Diese Arbeit wurde von der Austrian Research Foundation (FWF, gewähren N211-NAN DS) und die Österreichische Forschungsförderungsgesellschaft mbH (FFG, gewähren N200 zum DS) unterstützt worden. Die Autoren danken Claudia URL für hervorragende technische Unterstützung und Monica Farrell für die sprachliche Bearbeitung.

1. Ausgangsmaterial Beginnen Sie mit Thrombozyten-reichem Plasma (PRP) Einheiten durch Zytopherese oder aus Buffy Coats. 2. Sterilität zu überprüfen Für die Sterilität zu überprüfen eine Probe von 20 ml aus jedem PRP-Einheit durch die Übertragung der Lautstärke auf einem angeschlossenen kleinen Beutel (Baxter). Trennen Sie diese Tasche durch Schweißen. 3. Einfrieren von PRP-Einheiten Unmittelbar nach der Herstellung, frieren die PRP-Einheiten bis mindestens -20 ° C in der ursprünglichen Aufbewahrungstasche ohne weitere Manipulation. 4. Auftauen von HPL-Einheiten Wenn bakteriellen Tests negativ sind, Tauwetter menschlichen Thrombozyten-Lysat Einheiten, die heute als HPL-Einheiten bei 37 ° C (Wasserbad), bis das Eis Blutgerinnsel verschwinden. Nicht erwärmen die HPL. 5. Pooling von HPL-Einheiten Nehmen Sie sich mindestens 10 bis 15 aufgetaut HPL-Einheiten für eine Thrombozyten-Lysat-Pool, ein standardisiertes Produkt vorbereiten. Schließen Sie das HPL Beutel nacheinander auf die Zusammenlegung doppelten Beutel (MacoPharma) und übertragen Sie die HPL in diese beiden Taschen. Trennen Sie die leere HPL Beutel verschweißt. Holen Sie sich ein Gesamtvolumen von 3 bis 4 L von gepoolten menschlichen Thrombozyten-Lysat (PHPL) durch Mischen der Inhalt der beiden Tüten. Schließen Sie eine kleine Tasche (Baxter) und nehmen Sie eine Probe von 20 mL PHPL für Sterilität Überprüfung der gepoolten Produkt. Trennen Sie diese Tasche durch Schweißen. 6. Portionieren der PHPL Portion der PHPL geeignete Aliquots für die weitere Verarbeitung zu bekommen. Connect kleine Beutel (Baxter), um die Bündelung doppelten Beutel (Macopharma) und Transfer Volumen von bis zu 250 mL der kleinen Säcken zur Lagerung (Baxter). Trennen Sie die gefüllten Säcke durch Schweißen. 7. Re-Einfrieren der PHPL Aliquots Erhöhung des Anteils der Thrombozyten-Fragmentierung und die Menge des freigesetzten Wachstumsfaktoren durch einen weiteren Frost / Tau-Schritt. Frieren Sie den kleinen Beutel portioniert PHPL wieder bei mindestens -20 ° C. 8. Re-Auftau-und Portionierung des PHPL Aliquots Auftauen PHPL Beutel bei 37 ° C (Wasserbad). Übertragen Sie die Inhalte in 50 ml-Fläschchen (Falcons BD), indem das Rohr des Beutels mit einer sterilen Schere und Gießen der PHPL in das Fläschchen. Führen Sie diesen Schritt in einer laminaren Strömung Bank, um bakterielle oder Pilzinfektionen Kontamination zu vermeiden. 9. Entfernen von Thrombozyten-Fragmente Da Thrombozyten-Fragmente zur Aggregation neigen und kann Alloimmunisierung induzieren, entfernen Sie sie aus dem PHPL. Zentrifugieren Sie die PHPL Fläschchen daher bei 4.000 g (15 Minuten, 4 ° C). In einer laminaren Strömung Bank Pipette der Überstand Plasma in die Endlagerung Fläschchen (Falcons BD) und entsorgen Sie die Thrombozyten-Pellets zu vermeiden Fragmente in der Zellkultur. 10. Speicherung von PHPL Freeze-Aliquots von 50 ml Fläschchen PHPL wieder mindestens-20 ° C und lagern Sie sie zu Versuchszwecken verwendet. 11. Verwendung von PHPL in der Zellkultur Zunächst fügen Sie frei von Konservierungsmitteln Heparin auf die mittel-bis Gelbildung zu vermeiden. Tauwetter ein PHPL Aliquot bei 37 ° C und ergänzen das Kulturmedium durch Zugabe von 8 bis 10%. Medien MSC-Medium: Verwenden Sie 500 ml a-MEM, fügen Sie 56 ml des aufgetauten PHPL (siehe auch Hinweis 1 für weitere Details) und 2 IU / mL (= 224 ul der Stammlösung) der frei von Konservierungsmitteln Heparin (verhindert Koagulation des Mediums durch Verklumpung der das Fibrinogen im Plasma), um eine endgültige Konzentration von 10% PHPL. Zusätzlich fügen Penicillin (100U/mL) / Streptomycin (100μg/mL)-Lösung und 2 mM L-Glutamin (beide Sigma). Filter das Medium, durch ein 20 um-Porengröße Vakuum-Filter (Millipore). Beschriften Sie die Flasche entsprechend (Inhalt, Datum). ECFC-Medium: Verwenden Sie eine Flasche (500 ml) von EBM, fügen Sie die Zytokin-Aliquots, 56 ml PHPL, 10 IU / ml (= 1120μl Stammlösung) der frei von Konservierungsmitteln Heparin, Penicillin (100U/mL) / Streptomycin (100 g / ml)-Lösung und 2 mM L-Glutamin, die Basalmedium und Filter mit 20 ul-Porengröße Vakuum-Filter (Millipore). Beschriften Sie die Flasche entsprechend (Inhalt, Datum). <img src="/files/ftp_upload/1523/1523fig1.jpg" alt="Abbildung 1" /> Abbildung 1: Herstellung von Plättchen-reiches Plasma aus einer Vollblutspende von einem lokalen Blutbank oder anderen zugelassenen Anbieter. Nach Zentrifugation kann das Blut in Plasma, Buffy Coat und Erythrozyten getrennt werden. Vier Buffy-Coat-Einheiten, Blutgruppe O und eine Blutgruppe AB Plasma kann gebündelt vor der Zentrifugation der Blutplättchen reiches Plasma getrennt zu sein. Eine regelmäßige Qualitätskontrollen Plättchen-reiches Plasma-Einheit von etwa 300 ml enthalten sollte 1x10 9 Thrombozyten pro ml oder 3x10 11 platelets insgesamt. <img src="/files/ftp_upload/1523/1523fig2.jpg" alt="Abbildung 2" /><img src="/files/ftp_upload/1523/1523fig3.jpg" alt="Abbildung 3" />

