Supporto: NIDCD R01DC009801, P30DC010362, 5T32DC000040-17

Dichiarazione Etica: uso dei campioni tumorali umani in questo protocollo è stato approvato dalla University of Iowa Institutional Review Board (IRB). 1. Setup giorno prima Tumore Harvest <ol><li> Cappotto cellulari piastre di coltura: in una cappa sterile cultura, aggiungere abbastanza poli-L-ornitina (soluzione 0,01%) per coprire il fondo di una cultura polistirolo / plastica e / piatto, sostituire i coperchi, e lasciate riposare a temperatura ambiente per almeno 2 hr. Vetrini e vetrini possono essere sostituiti per polistirene per esperimenti che richiedono l&#39;imaging. </li><li> Dopo 2 ore, rimuovere la soluzione di poli-L-ornitina mediante aspirazione e permettono le piastre asciugare completamente in un sterili cappe cultura. Aggiungere la soluzione laminina abbastanza (10 mg / ml di Ca 2 + / Mg 2 + senza HBSS [HBSS-/ -]) per coprire completamente le piastre di coltura, sostituire le palpebre, poi o 1) posto in un luogo fresco 4 ° C frigorifero per una notte, o 2) inserire nel 37 ° C incubatore per almeno 2 ore. Se le piastresaranno memorizzati notte o più, avvolgere le piastre in involucro di plastica per evitare l&#39;evaporazione / essiccazione. </li></ol> 2. Impostazione del Giorno del Tumore Harvest <ol><li> Preparare Culture Media: media è fatta fresca del giorno di raccolta e di trasformazione tumorale, conservato a 4 ° C, e riscaldato a 37 ° C in incubatore immediatamente prima ai media aggiunte / modifiche. </li><li> Cultura schwannoma media è alta glucosio (4,5 mg / ml) DMEM (con rosso fenolo) con 0,1 mg / ml di penicillina e 0,1 mg / ml streptomicina; Mezzi N2 integrano diluizione finale 1:100; Insulina Bovini 5 mg / ml; Siero fetale bovino al 10% del volume totale. Filtrare tutti i media attraverso il filtro di 0,22 micron. </li><li> Preparare campioni Trasporti di raffreddamento: Manda un dispositivo di raffreddamento del ghiaccio pieno di 1-2 (a seconda della quantità di tumore per essere raccolto) 50 ml provette coniche sterili con 25 ml di HBSS con Ca 2 + / Mg 2 + (HBSS + / +) per sala operatoria per il trasferimento del campione tumorale e trasporto. </li><li> Preparare indivtubi tumore dissociazione idual - riempiono sterile 2 ml tondi tubi inferiori di 1 ml di HBSS + / + e posto sul ghiaccio. (Tubi sarà utilizzato per sharp dissezione / dissociazione di campioni tumorali). </li><li> Per dissociazione, preparare circa un tubo di dissociazione per ogni 0,5 cm 3 di tessuto raccolto; per esempio, un campione di tumore che misurano 1,0 x 1,0 x 1,0 cm (1 cm 3) richiederà due tubi + / + dissociazione HBSS. </li><li> La luce ultravioletta sterilizzazione: forbici Luogo di tessuto, piccole pinze, bisturi manico, 1x piatto normale Petri, P1000 pipette e puntali per pipette in una cappa sterile cultura, e lasciare sotto la luce ultravioletta per ~ 15 minuti prima dell&#39;arrivo e la trasformazione del tessuto tumorale. </li></ol> 3. Isolamento dei campioni e dei Trasporti <ol><li> Isolare campioni tumorali con la resezione chirurgica. </li><li> Porre immediatamente campioni tumorali nel HBSS ghiacciata + / + il più rapidamente possibile dopo la rimozione dal paziente. Una volta che tutti Tissue campioni vengono prelevati, i campioni di trasporto (su ghiaccio), dal teatro operativo al laboratorio per ulteriori elaborazioni. </li></ol> 4. Dissociazione tessuti e Trituration <ol><li> Seguente tecnica sterile norma in una cappa a flusso laminare, portare provetta con il campione tumorali nella cappa coltura, e lavare i campioni capovolgendo tubo e tumore 50x. Consentire frammenti di cadono sul fondo del tubo, e poi rimuovere HBSS + / +. Riempire con 30 ml HBSS + / +, ed agitare / invertire 50x di nuovo. Ripetere rimozione inversione / media per un totale di 4x. </li><li> Risospendere frammenti di tumore in 30 ml HBSS + / + per l&#39;ultima volta, poi versare la miscela in un piatto di Petri mm sterile100 e tumore separato in 0,5 centimetri 3 raggruppamenti. Se frammenti di tumore