgesät. ES-derived Nachkommen beginnen sichtbar zu werden an Tag 3 der Differenzierung, der mit ES-derived Nachkommen aussehen wie eine Ansammlung von Zellen mit einer definierten Grenze oder Anfang an einen Quirl Muster zu bilden. Hemangioblasts, die das gemeinsame mesodermalen Vorläufer für die Endothel-und hämatopoetischen Linien sind, sollte wie bis Quirle von Zellen an Tag 5 angehäuft. Für Co-Kultur Zeiträume von mehr als 5 Tage sollte ES-derived Nachkommen als Cluster von hämatopoetischen Zellen erscheinen. Zwei bis vier celled Cluster, an Tag 6 / 7, erscheinen soll und wachsen in größeren Zellverbänden von Tag 8 / 9 der Co-Kultur. Andere ES abgeleitet Nachkommen finden sich auch fest an die Anhänger Stromazellen Schicht gebunden. <img src="/files/ftp_upload/2559/2559fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Erfolglose ES-Zell-Differenzierung. Die Ergebnisse dieser Co-Kultur widerspiegeln unsachgemäße ES-Zell-Differenzierung. Beachten Sie, wie die ES-derived Nachkommen nicht in einem Quirl Muster am Tag 5 erscheinen. Das Fehlen von Zellen in einem Quirl Muster weist auf unsachgemäße hemangioblast Bildung. Wenn hemangioblast nicht richtig entwickeln zu tun, dann weiter Co-Kultur wird in verkümmerten hämatopoetischen Linie Differenzierung führen. Co-Kulturen mit unsachgemäße hemangioblast Bildung muss verworfen werden. <img src="/files/ftp_upload/2559/2559fig4.jpg" alt="Abbildung 4" /> Abbildung 4. mStrawberry OP9 Zellkulturen. (A) mStrawberry OP9 Zelle sind subkultiviert70-90% Konfluenz. (B) Für eine korrekte Differenzierung, sind ES-Zellen und ES-derived Nachkommen auf einen übermäßig konfluente Schicht von mStrawberry OP9 Zellen gelegt. <img src="/files/ftp_upload/2559/2559fig5.jpg" alt="Abbildung 5" /> Abbildung 5. Vertreter ES / mStrawberry OP9 Co-Kultur Strömungsprofil. Alle ES-derived Nachkommen sind mStrawberry negativen Zellen und kann Flow aus der mStrawberry OP9 Zellen sortiert werden. mStrawberry negativen Zelle Tor wurde mit einem traditionellen ES/OP9 Co-Kultur.

