Support: NIDCD R01DC009801, P30DC010362, 5T32DC000040-17

Ethikerklärung: Einsatz der menschlichen Tumorproben in diesem Protokoll wurde von der University of Iowa Institutional Review Board (IRB) genehmigt. 1. Setup-Tumor am Tag vor Ernte <ol><li> Coat Zellkulturplatten: In einem sterilen Kulturhaube, fügen Sie genug Poly-L-Ornithin (0,01% Lösung) auf den Boden einer Polystyrol / Kunststoff-Kultur gut / Schale bedecken, ersetzen Sie den Deckel, und lassen Sie sich bei Raumtemperatur für mindestens 2 Stunden. Glasobjektträger oder Deckgläser für Polystyrol für Experimente Bildgebung erfordern ersetzt werden. </li><li> Nach 2 Stunden, entfernen Sie die Poly-L-Ornithin-Lösung mit Saug-und damit die Platten vollständig in einem sterilen Kulturhauben trocknen. Fügen Sie genug Laminin-Lösung (10 g / ml in Ca 2 + / Mg 2 +-freiem HBSS [HBSS-/ -]), um die Kulturplatten vollständig zu bedecken, Deckel zu ersetzen, dann ist entweder 1) in kühlen 4 ° C Kühlschrank über Nacht oder 2) in 37 ° C-Inkubator für mindestens 2 Stunden. Wenn Plattenwird über Nacht oder länger aufbewahrt werden, wickeln die Platten in der Plastikverpackung, um Verdunstung / Austrocknung zu verhindern. </li></ol> 2. Setup-der Tag der Ernte Tumor <ol><li> Bereiten Kultur, Medien: Die Medien werden frisch am Tag der Tumor Ernte und Verarbeitung gemacht, bei 4 ° C gelagert und auf 37 ° C im Brutschrank unmittelbar vor der Medien Ergänzungen / Änderungen. </li><li> Schwannom Kulturmedien mit hohem Glucosegehalt (4,5 mg / ml) DMEM (mit Phenolrot) mit 0,1 mg / ml Penicillin und 0,1 mg / ml Streptomycin; N2 Medien ergänzen endgültigen Verdünnung 1:100; Rinder-Insulin 5 ug / ml; Fötales Rinderserum und 10% Volumen. Filtern Sie alle Medien durch 0,22 um-Filter. </li><li> Bereiten Probentransport Kühler: Senden Sie eine Eis gefüllt Kühler mit 1-2 (je nach Höhe der Tumor, geerntet werden) steriles 50 ml konische Röhrchen mit 25 ml HBSS mit Ca 2 + / Mg 2 + (HBSS + / +) in den Operationssaal für die Tumorprobentransfer und Transport. </li><li> Bereiten indiv.idual Tumor Dissoziation Röhren - füllen sterile 2 ml Rundbodenröhrchen mit 1 ml HBSS + / + und auf Eis. (Tubes für scharfe Dissektion / Dissoziation von Tumorproben verwendet werden). </li><li> Für Dissoziation, bereiten etwa eine Dissoziation Rohr für jeden 0,5 cm 3 gewonnene Gewebe; zum Beispiel, wird eine Tumorprobe beträgt 1,0 x 1,0 x 1,0 cm (1 cm 3) zwei HBSS + / + Dissoziation Rohre erfordern. </li><li> UV-Licht Sterilisation: Platz Gewebe Schere, kleine Zange, Skalpell Griff, 1x normale Petrischale, Pipette P1000 und Pipettenspitzen in einem sterilen Kultur Haube, und lassen unter UV-Licht für ~ 15 min vor Ankunft und Verarbeitung von Tumorgewebe. </li></ol> 3. Specimen Isolation und Verkehr <ol><li> Isolieren Tumorproben durch chirurgische Resektion. </li><li> Sofort legen Tumorproben in die eiskalte HBSS + / + so schnell wie möglich nach der Entnahme aus dem Patienten. Sobald alle tissue-Proben werden gesammelt, Transport-Proben (auf Eis) von der operativen Theater an das Labor zur weiteren Verarbeitung. </li></ol> 4. Tissue Dissoziation und Verreiben <ol><li> Folgende Standard sterilen Technik in einem Abzug mit laminarer Strömung, bringen konischen Rohr mit Tumorprobe in das Kulturhaube und Waschproben durch Umdrehen des Röhrchens und Tumor 50x. Lassen Fragmente auf den Boden der Röhre fallen, und entfernen Sie dann HBSS + / +. Refill mit 30 ml HBSS + / + und agitieren / invertieren 50x wieder. Wiederholen Inversion / media Entfernung für insgesamt 4x. </li><li> Re-suspend Tumorfragmente in 30 ml HBSS + / + zum letzten Mal, dann gießen Mischung in eine sterile100 mm Petrischale und separate Tumor in 0,5 cm 3 Gruppierungen. Wenn größere Tumorfragmente