<ol>
	<li>Nurden, A. T., Nurden, P., Sanchez, M., Andia, I., Anitua, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelets+and+wound+healing.">Platelets and wound healing.</a> Front Biosci. 13, 3532-3548 (2008).</li><li>Barrientos, S., Stojadinovic, O., Golinko, M. S., Brem, H., Tomic-Canic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+factors+and+cytokines+in+wound+healing.">Growth factors and cytokines in wound healing.</a> Wound Repair Regen. 16, 585-601 (2008).</li><li>Giacco, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombin-activated+platelets+induce+proliferation+of+human+skin+fibroblasts+by+stimulating+autocrine+production+of+insulin-like+growth+factor-1.">Thrombin-activated platelets induce proliferation of human skin fibroblasts by stimulating autocrine production of insulin-like growth factor-1.</a> FASEB J. 20, 2402-2404 (2006).</li><li>Kocaoemer, A., Kern, S., Kluter, H., Bieback, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+AB+serum+and+thrombin-activated+platelet-rich+plasma+are+suitable+alternatives+to+fetal+calf+serum+for+the+expansion+of+mesenchymal+stem+cells+from+adipose+tissue.">Human AB serum and thrombin-activated platelet-rich plasma are suitable alternatives to fetal calf serum for the expansion of mesenchymal stem cells from adipose tissue.</a> Stem Cells. 25, 1270-1278 (2007).</li><li>Kakudo, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation-promoting+effect+of+platelet-rich+plasma+on+human+adipose-derived+stem+cells+and+human+dermal+fibroblasts.">Proliferation-promoting effect of platelet-rich plasma on human adipose-derived stem cells and human dermal fibroblasts.</a> Plast Reconstr Surg. 122, 1352-1360 (2008).</li><li>Doucet, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+lysates+promote+mesenchymal+stem+cell+expansion:+A+safety+substitute+for+animal+serum+in+cell-based+therapy+applications.">Platelet lysates promote mesenchymal stem cell expansion: A safety substitute for animal serum in cell-based therapy applications.</a> J Cell Physiol. 205, 228-236 (2005).</li><li>Schallmoser, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+platelet+lysate+can+replace+fetal+bovine+serum+for+clinical-scale+expansion+of+functional+mesenchymal+stromal+cells.">Human platelet lysate can replace fetal bovine serum for clinical-scale expansion of functional mesenchymal stromal cells.</a> Transfusion. 47, 1436-1446 (2007).</li><li>Reinisch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+system+to+propagate+cord+blood-derived+multipotent+mesenchymal+stromal+cells+for+clinical+application.">Humanized system to propagate cord blood-derived multipotent mesenchymal stromal cells for clinical application.</a> Regen Med. 2, 371-382 (2007).</li><li>Schallmoser, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+large-scale+expansion+of+functional+mesenchymal+stem+cells+from+unmanipulated+bone+marrow+without+animal+serum+Tissue+Eng+Part.">Rapid large-scale expansion of functional mesenchymal stem cells from unmanipulated bone marrow without animal serum Tissue Eng Part.</a> C Methods. 14, 185-196 (2008).</li><li>Lange, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+and+safe+expansion+of+human+mesenchymal+stromal+cells+in+animal+serum-free+medium+for+transplantation+and+regenerative+medicine.">Accelerated and safe expansion of human mesenchymal stromal cells in animal serum-free medium for transplantation and regenerative medicine.</a> J Cell Physiol. 213, 18-26 (2007).</li><li>Reinisch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+large-scale+expanded+endothelial+colony-forming+cells+function+in+vitro+and+in+vivo.">Humanized large-scale expanded endothelial colony-forming cells function in vitro and in vivo.</a> Blood. 113, 6716-6725 (2009).</li><li>Bernardo, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+in+vitro+expansion+of+human+multipotent+mesenchymal+stromal+cells+for+cell-therapy+approaches:+further+insights+in+the+search+for+a+fetal+calf+serum+substitute.">Optimization of in vitro expansion of human multipotent mesenchymal stromal cells for cell-therapy approaches: further insights in the search for a fetal calf serum substitute.</a> J Cell Physiol. 211, 121-130 (2007).