1. Protocollo da utilizzare Multi-fiasche per colture cellulari <ol><li> Preparazione dei vasi e l&#39;aggiunta di sospensione cellulare <ol><li> Preparare la crescita media delle cellule, se necessario. </li><li> Line up multi-Flask verticalmente su un lato con il tappo rivolto verso l&#39;alto sul piano di lavoro (cappa a flusso laminare sterile). <ol><li> Questi contenitori sono disponibili in 3 e 5 strati formati e utilizzare un simile flusso di lavoro come un modello T-175 pallone. </li></ol></li><li> Allentare e rimuovere i tappi. Aggiungi quantità richiesta di pre-riscaldato mezzo in fiasco con una pipetta da 50 o 100 ml o per infusione. <ol><li> Pipette che sono ≤ 10 ml può raggiungere il fondo del recipiente in cui Multi-fiasche sono posti verticalmente con tappo rivolto verso l&#39;alto. Pipette ≥ 10 ml può raggiungere in nave subito dopo il logo sul Multi-Flask, fornendo così un portale comodo per creare maggiori volumi di supporti per nave. </li></ol></li><li> Per evitare bolle di media, permettono flusso di liquido di flow lungo la parete interna della Multi-Flask coperchio (logo-side). </li><li> Aggiungi sospensione cellulare da uno stock di cellule concentrate in terreno di crescita attraverso lo strato superiore con una pipetta 10 ml e fiaschi cap. Suggerimenti: - Trasporto Multi-fusti a un carrello al sito incubatore ed eseguire passaggi rimanenti. - La densità di semina delle cellule varia a seconda del tipo di cellula, medio e durata bisogno di cultura. Iniziare con la densità di semina e il volume dei media a quello usato nello standard T-175 palloni e moltiplicare per 3 o 5 a seconda della Multi-Flask formato utilizzato. </li></ol></li><li> Miscelazione di cellule <ol><li> Mix posizione: Tenere il Multi-Flask verticale con il logo rivolto verso di voi e girare in senso antiorario ad un angolo di 45 ° con porta mix rivolto verso l&#39;alto. </li><li> Tenendo lo stesso angolo, leggermente inclinare Multi-Flask da davanti a dietro (collo lontano da voi) fino liquido nello strato superiore scarichi completamente downwards attraverso la porta mix. Perno sul mix di babordo. </li><li> Allo stesso modo, dolcemente rocce multi-Flask da dietro in avanti (collo verso di voi) fino a metà scarichi completamente dallo strato basso verso l&#39;alto attraverso la porta mix. Ripetere i punti 1.2.2 e 1.2.3 ancora una volta per garantire la corretta miscelazione. </li><li> Portare Multi-Flask in posizione mix (Fase 1.2.1) e passare al punto 1,3 </li><li> In alternativa, la sospensione di cellule possono essere preparati esternamente dalla Multi-Flask e la sospensione cellulare possono essere aggiunti alla nave usando una pipetta o da dolci versando. <ol><li> Porto mix permette in-vaso di miscelazione delle cellule con i media ed elimina la necessità di fare grandi volumi di sospensioni cellulari esternamente. </li><li> Consente inoltre l&#39;equalizzazione dei media attraverso gli strati della Multi-Flask </li></ol></li></ol></li><li> Equilibrio del fluido <ol><li> Dopo la miscelazione / aggiunta di sospensione cellulare, luogo multi-Flask in verticale su una superficie di lavoro piana per pareggiare il volume liquido equazionelly in tutti gli strati. </li></ol></li><li> Liquido partizione in ogni strato <ol><li> Tenere premuto il Multi-Flask con il logo rivolto verso l&#39;alto e ruotare in senso orario ad un angolo di 45 ° per partizionare il liquido in ogni livello. Suggerimento: - Per ottenere i migliori risultati, è importante utilizzare una superficie piana di lavoro per i passaggi 1,3-1,4 </li></ol></li><li> Trasporti <ol><li> Una volta che i supporti contenenti sospensione cellulare viene partizionato, il trasporto multi-Flask allo stesso angolo di 45 ° (in senso orario) come al punto 1.4.1 con il supporto di distanza dal porto mix in incubatrice. Questa posizione è adatta anche quando si rimuove fiaschi da incubatore per la visualizzazione su un microscopio. </li></ol></li><li> Posizionamento Multi-Flask su incubatore scaffale <ol><li> Tenendo Multi-Flask al 45 ° (in senso orario, lontano dal mix-port), ruotare delicatamente verso il basso orizzontalmente sulla superficie incubatore (utilizzando l&#39;angolo lontano dalla porta come un mixpivot). Lay Multi-Fiasca con il logo rivolto verso l&#39;alto. <ol><li> Falcon Multi-Flask progettazione consente ai pescherecci di essere impilati e li tiene in posizione da una nervatura robusta accatastamento. </li></ol></li></ol></li><li> Distribuzione di cellule e reagenti <ol><li> Dopo aver posizionato Multi-Flask piatta sulla superficie di lavoro, dolcemente dondolare avanti e indietro e da lato a lato per distribuire le cellule in modo uniforme su superfici cultura facendo attenzione a non versare liquidi di ogni strato. Stack fiaschi. <ol><li> Questo Flask è fatta di materiale otticamente trasparente e celle l&#39;ultimo strato possono essere facilmente visualizzati su un microscopio. </li></ol></li></ol></li><li> Mezzi di scambio <ol><li> Aspirare mentre inclinando il Multi-Flask a sinistra (mix porta-side) con il logo rivolto verso l&#39;alto. </li><li> Poi inclinazione, il Multi-Flask a destra, continuando ad aspirare tutti i media residua. </li><li> Aggiungi quantità appropriata di media e seguire i punti da 1,3-1,7 </li></ol></li></ol> 2.La raccolta delle cellule da Multi-fiasche <ol><li> Dissociare le cellule, in fila Multi-Flasks verticale e portare a ciascuno di Mix posizione (punto 1.2.1). Capovolgere delicatamente palloni con il collo rivolto verso di voi in modo da consentire ai media di scarico per lo strato superiore della Multi-Flask. </li><li> Inserire una punta di 5 o 10 ml aspirare attraverso il collo fino a raggiungere il livello del liquido nei pressi del porto mix. Aspirare il terreno esausto. </li><li> Aggiungi dissociazione reagente / tampone di lavaggio e portare a Mix posizione come nel passo 1.2.1, quindi procedere e seguire i passaggi 1,3-1,7. Neutralizzare con mezzo di crescita / neutralizzazione e versare sospensione di cellule in una provetta di ricezione o invertire fiasco tale che le cellule di scarico per lo strato superiore della nave e raccogliere le cellule con una pipetta 10 ml. Suggerimento: per favorirne il recupero di cellule o reagenti, portare Multi-pallone per mescolarne posizione (punto 1.2.1), invertire con il collo verso l&#39;operatore per permettere il drenaggio completo dei media da al<html> strati l per lo strato superiore. Poi, inclinazione Multi-Flask in senso orario per angolo di 45 ° (lontano dal porto mix), mentre il Multi-Flask rimane invertita. Utilizzare una pipetta (1-10 ml) per raccogliere tutti i reagenti rimasti. </li></ol> 3. Consigliato volume di lavoro in Falcon Multi-fiasche La crescita dei media 3-Layer: 75-150 ml per Multi-Flask 5-Layer: 125-250 mL per Multi-Flask Dissociazione agente 3-Layer: ≥ 15 ml per ogni Multi-Flask 5-Layer: ≥ 25 ml per Multi-Flask Suggerimento: Inizia con volume medio usato in standard T-175 palloni e moltiplicare per 3 o 5 volte a seconda della multi-formato Flask valutata in modo che mL per unità di superficie rimane la stessa. 