Biz U. Ganapati mStrawberry OP9 hücre hattı kurulması ve test için teşekkür ederiz. Ayrıca, ko-kültür ile H. Shafifor ona yardım teşekkür ederim. HP, Ulusal Kalp, Akciğer ve Kan Enstitüsü (F31HL087714) (HP) bir arkadaş grubu tarafından desteklenmektedir. Bu çalışmanın içeriği yalnızca yazarın sorumluluğundadır ve Ulusal Kalp, Akciğer ve Kan Enstitüsü ve Ulusal Sağlık Enstitüleri (NIH) mutlaka resmi görüşlerini temsil etmemektedir. UCLA Sitometrisi Core Tesis NIH (CA-16.042 ve AI-28.697), Jonsson Kanser Merkezi, UCLA AIDS Enstitüsü ve UCLA Tıp Okulu tarafından desteklenmektedir.

1. ES hücrelerinin hazırlanması <ol><li> Özellikle hücre hattı için standart protokole göre, Kültür ES hücrelerinin. Gün 0 mStrawberry OP9 hücreleri üzerine plakası için yeterli ES hücrelerinin hazırlamak için emin olun (ES hücreleri Gün 0 cm 2 başına 1x10 ^ 3 hücre kaplama olacak) </li></ol> 2. OP9 hücrelerin hazırlanması <ol><li> Büyüyen mStrawberry OP9 hücreleri önce OP9 büyüme ortamı hazırlamak OP-9 Hücre Kültürü Orta <table border="1"><tbody><tr><td></td><td> Hisse senedi </td><td> Final kons. </td><td> Hacim (ml) </td></tr><tr><td> aMEM </td><td></td><td></td><td> 39,5 </td></tr><tr><td> FBS (OP-9 test) </td><td> % 100 </td><td> % 20 </td><td> 10 </td></tr><tr><td> L-glutamin </td><td> 100X (200mm) </td><td> 2mm </td><td> 0,5 </td></tr><tr><td></td><td></td><td></td><td> 50ml </td></tr></tbody></table> 4 ° C ve bir hafta içinde hazırlık Mağaza </li><li> MStrawberry OP9 hücreleri korumak için, medya, 2 günde değişti olmalıdır ve hücreler (% confluency 70-90 altkültürü) confluency ulaşan hücrelere önce pasajlandı olmalıdır. bu adipositlere farklılaşma sonucu olarak mStrawberry OP9 hücre kültürlerinde% 90 confluency hiç ötesinde büyüdü. Şekil 3&#39;e bakın. <ol class="lalpha"><li> PBS (veya tripsin-EDTA ile PBS + 1x% 0.1 ile 1x) ile 2X yıkama hücreleri tarafından Altkültür hücreleri </li><li> 37 ° önceden ısıtılmış% 0.1 tripsin-EDTA ° C 5 dakika boyunca (veya hücreleri ayırarak kadar) </li><li> Hücreleri müstakil, OP-9 büyüme orta ekleyin. 50ml konik tüp hücreleri aktarın. </li><li> Oda sıcaklığında 5 dakika boyunca 300 xg&#39;de Pelet hücreleri supernatant atın. </li><li> Taze mStrawberry OP9 büyüme ortamı içinde süspanse edin hücreleri </li><li> 10 4 hücre / cm 2, en az yoğunlukta Plaka hücreleri </li><li> Grow için gerekli ko-kültür ES hücreleri (0. Gün, Gün 5, ya da deney 8.Gün / 9) gün önce 3 gün başlayan confluency mStrawberry OP9 hücreleri </li></ol></li></ol> 3. ES / mStrawberry OP9 Co-kültür <ol><li> Co-kültür farklılaşmasını ortamı hazırlayın <table border="1"><tbody><tr><td></td><td> Hisse senedi </td><td> Son </td><td> Hacim (ml) </td></tr><tr><td> aMEM </td><td></td><td></td><td> 39,5 </td></tr><tr><td> FBS </td><td> % 100 </td><td> % 20 </td><td> 10 </td></tr><tr><td> L-glutamin </td><td> 200mm </td><td> 2mm </td><td> 0,5 </td></tr><tr><td></td><td></td><td></td><td> 50ml </td></tr></tbody></table></li><li> mStrawberry OP9 hücreleri (Gün -3) <ol class="lalpha"><li> DAİMA Gün -3 OP9 hücreleri konfluent ko-kültür Günü 0 mStrawberry OP9 hücrelerinin katmanları var, böylece Altkültür. Bkz: Şekil 4b. Gün 0 &#39;confluency için 1x10 4 / cm 2 Levha mStrawberry OP9 hücreleri. Gün 5 ve eş-kültür devamı için 8 / 9 Gün altkültürü ekstra hücrelere emin olun. </li></ol></li><li class="jove_content"> ES / mStrawberry OP9 ko-kültür Set-Up (Gün 0) <ol class="lalpha"><li class="jove_content"> 2x ES hücreler PBS ile yıkayın </li><li> Önceden ısıtılmış% 0.25 &#39;lik tripsin-EDTA, inkübasyon ES hücrelerinin Trypsinize 5min @ 37 ° C </li><li> Co-kültür farklılaşmasını medya hücreleri ekle </li><li> Pelet ES hücreleri 400xg az, 5-7dk, RT. Süpernatant atın </li><li> Co-kültür farklılaşmasını medyada yeniden süspanse hücre </li><li> MStrawberry OP9 hücreleri medya kaldırma </li><li> MStrawberry OP9 şişeler Co-kültür farklılaşmasını Medya ekle </li><li> MStrawberry OP9 hücreleri balona Plaka 1x10 3 ES hücre / cm 2. 37 ° hücreleri ° C&#39;de,% 5 CO 2 nem ile. </li></ol></li><li> mStrawberry OP9 hücreleri (2. Gün) <ol class="lalpha"><li> Altkültür mStrawberry OP9 konfluent ko-kültür 5. Gün mStrawberry OP9 hücrelerinin katmanları var. 5. Gün confluency için 1x10 4 / cm 2 Levha mStrawberry OP9 hücreleri (adım 3.2 &#39;deki gibi). Gün 8 / 9, gerekirse (adım 2.2 &#39;de olduğu gibi) için altkültürü ekstra hücrelere emin olun. </li></ol></li><li> ES / mStrawberry OP9 ko-kültür (Gün 3) <ol class="lalpha"><li> Co-kültür dikkatlice orta kaldırmak ve Co-kültür farklılaşması orta taze ile değiştirin </li></ol></li><li> ES / mStrawberry OP9 ko-kültür (5. Gün) <ol class="lalpha"><li> PBS ile 2x ortak kültürler dikkatlice yıkayın </li><li> Önceden ısıtılmış% 0.25 tripsin-EDTA içeren hücreleri Trypsinize </li><li> Co-kültür farklılaşmasını medya ile şişeyi kapalı hücreler yıkayın </li><li> 400xg Pelet hücreleri, 7dk, RT supernatant atın </li><li>Hücrelere Co-kültür farklılaşmasını Medya ekle hücreleri tekrar süspansiyon haline getirin ve tek hücre süspansiyonu