</li><li>Carrancio, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+mesenchymal+stem+cell+expansion+procedures+by+cell+separation+and+culture+conditions+modification.">Optimization of mesenchymal stem cell expansion procedures by cell separation and culture conditions modification.</a> Exp Hematol. 36, 1014-1021 (2008).</li><li>Muller, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+lysate+as+a+serum+substitute+for+2D+static+and+3D+perfusion+culture+of+stromal+vascular+fraction+cells+from+human+adipose+tissue.">Platelet lysate as a serum substitute for 2D static and 3D perfusion culture of stromal vascular fraction cells from human adipose tissue.</a> Tissue Eng Part A. 15, 869-875 (2009).</li><li>Anitua, E., Sanchez, M., Orive, G., Andia, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+impact+of+the+preparation+rich+in+growth+factors+(PRGF)+in+different+medical+fields.">The potential impact of the preparation rich in growth factors (PRGF) in different medical fields.</a> Biomaterials. 28, 4551-4560 (2007).</li><li>Anitua, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+and+novel+applications+for+platelet-rich+fibrin+therapies.">New insights into and novel applications for platelet-rich fibrin therapies.</a> Trends Biotechnol. 24, 227-234 (2006).</li><li>Martinez-Zapata, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+and+safety+of+the+use+of+autologous+plasma+rich+in+platelets+for+tissue+regeneration:+a+systematic+review.">Efficacy and safety of the use of autologous plasma rich in platelets for tissue regeneration: a systematic review.</a> Transfusion. 49, 44-56 (2009).</li><li>Weibrich, G., Kleis, W. K., Hafner, G., Hitzler, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+factor+levels+in+platelet-rich+plasma+and+correlations+with+donor+age,+sex,+and+platelet+count.">Growth factor levels in platelet-rich plasma and correlations with donor age, sex, and platelet count.</a> J Craniomaxillofac Surg. 30, 97-102 (2002).</li><li>Frechette, J. P., Martineau, I. &. a. m. p. ;. a. m. p., Gagnon, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-rich+plasmas:+growth+factor+content+and+roles+in+wound+healing.">Platelet-rich plasmas: growth factor content and roles in wound healing.</a> J Dent Res. 84, 434-439 (2005).</li><li>Seghatchian, J., Krailadsiri, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+platelet+storage+lesion.">The platelet storage lesion.</a> Transfus Med Rev. 11, 130-144 (1997).</li><li>Selvaggi, T. A., Walker, R. E., Fleisher, T. 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In einigen Regionen platelet rich plasma (PRP) kann von Buffy-Coats sonst, ein Abfallprodukt der gepackten roten Blutkörperchen aus getestet Blutspenden (Abbildung 1) erreicht werden. Optimal ist PRP sofort für die weitere Bearbeitung von PHPL verwendet, wie in veralteten Thrombozytenkonzentraten die Verfügbarkeit von Wachstumsfaktoren können durch Thrombozyten-Lagerung Läsionen und Abbau 20 reduziert werden. Es wird ferner empfohlen, PRP durch Anpassung Thrombozyten der Blutgruppe O mit Plasma der Blutg...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Double bag (2 x 3.5L)</td>
<td>&nbsp;</td>
<td>MacoPharma</td>
<td>VDL 8000XQ</td>
<td>originally for ascites puncture</td>
</tr>
<tr>
<td>50 mL Falcon tube</td>
<td>&nbsp;</td>
<td>Falcon BD</td>
<td>2098</td>
<td>&nbsp;</td>
<tr>
<td>50 mL Stripettes</td>
<td>&nbsp;</td>
<td>Costar</td>
<td>4501</td>
<td>&nbsp;</td>
<tr>
<td>Plasma bags (600mL)</td>
<td>&nbsp;</td>
<td>Baxter</td>
<td>R4R2021</td>
<td>&nbsp;</td>
<tr>
<td>Sterile tubing welder</td>
<td>&nbsp;</td>
<td>Terumo</td>
<td>TSCD-II</td>
<td>&nbsp;</td>
<tr>
<td>Welding equipment </td>
<td>&nbsp;</td>
<td>Fresenius Kabi</td>
<td>Compo Seal</td>
<td>&nbsp;</td>
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<td>Water bath</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Sterile scissors</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Clamps</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Centrifuge</td>
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preparation of pooled human platelet lysate (phpl) as an efficient supplement for animal serum-free human stem cell cultures