auftreten, verwenden Sie die Schere oder Skalpell Gewebe (# 11 oder # 15 Klinge) in kleinere Stücke schneiden. </li><li> Verwenden Sie die Zange, um die 0,5 cm 3 Gewebe Gruppen in den einzelnen HBSS + / + gefüllten, 2 ml-Rundkegelal Röhren, die alle auf dem Eis. Verwenden Sie die Schere, um Gewebe Tumorfragmente in Unter 1 mm-Fragmente der Aussetzung Hackfleisch sollte eine &quot;Schneekugel&quot; Aussehen (Abbildung 1) zu haben. Blatt vor den Tumor nur bis die Mehrheit der Fragmente Unter 1 mm; nicht &quot;over-mince&quot; die Tumorproben. Zeigen vollständig zerkleinerte Gewebe Rohr zurück auf dem Eis, und wiederholen Sie mit allen zusätzlichen Probenröhrchen. </li><li> Übertragen Sie alle fragmentierten Tumorproben in einem einzigen 15 ml konischen Röhrchen. Ein P1000 Pipette mit einem Schnitt / geändert sterile Spitze eignet sich gut für diesen Schritt. Sie besonders HBSS + / + zu spülen / spülen Sie die 2 ml Röhrchen Dissoziation sicherstellen, dass alle Tumorprobe wurde in 15-ml-Röhrchen gesammelt. Spin down-Gemisch in Zentrifuge bei 23 ° C, bei 73 xg für 5 min. </li><li> Abzusaugen HBSS + / +, so dass die Zellpellet. Re-suspend-Fragmente in 1:1-Gemisch von 0,25% Trypsin: 0,2% Kollagenase (in HBSS-/ gelöst -) - fügen gerade genug, um in Tumorfragmente halten engen suspension. Ersetzen Schraubverschluss, und in 37 ° C-Inkubator für 30 min. Man schüttelt Zellgemisch mindestens einmal während der Inkubation, um Fragmente in Suspension zu halten. Legen Sie das Schwannom Kulturmedien in den Brutschrank, um es in dieser Zeit zu wärmen auch. </li><li> Nach der Inkubation werden 100 ul FBS zu jedem Röhrchen, um das Trypsin zu inaktivieren, und dann Zentrifuge Probe bei 23 ° C, 73 × g für 5 min. Mit einer Pipette vorsichtig abzusaugen, so viel wie möglich, ohne überstehende Zellpellet zu stören. Hinzufügen des erwärmten Kulturmedien (N2/insulin/5% FBS) zu den Tumorfragmente zu einem endgültigen Verhältnis von 1:2 Tumorfragmente: Kulturmedien (z. B. wenn es 1 ml Tumorfragmente, fügen in 2 ml warmem Medien). </li><li> Bereiten Sie für die Tumor Verreiben durch Schneiden der Spitze weg von einer P1000 Pipettenspitze zu einem Durchmesser von etwa 1-2 mm. Langsam verreiben die Zellsuspension mit immer kleiner und kleiner Durchmesser Pipettenspitzen, bis Sie einfach pipettieren der Lösung durch eine unaltEred P1000 Pipettenspitze. Unmittelbar auf eine kleinere Spitze zu bewegen, sobald man leicht pipettieren den Tumor-Lösung durch die Spitze - nicht über die Probe verreiben. Wenn ein einzelnes größeres Stück Gewebeblöcke Aspiration während Verreiben, Antippen der Pipettenspitze an der Unterseite des konischen Rohrs ermöglicht oft fort Verreiben. </li></ol> 5. Zellüberzug <ol><li> Ein 0,5 mm 3-Fragment von Gewebe ist genug, um 1 x 100 mm-Schale, oder 4 x Dias 8-well/4-well Saatgut. Kurz vor der Zugabe der Medien / Zellgemisch, entfernen Sie das Laminin-Lösung von den Kulturplatten, aber nicht die Platten trocknen lassen. </li><li> Für jede 100-mm-Schale, die Auflösung der Tumor-Fragment in 10 ml Kulturmedium erwärmt. </li><li> Für jede Vertiefung einer 4-gut rutschen, 750 ul erwärmt Kulturmedien (3 ml für die ganze Folie). </li><li> Für jede Vertiefung eines 8-Well-rutschen, 400 ul erwärmt Kulturmedien (3,2 ml für die ganze Folie). </li><li> Die Kulturen in 37 &amp; inkubieren# 160; ° C, 100% Luftfeuchtigkeit, 6% CO 2. Lassen Sie die Kulturen allein für mindestens 36 Stunden, und ändern Sie dann die Medien, wenn die Lösung vergilbt (sauer). Die Medien bei 72 h Ansonsten ändern, dann alle 2-3 Tage oder wenn die Medien zu sauer. Wenn das Medium sauer wird täglich für mehrere Tage in einer Reihe, das ist in der Regel ein Hinweis auf einen möglichen Bakterien-oder Hefe-Verunreinigung. </li></ol>