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Proper Differenzierung von ES-Zellen auf konfluente Schicht von mStrawberry OP9 Zellen führt zur Produktion von Flk1 + hemangioblasts am Tag 5 der Co-Kultur. Hemangioblasts sollte wie bis Quirle von Zellen, wie in Abbildung 1 beobachtet angehäuft. Für den Rest der Co-Kultur sollte sichtbar ES-derived Nachkommen als Cluster von hämatopoetischen Zellen (Abbildung 1) erscheinen. Am Tag 6 / 7 zwei vor vier Zellhaufen erscheinen soll und wachsen in größeren Zellverbänden (beide haftend und in Suspension) von Day 8 / 9 der Co-Kultur. Andere ES abgeleitet Nachkommen finden sich auch nahtlos in die adhärenten Stromazellen Schicht gebunden. Obwohl nicht im Protokoll aufgeführt sind, ist es entscheidend, Pre-Test der FBS in der ES Wachstumsmedium, die mStrawberry OP9 Wachstum Medien und die ES / mStrawberry OP9 Differenzierung Medien eingesetzt. Richtig getestet Serum sollte richtiges Wachstum von mStrawberry OP9 Zellen, sowie, korrekte Differenzierung von ES-Zellen in Co-Kultur bis Tag 5 der Differenzierung zu gewährleisten. Als Alternative oder zusätzlich zu mStrawberry OP9 Zellen können OP9 Zellen bestrahlt, bei 8000 rad sein, vor der Aussaat bei Konfluenz (7.8x10 4 Zellen / cm 2). Die Bestrahlung von Zellen erlaubt OP9 für die Beseitigung der wiederholten Neuplattierungen Schritte, sowie, ermöglicht fast vollständige Beseitigung der alten, verunreinigen OP9 Zellen aus Downstream-Assays. OP9 Zellen wurden entwickelt, um mStrawberry Protein, das durch Transduktion OP9 Zellen mit 3ug/mL eines lentiviralen Vektor, mStrawberry unter der Kontrolle eines CMV-Promotors (pRRLsin-CMV-Vektor, UCLA-Vektor-Kern) zu äußern. Standard ES/OP9 Co-Kultur-Protokolle beinhalten die Vermittlung von ES-Zellen auf eine konfluente Schicht OP9 Zellen, sowie eine Tag 5 Neuplattierungen Schritt zur Reduzierung alt, verunreinigen OP9 Zellen aus Zellpassage. Darüber hinaus sind nur hämatopoetischen Zellen in Suspension gefunden für die Bewertung von ES-derived Nachkommen entfernt. Doch sowohl mit einem Neuplattierungen Schritt und die Ernte von nur Suspensionszellen eine potentiell vermisst eine beträchtliche Anzahl von Entwicklungsländern ES-abgeleitete hämatopoetische Zellen. Dieses Problem wird es wichtig, Adresse wenn man versucht, hämatopoetischen Defekte mit KO-ES-Linien verbunden zu charakterisieren. Um dieses Problem zu umgehen unserem Labor verwendet ein modifiziertes Co-Kultur, durch den Einsatz von mStrawberry-exprimierenden OP9 Zellen. Dadurch ist gewährleistet, dass alle ES Nachkommen der anhaltenden Co-Kultur sind an Tag 5 übertragen, und für die Ernte aller ES-derived Nachkommen für Downstream-Assays. Diese Änderung stellt eine nützliche Tools in der Evaluierung und Charakterisierung von hämatopoetischen Nachkommen von Mensch und Maus-ES-Linien abgeleitet. Das ES-mStrawberry OP9 Co-Kultur-Modell fasst die verschiedenen Stufen primitive und definitive Hämatopoese. Bei der Untersuchung der frühesten Stadien der hämatopoetischen Entwicklung, nutzen viele Menschen die BL-CFC (Blast-wie koloniebildenden Zellen)-Assay, der die Entwicklung der hemangioblast aus der ES-Zellen zu beurteilen. Während die BL-CFC-Assay ist ein nützliches Werkzeug bei der Untersuchung von frühen hämatopoetischen Entwicklung, weist die mStrawberry-ES Co-Kultur-Systeme zu erhöhen Dienstprogramm, wie es für die Beurteilung der hemangioblast sowie mehr erwachsene hämatopoetischen Linien ermöglicht. Frühere Arbeiten mit traditionellen OP9/ES und EB Differenzierung Protokolle hat gezeigt, dass zusätzliche Faktoren vielleicht notwendig, wie die Überexpression von HOXB4, um daraus langfristig repopulating hämatopoetischen Stammzellen in vitro. Zusätzliche Arbeit ist zu prüfen, ob mStrawberry negativ, sortiert ES-derived Bevölkerung wahr hämatopoetischen Stammzellen enthalten.