4. Rappresentante dei risultati: 1. Disegno di Falcon Multi-Flask files/ftp_upload/3418/3418fig1.jpg &quot;/&gt; . Figura 1: Multi-Flask vasi coltura cellulare sono disponibili in 3 - e 5-strato formato stackable di scale-up delle cellule fornendo 525 e 875 cm 2 di superficie di crescita, rispettivamente. Pipetta di accesso facilita aggiunta e la rimozione delle cellule e reagenti in e fuori della nave. Presenza di mix-port consente una rapida in-vaso di miscelazione ed equalizzazione dei media in tutti gli strati della Multi-Flask. 2. Yield cellulare con Multi-Flask: Questi vasi sono disponibili in 3 strati e 5 strati formati che corrispondono a 3 e 5 volte la superficie di T-175 fiaschi. Di conseguenza, ≥ 3 e 5 volte (130 ± 6,8 x 10 6 e 218 ± 23,6 x 10 6 cellule, rispettivamente) il numero di BHK-21 (bambino renali di criceto), le cellule sono state coltivate e recuperati da Multi-Flask rispetto al T-175 fiaschi (43,2 ± 3,5 x 10 6 cellule; Fig.2a). Produzione di cellule per usuperficie nit era equivalente a 3 - e 5-layer multi-Palloni e T-175 palloni per BHK-21, LNCaP (adenocarcinoma prostatico umano linea cellulare), Hep-G2 (linea di cellule di epatocarcinoma umano), Ecopack 2-293 (umano linea cellulare di rene) in coltura per un periodo di 48-96h (Fig.2b) in terreni di coltura (35 ml per strato), come raccomandato dal fornitore cellulare (ATCC, Sigma e / o Clontech). Le cellule sono state elencate in un sistema automatizzato Vi-CELL contatore (1). <img alt="Figura 2A" src="/files/ftp_upload/3418/3418fig2A.jpg" /> Figura 2A: tre e cinque volte il numero di cellule BHK-21 sono stati coltivati ​​e recuperati da 3 - e 5-strato Multi-fiasche rispetto al T-175 fiaschi. Rendimento atteso è stata determinata utilizzando rendimenti medi dal controllo delle cellule T-175 fiaschi moltiplicato per tre volte e cinque per il 3 - e 5-strato Multi-fiasche rispettivamente (n = 4 flaconi / formato). <img alt="Figura 2B" src="/files/ftp_upload/3418/3418fig2B.jpg" /><br/ Figura&gt; 2B: resa celle per cm 2 è stato equivalente a 3 - e 5-layer multi-Palloni e T-175 palloni per BHK-21, LNCaP, HepG2 e EcoPack2-293 cellule. Ogni barra rappresenta la media di 4-6 fiaschi. BHK-21 (11.000 cells/cm2) sono state coltivate per 72 ore, le cellule LNCaP (20.000 cells/cm2) e EcoPack2-293 cellule (~ 35.000 cells/cm2) sono state coltivate per 96 ore e cellule HepG2 (25.000 cellule / cm 2 ) sono state coltivate per 48 ore prima del raccolto. 3. Distribuzione media tra strati di multi-Flask Terreno di coltura cellulare (DMEM, Invitrogen) è stato aggiunto a 5 strati multi-Palloni (250 nave mL/5-layer) e suddiviso in strati secondo il protocollo sopra descritto. Distribuzione dei media è stata misurata mediante fori in ogni strato e media pompato fuori da singoli livelli. Peso del liquido recuperato da ogni strato è risultata essere relativamente uniforme da un livello all&#39;altro come mostrato in Fig. 3. Essi sono i seguenti: 510,8 ± 0,73, 50,3 ± 0,58, 50,21 ± 0,13, 49,88 ± 0,35, 49,45 ± 0,37 (gm). <img alt="Figura 3" src="/files/ftp_upload/3418/3418fig3.jpg" /> Figura 3. Uniforme distribuzione dei media in ciascuno dei cinque strati di un 5-strato Multi-Flask. Terreno di coltura cellulare è stato aggiunto al Multi-Palloni (250 nave ml/5-layer), equilibrato e diviso per singoli livelli. Il supporto è stato pompato fuori attraverso fori praticati nei singoli strati e il peso del liquido è stato registrato da ogni strato (n = 6 bottiglie). 4. Distribuzione tra gli strati di celle multi-Flask Le celle possono essere aggiunti e mescolati all&#39;interno del Multi-Flask. Abbiamo simulato la distribuzione di cellule tra gli strati di multi-Flask utilizzando perline (10μm; Polysciences Inc.) di dimensioni simili a celle. Sospensione tallone è stato aggiunto in Multi-Flask nave con una pipetta 10 ml attraverso lo strato superiore e mescolato con i media nel vaso come discribed nel protocollo di cui sopra. Distribuzione tallone è stata misurata mediante fori in ogni strato e fluido contenente sospensione tallone è stata pompata fuori dai singoli livelli. Concentrazione tallone recuperato da ogni strato è stato letto su un contatore Coulter e registrati. Qui sotto sono equivalenti distribuzioni tallone inter-strato a 3 strati multi-Palloni (Fig.4A). Il mix-porta consente una distribuzione omogenea di cellule e reagenti tra Flask multi-strati. <img alt="Figura 4A" src="/files/ftp_upload/3418/3418fig4A.jpg" /> Figura 4A: Bead distribuzione in ognuno dei tre strati di un 3-strato Multi-Flask. Una sospensione di perline (3,6 x10 6 / ml) è stato aggiunto al medio erogato in Multi-Palloni (sospensione perle: il volume dei media è di 1:10, vol: vol) e misti, seguiti da fasi di separazione e di equilibrio utilizzando il protocollo descritto. Concentrazione tallone recuperato da ogni strato (medio pompato attraverso fori praticati su ogni livello) è stata letta su un conte Coulterer e registrati. Qui sotto sono equivalenti distribuzioni tallone inter-layer 3 - strato Multi-Palloni (n = 5 palloni) Mostrate di seguito sono immagini rappresentative di Ecopack 2-293 pattern di colorazione delle cellule cresciute a confluenza&gt; 80% sul 3-strati multi-fiasche in terreni di coltura integrata. Monostrati cellulari sono stati fissati e colorati con cristallo viola e multi-Flask strati sono stati poi tagliati e immagini digitalizzate (2). Nota, patterning cellulare è stata omogenea su tutti i livelli di Multi-Flask (Fig.4b). Risultati simili sono stati ottenuti con più tipi di cellule valutato (dati non riportati). <img alt="Figura 4B" src="/files/ftp_upload/3418/3418fig4B.jpg" /> Figura 4B: Questa figura illustra la crescita omogenea delle cellule tra gli strati di multi-fiaschi. Ecopack-2-293 cellule cresciute a confluenza&gt; 80% in 3-strati multi-Palloni e T-175 sono state fissate e colorate con violetto cristallo. Multi-Flask navi sono state tagliate e ogni strato colorato è stato scansionato. 5 Alimentazione aria in Multi-Flask.: Analisi dei mezzi di comunicazione speso utilizzando BioProfile FLEX analizzatore (3,4) (Nova Biomedical) da EcoPack2-293 cellule in coltura per 96 ore hanno rivelato alcuna differenza di saturazione dell&#39;aria di cellule cresciute in 5 strati multi-fiasche vs T-175 flaconi (81,03 ± 1,9 vs 83,4 ± 5,8% ambient O 2). <img alt="Figura 5" src="/files/ftp_upload/3418/3418fig5.jpg" /> Figura 5: Aria di saturazione (% ambient O 2) di media spesi erano simili in pre-mixed media da 5 strati multi-fiasche vs T-175 fiaschi. EcoPack2-293 cellule sono state seminate ad una densità di 35.000 cellule / cm 2 e coltivate per 96 ore prima analisi dei media (n = 3 palloni).