Platelet derived growth Faktoren haben gezeigt, dass die Zellproliferation effizient stimulieren<em&gt; In vivo</em<sup&gt; 1,2</sup&gt; Und<em&gt; In-vitro-</em&gt;. Dieser Effekt wurde für mesenchymale Stromazellen (MSCs), Fibroblasten und Endothelzellen Kolonie-bildenden Zellen mit Thrombozyten durch Thrombin aktiviert gemeldet<sup&gt; 3-5</sup&gt; Oder durch Frost / Tau-Zyklen lysiert<sup&gt; 6-14</sup&gt; Vor der Thrombozyten releasate ist das Zellkulturmedium aufgenommen. Die trophische Wirkung von thrombozytären Wachstumsfaktoren hat bereits in mehreren Studien für das Tissue Engineering und der regenerativen Therapie getestet.<sup&gt; 1,15-17</sup&gt; Unterschiedliche Effizienz gilt als zumindest teilweise durch individuell unterschiedliche Konzentrationen von Wachstumsfaktoren werden<sup&gt; 18,19</sup&gt; Und einem aktuellen Mangel an standardisierten Protokollen für Thrombozyten-Vorbereitung.<sup&gt; 15,16</sup&gt; Dieses Protokoll stellt ein praktikables Verfahren zu einem Pool von menschlichen Thrombozyten-Lysat (PHPL) von routinemäßig produziert platelet rich plasma (PRP) von 40 bis 50 einzelnen Blutspenden gewonnen zu generieren. Durch mehrere Frost / Tau-Zyklen der Thrombozyten-Membranen sind beschädigt und Wachstumsfaktoren sind effizient in das Plasma freigesetzt. Schließlich sind die Thrombozyten-Fragmenten durch Zentrifugation umfangreiche Aggregatbildung zu vermeiden und zum Abbau potenzieller Antigene entfernt. Die Umsetzung der PHPL in Standard-Kultur-Protokolle stellt ein viel versprechendes Instrument für die weitere Entwicklung von Zelltherapeutika propagiert in einem Tier-Protein-freies System.