<ol>
	<li>Arthurs, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+treatment+modalities+for+vestibular+schwannoma.">A review of treatment modalities for vestibular schwannoma.</a> Neurosurg Rev. 34 (3), 265-277 (2011).</li><li>Evans, G. R., Lloyd, S. K., Ramsden, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurofibromatosis+type+2.">Neurofibromatosis type 2.</a> Adv Otorhinolaryngol. 70, 91-98 (2001).</li><li>Fong, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+biology+and+novel+treatments+of+vestibular+schwannomas.">The molecular biology and novel treatments of vestibular schwannomas.</a> J Neurosurg. 115 (5), 906-914 (2011).</li><li>Jacob, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+validation+of+AR42,+a+novel+histone+deacetylase+inhibitor,+as+treatment+for+vestibular+schwannomas.">Preclinical validation of AR42, a novel histone deacetylase inhibitor, as treatment for vestibular schwannomas.</a> Laryngoscope. 122 (1), 174-189 (2012).</li><li>Evans, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurofibromatosis+2+[Bilateral+acoustic+neurofibromatosis,+central+neurofibromatosis.">Neurofibromatosis 2 [Bilateral acoustic neurofibromatosis, central neurofibromatosis.</a> NF2, neurofibromatosis type II]. Genet Med. 11 (9), 599-610 (2009).</li><li>Anniko, M., Noren, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Human+Acoustic+Neurinoma+in+Organ-Culture+.1.+Methodological+Aspects.">The Human Acoustic Neurinoma in Organ-Culture .1. Methodological Aspects.</a> Acta Oto-Laryngologica. 91 (1-2), 47-53 (1981).</li><li>Manent, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+cell+sorting+for+enriching+Schwann+cells+from+adult+mouse+peripheral+nerves.">Magnetic cell sorting for enriching Schwann cells from adult mouse peripheral nerves.</a> J Neurosci Methods. 123 (2), 167-173 (2003).</li><li>Nair, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+cultures+of+human+vestibular+schwannoma:+selective+growth+of+schwannoma+cells.">Primary cultures of human vestibular schwannoma: selective growth of schwannoma cells.</a> Otol Neurotol. 28 (2), 258-263 (2007).</li><li>Baur, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin+promotes+differentiation,+adhesion+and+proliferation+of+cell+cultures+derived+from+human+acoustic+nerve+schwannoma.">Laminin promotes differentiation, adhesion and proliferation of cell cultures derived from human acoustic nerve schwannoma.</a> Acta Otolaryngol. 115 (4), 517-521 (1995).</li><li>Cravioto, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+neurinoma+in+tissue+culture.">Experimental neurinoma in tissue culture.</a> Acta Neuropathol. 21 (2), 154-164 (1972).</li><li>Kaasinen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+of+acoustic+neuroma--methodological+aspects.">Culturing of acoustic neuroma--methodological aspects.</a> Acta Otolaryngol Suppl. 520 Pt 1, 25-26 (1995).</li><li>Kredel, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Culture+of+Intracranial+Tumors+with+a+Note+on+the+Meningiomas.">Tissue Culture of Intracranial Tumors with a Note on the Meningiomas.</a> Am J Pathol. 4 (4), 337-340 (1928).</li><li>Murray, M. R., Stout, A. P., Bradley, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schwann+cell+versus+fibroblast+as+the+origin+of+the+specific+nerve+sheath+tumor:+Observations+upon+normal+nerve+sheaths+and+neurilemomas+in+vitro.">Schwann cell versus fibroblast as the origin of the specific nerve sheath tumor: Observations upon normal nerve sheaths and neurilemomas in vitro.</a> Am J Pathol. 16 (1), 41-60 (1940).</li><li>Cravioto, H., Lockwood, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+behavior+of+acoustic+neuroma+in+tissue+culture.">The behavior of acoustic neuroma in tissue culture.</a> Acta Neuropathol. 12 (2), 141-157 (1969).</li><li>Lee, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+epigenetic+alterations+of+the+NF2+gene+in+sporadic+vestibular+schwannomas.">Genetic and epigenetic alterations of the NF2 gene in sporadic vestibular schwannomas.</a> PLoS One. 7 (1), 30418 (2012).</li><li>Wippold, F. J., 2nd, , et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropathology+for+the+neuroradiologist:+Antoni+A+and+Antoni+B+tissue+patterns.">Neuropathology for the neuroradiologist: Antoni A and Antoni B tissue patterns.</a> AJNR Am J Neuroradiol. 28 (9), 1633-1638 (2007).</li><li>Mennel, H. 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Interessenkonflikte offen zu legen.