OP-9 Zellen Wartung Medium Lager-Komponenten <table border="1"><tbody><tr><td> Name des Reagenz </td><td> Firma </td><td> Catalog Number </td><td> Kommentare </td></tr><tr><td> aMEM </td><td> Invitrogen </td><td> 12571-063 </td><td></td></tr><tr><td> FBS </td><td> Omega Scientific </td><td> FB01 </td><td> Pre-Tested </td></tr><tr><td> L-Glutamin </td><td> CellGro </td><td> 25 bis 005-CI </td><td> 200 nM </td></tr><tr><td> Trypsin-EDTA </td><td> Stem Cell Technologies </td><td> 07901 </td><td></td></tr></tbody></table> ES/OP-9 Zelldifferenzierung Medium Lager-Komponenten <table border="1"><tbody><tr><td> Name des Reagenz </td><td> Firma </td><td> Catalog Number </td><td> Kommentare </td></tr><tr><td> aMEM </td><td> Invitrogen </td><td> 12571-063 </td><td></td></tr><tr><td> FBS </td><td> Hyclone </td><td> SH30070 </td><td> Pre-Tested </td></tr><tr><td> L-Glutamin </td><td> CellGro </td><td> 25 bis 005-CI </td><td> 200 mM </td></tr><tr><td> PBS (ohne Ca 2 + und Mg 2 +) </td><td> CellGro </td><td> 21 bis 031-CV </td><td></td></tr><tr><td> Trypsin-EDTA </td><td> Stem Cell Technologies </td><td> 07901 </td><td></td></tr></tbody></table>

modified es / op9 co-culture protocol provides enhanced characterization of hematopoietic progeny

Die in vitro Differenzierung von ES-Zellen zu einer hämatopoetischen Zelle Schicksal ist nützlich, wenn das Studium Zellpopulationen, die schwer zu in vivo und zur Charakterisierung der frühesten Gene in Blutbildung beteiligt sind, ohne sich mit embryonalen Letalitäten viel zuzugreifen. Die ES/OP9 Co-Kultur-System wurde ursprünglich entwickelt, um hämatopoetische Nachkommen zu produzieren, ohne über die Produktion von Makrophagen, die OP9 stromalen Zelllinie, die aus der Schädeldecke des Osteopetrose mutierten Mäusen, die funktionelle M-CSF Mangel abgeleitet ist. Die in vitro ES/OP9 Co-Kultur-System kann verwendet werden, um frühzeitig hämatopoetischen Entwicklung rekapitulieren werden. Wenn auf OP9 Stromazellen kultiviert, differenzieren ES-Zellen in Flk-1 + hemangioblasts, hämatopoetischen Vorläuferzellen und schließlich reifen, terminal differenzierten Linien. Die Standard-ES/OP9 Co-Kultur-Protokoll beinhaltet die Vermittlung von ES-Zellen auf eine konfluente Schicht von OP9 Zellen, sowie periodische Neuplattierungen Schritte zur Entfernung alter, verunreinigen OP9 Zellen. Darüber hinaus beinhalten aktuelle Protokolle Auswertung nur die blutbildenden Zellen in Suspension und sind nicht für die Bewertung von ES-derived Nachkommen an jedem Tag der Differenzierung optimiert. Doch mit Neuplattierungen Schritte und die Ernte von nur Suspensionszellen eine potentiell fehlt ein großer Teil der ES-derived Nachkommen und Entwicklung hämatopoetischen Zellen. Dieses Problem wird es wichtig, Adresse wenn man versucht, hämatopoetischen Defekte mit KO-ES-Linien verbunden zu charakterisieren. Hier beschreiben wir eine modifizierte ES / mStrawberry OP9 Co-Kultur, die für die Beseitigung der Verunreinigung OP9 Zellen aus Downstream-Assays ermöglicht. Diese Methode ermöglicht die komplette Auswertung aller ES-derived Nachkommen an allen Tagen der Co-Kultur, was zu einer hämatopoetischen Differenzierung Muster, die mehr entspricht direkt der hämatopoetischen Differenzierung Muster innerhalb der Embryo beobachtet.

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Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny

mStrawberry OP9 Zellen ermöglichen vollständige Bewertung aller ES-derived Nachkommen von Co-Kultur.

Geändert ES / OP9 Co-Kultur-Protokoll bietet eine verbesserte Charakterisierung von hämatopoetischen Progeny

The in vitro differentiation of ES cells towards a hematopoietic cell fate is useful when studying cell populations that are difficult to access in vivo ...

