1. Protokol, hücre kültürü için Multi-şişeler kullanmak için <ol><li> Gemilerin hazırlanması ve hücre süspansiyonu ekleyerek <ol><li> Gerektiği gibi hücre büyüme ortamı hazırlayın. </li><li> Multi-Flask dikey çalışma yüzeyi (steril laminer akış kaput) yukarı bakacak şekilde kapağı ile yan hizaya getirin. <ol><li> Bu şişeler 3 ve 5-katlı formatları ve T-175 şişesi de benzer bir iş akış deseni kullanabilirsiniz. </li></ol></li><li> Gevşetin ve kapaklarını çıkarın. 50 veya 100 mL pipet kullanarak balona ya da dökerek önceden ısıtılmış orta gerekli miktarda ekleyin. <ol><li> Multi-şişeler kapağı yukarı bakacak şekilde dik olarak yerleştirilir ≤ 10 ml geminin alt ulaşabilirsiniz Pipetler . Pipetler ≥ 10ml Çok Flask logo geçmiş, böylece damar medyanın büyük birimler eklemek için uygun bir portal sağlayan damar içine hemen ulaşabilirsiniz. </li></ol></li><li> Orta köpüren önlemek için, sıvı akışı fl izinÇok Flask kapak (logo tarafında) iç duvar boyunca ow. </li><li> 10 ml pipet ve kap şişeler kullanarak üst katmanı sayesinde büyüme ortama konsantre bir hücre stokunun hücre süspansiyonu ekleyin. İpuçları: - Ulaştırma inkübatör sitesine sepeti Multi-şişeler ve geri kalan adımları uygulayın. - Hücre ekim yoğunluğu, hücre tipi, orta ve kültür süresine ihtiyaç bağlı olarak değişecektir . Standart T-175 şişelerde kullanılan tohum yoğunluğu ve medya hacmi ile başlayın ve biçimi kullanılır Multi-Flask bağlı olarak 3 veya 5 ile çarpın. </li></ol></li><li> Hücre karıştırma <ol><li> Pozisyonu logosu bakacak şekilde dik tutun ve Multi-Flask sayacı 45 saat yönünde çevirin ° açı size dönük olacak şekilde karışımı bağlantı noktası ile karıştırın. </li><li> Aynı açıyla tutarak yavaşça üst tabaka sıvı kadar önden arkaya doğru (sizden uzağa boyun) Multi-Flask eğim tamamen downwar dreneds karışımı bağlantı noktası üzerinden. Karışımı port tarafı üzerinde özetleyin. </li><li> Aynı şekilde, orta karışımı bağlantı noktası üzerinden alt tabakadan üste doğru tamamen drene kadar nazikçe geri ön (size karşı boyun) Multi-Flask kaya. Adımlar 1.2.2 ve düzgün bir karışım sağlamak için 1.2.3 bir kez daha tekrarlayın. </li><li> Karışım pozisyonlar (Adım 1.2.1) Multi-Flask getirin ve 1.3 adıma geçin </li><li> Alternatif olarak, hücre süspansiyonu Multi-Flask dışarıdan hazırlanan ve hücre süspansiyonu bir pipet kullanarak gemi veya yumuşak dökerek eklenebilir. <ol><li> Mix bağlantı noktası medya ile hücrelerin damar mix yapma imkanı verir ve dışarıdan büyük miktarda hücre süspansiyonları yapma ihtiyacını ortadan kaldırır . </li><li> Ayrıca, Multi-Flask katmanları arasında medya eşitleme sağlar </li></ol></li></ol></li><li> Sıvı, Denge <ol><li> Karıştırma / hücre süspansiyonu ekledikten sonra, yer Multi-Flask dikey düz bir çalışma yüzeyi üzerindeki sıvı hacim denklemleri eşitlemek içinlly tüm katmanları. </li></ol></li><li> Her bir tabaka haline Bölme sıvı <ol><li> Multi-Flask logosu ile size dönük olacak şekilde tutun ve katmanların her birine bir sıvı bölümü ile 45 ° açı saat yönünde çevirin. İpucu: - En iyi sonuç için düz bir çalışma yüzeyi, Adımlar 1,3-1,4 için kullanmak önemlidir. </li></ol></li><li> Taşıma <ol><li> Bir zamanlar medya hücre süspansiyonu içeren nakil Multi-Flask aynı 45 ° açı (saat yönünde) inkübatör içine karışımı bağlantı uzak medya ile Adım 1.4.1 olarak bölümlendirilmiş olduğundan emin olun. Bu pozisyon, bir mikroskop görüntülemek için inkübatör şişeler çıkarırken de uygundur. </li></ol></li><li> Inkübatör raf üzerine Multi-balon yerleştirilmesi <ol><li> Multi-Flask Holding 45 ° açı (saat yönünde, uzak mix-port), yavaşça bir karışımı portu uzak köşesinde kullanarak (inkübatör yüzeye yatay aşağı döndürmekPivot). Logo yukarı bakacak şekilde Multi-Flask yatırın. <ol><li> Falcon Multi-Flask tasarım damarları yığılmış olmasını sağlar ve onları sağlam bir istifleme kaburga yerde tutar. </li></ol></li></ol></li><li> Hücre ve reaktifler Dağılımı <ol><li> Çalışma yüzeyinin düz Multi-Flask yerleştirdikten sonra hafifçe ileri geri ve yan yana rock