Watch this Scientific Journal Video about Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures at JoVE.com

Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Menschliche Thrombozyten-Lysat ist eine reiche Quelle von Wachstumsfaktoren und eine starke Ergänzung in der Zellkultur. Dieses Protokoll stellt den Prozess der Vorbereitung einen großen Pool an menschlichen Thrombozyten-Lysat ausgehend von platelet rich plasma, Durchführung mehrerer Gefrier-Auftau-Zyklen und zum Abbau der Thrombozyten-Fragmente.

Vorbereitung der gepoolten menschlichen Thrombozyten-Lysat (PHPL) als effiziente Ergänzung zum tierischen Serum-freien menschlichen Stammzell-Kulturen

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for ...

preparation-pooled-human-platelet-lysate-phpl-as-an-efficient

Research

JoVE Journal

Biology

Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

25.1K Aufrufe.  Medical University of Graz, Austria. Menschliche Thrombozyten-Lysat ist eine reiche Quelle von Wachstumsfaktoren und eine starke Ergänzung in der Zellkultur. Dieses Protokoll stellt den Prozess der Vorbereitung einen großen Pool an menschlichen Thrombozyten-Lysat ausgehend von platelet rich plasma, Durchführung mehrerer Gefrier-Auftau-Zyklen und zum Abbau der Thrombozyten-Fragmente.

Serie: Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

Maus Epidermal Neural Crest Stem Cell (EPI-NCSC) Kulturen

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

Forschung

Biologie

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.
An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.
In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

Vorbereitung der Aplysia Sensory-Motor neuronalen Zellkulturen

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons.  The neurons are large (up ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

Isolation und tierisches Serum Freie Expansion der menschlichen Nabelschnur Derived mesenchymale Stromazellen (MSCs) und Endothelial Colony Forming Progenitorzellen (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Differenzierung von embryonalen Stammzellen in Oligodendrozyten Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

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 ...

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity4. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.

Despite ongoing efforts to transition cultures to feeder-free conditions, the derivation and culture of human embryonic stem cells (hESC) remain largely dependent on co-cultures with mouse embryonic feeders (MEFs). Here, we show a novel methodology for rapidly removing feeders from hESC cultures prior to experimentation.

Schnelle Fibroblast Entfernung aus High Density humanen embryonalen Stammzellen Kulturen

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Naive Adult Stem Cells Isolation von primären humanen Fibroblasten Kulturen

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

Effiziente Erzeugung Menschen induzierten pluripotenten Stammzellen aus menschlichen Körperzellen mit Sendai-Virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Alternative Kulturen für menschlichen pluripotenten Stammzellen Produktion, Wartung und Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Efficient and Consistent Generation of Retinal Pigment Epithelium/Choroid Flatmounts from Human Eyes for Histological Analysis

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and vision. Proteins on the RPE apical surface are tightly associated with proteins on the photoreceptor outer segment surface, making it difficult to consistently separate the RPE from the photoreceptors/retina. We developed a method to efficiently separate the retina from the RPE of human eyes to generate complete RPE/choroid and retina flatmounts for separate cellular analysis of the photoreceptors and RPE cells. An intravitreal injection of a high-osmolarity solution of D-mannitol, a sugar not transported by the RPE, induced the separation of the RPE and retina across the entire posterior chamber without causing damage to the RPE cell junctions. No RPE patches were observed attached to the retina. Phalloidin labeling of actin showed RPE shape preservation and allowed morphometric analysis of the entire epithelium. An artificial intelligence (AI)-based software was developed to accurately recognize and segment the RPE cell borders and quantify 30 different shape metrics. This dissection method is highly reproducible and can be easily extended to other animal models.

We describe a method to efficiently separate retinal pigment epithelium (RPE) from the retina in human eyes and generate whole RPE/choroid flatmounts for histological and morphometric analyses of the RPE.

Effiziente und konsistente Generierung von retinalen Pigmentepithel-/Aderhaut-Flatmounts aus dem menschlichen Auge für die histologische Analyse

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and ...