sağlamak için 21-gauge bluntended iğne kullanın </li><li> Hemasitometre kullanarak hücreleri saymak. Gün 0 ES hücre sayısı 100-150 kat artış olması gerekir. </li><li> Ynt tohumu 6.48x10 4 / cm 2 - birleşmiş, mStrawberry OP9 hücreler yeni bir katmana 7.76x10 4 / cm 2 ko-kültür hücreleri </li><li> 5. Gün Tahliller-ko-kültür hücreleri Kalan flow sitometri analizi, cytopreps (30K hücreleri), ChIP testleri, vb. </li></ol></li><li> ES / mStrawberry OP9 ko-kültür (Gün 8 / 9 ve 12 Gün) <ol class="lalpha"><li> Şişeyi ayırt medyayı çıkartın ve saklayın. Süspansiyon hematopoietik hücreleri çıkarmak için PBS ile yıkayın tek tabaka 1x hematopoetik hücreler dışarı atmak. </li><li> Önceden ısıtılmış% 0.25 tripsin-EDTA ile yapışık hücrelere Trypsinize </li><li> ES farklılaşma medya ile balon kapalı hücre yıkayın ve süspansiyon = bulunan hematopoietik hücreleri eklemek </li><li> 400xg Pelet hücreleri, 7dk, RT supernatant atın </li><li> Co-kültür farklılaşmasını medya veya PBS hücrelere ekleyin. Hücreleri tekrar süspansiyon ve hemasitometre kullanarak tek hücre süspansiyonu Sayısı hücrelerin sağlamak için 21-gauge bluntended iğne kullanın. </li><li> Co-kültür, 12 gün sonra tekrar yeni bir katman üzerine Gün 8 ya da 9 birleşik, mStrawberry OP9 hücreleri yeniden tohumlama ko-kültür hücreleri tarafından daha olgun hematopoietik atalarıdır değerlendirmek ve 14 gün süreyle devam edilebilir. Ynt tohumu gün tahlil için gerekli hücre kurtarma dayalı optimize edilmiş hücre sayısı. </li></ol></li><li> ES-kökenli döl (Gün 1-14) değerlendirilmesi. ES-türetilmiş döl mStrawberry negatif hücreler (ES-türetilmiş döl) sıralama akış eş-kültür her günü değerlendirilebilir. <ol class="lalpha"><li> Şişeyi ayırt medyayı çıkartın ve saklayın. PBS ile yıkayın tek tabaka 1x süspansiyon hematopoietik hücreleri çıkarmak için. Hematopoetik hücreler dışarı atmak değil. </li><li> Önceden ısıtılmış% 0.25 tripsin-EDTA ile yapışık hücrelere Trypsinize </li><li> Co-kültür farklılaşmasını medya ile yıkayın ve şişeyi kapalı hücre süspansiyon bulunan hematopoietik hücrelere </li><li> 400xg Pelet hücreleri, 7dk, RT. Süpernatant atın </li><li> 21-gauge bluntended iğne sayısı ile yeniden süspanse hücreleri </li><li> ES-kaynaklı döl (mStrawberry negatif hücreler) mStrawberry olumlu OP9 hücreleri sıralanır akışı olabilir. ES-kaynaklı döl sonra flow sitometri analizi, cytopreps, ChIP testleri, vb ile karakterize edilebilir </li></ol></li></ol> 4. Temsilcisi Sonuçlar: <img src="/files/ftp_upload/2559/2559fig1.jpg" alt="Şekil 1" /> Şekil 1. ES / mStrawberry OP9 Co-kültür Sistemi laboratuar model hematopoetik geliştirme in vitro ES hücre ko-kültür sistemi kullanır. Konfluent mStrawberry OP9 hücrelerinin bir katman üzerine yerleştirildiği zaman, bu ko-kültür sisteminde, ES hücreleri, gün5 hemangioblasts içine ayırt. Co-kültür ilerledikçe, hematopoietik öncüleri terminal farklılaşmış hematopoetik soy 14. Gün görünür, Gün 8 mevcuttur. Gerekli de mStrawberry OP9 hücrelerinin yeni konfluent katmanları üzerine ES-kökenli döl Pasajlanması zaman Oklar farklılaşma gün göstermektedir. <img src="/files/ftp_upload/2559/2559fig2.jpg" alt="Şekil 2" /> Şekil 2. Uygun ES hücre Farklılaşma. Embriyonik kök hücrelerin, Gün 0 &#39;mStrawberry OP9 konfluent tabakası üzerine numaralı seribaşı. ES-türetilmiş döl ES-kökenli döl tanımlanmış bir sınır ile bir hücre kümesi gibi görünen veya bir girdap desen oluşmaya başlayan farklılaşma 3. Gün görünür hale başlar. Gün 5 hücre loca yığılmış olarak endotel ve hematopoetik soy için ortak mezodermal habercisi Hemangioblasts, görünmelidir. Co-kültür beş gün daha uzun süreler için, ES-türetilen döl hematopoetik hücre kümeleri gibi görünmelidir. Iki ila dört hücreli kümeler, 6 / 7 Gün görünmelidir ve co-kültür Günü 8 / 9 tarafından büyük bir hücre kümeleri içine büyümek. Diğer ES türetilen döl sıkıca yapışık stromal hücre katmanı bağlı bulunmaktadır. <img src="/files/ftp_upload/2559/2559fig3.jpg" alt="Şekil 3" /> Şekil 3. Bu ko-kültür Başarısız ES hücre farklılaşması. Sonuçları uygunsuz ES hücre farklılaşması yansıtmaktadır. ES-kaynaklı döl 5. Gün bir girdap desen görünmüyor nasıl unutmayın. Bir girdap desen hücrelerin yokluğunda yanlış hemangioblast oluşumu gösterir. Hemangioblast düzgün geliştirmek değilseniz, sonra ko-kültür bodur hematopoetik soy farklılaşma neden olacaktır. Uygunsuz hemangioblast oluşumu ile birlikte kültürler atılmalıdır. <img src="/files/ftp_upload/2559/2559fig4.jpg" alt="Şekil 4" /> Şekil 4. mStrawberry OP9 hücre kültürleri (A) mStrawberry OP9 hücre pasajlandı.Confluency 70-90%. (B) uygun farklılaşması için, ES hücreleri ve ES-türetilen döl mStrawberry OP9 hücrelerinin aşırı konfluent bir tabaka üzerine yerleştirilir. <img src="/files/ftp_upload/2559/2559fig5.jpg" alt="Şekil 5" /> Şekil 5. Temsilcisi ES / mStrawberry OP9 Co-kültür akış profili. ES-kökenli döl mStrawberry negatif hücreler ve mStrawberry OP9 hücrelerden sıralaması akışı olabilir. mStrawberry negatif hücre kapısı geleneksel bir ES/OP9 ko-kültür kullanılarak tespit edildi.