ve her katman sıvı dökmemeye özen kültür yüzeylerinden eşit hücrelerini dağıtmak için. Matara yığını. <ol><li> Bu Flask optik olarak şeffaftır malzemeden üretilmiş olup, son kat hücreleri mikroskop altında kolayca görülebilir. </li></ol></li></ol></li><li> Medya döviz <ol><li> Aspire ederken logosu ile size dönük olacak şekilde sola doğru eğerek Multi-Flask (mix port tarafı). </li><li> Sonra eğme, sağa Multi-Flask, artık tüm medya aspire devam etmektedir. </li><li> Ekleyin ve uygun miktarda medya Adımlar 1,3-1,7 izleyin </li></ol></li></ol> 2.Multi-şişeler Hasat hücrelerle <ol><li> Hücreleri, çizgi Multi-şişeler dikey ilgimiz getirmek için her biri pozisyonlar (Adım 1.2.1) karıştırın. Şişeler medya Multi-Flask üst tabakası drenaj izin verecek şekilde boyun size bakacak şekilde yavaşça çevirin. </li><li> Karışımı limanı yakınlarında sıvı seviyesine ulaşana kadar boyun aracılığıyla 5 veya 10 ml aspire ucu yerleştirin. Bitkin orta aspire. </li><li> Disosiasyon reaktif / yıkama tamponu ekleyin ve Adım 1.2.1 &#39;deki gibi pozisyon karıştırın getirmek, sonra devam edin ve aşağıdaki adımları 1,3-1,7 uygulayın. Büyüme orta / nötralize edici ajan ile nötralize hücre süspansiyonu ve bir tüp içine dökün VEYA hücreleri için drenaj şişesi gibi ters Geminin en üst tabakası ve 10 ml pipet kullanarak hücreleri toplamak. İpucu: al medya tam drenaj izin vermek için hücreleri veya reaktiflerin kurtarma geliştirmek pozisyonlar (Adım 1.2.1) karıştırmak için Multi-Flask getirmek, boyun operatöre doğru ters<html> l katmanları üst tabakası. Multi-Flask ters kalırken, eğim Multi-Flask 45 ° açı (karışımı portu uzak) için saat yönünde. Kalan tüm reaktifler toplamak için bir pipet (1-10 ml) kullanın. </li></ol> 3. Falcon Multi-şişeler Önerilen çalışma hacmi Büyüme medya 3-Layer: Multi-Flask başına 75-150 ml 5-Layer: Multi-Flask başına 125-250 ml Disosiyasyon ajanı 3-Layer: Multi-Flask başına ≥ 15 ml 5-Layer: Multi-Flask başına ≥ 25 ml İpucu: standart T-175 matara kullanılan orta hacimli ile başlayın ve 3 veya 5 kez birim yüzey alanı başına bu mL aynı kalacak şekilde değerlendirilir Multi-Flask formatına bağlı ile çarpın. 4. Temsilcisi Sonuçlar: Falcon Multi-Flask 1. Tasarım files/ftp_upload/3418/3418fig1.jpg &quot;/&gt; Şekil 1: Multi-Flask Hücre Kültürü gemileri, 3 - 5-katmanlı ve ölçek sırasıyla 525 ve 875 cm 2 büyüme yüzey alanı sağlayan hücreleri istiflenebilir formatı . Pipet erişim ek ve içine ve dışına geminin hücreleri ve reaktiflerin çıkarılmasını kolaylaştırır. Mix-port varlığı, Multi-Flask tüm katmanlar arasında medya-damar hızlı karıştırma ve dengeleme sağlar. 2. Hücre Verimi Multi-Flask: Bu gemiler, 3-katmanlı ve 5 katlı formatları 3&#39;e uygun ve T-175 şişe 5 kat yüzey alanı mevcuttur. Buna göre, ≥ 3 ve 5 kez (130 ± 6.8 x 10 6 ve 218 ± 23.6 x 10 6 hücre), BHK-21 numarası (bebek Hamster böbrek) hücreler, T-175 kıyasla büyümüş ve Multi-Flask kurtarıldı matara (43.2 ± 3.5 x 10 6 hücre; Fig.2A). U ortalama Hücre verimnit yüzey alanı 3 eşit miktarda idi - ve 5-katmanlı Multi-şişeler ve BHK-21 T-175 matara, LnCAP (insan prostat adenokarsinom hücre hattı), Hep-G2 (insan hepatokarsinoma hücre hattı), Ecopack 2-293 (insan böbrek hücre hattı) 48-96h hücre satıcısı (ATCC, Sigma ve / veya Clontech) tarafından önerilen büyüme ortamı (katman başına 35 ml) bir süre (Fig.2B) kültüre. Hücreler bir otomatik Vi-HÜCRE sayacı (1) numaralandırılır. <img alt="Şekil 2A" src="/files/ftp_upload/3418/3418fig2A.jpg" /> Şekil 2A: BHK-21 hücre sayısı üç ve beş kez büyüdü ve kurtarılan 3 - ve 5-katmanlı Multi-şişeler T-175 matara. Beklenen verim ortalama hücre verim kontrolü kullanılarak tespit edilmiştir T-175, üç ve beş kez 3 ile çarpılır şişeler ve 5 katmanlı Multi-şişeler sırasıyla (n = 4 matara / format). <img alt="Şekil 2B" src="/files/ftp_upload/3418/3418fig2B.jpg" /><br/&gt; Şekil 2B: cm2 başına Hücre verim, eşdeğer 3 - ve 5-katmanlı Multi-şişeler ve BHK-21, LnCAP, HepG2 ve EcoPack2-293 hücre için T-175 şişeler . Her çubuk, 4 ila 6 şişe anlamına temsil eder. BHK-21 hücreleri (11.000 cells/cm2), 72 saat, LnCAP hücreleri (20.000 cells/cm2) ve EcoPack2-293 hücrelerinde (~ 35.000 cells/cm2) için kültüre edildi 96 saat ve HepG2 hücreleri (25.000 hücre / cm 2 kültüre edildi ) 48 saat için hasattan önce kültüre edildi. 