<ol>
	<li>Szabo, S. E., Monroe, S. L., Fiorino, S., Bitzan, J., Loper, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+an+automated+instrument+for+viability+and+concentration+mesurements+of+cryopreserved+hematopoietic+cells.">Evaluation of an automated instrument for viability and concentration mesurements of cryopreserved hematopoietic cells.</a> Lab. Hematol. 10, 109-109 (2004).</li><li>Bonnekoh, B., Wevers, A., Jugert, F., Merk, H., Mahrle, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+growth+assay+for+epidermal+cell+cultures+by+their+crystal+violet+binding+capacity.">Colorimetric growth assay for epidermal cell cultures by their crystal violet binding capacity.</a> Arch. Dermatol. Res. 281, 487-487 (1989).</li><li>Sengupta, N., Rose, S. T., Morgan, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+Flux+analysis+of+CHO+cell+metabolism+in+the+late+non-growth+phase.">Metabolic Flux analysis of CHO cell metabolism in the late non-growth phase.</a> Biotechnol. Bioeng. 108, 83-83 (2010).</li><li>Zhu, M. M., Goyal, A., Rank, D. L., Gupta, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+elevated+pCO2+and+osmolality+on+growth+of+cells+and+production+of+antibody&#x2013;fusion+protein+B1:+a+case+study.">Effects of elevated pCO2 and osmolality on growth of cells and production of antibody&#x2013;fusion protein B1: a case study.</a> Biotechnol. Prog. 21, (2005).</li><li>Ryan, J. M., Sharf, B. B., Cristofalo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+culture+medium+volume+on+cell+density+and+life-span+of+human+diploid+fibroblasts.">The influence of culture medium volume on cell density and life-span of human diploid fibroblasts.</a> Exp Cell Res. 91, 389-389 (2004).</li><li>Flaherty, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Tips and Techniques for enhancing your cell culture. , (2009).</li><li>Sanyal, S., Abraham, E. J., Slater, K., Cai, A., Lacey, B., Hartshorn, C., Flaherty, P., McHugh, B., Qian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Scaling up cells in BD Falcon Cell Culture Multi-Flasks. , (2011).</li><li>DiPersio, C. M., Jackson, D. A., Zaret, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extracellular+matrix+coordinately+modulates+liver+transcription+factors+and+hepatocyte+morphology.">The extracellular matrix coordinately modulates liver transcription factors and hepatocyte morphology.</a> Mol. Cell. Biol. 11, 4405-4405 (1991).</li><li>Grose, R., Hutter, C., Bloch, W., Thorey, I., Watt, F. M., Fassler, R., Brakebusch, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+crucial+role+of+&#x03B2;1+integrins+for+keratinocyte+migration+in+vitro+and+during+cutaneous+wound+repair.">A crucial role of &#x03B2;1 integrins for keratinocyte migration in vitro and during cutaneous wound repair.</a> Dev. 129, 2303-2303 (2002).</li></ol>