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Serie: Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny

Freezing Human ES Cells

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove residual media that could otherwise quench the Trypsin reaction.  Warmed 0.05% Trypsin-EDTA is then added to cover the cells, and the plate allowed to incubate for up to 5 mins at room temperature.  During this time cells can be observed rounding, and colonies lifting off the plate surface.  Gentle repeated pipetting will remove cells and colonies from the plate surface.  Trypsinized cells are placed in a standard conical tube containing pre-warmed hES cell media to quench remaining trypsin, and then spun.  Cells are resuspended growth media at a concentration of approximately one million cells in one mL of media, a concentration such that one frozen aliquot is sufficient to resurrect a culture on a 10cm plate.  After cells are adequately resuspended, ice cold freezing media is added at equal volume.  Cell suspensions are mixed thoroughly, aliquoted into freezing vials, and allowed to slowly freeze to -80C over 24 hours.  Frozen cells can then moved to the vapor phase of liquid nitrogen for long term storage, or remain at -80 for approximately six months.

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.

Einfrieren menschlicher ES-Zellen

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove ...

Forschung

Biologie

Human ES cells: Starting Culture from Frozen Cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator.  A frozen vial of cells from long term liquid nitrogen storage or a -80C freezer is sourced and quickly submerged in a 37C water bath for quick thawing.  Cells in freezing media are then removed from the vial and placed in a large volume of HuES media.  The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate.  Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate.  It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate.  The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.

Humane ES-Zellen: Starting Kultur aus eingefrorenen Zellen

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

ES Cell-derived Neuroepithelial Cell Cultures

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In general, the differentiation of ES cells follows the concept to first generate immature progenitor cells, which then can be propagated and differentiated into mature cellular phenotypes. This also applies for ES cell-derived neurogenesis, in which the development of neural cells follows two major steps: First, the derivation and expansion of immature neuroepithelial precursors and second, their differentiation into mature neural cells. A common method to produce neural progenitors from ES cells is based on embryoid body (EB) formation, which reveals the differentiation of cells from all germ layers including neuroectoderm. An alternative and more efficient method to induce neuroepithelial cell development uses stromal cell-derived inducing activity (SDIA), which can be achieved by co-culturing ES cells with skull bone marrow-derived stromal cells (1). Both, EB formation and SDIA, reveal the development of rosette-like structures, which are thought to resemble neural tube- and/or neural crest-like progenitors. The neural precursors can be isolated, expanded and further differentiated into specific neurons and glia cells using defined culture conditions. Here, we describe the generation and isolation of such rosettes in co-culture experiments with the stromal cell line MS5 (2-5).

Derivation of neuroepithelial precursors from embryonic stem (ES) cells using stromal cell-derived inducing activity (SDIA).

ES-Zellen abgeleiteten neuroepithelialen Zellkulturen

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In ...

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

Vermehrung von humanen embryonalen Stammzellen (ES-Zellen)

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Eine Neuronale und Astrocyte Co-Kultur-Assay für High Content Analyse der Neurotoxizität

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

Ein Multi-Kompartiment-ZNS-Neuron-Glia-Co-Kultur mikrofluidischen Plattform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

Mikrofluidik-Co-Kultur von Epithelzellen und Bakterien für die Untersuchung Löslich Signal-vermittelte Wechselwirkungen

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

Isolierung von Brustepithelzellen aus dreidimensionalen Mixed-Zelle Spheroid Co-Kultur

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a &#8216;targeting&#8217; vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term &#8216;subcloning plus insertion&#8217;, which can be used to generate custom gene targeting vectors rapidly.

Subklonierung Plus-Insertion (SPI) - A Novel Recombineering Verfahren zur schnellen Bau der Gene Targeting Vektoren

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

Entwicklung eines Insert Co-Kultursystem von zwei Cellular Typen in der Abwesenheit von Zell-Zell-Kontakt

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...