3. Multi-Flask katmanları arasında Medya dağıtım Hücre kültürü, orta (DMEM; Invitrogen) 5 katmanlı Çok şişeler (250 mL/5-layer gemi) eklenir ve protokol yukarıda açıklanan göre katmanlara bölünmüştür. Medya dağıtım bireysel katmanları dışarı pompalanır her katmanı ve medya üzerinde herhangi bir delik ile ölçüldü. Şekil 3&#39;te gösterildiği gibi katman katman nispeten homojen olması için her katman kurtarıldı sıvı ağırlığı bulundu. Bunlar aşağıdaki gibidir: 510,8 ± 0.73, 50.3 ± 0.58, 50.21 ± 0,13, 49,88 ± 0,35, 49,45 ± 0,37 (gr). <img alt="Şekil 3" src="/files/ftp_upload/3418/3418fig3.jpg" /> Şekil 3, her beş kat 5 kat Multi-Flask Tekdüzen medya dağıtım . Hücre kültürü ortamı dengelenmiş ve bireysel katmanların bölümlenmiş Multi-şişeler (250 ml/5-layer gemi) eklendi. Medya bireysel katmanların ve sıvı ağırlığı içine delinmiş deliklerden dışarı pompalanır her katmanı (n = 6 şişeler) kaydedildi. 4. Multi-Flask katmanları arasında Hücre dağıtım Hücreler Multi-Flask içinde eklenir ve karıştırılır. Biz boncuk (10μm, PolySciences Inc.) Kullanarak Multi-Flask katmanları arasında hücre simüle dağıtım boyutu hücrelere benzer. Boncuk süspansiyon üst tabaka ile 10 ml pipet kullanarak Multi-Flask damar içine eklenir ve azalan gibi damar medya ile karıştırılırYukarıdaki protokol ribed. Boncuk boncuk süspansiyonu bireysel katmanların dışarı pompalanır oldu içeren her katmanı ve sıvı delik dağıtımı ile ölçüldü. Her katman kurtarıldı Boncuk konsantrasyonu bir Coulter Sayaç okuma ve kaydedildi. 3-katmanlı Çoklu şişeler (Fig.4A) eşdeğer arası katmanlı boncuk dağılımları aşağıda gösterilmiştir. Mix-port, Multi-Flask katmanları arasında hücreler ve reaktiflerin homojen dağılımını sağlar. <img alt="Şekil 4A" src="/files/ftp_upload/3418/3418fig4A.jpg" /> Şekil 4A: 3-katmanlı bir Multi-Flask üç katmandan her Boncuk dağıtım . (Vol, Medya ses seviyesini 01:10: boncuk süspansiyonu hacim) boncuk süspansiyon (3,6 x10 6 / ml), Multi-şişeler içine dağıtılır orta eklendi ve karışık, dengeleme ve açıklanan protokolünü kullanarak bölümü adımları takip . Her bir katman (orta her katman üzerinde delikler aracılığıyla pompalanan) kurtarıldı Boncuk konsantrasyonu Coulter Sayım okunduer ve kaydedildi. Katmanı Multi-şişeler (n = 5 şişeler) - 3 arası katmanlı eşdeğer boncuk dağılımları aşağıda gösterilmiştir Aşağıda gösterildiği desteklenmiş büyüme medya 3-katmanlı Multi-şişeler&gt;% 80 izdiham yetiştirilen hücreler Ecopack 2-293 boyanma temsilcisi görüntüleri. Hücre mono tabakaları sabit ve kristal viole ile boyandı ve Multi-Flask katmanlar daha sonra kesildi ve görüntü (2) taranır . Not hücre desenlendirme Multi-Flask (Fig.4B) tüm katmanları üzerinde homojen oldu. Birden fazla hücre tipleri değerlendirilir (veriler gösterilmemiştir) ile benzer sonuçlar elde edildi. <img alt="Şekil 4B" src="/files/ftp_upload/3418/3418fig4B.jpg" /> Şekil 4B: Bu rakam, Multi-Flasks katmanları arasında homojen hücre büyüme göstermektedir . 3-katmanlı Multi-şişeler ve T-175&gt;% 80 izdiham büyüdü Ecopack-2-293 hücre sabit ve kristal viole ile boyandı. Multi-Flask damarları kesilmiştir ve her vitray tabaka tarandı. 5. Çoklu-Flask Hava kaynağı. BioProfile FLEX analizörü (3,4) (Nova Biyomedikal) 96 saat için EcoPack2-293 hücre kültürü kullanarak harcanan medya analizi, Multi-5-katmanlı şişeler vs T-175 matara (81,03 yetiştirilen hücreler hava doygunluk fark olmadığını ± 1.9 vs 83.4 ± 5.8% ortam O 2). <img alt="Şekil 5" src="/files/ftp_upload/3418/3418fig5.jpg" /> Şekil 5: Hava harcanan medya saturasyonu (% ortam O 2) 5-katlı Multi-şişeler vs T-175 matara önceden karıştırılmış medya benzer. EcoPack2-293 hücre yoğunluğu 35.000 hücre / cm 2 numaralı seribaşı ve medya analizi (n = 3 şişeler) önce 96 saat kültüre edildi.