Tutti gli autori sono dipendenti di Becton Dickinson, Segmento Biosciences, Discovery unità mediche di laboratorio.

Lo studio attuale dimostra l&#39;aumento di produttività che di design Multi-Flask offre ai ricercatori. Mentre è importante seguire la procedura descritta in precedenza per ottenere prestazioni ottimali durante l&#39;utilizzo multi-Palloni, ci sono pochi passaggi critici in questo protocollo che sono ritenute più essenziali. Questi includono (i) la miscelazione di cellule e reagenti mediante la porta miscela all&#39;interno del serbatoio (ii) il trasporto multi-pallone in un angolo di 45 ° in senso orario dopo il partizionamento del fluido in ciascuno dei livelli (iii) che multi-Flask messaggio piatto partizionamento in incubatrice. L&#39;uso corretto di Multi-Flask porta alla produzione di una popolazione di cellule omogenee all&#39;interno di ciascuna nave che viene colto in un ambiente che è congruente al T-175 palloni (5). Questi vasi forniscono cellule 3 e 5 volte di più in un ingombro simile a quella del T-175 pallone. La nave 3 e 5 strati fornisce 525 e 875 centimetri 2 di area di crescita, rispettivamente, e offrono spazio e del lavoro savings agli utenti. Cultura trattamento del tessuto di superficie è paragonabile a fiaschi di serie consentendo così di scale-up senza la necessità di ri-ottimizzazione delle condizioni di coltura esistente o compromettere la qualità, omogeneità o prestazioni delle celle (6, 7). Questo prevede anche la comparabilità con i dati precedentemente raccolti. Questi vasi possono essere anche rivestiti con i reagenti come il collagene, fibronectina, poli-D-lisina per fornire un substrato specializzata per l&#39;attacco, la crescita e la differenziazione di alcuni tipi di cellule come gli epatociti 8, kertainocytes 9, le cellule staminali coltivate in serum-free dei media formulazioni. Soluzioni di rivestimento può essere rimosso con un minimo di ritenzione di liquido residuo in modo da ridurre lo spreco di reagenti preziosi così come la rimozione efficace di soluzione di rivestimento prima della coltura delle cellule. La raccomandata volume ottimale dei media per le cellule in cultura multi-fiaschi gamma ,142-0,287 ml / cm 2, che si traducono in 25-50 ml per strato. A differenza di un altro fiasco multistrato, til suo prodotto offre un mix-porto la nave, che consente una rapida in-vaso di miscelazione e distribuzione uniforme delle cellule e reagenti all&#39;interno e tra gli strati di ogni nave. Diverse linee cellulari, colture primarie e cellule staminali sono stati scalati in modo efficiente utilizzando Multi-fiaschi. Questi vasi sono particolarmente vantaggiosa in applicazioni che richiedono un gran numero di tali cellule come in high throughput screening, produzione di vaccini, trasfezioni vettori virali e terapia cellulare. Risparmio di tempo, spazio, lavoro e produzione di rifiuti ridotti sono vincite chiave del Multi-Flask contro le culture tradizionali in monostrato navi. Siamo in grado di cultura circa tre volte il numero di cellule raccolte da 5, T-175 palloni nello stesso spazio con 3, 5-strato Multi-fiaschi. Questo vantaggio in risparmio di spazio non si limita solo al T-175, ma può essere esteso anche alle altre navi: una norma apparato bottiglia a rulli che si inserisce in case comuni incubatori di laboratorio ~ 4 rolbottiglie ler (2200 ml) ciascuna delle quali fornisce 850 centimetri 2 di superficie. Nella stessa zona, ~ 20, a 5 strati multi-fiasche possono essere alloggiati fornendo così a cinque volte la superficie di crescita per le cellule della cultura. Inoltre, con un aumento della consapevolezza &quot;Go-Green&quot;, le opzioni per ridurre la produzione di rifiuti è altamente auspicabile. A tal proposito, c&#39;è un 38% (5, T-175 palloni pesano ~ 640g, mentre uno, a 5 strati multi-Flask ~ pesa 400g) diminuzione della produzione di rifiuti usando Multi-fiasche rispetto al T-175 palloni e questi vantaggi portano a diminuire in stoccaggio dei rifiuti ei costi di smaltimento e di tradursi in un risparmio economico per l&#39;utente.