più grandi sono incontrati, usare le forbici di tessuto o bisturi (# 11 o # 15 lame) per tagliare in pezzi più piccoli. </li><li> Utilizzare le pinze per collocare i 0,5 centimetri 3 gruppi tissutali nella persona HBSS + / + compilato, 2 ml rotondo fondo conicotubi al, tutte su ghiaccio. Usare le forbici tessuti per sminuzzare frammenti di tumore in sub-1 millimetro frammenti, la sospensione dovrebbe avere un aspetto &#39;snowglobe&#39; (figura 1). Tritare il tumore solo fino alla maggior parte dei frammenti sono sub-1 mm; non &#39;over-trita&#39; i campioni di tumore. Posizionare il tubo di tessuto completamente macinata di nuovo sul ghiaccio, e ripetere con tutte le provette dei campioni supplementari. </li><li> Trasferire tutti i campioni tumorali frammentati in un unico tubo da 15 ml. Una pipetta P1000 con un taglio / modificato punta sterile funziona bene per questo passo. Utilizzare supplementare HBSS + / + per svuotare / lavare i 2 tubi ml di dissociazione per assicurarsi che tutti i campioni di tumore è stato raccolto in 15 ml di tubo. Centrifugare miscela in centrifuga a 23 ° C, a 73 xg per 5 min. </li><li> Aspirare off HBSS + / +, lasciando il pellet. Re-sospendere frammenti in miscela 1:1 di 0,25% tripsina: 0.2% collagenasi (sciolto in HBSS-/ -) - aggiungere quel tanto che basta per tenere frammenti di tumore in stretto suspension. Sostituire il tappo a vite, e il luogo nel 37 ° C incubatore per 30 min. Agitare la miscela di cellule almeno una volta durante l&#39;incubazione per mantenere i frammenti in sospensione. Posizionare la cultura dei media Schwannoma nell&#39;incubatrice per riscaldarla in questo tempo pure. </li><li> Dopo l&#39;incubazione, aggiungere 100 microlitri FBS a ciascuna provetta per inattivare la tripsina, e poi campione centrifugare a 23 ° C, 73 xg per 5 min. Usando una pipetta, accuratamente aspirazione fuori il più possibile surnatante senza disturbare pellet di cellule. Aggiungere i terreni di coltura riscaldato (N2/insulin/5% FBS) ai frammenti di tumore a un rapporto finale di 1:2 frammenti di tumore: coltura (per esempio, se vi è 1 ml di frammenti di tumore, aggiungi in 2 ml di riscaldato media). </li><li> Prepararsi per tumore trituration tagliando la punta di una punta P1000 pipetta ad un diametro di circa 1-2 mm. Lentamente triturare la sospensione cellulare con punte per pipette di diametro progressivamente sempre più piccoli, fino a quando si può facilmente pipetta la soluzione attraverso un unaltEred P1000 punta di pipetta. Passare immediatamente a una punta più piccola, una volta si può facilmente pipetta la soluzione tumore attraverso la punta - non più di triturare il campione. Se un unico pezzo più grande di blocchi di tessuto aspirazione durante triturazione, toccando la punta della pipetta sul fondo della provetta conica permette spesso continua triturazione. </li></ol> 5. Cella di placcatura <ol><li> Uno 0,5 millimetri 3 frammento di tessuto è sufficiente per seminare 1 x 100 mm piatto, o 4 x diapositive 8-well/4-well. Poco prima di aggiungere alla miscela media / cellulare, rimuovere la soluzione laminina dalle piastre di coltura, ma non lasciare le piastre a secco. </li><li> Per ogni piatto 100 mm, sciogliere il frammento tumore in 10 ml di terreni di coltura riscaldato. </li><li> Per ciascun pozzetto di una slitta 4 pozzetti, utilizzare 750 microlitri Terreni di coltura riscaldata (3 ml per l&#39;intero vetrino). </li><li> Per ogni ben di uno scivolo 8-bene, usare 400 microlitri di media cultura riscaldato (3.2 ml per tutta la diapositiva). </li><li> Incubare le culture nel 37 &amp;# 160; ° C, umidità del 100%, 6% di CO 2. Lasciare le colture da solo per almeno 36 ore, e quindi modificare il supporto se la soluzione è ingiallito (acido). In caso contrario, cambiare il supporto a 72 ore, poi ogni 2-3 giorni o quando i media diventano acide. Se il supporto diventa acido giorno per diversi giorni di fila, questo è generalmente un segno di potenziale contaminazione batterica o di lievito. </li></ol>