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Tüm yazarlar Becton Dickinson, Biosciences Segment Discovery Laboratuvar Birimi çalışanları.

Bu çalışmada, Multi-Flask tasarım araştırmacılara sunduğu verimlilik artış göstermektedir. Multi-şişeler kullanarak optimum performans için yukarıda belirtilen adımları takip etmek önemli olmakla birlikte, bu protokolü en önemli gördüğü bazı kritik adımlar vardır. Bu bölünme Multi-Flask düz yazılan döşeme katmanları (iii) her birine sıvı bölümleme sonra saat yönünde 45 ° &#39;lik açıyla Multi-Flask taşıyan gemi, (ii) içinde karıştırın bağlantı noktasını kullanarak hücrelerin ve reaktifler karıştırma (i) inkübatör. Multi-Flask doğru kullanımı (5) T-175 şişeler için birbiriyle uyumlu bir ortamda kültüre her geminin içinde homojen bir hücre popülasyonu üretimine yol açar. Bu gemiler, T-175 şişesi de benzer bir ayak izi 3 ve 5 kat daha fazla hücre sağlar. 3 ve 5 katlı gemi, sırasıyla 525 ve 875cm 2, büyüme alanı sağlar ve alanı ve işçilik &#39;ın ikisinin de teklifkullanıcılara avings. Doku Kültürü Yüzey İşleme, böylece gerek kalmadan ölçek sağlayan standart şişeler ile karşılaştırılabilir yeniden optimize mevcut kültür koşulları, kalite, homojenlik veya hücreleri (6, 7) performans ödün. Bu aynı zamanda daha önce toplanan verileri ile karşılaştırılabilir sağlar. Bu gemiler aynı zamanda kollajen gibi reaktifler ile kaplı olabilir, hepatositler 8, kertainocytes 9, serum serbest medya yetiştirilen kök hücreler gibi bazı hücre tiplerinin eki, büyüme ve farklılaşma için özel bir alt tabaka sağlamak için poli-D-lizin, fibronektin formülasyonlar. Kaplama çözümler hücreleri kültür öncesinde kaplama çözümü etkili bir şekilde kaldırılması gibi değerli reaktiflerin israfını azaltmak amacıyla minimal rezidüel sıvı retansiyonu ile kaldırılabilir. Multi-şişeler aralığı 25-50 ml katman başına çevirmek 0,142-0,287 ml / cm 2 kültür hücreleri önerilen en iyi medya hacmi. Çok katmanlı bir başka balon, t aksinekendi ürün sağlayan damar-damar karıştırma her bir geminin içinde ve katmanlar arasındaki hücreler ve reaktiflerin tekdüze dağılım olarak aynı zamanda hızlı bir karışımı-port sunuyor. Çeşitli hücre hatları, primer kültürlerinde ve kök hücre Multi-şişeler ile verimli bir şekilde büyütülüyor edilmiştir. Bu gemiler, yüksek kapasiteli tarama, aşı üretimi, viral vektör transfections ve hücre tedavisi gibi hücre çok sayıda talep uygulamalarında özellikle avantajlıdır. Zaman, mekan, emek ve daha az atık üretimi Tasarruf tek katmanlı gemiler geleneksel kültürlere karşı Multi-Flask önemli kazançlar. Biz kültürü 5 hasat hücrelerin sayısı yaklaşık üç kat, T-175, 3 kullanarak aynı alanda bulunan şişeler, 5-katlı Multi-şişeler. Bu avantaj, yer tasarrufu T-175, sadece bunlarla sınırlı değildir, ancak aynı zamanda diğer gemilere uzatılabilir: ortak laboratuar inkübatörler evler sığar bir standart silindir şişe aparatı ~ 4 rol850cm 2 yüzey alanı sağlar ler şişeleri her biri (2200 ml). Aynı bölgede, ~~ 20, 5-katlı Multi-şişeler böylece kültür hücreleri beş kat büyüme yüzey sağlayarak yerleştirilebilir. Ayrıca, &quot;Go Green&quot; farkındalık bir artış ile, atık oluşumunu azaltmak için seçenekleri son derece arzu edilir. Bu bağlamda,% 38, T-175 matara ve bu avantajlar ile karşılaştırıldığında Multi-şişeler kullanarak atık üretiminin azalması kurşun (5, T-175 matara, biri 5 katlı Multi-Flask ~ 400g ağırlığında iken ~ 640g ağırlığında) var atık depolama ve bertaraf maliyetleri ve kullanıcıya ekonomik tasarruf sonucu meydana gelen azalma.