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Eş-kültür Gün 5 Flk1 + hemangioblasts üretiminde mStrawberry OP9 hücreleri sonuçları konfluent katman üzerinde ES hücrelerinin doğru farklılaşma. Hemangioblasts Şekil 1&#39;de görüldüğü gibi hücre loca gibi yığılı görünmelidir. Co-kültür geri kalanı için, görünür ES-türetilen döl hematopoetik hücre kümeleri (Şekil 1) olarak görünmelidir. Gün6 7 / 03:58 hücre kümeleri görünmelidir ve co-kültür Günü 8 / 9 büyük bir hücre kümeleri (yapışık ve süspansiyon) içine büyümeye. Diğer ES türetilen döl sıkıca yapışık stromal hücre katmanı içinde bağlı bulunmaktadır. Protokolde yer olmasa da, FBS ES büyüme ortamı, mStrawberry OP9 büyüme ortamı ve ES / mStrawberry OP9 farklılaşma medya kullanılan ön test için kritik öneme sahiptir. Düzgün serum ko-kültür uygun mStrawberry OP9 hücrelerinin büyüme yanı sıra, ES hücreleri düzgün farklılaşma farklılaşma Gün 5 kadar emin olmalıdır test edilmiştir. MStrawberry OP9 hücrelere ek olarak, alternatif olarak veya OP9 hücreleri (7.8x10 4 hücre / cm 2) confluency ekim öncesinde, 8000 rad, ışınlanmış olabilir. OP9 hücreleri ışınlanması downstream deneyleri OP9 hücre kirlenmesine neden olan, neredeyse tamamen ortadan kaldırılması için eski sağlar yanı sıra, tekrarlanan replating adımları ortadan kaldırılması için izin verir. OP9 hücreleri CMV organizatörü (pRRLsin CMV vektör, UCLA vektör çekirdek) kontrolü altında bir lentiviral vektör ifade mStrawberry 3ug/mL OP9 hücreleri uyum mStrawberry protein ifade etmek için tasarlanmıştır. Standart ES/OP9 ko-kültür protokolleri yanı sıra, OP9 hücreleri hücre geçit kirlenmesine, eski azaltmak amacıyla, bir konfluent katmanı OP9 hücreleri üzerine bir gün 5 replating adım ES hücrelerinin yerleştirilmesi gerektirecektir. Ayrıca, sadece süspansiyon bulundu hematopoetik hücreler ES-kökenli döl değerlendirilmesi için kaldırılır. Ancak, bir replating sadece süspansiyon hücrelerinin adım ve hasat ile bir potansiyel, gelişmekte olan önemli sayıda ES-kaynaklı hematopoetik hücreler özlüyor. Bu sorun, nakavt ES hatları ile ilgili hematopoetik kusurları karakterize etmek için çalışırken ele almak önem kazanmaktadır. Bu sorunu aşmak için amacıyla laboratuvar OP9 hücreleri mStrawberry ifade kullanımı yoluyla değiştirilmiş bir ko-kültür kullanılır. Bu tüm ES döl 5 gün devam eden ko-kültür transfer olmasını sağlar, aşağı testleri için tüm ES-türetilen döl hasat için. Bu değişiklik, değerlendirme ve karakterizasyonu, insan ve fare ES hatları türetilen hematopoietik döl yararlı araçlar sağlar. Çeşitli aşamalarında, ilkel ve kesin hematopoez Bu ES-mStrawberry OP9 ko-kültür modeli özetlediği. Hematopoetik gelişiminin erken evrelerinde incelerken, birçok kişi, ES hücre hemangioblast gelişimini değerlendirmek, BL-CFC (koloni oluşturan hücre Patlamaya gibi) testi kullanır. BL-CFC testinin erken hematopoetik gelişimi araştıran yararlı bir araçtır hemangioblast değerlendirilmesi yanı sıra, daha yetişkin hematopoetik soy sağlar mStrawberry-ES, ko-kültür sistemleri artış yardımcı programı sergiler. Için, HoxB4, aşırı ekspresyonunun gibi, belki de gerekli ek faktörler hematopoietik kök hücrelerinin in vitro repopulating uzun vadeli elde etmek için, geleneksel OP9/ES ve EB farklılaşma protokolleri kullanarak Önceki iş göstermiştir. Ek iş mStrawberry negatif, ES-kökenli nüfus sıralaması doğru hematopoietik kök hücreleri içeren olup olmadığını değerlendirmek için gereklidir.