<table border="1"><tbody><tr><td> Nome del reagente / attrezzature </td><td> Azienda </td><td> Catalogo # </td><td> Commenti </td></tr><tr><td> 3-strato di tessuto Cultura trattati con 525 centimetri 2 </td><td> BD Biosciences </td><td> 353143 </td><td rowspan="21"> <a href="http://www.bdbiosciences.com/cellculture/multiflasks/index.jsp">BD Biosciences Colture Cellulari - BD Falcon Multi-fiasche</a> </td></tr><tr><td> 5-strato di tessuto Cultura trattati con 875 centimetri 2 </td><td> BD Biosciences </td><td> 353144 </td></tr><tr><td> 100ml pipetta </td><td> BD Biosciences </td><td> 357600 </td></tr><tr><td> 10 ml pipetta </td><td> BD Biosciences </td><td> 357551 </td></tr><tr><td> 5 ml aspirazione pipetta </td><td> BD Biosciences </td><td&gt; 357501 </td></tr><tr><td> 50 ml di tubo in polipropilene conica </td><td> BD Biosciences </td><td> 352070 </td></tr><tr><td> T-175 fiasco Tissue Culture-trattati </td><td> BD Biosciences </td><td> 353028 </td></tr><tr><td> Grammo di cristallo viola </td><td> BD </td><td> 212525 </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 11885 </td></tr><tr><td> GMEM </td><td> Sigma </td><td> G5154 </td></tr><tr><td> RPMI-1640 </td><td> ATCC </td><td> 30-2001 </td></tr><tr><td> MEM </td><td> ATCC </td><td> 30-2003 </td></tr><tr><td> Tripsina-EDTA </td><td> Lonza </td><td> CC-5012 </td></tr><tr><td> Siero fetale bovino </td><td> Invitrogen </td><td> 16000-044 </td></tr><tr><td> Triptosio fosfato brodo </td><td> Sigma </td><td> T8159 </td&gt; </tr><tr><td> BHK-21 </td><td> Sigma </td><td> 85011433 </td></tr><tr><td> Hep-G2, LNCaP </td><td> ATCC </td><td></td></tr><tr><td> Ecopack 2-293 </td><td> Clontech </td><td></td></tr><tr><td> Paraformaldeide </td><td> Electron Microscopy Sciences </td><td> 15710 </td></tr><tr><td> Polistirolo Bead </td><td> Polysciences Inc. </td><td> 24628-20 </td></tr><tr><td> Bio-Flex Profile Analyzer </td><td> Nova Biomedical </td><td></td></tr></tbody></table>