Geschichte der In-vitro-Kulturen Schwannom Die Bemühungen um die In-vitro-Kulturen zu etablieren begann Schwannom weniger Jahrzehnte nach der ersten Akustikusneurinom offenen chirurgischen Resektion erfolgte im Jahre 1894 12. Die erste aufgezeichnete Kulturen waren von Kredel in den 1920er Jahren, der erfolglos versuchte, Hackfleisch Tumor durch &quot;hanging drop-Methode&quot; 12 wachsen . Später Drs. Murray und Stout veröffentlich...

Vestibuläre Schwannome (VSS) stellen Schwann-Zellen (SC) Tumoren des Nervus vestibularis, Kompromisse 10% aller intrakraniellen Neoplasmen 1-3. VSs treten entweder sporadisch oder familiär (Neurofibromatose Typ 2, NF2) Formen, die beide mit Inaktivierung von Defekten in der NF2 Tumorsuppressor verbunden Gen. Die Behandlung ist in der Regel für VSS chirurgische Resektion oder Radiochirurgie, aber die Morbidität dieser Verfahren beinhaltet Taubheit, Gesichts Neuropathien, Rückenmarksflüssigkeit auslaufen, Ungleichgewicht, und ein erneutes Tumorwachstum. Außerdem werden nicht alle Patienten sind akzeptabel chirurgischen oder strahlen Kandidaten. Solche erheblichen Morbidität sowie ein Mangel an alternativen Therapien hat die Untersuchung in die einzigartige molekulare Biologie der VSs in der Hoffnung, die Entwicklung neuer Behandlungen 3,4 angetrieben. Zellkultur ermöglicht eine schnelle, einfache und gründliche Analyse der molekularen und zellulären Verhalten und Screening von potenziellen therapeutischen comPfund. Historisch sind der fehlende Zugang zu frischen Gewebeproben und der Tatsache, dass Schwannom Zellen nicht verewigt deutlich die Verwendung von Primärkulturen zur Untersuchung der Tumorgenese Schwannom behindert. Um das begrenzte Angebot an Primärkulturen zu überwinden, wurde die verewigt HEI193 VS Zelllinie durch Transduktion mit HPV-Onkogene E6 und E7 5 erzeugt. Diese onkogene Transduktion eingeführt signifikanten molekularen und phänotypischen Veränderungen der Zellen, die ihre Verwendung als Modell für menschliche Tumoren Schwannom begrenzen. SC Kulturen aus transgenen Mauslinien, die eine funktionelle NF2-Gen fehlt abgeleitet sind eine weitere Alternative zu NF2 abhängige SC Tumorgenese in vitro zu untersuchen. Diese Kulturen allerdings nicht die heterogene Natur der menschlichen VSs oder Konto für den menschlichen bestimmtes Verhalten zu rekapitulieren. Die meisten bisherigen VS Kulturtechniken benötigt relativ lange Bearbeitungszeiten und komplizierte selektiven Kulturtechniken 8.6.Hier präsentieren wir Ihnen eine einfache, reproduzierbare Protokoll für VS primären Zellkultur mit Komplettbearbeitung in unter 3 Stunden, mit 95% Reinheit, wie Tumorzellen durch Immunfärbung bestimmt.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Poly-L-Ornithine, 0.01% Solution</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">P4957</td>
			<td style="width:167px;">Cell Culture Surface Treatment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Laminin Mouse Protein&#160;</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">23017-015 / L2020</td>
			<td>Cell Culture Surface Treatment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">0.2% Collagenase (dissolved in HBSS -/-)</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">C2674</td>
			<td>Dissociation Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.25% Trypsin</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">25200-056</td>
			<td>Dissociation Reagent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TPS 100mm Round Culture Dish</td>
			<td style="width:71px;">Midwest Scientific</td>
			<td style="width:119px;">TP93100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Permanox 4 well slide</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">1256521</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Permanox 8 well slide</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">1256522</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Suction Filter Flask</td>