<table border="1"><tbody><tr><td> Reaktif / Ekipman Adı </td><td> Şirket </td><td> Katalog # </td><td> Yorumlar </td></tr><tr><td> 3-katmanlı Doku Kültürü ile tedavi edilen 525cm 2 </td><td> BD Biosciences </td><td> 353143 </td><td rowspan="21"> <a href="http://www.bdbiosciences.com/cellculture/multiflasks/index.jsp">BD Biosciences Hücre Kültürü - BD Falcon Multi-şişeler</a> </td></tr><tr><td> 5-katmanlı Doku Kültürü ile tedavi edilen 875cm 2 </td><td> BD Biosciences </td><td> 353144 </td></tr><tr><td> 100ml pipet </td><td> BD Biosciences </td><td> 357600 </td></tr><tr><td> 10 ml pipet </td><td> BD Biosciences </td><td> 357551 </td></tr><tr><td> 5 ml pipetle aspire </td><td> BD Biosciences </td><td&gt; 357.501 </td></tr><tr><td> 50 ml Polipropilen konik boru </td><td> BD Biosciences </td><td> 352070 </td></tr><tr><td> T-175 şişesi Doku Kültürü ile tedavi edilen </td><td> BD Biosciences </td><td> 353028 </td></tr><tr><td> Gram Kristal menekşe </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> Tripsin-EDTA </td><td> Lonza </td><td> CC-5012 </td></tr><tr><td> Fetal sığır serumu </td><td> Invitrogen </td><td> 16000-044 </td></tr><tr><td> Tryptose Fosfat broth </td><td> Sigma </td><td> T8159 </td&gt; </tr><tr><td> BHK-21 hücre </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> Paraformaldehit </td><td> Elektron Mikroskopi Bilimleri </td><td> 15710 </td></tr><tr><td> Polistiren Boncuk </td><td> PolySciences Inc. </td><td> 24628-20 </td></tr><tr><td> Bio-Profil Flex Analyzer </td><td> Nova Biyomedikal </td><td></td></tr></tbody></table>