scale-up of mammalian cell culture using a new multilayered flask

Un numero crescente di cellulari basati su applicazioni richiedono un gran numero di cellule. L&#39;utilizzo di singolo strato T-boccette, che sono adeguati durante l&#39;espansione di piccole dimensioni, può diventare ingombrante, laborioso e richiede molto tempo quando un gran numero di cellule sono obbligatori. Per rispondere a questa esigenza, le prestazioni di un nuovo multi-layered nave coltura cellulare per facilitare la scala facile da cellule stratificate da sola T-palloni saranno discussi. I palloni testati sono disponibili in 3 - e 5-strato formato e consentire il recupero della cultura e completo di tre e cinque volte il numero di cellule, rispettivamente, rispetto al T-175 fiaschi. Una caratteristica fondamentale del BD Multi-Flask è un mix / equilibrio porta che permette una rapida in-vaso di miscelazione e distribuzione uniforme delle cellule e reagenti all&#39;interno e tra gli strati di ogni nave e produrre cellule che possono essere coltivate in un ambiente che è congruente al T-175 fiaschi. Il design di questi multi-fiasche consente inoltre di caccesso pipetta onvenient per l&#39;aggiunta di reagenti e le cellule direttamente nelle fiasche e recupero efficiente di cellule preziose e reagenti e riduce il rischio di contaminazione a causa della colata. Per le applicazioni dove è preferibile versare sopra pipettaggio, il design permette di minima ritenzione di liquidi residui in modo da ridurre gli sprechi di preziose cellule e reagenti.