<ol>
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Storia delle culture in vitro schwannoma a Sforzi per stabilire in colture in vitro schwannoma iniziato solo pochi decenni dopo la neuroma acustico iniziale resezione chirurgica aperta si è verificato nel 1894 12. Le prime colture registrate erano di Kredel nel 1920, che hanno tentato senza successo di crescita del tumore macinate dal &quot;metodo goccia appeso&quot; 12 . Più tardi, Drs. Murray e Stout pubblicato la...

Schwannoma vestibolare (VSS) rappresentano cellule di Schwann (SC) i tumori del nervo vestibolare, compromettendo il 10% di tutte le neoplasie intracraniche 1-3. Vss verifica in (neurofibromatosi di tipo 2, NF2) forma sporadica o familiare, entrambe associate a difetti inattivazione nel tumore soppressore NF2 gene. Il trattamento per VSS è generalmente la resezione chirurgica o la radiochirurgia, ma la morbosità di tali procedure comprende sordità, neuropatie del viso, perdita di liquido spinale, squilibrio, e la ricrescita del tumore 1. Inoltre, non tutti i pazienti sono candidati per la chirurgia o radiazioni accettabili. Tale significativa morbilità, nonché una mancanza di terapie alternative ha spinto indagine sulla biologia molecolare unico di VSS nella speranza di sviluppare nuovi trattamenti 3,4. Coltura cellulare permette una rapida, facile, e approfondita analisi del comportamento molecolare e cellulare e lo screening del potenziale terapeutico comsterline. Storicamente, la mancanza di accesso ai campioni di tessuto fresco e il fatto che le cellule schwannoma non sono immortalati hanno ostacolato in modo significativo l&#39;uso di colture primarie per le indagini di schwannoma tumorigenesi. Per superare la limitata offerta di colture primarie, la linea cellulare HEI193 VS immortalata è stato generato da trasduzione con HPV oncogeni E6 ed E7 5. Questo trasduzione oncogenica introdotto significative alterazioni molecolari e fenotipici alle cellule, che ne limitano l&#39;uso come modello per i tumori schwannoma umani. Culture SC derivate da linee di topi transgenici che mancano di un gene NF2 funzionale rappresentano un&#39;altra alternativa per indagare NF2-dipendente tumorigenesi SC in vitro. Queste culture però non riescono a riepilogare la natura eterogenea del Vss umano o di un conto per il comportamento umano specifico. La maggior parte delle precedenti tecniche di coltura VS richiesti tempi relativamente lunghi di lavorazione e complicate tecniche di coltura selettivi 6-8.Qui vi presentiamo un protocollo semplice e riproducibile per primario VS coltura cellulare con l&#39;elaborazione completa in meno di 3 ore, con il 95% di purezza delle cellule tumorali come determinato da immunoistochimica.