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

Giderek artan sayıda hücre tabanlı uygulamalar çok sayıda hücre gerektirir. Hücrelerin çok sayıda gerektiğinde, küçük ölçekli genişleme sırasında yeterli tek katmanlı T-matara, kullanımı, hantal, zahmetli ve zaman alıcı olabilir. Bu ihtiyacı karşılamak için, yeni bir çok katmanlı hücre kültürü geminin performansını kolay ölçekli T-şişeler tek katmanlı hücre kolaylaştırmak için ele alınacaktır. T-175 matara göre 5 kat formatı ve sırasıyla kültür ve tam üç kurtarma ve hücre sayısı beş kat etkinleştirmek - 3 test şişeler mevcuttur. BD Multi-Flask önemli bir özelliği de, damar karıştırma her geminin katmanları içinde ve arasında hücreler ve reaktiflerin yanı sıra tekdüze dağılım ve sürekli olduğu bir ortamda kültüre olabilir hücreleri üretmek hızlı sağlar mix / dengeleme bağlantı noktası T-175 şişeler için uyum. C için bu Multi-şişeler tasarımı da sağlaronvenient pipet şişeler doğrudan reaktifler ve hücreler ekleyerek değerli hücreleri ve reaktiflerin yanı sıra etkin kurtarma için erişim ve dökme için bağlı kontaminasyon riskini azaltır. Dökme pipetleme tercih edilir uygulamalar için, tasarım, değerli hücreleri ve reaktiflerin israfını azaltmak amacıyla minimal rezidüel sıvı retansiyonu sağlar.