Watch this Scientific Journal Video about Scale-Up of Mammalian Cell Culture using a New Multilayered Flask at JoVE.com

Scale-Up of Mammalian Cell Culture using a New Multilayered Flask

Cellule svolgono un ruolo fondamentale e crescente nella ricerca, e la scoperta e lo sviluppo di nuove terapie. Grazie a questa crescente necessità di un maggior numero di cellule abbiamo bisogno di modi più efficienti ed efficaci per la coltivazione e la raccolta delle cellule attaccamento dipendente. Un fiasco multistrato con le giuste caratteristiche può servire a questo scopo.

Scale-up di colture cellulari di mammifero utilizzando un pallone nuovo multistrato

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale ...

scale-up-of-mammalian-cell-culture-using-a-new-multilayered-flask

Research

JoVE Journal

Biology

37.5K Views.  BD Biosciences. Cellule svolgono un ruolo fondamentale e crescente nella ricerca, e la scoperta e lo sviluppo di nuove terapie. Grazie a questa crescente necessità di un maggior numero di cellule abbiamo bisogno di modi più efficienti ed efficaci per la coltivazione e la raccolta delle cellule attaccamento dipendente. Un fiasco multistrato con le giuste caratteristiche può servire a questo scopo.

Video: Scale-Up of Mammalian Cell Culture using a New Multilayered Flask

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

A lungo termine Imaging cellule di mammifero utilizzando Wide-Field Microscopia

Ricerca

Biologia

Ole Isacson: Development of New Therapies for Parkinson's Disease

Ole Isacson gives a concise overview of Parkinsons's disease, its causes, therapeutic strategies, and advances in Parkinson's research.

Ole Isacson: sviluppo di nuove terapie per la malattia di Parkinson

Development of New Therapeutic Applications Using Microfluidics

Sviluppo di nuove applicazioni terapeutiche Utilizzando Microfluidica

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

Metodo per la Cultura di Chick primi embrioni ex vivo (Nuova Cultura)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

Analisi della posizione ciclo cellulare nelle cellule di mammifero

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Determination of Mammalian Cell Counts, Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A critical breakthrough in cell/particle counting technologies was the development of the Coulter technique by Wallace Coulter over 50 years ago. The technique involves the application of an electric field across a micron-sized aperture and hydrodynamically focusing single particles through the aperture. The resulting occlusion of the aperture by the particles yields a measurable change in electric impedance that can be directly and precisely correlated to cell size/volume. The recognition of the approach as the benchmark in cell/particle counting stems from the extraordinary precision and accuracy of its particle sizing and counts, particularly as compared to manual and imaging based technologies (accuracies on the order of 98% for Coulter counters versus 75-80% for manual and vision-based systems). This can be attributed to the fact that, unlike imaging-based approaches to cell counting, the Coulter Technique makes a true three-dimensional (3-D) measurement of cells/particles which dramatically reduces count interference from debris and clustering by calculating precise volumetric information about the cells/particles. Overall this provides a means for enumerating and sizing cells in a more accurate, less tedious, less time-consuming, and less subjective means than other counting techniques6.
Despite the prominence of the Coulter technique in cell counting, its widespread use in routine biological studies has been prohibitive due to the cost and size of traditional instruments. Although a less expensive Coulter-based instrument has been produced, it has limitations as compared to its more expensive counterparts in the correction for "coincidence events" in which two or more cells pass through the aperture and are measured simultaneously. Another limitation with existing Coulter technologies is the lack of metrics on the overall health of cell samples. Consequently, additional techniques must often be used in conjunction with Coulter counting to assess cell viability. This extends experimental setup time and cost since the traditional methods of viability assessment require cell staining and/or use of expensive and cumbersome equipment such as a flow cytometer.
The Moxi Z mini automated cell counter, described here, is an ultra-small benchtop instrument that combines the accuracy of the Coulter Principle with a thin-film sensor technology to enable precise sizing and counting of particles ranging from 3-25 microns, depending on the cell counting cassette used. The M type cassette can be used to count particles from with average diameters of 4 - 25 microns (dynamic range 2 - 34 microns), and the Type S cassette can be used to count particles with and average diameter of 3 - 20 microns (dynamic range 2 - 26 microns). Since the system uses a volumetric measurement method, the 4-25 microns corresponds to a cell volume range of 34 - 8,180 fL and the 3 - 20 microns corresponds to a cell volume range of 14 - 4200 fL, which is relevant when non-spherical particles are being measured. To perform mammalian cell counts using the Moxi Z, the cells to be counted are first diluted with ORFLO or similar diluent. A cell counting cassette is inserted into the instrument, and the sample is loaded into the port of the cassette. Thousands of cells are pulled, single-file through a "Cell Sensing Zone" (CSZ) in the thin-film membrane over 8-15 seconds. Following the run, the instrument uses proprietary curve-fitting in conjunction with a proprietary software algorithm to provide coincidence event correction along with an assessment of overall culture health by determining the ratio of the number of cells in the population of interest to the total number of particles. The total particle counts include shrunken and broken down dead cells, as well as other debris and contaminants. The results are presented in histogram format with an automatic curve fit, with gates that can be adjusted manually as needed.
Ultimately, the Moxi Z enables counting with a precision and accuracy comparable to a Coulter Z2, the current gold standard, while providing additional culture health information. Furthermore it achieves these results in less time, with a smaller footprint, with significantly easier operation and maintenance, and at a fraction of the cost of comparable technologies.