			<td style="width:71px;">Midwest Scientific</td>
			<td style="width:119px;">TP99500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">15ml Conical Tube</td>
			<td style="width:71px;">Midwest Scientific</td>
			<td style="width:119px;">TP91015</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">50ml Conical Tube</td>
			<td style="width:71px;">Midwest Scientific</td>
			<td style="width:119px;">TP91050</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">2mL Round Bottom Tube</td>
			<td style="width:71px;">USA Scientific</td>
			<td style="width:119px;">1620-2700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Small Scissor</td>
			<td style="width:71px;">FST</td>
			<td style="width:119px;">14058-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Small Forceps</td>
			<td style="width:71px;">FST</td>
			<td style="width:119px;">11251-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scalpel Handle</td>
			<td style="width:71px;">FST</td>
			<td style="width:119px;">10004-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">#11 Scalpel Blade</td>
			<td style="width:71px;">Roboz</td>
			<td style="width:119px;">RS-9801-11</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Non-Tissue Culture 100mm Round Petri Dish</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">50-820-904</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">P1000 Pipetteman</td>
			<td style="width:71px;">Bioexpress</td>
			<td style="width:119px;">P3963-1000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Serological Pipetteman</td>
			<td style="width:71px;">Bioexpress</td>
			<td style="width:119px;">R3073-2P</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sterile, Non-Filtered P1000 Pipette Tips</td>
			<td style="width:71px;">Midwest Scientific</td>
			<td style="width:119px;">TD1250R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Insulated Ice Cooler</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Culture Hood&#160;</td>
			<td style="width:71px;">Baker</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Centrifuge&#160;</td>
			<td style="width:71px;">Eppendorf -5810R</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hanks Balanced Salt Solution (HBSS) +/+ (w/ Ca2+, Mg2+)&#160;</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">24020-117</td>
			<td>Schwannoma Culture and Media Components</td>
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			<td height="42" style="height:42px;width:216px;">Hanks Balanced Salt Solution (HBSS) -/- (w/out Ca2+, Mg2+)</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">14170-112</td>
			<td>Schwannoma Culture and Media Components</td>
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		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Dulbecco&#8217;s Modified Eagle Medium (DMEM), High Glucose, w/ Phenol Red</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">11965-092</td>
			<td>Schwannoma Culture and Media Components</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">N2 Supplement</td>
			<td style="width:71px;">Gibco</td>
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			<td>Schwannoma Culture and Media Components</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">26140-079</td>
			<td>Schwannoma Culture and Media Components</td>
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			<td height="21" style="height:21px;width:216px;">Penicillin - Streptomycin</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">15140-163</td>
			<td>Schwannoma Culture and Media Components</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bovine Insulin (1mg/ml 200x)</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">I6634</td>
			<td>Schwannoma Culture and Media Components</td>
		</tr>
	</tbody>
</table>