OP-9 Hücre Bakım Orta Stok Bileşenleri <table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog Numarası </td><td> Yorumlar </td></tr><tr><td> aMEM </td><td> Invitrogen </td><td> 12571-063 </td><td></td></tr><tr><td> FBS </td><td> Omega Bilimsel </td><td> FB01 </td><td> Önceden-Tested </td></tr><tr><td> L-glutamin </td><td> Cellgro </td><td> 25-005-CI </td><td> 200 nm </td></tr><tr><td> Tripsin-EDTA </td><td> Stem Hücre Teknolojileri </td><td> 07901 </td><td></td></tr></tbody></table> ES/OP-9 Hücre Farklılaşma Orta Stok Bileşenleri <table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog Numarası </td><td> Yorumlar </td></tr><tr><td> aMEM </td><td> Invitrogen </td><td> 12571-063 </td><td></td></tr><tr><td> FBS </td><td> Hyclone </td><td> SH30070 </td><td> Önceden-Tested </td></tr><tr><td> L-glutamin </td><td> Cellgro </td><td> 25-005-CI </td><td> 200mm </td></tr><tr><td> PBS (+ ve Mg 2 + Ca 2 olmadan) </td><td> Cellgro </td><td> 21-031-CV </td><td></td></tr><tr><td> Tripsin-EDTA </td><td> Stem Hücre Teknolojileri </td><td> 07901 </td><td></td></tr></tbody></table>