The Moxi Z miniature automated cell counter is a novel instrument that combines the Coulter Principle with patented thin-film sensor technology and a proprietary software algorithm to perform sizing and counting of a broad size range of particles as well as to determine the overall health of monodisperse mammalian cell cultures. This protocol describes the use of this instrument for counting and assessing the health of cell cultures.

Determinazione del conteggio delle cellule dei mammiferi, le dimensioni delle cellule e la salute delle cellule utilizzando il Moxi Z Mini contaglobuli automatizzato

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A ...

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

Preparazione di Rat Siero Adatto per mammiferi tutta la cultura Embrione

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for ...

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

Diretto Protein Consegna a cellule di mammifero Utilizzando Cell-permeabile Cys<sub&gt; 2</sub&gt; -Il suo<sub&gt; 2</sub&gt; domini zinc-finger

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...

Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus

The insect fat body plays a central role in insect metabolism and nutrient storage, mirroring functions of the liver and fat tissue in vertebrates. Insect fat body tissue is usually distributed throughout the insect body. However, it is often concentrated in the abdomen and attached to the abdominal body wall.
The mosquito fat body is the sole source of yolk proteins, which are critical for egg production. Therefore, the in vitro culture of mosquito fat body tissues represents an important system for the study of mosquito physiology, metabolism, and, ultimately, egg production. The fat body culture process begins with the preparation of solutions and reagents, including amino acid stock solutions, Aedes physiological saline salt stock solution (APS), calcium stock solution, and fat body culture medium. The process continues with fat body dissection, followed by an experimental treatment. After treatment, a variety of different analyses can be performed, including RNA sequencing (RNA-Seq), qPCR, Western blots, proteomics, and metabolomics.
In our example experiment, we demonstrate the protocol through the excision and culture of fat bodies from the yellow fever mosquito, Aedes aegypti, a principal vector of arboviruses including dengue, chikungunya, and Zika. RNA from fat bodies cultured under a physiological condition known to upregulate yolk proteins versus the control were subject to RNA-Seq analysis to demonstrate the potential utility of this procedure for investigations of gene expression.

Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus

The fat body is the central metabolic organ in insects. We present a live organ culture system that enables the user to study the responses of isolated fat body tissue to various stimuli.

Sistema di coltura di organo del grasso corporeo in Aedes Aegypti, vettore di Virus di Zika

The insect fat body plays a central role in insect metabolism and nutrient storage, mirroring functions of the liver and fat tissue in vertebrates. Insect ...

Anaerobic Growth and Maintenance of Mammalian Cell Lines

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that the incubation in normoxic (5% CO2 in air) or hypoxic (low oxygen levels) conditions followed by an anoxic incubation (an absence of free oxygen) results in limited viability (2&#8211;3 days). A novel methodology was developed that enables an anoxic cell cultivation (for at least 17 days; the maximum time tested). The most critical aspect of this methodology is to ensure that no oxygen is introduced into the system. This is obtained by the degassing of media, and by flushing tubes, dishes, flasks, and pipettes with an anaerobic gas mixture (H2, CO2, N2) followed by permitting the materials to equilibrate to the anoxic (non-oxygen) environment prior to usage.&#160;Additional care must be exercised when acquiring photomicrographs to ensure that the micrographs obtained do not contain artifacts. In the absence of oxygen, cell morphology is significantly altered. Two distinct morphotypes are noted for all anaerobically-grown cells. The ability to grow and maintain mammalian cells in the absence of oxygen can be applied to the analysis of cell physiology, polymicrobial interactions, and the characterization of biosynthetic pathways for novel cancer drug development.

Here, we describe a new method that enables the anaerobic long-term cultivation of established cell lines. The maximum survival time that was tested is 17 days. This method is suitable for the testing of cytotoxic agents and exploring the physiology of anoxically replicating cells.

Crescita anaerobica e la manutenzione di linee cellulari di mammifero

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that ...