<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">N2 Supplement</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">17502-048</td>
			<td>Schwannoma Culture and Media Components</td>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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

Schwannoma vestibolare (VSS) rappresentano cellule di Schwann (SC) i tumori del nervo vestibolare, compromettendo il 10% di tutte le neoplasie intracraniche. Vss verifica in (neurofibromatosi di tipo 2, NF2) forma sporadica o familiare, entrambe associate a difetti inattivazione nel tumore soppressore NF2 gene. Il trattamento per VSS è generalmente la resezione chirurgica o la radiochirurgia, ma la morbosità di tali procedure ha spinto indagini trattamenti meno invasivi. Storicamente, la mancanza di accesso ai campioni di tessuto fresco e il fatto che le cellule schwannoma non sono immortalati hanno ostacolato in modo significativo l&#39;uso di colture primarie per le indagini di schwannoma tumorigenesi. Per superare la limitata offerta di colture primarie, la linea cellulare HEI193 VS immortalata è stato generato da trasduzione con HPV oncogeni E6 ed E7. Questo trasduzione oncogenica introdotto significative alterazioni molecolari e fenotipici alle cellule, che limitano il loro uso come modello per s umanitumori chwannoma. Abbiamo quindi illustrare un protocollo riproducibile semplificata per cultura umana primaria VS cellule. Questa tecnica permette facilmente masterizzato per indagini molecolari e cellulari che ricapitolano più accuratamente la complessità della malattia VS.

Corretta frammentazione tumore è essenziale per la semina ottimale e escrescenza nelle piastre di coltura tissutale (Figura 1). Identificazione di cellule schwannoma durante i primi giorni dopo placcatura è spesso difficile, a causa oscurando quantità di detriti cellulari e globuli rossi. Con il 4 ° o 5 ° giorno, estensioni delle cellule vicino frammenti di tumore aderenti creeranno riconoscibile &#39;allacciatura&#39; modelli tra i detriti. Successive modifiche di supporto ge...

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

Primary Culture of Human Vestibular Schwannomas

Vestibolare Schwannomi (VSS) sono tumori non maligni della cella (SC) di origine Schwann, associati a mutazioni del NF2 gene soppressore del tumore. Riportiamo un protocollo efficace e riproducibile per uso umano primario VS coltura cellulare che permette di manipolazione sperimentale molecolare e cellulare e di analisi e ricapitola la natura eterogenea delle malattie umane.

Cultura primario di Human vestibolare Schwannomi

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

12.8K Views.  University of Iowa Hospitals and Clinics.  Vestibolare Schwannomi (VSS) sono tumori non maligni della cella (SC) di origine Schwann, associati a mutazioni del NF2 gene soppressore del tumore. Riportiamo un protocollo efficace e riproducibile per uso umano primario VS coltura cellulare che permette di manipolazione sperimentale molecolare e cellulare e di analisi e ricapitola la natura eterogenea delle malattie umane. 

Video: 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.

Pelle Punch Biopsia Espianto Cultura per la derivazione di fibroblasti umani primari

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

Ricerca

Medicina

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 organoide Cultura di primari tumori del colon mouse

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.

Isolamento, Cultura e Imaging di Human Fetal pancreatici gruppi di cellule

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.

A Three-dimensional Tissue Culture modello per studiare Primario Osso Osseo e le sue Malignità

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

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as &#8220;the scraping method&#8221; and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed &#8220;the trypsin method.&#8221; This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients.&#160;

Due metodi per stabilire primarie cellule umane endometriale stromali da isterectomia campioni

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

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.

Tridimensionale alginato-tallone coltura di cellule umane adenoma ipofisario

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.

Isolamento e coltura di cellule endoteliali primarie dalle arterie e vene Canine

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.

Isolamento di cellule deciduali umane primarie dalle membrane fetali del termine intempestivo della placenta

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.

Squilibri muscolari: Test e formazione forza eccentrica funzionale del tendine del ginocchio nelle popolazioni di atletiche

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.

Labyrinthectomy chirurgica del ratto per studiare il sistema vestibolare

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