modified es / op9 co-culture protocol provides enhanced characterization of hematopoietic progeny

In vivo ve embriyonik lethalities ile uğraşmak zorunda kalmadan, hematopoez dahil erken genleri tanımlamak için ulaşılması zor olan hücre popülasyonlarının çalışılırken, hematopoetik hücre kaderinin doğru ES hücrelerinin in vitro farklılaşması yararlıdır . OP9 stromal hücre hattı M-CSF fonksiyonel eksikliği osteopetrosis mutant farelerin calvaria türetilen ES/OP9 ko-kültür sisteminin ilk olarak makrofajların aşırı üretim olmadan, hematopoetik döl üretmek için dizayn edilmiştir. In vitro ES/OP9 ko-kültür sisteminde erken hematopoetik gelişimi özetlemek amacıyla kullanılabilir. OP9 stromal hücrelerinin kültüre, ES hücreleri FLK-1 + hemangioblasts, hematopoetik atalarıdır ve nihayet olgun, terminal farklılaşmış soy farklılaşırlar. Standart ES/OP9 ko-kültür protokolü OP9 hücrelerinin konfluent tabakası üzerine ES hücrelerinin yerleştirme gerektirir; OP9 hücreleri kirlenmesine neden olan, eski kaldırmak için yanı sıra, periyodik replating adımları. Ayrıca, mevcut protokolleri sadece süspansiyon bulundu hematopoetik hücrelerin değerlendirilmesi ve farklılaşma her gün ES-kökenli döl değerlendirilmesi için optimize edilmiş değildir. Ancak, adımları ve sadece süspansiyon hücrelerinin hasat replating bir potansiyel ES-kökenli döl ve hematopoietik hücreleri gelişmekte olan büyük bir bölümünü özlüyor. Bu sorun, nakavt ES hatları ile ilgili hematopoetik kusurları karakterize etmek için çalışırken ele almak önem kazanmaktadır. Burada downstream deneyleri OP9 hücre kirlenmesine neden ortadan kaldırılması için izin veren bir değiştirilmiş ES / mStrawberry OP9 ko-kültür, açıklar. Bu yöntem, embriyo içinde gözlenen hematopoetik farklılaşma desen daha doğrudan karşılık gelen bir hematopoetik farklılaşma desen, tam bir değerlendirme için, eş-kültür her gün tüm ES-türetilen döl verir.

Watch this Scientific Journal Video about Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny at JoVE.com

Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny

mStrawberry OP9 hücreleri ko-kültür tüm ES-türetilen döl tam değerlendirme için izin verir.