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

Hücreler enstrümantal bir araştırma ve artan bir rol oynar ve yeni terapötikler keşfi ve geliştirilmesi. Eklenti bağımlı hücreler büyüyen ve hasat için daha verimli ve etkin şekilde hücreler daha fazla sayıda bu artan ihtiyacı. Doğru özellikleri ile çok katmanlı bir şişenin bu amaca hizmet edebilir.

Yeni çok katmanlı bir balon kullanarak Memeli Hücre Kültürü Ölçeği-Up

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

Nanyang Technological University

Research

JoVE Journal

Biology

37.5K Görüntüleme.  BD Biosciences. Hücreler enstrümantal bir araştırma ve artan bir rol oynar ve yeni terapötikler keşfi ve geliştirilmesi. Eklenti bağımlı hücreler büyüyen ve hasat için daha verimli ve etkin şekilde hücreler daha fazla sayıda bu artan ihtiyacı. Doğru özellikleri ile çok katmanlı bir şişenin bu amaca hizmet edebilir.

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.

Geniş Alan Mikroskop kullanarak Uzun vadeli Görüntüleme Memeli Hücreleri

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Laser Capture Microdissection of Mammalian Tissue

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.

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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.

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Determining Cell Number During Cell Culture using the Scepter Cell Counter

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and accuracy. Cell counts are important for monitoring cell health and proliferation rate, assessing immortalization or transformation, seeding cells for subsequent experiments, transfection or infection, and preparing for cell-based assays. It is important that cell counts be accurate, consistent, and fast, particularly for quantitative measurements of cellular responses.
Despite this need for speed and accuracy in cell counting, 71% of 400 researchers surveyed1 who count cells using a hemocytometer. While hemocytometry is inexpensive, it is laborious and subject to user bias and misuse, which results in inaccurate counts. Hemocytometers are made of special optical glass on which cell suspensions are loaded in specified volumes and counted under a microscope. Sources of errors in hemocytometry include: uneven cell distribution in the sample, too many or too few cells in the sample, subjective decisions as to whether a given cell falls within the defined counting area, contamination of the hemocytometer, user-to-user variation, and variation of hemocytometer filling rate2.
To alleviate the tedium associated with manual counting, 29% of researchers count cells using automated cell counting devices; these include vision-based counters, systems that detect cells using the Coulter principle, or flow cytometry1. For most researchers, the main barrier to using an automated system is the price associated with these large benchtop instruments1.
The Scepter cell counter is an automated handheld device that offers the automation and accuracy of Coulter counting at a relatively low cost. The system employs the Coulter principle of impedance-based particle detection3 in a miniaturized format using a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display. The disposable tip is engineered with a microfabricated, cell- sensing zone that enables discrimination by cell size and cell volume at sub-micron and sub-picoliter resolution. Enhanced with precision liquid-handling channels and electronics, the Scepter cell counter reports cell population statistics graphically displayed as a histogram.

The Scepter Cell Counter is a handheld automated device that can be used to count cells, monitor cell diameter and volume, and be used to check the health and quality of cellular populations from one culture to the next.

Hücre Kültürü sırasında Hücre Sayısını Belirleme Scepter Hücre Sayıcı

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and ...

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.

Memeli Hücreler, Hücre Döngüsü Pozisyon Analizi

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.

Moxi Z Mini Otomatik Hücre Sayacı Kullanımı Memeli Hücre Sayımı, Hücre Boyutu ve Hücre Sağlık Belirlenmesi

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.

Memeli Tüm Embriyo Kültürü için uygun Rat Serum hazırlanması

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.

Hücre geçirgen Cys kullanılarak memeli hücrelerinde doğrudan protein teslim<sub&gt; 2</sub&gt; His<sub&gt; 2</sub&gt; Çinko-parmak Alanlar

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 ...