primary culture of human vestibular schwannomas

Vestibuläre Schwannome (VSS) stellen Schwann-Zellen (SC) Tumoren des Nervus vestibularis, Kompromisse 10% aller intrakraniellen Neoplasmen. VSs treten entweder sporadisch oder familiär (Neurofibromatose Typ 2, NF2) Formen, die beide mit Inaktivierung von Defekten in der NF2 Tumorsuppressor verbunden Gen. Die Behandlung ist in der Regel für VSS chirurgische Resektion oder Radiochirurgie, aber die Morbidität dieser Verfahren hat Ermittlungen in weniger invasive Behandlungen angetrieben. Historisch sind der fehlende Zugang zu frischen Gewebeproben und der Tatsache, dass Schwannom Zellen nicht verewigt deutlich die Verwendung von Primärkulturen zur Untersuchung der Tumorgenese Schwannom behindert. Um das begrenzte Angebot an Primärkulturen zu überwinden, wurde die verewigt HEI193 VS Zelllinie durch Transduktion mit HPV-Onkogene E6 und E7 erzeugt. Diese onkogene Transduktion eingeführt signifikanten molekularen und phänotypischen Veränderungen der Zellen, die ihre Verwendung als Modell für menschliche s begrenzenchwannoma Tumoren. Wir stellen daher eine vereinfachte, reproduzierbare Protokoll für die Kultur von primären humanen Zellen VS. Diese leicht zu beherrschen Technik ermöglicht molekularen und zellulären Untersuchungen, genauer rekapitulieren die Komplexität der VS Krankheit.

Korrekte Tumor Fragmentierung ist für die optimale Aussaat und Vermehrung in Gewebekulturschalen (Abbildung 1). Identifizierung von Schwannom Zellen während der ersten Tage nach der Plattierung ist oft schwierig, aufgrund verdeckt Mengen von Zelltrümmern und roten Blutkörperchen. Durch die 4. oder 5. Tag, wird in der Nähe von adhärenten Zellforttumorfragmente erkennbar erstellen &quot;Schnürung&quot; Muster unter den Trümmern. Nachträgliche Änderungen in der Regel entfern...

Watch this Scientific Journal Video about Primary Culture of Human Vestibular Schwannomas at JoVE.com

Primary Culture of Human Vestibular Schwannomas

Vestibuläre Schwannome (VSS) nicht-malignen Tumoren von Schwann-Zellen (SC) Ursprungs, mit Mutationen im NF2 Tumorsuppressor-Gen assoziiert ist. Wir berichten über einen reproduzierbaren, effizienten Protokoll für primäre humane VS Zellkultur, die für die molekulare und zelluläre experimentelle Manipulation und Analyse ermöglicht und rekapituliert die heterogene Natur der menschlichen Krankheit.

Primäre Kultur der Menschen vestibuläre Schwannome

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in ...

primary-culture-of-human-vestibular-schwannomas

Research

JoVE Journal

Medicine

12.7K Aufrufe.  University of Iowa Hospitals and Clinics.  Vestibuläre Schwannome (VSS) nicht-malignen Tumoren von Schwann-Zellen (SC) Ursprungs, mit Mutationen im NF2 Tumorsuppressor-Gen assoziiert ist. Wir berichten über einen reproduzierbaren, effizienten Protokoll für primäre humane VS Zellkultur, die für die molekulare und zelluläre experimentelle Manipulation und Analyse ermöglicht und rekapituliert die heterogene Natur der menschlichen Krankheit. 

Serie: Primary Culture of Human Vestibular Schwannomas

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease1. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms2 and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.

The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.

Haut Stanzbiopsie Explantatkultur für die Ableitung des primären humanen Fibroblasten

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

Forschung

Medizin

In vitro Organoid Culture of Primary Mouse Colon Tumors

Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. 1, which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells.
The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.

In vitro Organoid Culture of Primary Mouse Colon Tumors

A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not.

In-vitro-Kultur von Organoide Primäre Maus Kolontumoren

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

Isolierung, Kultur und Imaging von humanen fötalen Pankreas-Zellhaufen

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

Eine dreidimensionale Zellkulturmodell zu primären humanen Knochenmark und seine Malignome Studieren

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

Dreidimensionale Alginat-Perle Kultur der Menschen Hypophysenadenom Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

Isolierung und Kultur von primären Endothelzellen von Canine Arterien und der

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

Isolation von primären menschlichen Deziduale Zellen aus der fetalen Membranen der Begriff Plazentas

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...

Muscle Imbalances: Testing and Training Functional Eccentric Hamstring Strength in Athletic Populations

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite of these eccentric hamstring actions are concentric quadriceps actions, where the larger and likely stronger quadriceps straighten the knee. Therefore, to stabilize the lower limbs during movement, the hamstrings must eccentrically combat against the strong knee-straightening torque of the quadriceps. As such, eccentric hamstring strength expressed relative to concentric quadricep strength is commonly referred to as the &#34;functional ratio&#34; as most movements in sports require simultaneous concentric knee extension and eccentric knee flexion. To increase the strength, resiliency, and functional performance of the hamstrings, it is necessary to test and train the hamstrings at different eccentric speeds. The main purpose of this work is to provide instructions for measuring and interpreting eccentric hamstring strength. Techniques for measuring the functional ratio using isokinetic dynamometry are provided and sample data will be compared. Additionally, we briefly describe how to address hamstring strength deficiencies or unilateral strength differences using exercises that specifically focus on increasing eccentric hamstring strength.