Modifiye ES / OP9 Co-Kültür Protokolü Hematopoetik Döl Geliştirilmiş Karakterizasyonu sağlar.

The in vitro differentiation of ES cells towards a hematopoietic cell fate is useful when studying cell populations that are difficult to access in vivo ...

modified-es-op9-co-culture-protocol-provides-enhanced

Research

JoVE Journal

Biology

20.6K Görüntüleme.  University of California, Los Angeles. mStrawberry OP9 hücreleri ko-kültür tüm ES-türetilen döl tam değerlendirme için izin verir.

Video: Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny

Freezing Human ES Cells

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove residual media that could otherwise quench the Trypsin reaction.  Warmed 0.05% Trypsin-EDTA is then added to cover the cells, and the plate allowed to incubate for up to 5 mins at room temperature.  During this time cells can be observed rounding, and colonies lifting off the plate surface.  Gentle repeated pipetting will remove cells and colonies from the plate surface.  Trypsinized cells are placed in a standard conical tube containing pre-warmed hES cell media to quench remaining trypsin, and then spun.  Cells are resuspended growth media at a concentration of approximately one million cells in one mL of media, a concentration such that one frozen aliquot is sufficient to resurrect a culture on a 10cm plate.  After cells are adequately resuspended, ice cold freezing media is added at equal volume.  Cell suspensions are mixed thoroughly, aliquoted into freezing vials, and allowed to slowly freeze to -80C over 24 hours.  Frozen cells can then moved to the vapor phase of liquid nitrogen for long term storage, or remain at -80 for approximately six months.

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.

Donma İnsan ES Hücreler

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove ...

Biyoloji

Human ES cells: Starting Culture from Frozen Cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator.  A frozen vial of cells from long term liquid nitrogen storage or a -80C freezer is sourced and quickly submerged in a 37C water bath for quick thawing.  Cells in freezing media are then removed from the vial and placed in a large volume of HuES media.  The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate.  Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate.  It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate.  The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.

Donmuş Hücreler itibaren Kültür: İnsan Embriyonik kök hücrelerin

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

ES Cell-derived Neuroepithelial Cell Cultures

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In general, the differentiation of ES cells follows the concept to first generate immature progenitor cells, which then can be propagated and differentiated into mature cellular phenotypes. This also applies for ES cell-derived neurogenesis, in which the development of neural cells follows two major steps: First, the derivation and expansion of immature neuroepithelial precursors and second, their differentiation into mature neural cells. A common method to produce neural progenitors from ES cells is based on embryoid body (EB) formation, which reveals the differentiation of cells from all germ layers including neuroectoderm. An alternative and more efficient method to induce neuroepithelial cell development uses stromal cell-derived inducing activity (SDIA), which can be achieved by co-culturing ES cells with skull bone marrow-derived stromal cells (1). Both, EB formation and SDIA, reveal the development of rosette-like structures, which are thought to resemble neural tube- and/or neural crest-like progenitors. The neural precursors can be isolated, expanded and further differentiated into specific neurons and glia cells using defined culture conditions. Here, we describe the generation and isolation of such rosettes in co-culture experiments with the stromal cell line MS5 (2-5).

Derivation of neuroepithelial precursors from embryonic stem (ES) cells using stromal cell-derived inducing activity (SDIA).

ES Hücre türetilen nöroepitelyal Hücre Kültürleri

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In ...

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

İnsan Embriyonik Kök Hücre (ES) yayılması

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Nörotoksisite Yüksek İçerik Analizi Nöronal ve Astrosit Hücre Co-Kültür Testi

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

Çok bölmesi MSS Nöron glia Co-kültür mikroakışkan Platformu

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

Çözünür Sinyal aracılı Etkileşimleri Araştırılması için Epitel Hücreleri ve Bakteriler mikroakışkan Co-kültür

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

Üç boyutlu Karışık-hücre Sfero Co-kültürden Meme Epitel Hücrelerinin İzolasyonu

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a &#8216;targeting&#8217; vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term &#8216;subcloning plus insertion&#8217;, which can be used to generate custom gene targeting vectors rapidly.

Alt klonlama Artı Ekleme (SPI) - Gen Hedefleme Vektörler Rapid İnşaat için Yeni Bir Yöntem Recombineering

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

Hücre-Hücre İletişim Yokluğunda iki Hücresel Türleri Ekleme Co-kültür Sisteminin Geliştirilmesi

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...