The hamstrings are a group of muscles that are sometimes problematic for athletes, resulting in soft tissue injury in the lower limbs. To prevent such injuries, functional training of the hamstrings requires intensive eccentric contractions. Additionally, hamstring function should be tested in relation to quadricep function at different contraction speeds.

Muskel-Ungleichgewichte: Testen und trainieren funktionale exzentrische Muskelfaserriss Stärke in Athletic Populationen

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite ...

Surgical Labyrinthectomy of the Rat to Study the Vestibular System

To study the vestibular system or the vestibular compensation process, a number of methods have been developed to cause vestibular damage, including surgical or chemical labyrinthectomy and vestibular neurectomy. Surgical labyrinthectomy is a relatively simple, reliable, and rapid method. Here, we describe the surgical technique for rat labyrinthectomy. A postauricular incision is made under general anesthesia to expose the external auditory canal and the tympanic membrane, after which the tympanic membrane and the ossicles are removed without the stapes. The stapes artery, which is located between the stapes and the oval window, is a vulnerable structure and must be preserved to obtain a clear surgical field. A hole to fenestrate the vestibule is made with a 2.1-mm drill bur superior to the stapes. Then, 100% ethanol is injected through this hole and aspirated several times. Meticulous dissection under a microscope and careful bleeding control are essential to obtain reliable results. Symptoms of vestibular loss, such as nystagmus, head tilting, and a rolling motion, are seen immediately after surgery. The rotarod or rotation chair test can be used to objectively and quantitatively evaluate the vestibular function.

This protocol describes the surgical labyrinthectomy of a rat, which is a useful method for studying the vestibular system.

Chirurgische Labyrinthectomy der Ratte das vestibuläre System untersuchen

To study the vestibular system or the vestibular compensation process, a number of methods have been developed to cause vestibular damage, including ...

Characterization of a Novel Human Organotypic Retinal Culture Technique

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal pigment epithelium-choroid (RPE-choroid) and sclera, the integrity of the fragile retina can easily be compromised. The aim of this study was to develop a novel HORC model that contains the retina, RPE-choroid and sclera to maintain retinal integrity when culturing retinal explants.
After cutting circumferentially along the limbus to remove iris and lens, four deep incisions were made to flatten the eyecup. In contrast to previous HORC protocols, a trephine was used to cut through not only the retina but also the RPE-choroid and sclera. The resultant triple-layered explants were cultured for 72 h. Hematoxylin and Eosin staining (H&#38;E) was used to assess anatomical structures and retinal explants were further characterized by immunohistochemistry (IHC) for apoptosis, M&#252;ller cell integrity and retinal inflammation. To confirm the possibility of disease induction, explants were exposed to high glucose (HG) and pro-inflammatory cytokines (Cyt), to mimic diabetic retinopathy (DR). The Luminex magnetic bead assay was used to measure DR-related cytokines released into the culture medium.
H&#38;E staining revealed distinct retinal lamellae and compact nuclei in retinal explants with the underlying RPE-choroid and sclera, while retinas without the underlying structures exhibited reduced thickness and severe nuclei loss. IHC results indicated absence of apoptosis and retinal inflammation as well as preserved M&#252;ller cell integrity. The Luminex&#160;assays showed significantly increased secretion of DR-associated pro-inflammatory cytokines in retinal explants exposed to HG + Cyt relative to baseline levels at 24 h.
We successfully developed and characterized a novel HORC protocol in which retinal integrity was preserved without apoptosis or retinal inflammation. Moreover, the induced secretion of DR-associated pro-inflammatory biomarkers when exposing retinal explants to HG + Cyt suggests that this model could be used for clinically translatable retinal disease studies.

This study aims to develop a novel human organotypic retinal culture (HORC) model that prevents compromising retinal integrity during explant handling. This is achieved by culturing the retina with the overlying vitreous and the underlying retinal pigment&#160;epithelium-choroid (RPE-choroid) and sclera.

Charakterisierung einer neuartigen humanen organotypischen Netzhautkulturtechnik

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal ...