Name: culturing and applications of rotating wall vessel bioreactor derived 3d epithelial cell models
Uploaded: 2012-04-03T13:00:00
Description: Watch this Scientific Journal Video about Culturing and Applications of Rotating Wall Vessel Bioreactor Derived 3D Epithelial Cell Models at JoVE.com

Авторы хотели бы поблагодарить Брук Хельм ее техническую экспертизу и Андрей Ларсена за его анализа белков. Эта работа была частично финансируется Альтернативы развития исследований Фонда (MMHK) грант NIH и NIAID передаваемых половым путем и противомикробных средств совместной научно-исследовательский центр IU19 AI062150-01 (MMHK). Мы выражаем глубокую признательность биологии размножения для повторного использования цифр.

Все шаги должны быть выполнены в соответствии BSL-2 в условиях ламинарном боксе. 1. Подготовка STLV биореактора <ol><li> Соберите STLV биореактора в соответствии с протоколом производителя и выполнять детоксикации протоколов для обеспечения стерильности в биореактор. Обложка о...

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Использование технологии RWV биореактор, представленные здесь может дать исследователям возможность для продвижения своей нынешней системе культуры клеток на более физиологически соответствующих органотипической модели культуры клеток. Ячейка RWV биореактор культуры система обеспе?...

<table border="1"><tbody><tr><td> Название реагента </td><td> Компания </td><td> Номер по каталогу </td><td> Комментарии </td></tr><tr><td> Alexa Fluor 488 </td><td> Invitrogen </td><td> A21131 </td><td> Используется в разведении 1:500 </td></tr><tr><td> FACSDiva </td><td> BD </td><td></td><td> Проточный цитометр </td></tr><tr><td> β-антитело tublin </td><td> Calbiochem </td><td> 654162 </td><td> Используется в разведении 1:5000 </td></tr><tr><td> Bio-Plex 2000 </td><td> BioRad </td><td> 171-000205 </td><td> v5 программного обеспечения </td></tr><tr><td> Биореактор и компоненты </td><td> Synthecon </td><td> РКЦ-4 </td><td></td></tr><tr><td> Сотовые фильтр </td><td> BD Сокол </td><td> 352340 </td><td> Размер пор 40 мкм </td></tr><tr><td> Конические трубки (50 мл) </td><td> Гранулирование </td><td> 5-538-60 </td><td></td></tr><tr><td> Покровные </td><td> VWR </td><td> 48366067 </td><td></td></tr><tr><td> Цитокинов шарик комплекты массив </td><td> BioRad </td><td> Пользовательские человека комплект </td><td></td></tr><tr><td> Cytodex бисера </td><td> Сигма </td><td> C3275 </td><td></td></tr><tr><td> DPBS </td><td> Гибко </td><td> 14190 </td><td></td></tr><tr><td> ЭДТА </td><td> Сигма </td><td> ED-500G </td><td> Этилендиаминтетрауксусной кислоты </td></tr><tr><td> Эпителиальные специфических антител (ЕКА) </td><td> Chemicon </td><td> CBL251 </td><td> Используется в 1:50 разбавления </td></tr><tr><td> Эмбриональной телячьей сыворотки (FBS) </td><td> Гибко </td><td> 10438 </td><td> Тепло инактивированная </td></tr><tr><td> HARV (одноразовые) </td><td> Synthecon </td><td> D-405 </td><td></td></tr><tr><td> Соляная кислота </td><td> Сигма </td><td> 258148 </td><td> 37% </td></tr><tr><td> Involucrin антител </td&gt; <td> Сигма </td><td> Я 9018 </td><td></td></tr><tr><td> Микроскопа </td><td> VWR </td><td> 16004-368 </td><td></td></tr><tr><td> МТТ реагент </td><td> MP Biomedicals, ООО </td><td> 194592 </td><td> 3 - (4,5-Dimethylthiazolyl 1-2) -2,5-дифенил тетразолия метила </td></tr><tr><td> MUC1 антител (микроскопия) </td><td> Санта-Крус </td><td> Sc-7313 </td><td> Используется в 1:50 разбавления </td></tr><tr><td> MUC1 антител (проточная цитометрия) </td><td> BD Pharmingen </td><td> 559774 </td><td> Также называется CD227, использовать 20 мкл на тест </td></tr><tr><td> Параформальдегид </td><td> Электронная микроскопия наук </td><td> 15710 </td><td> Препарат до 4% в DPBS </td></tr><tr><td> Петри (маленький) </td><td> BD Сокол </td><td> 353002 </td><td></td></tr><tr><td> Полистирол трубы с фильтром </td><td> BD Сокол </td><td> 352235 </td><td></td></tr><tr><td&gt; Полистирол проточной </td><td> BD Сокол </td><td> 352058 </td><td></td></tr><tr><td> PR-антитело </td><td> DAKO </td><td> M3569 </td><td> Используется в разведении 1:100 </td></tr><tr><td> Продлить Золото </td><td> Invitrogen </td><td> P36931 </td><td> Монтаж средств массовой информации с DAPI </td></tr><tr><td> RNeasy комплект мини </td><td> Qiagen </td><td> 74903 </td><td></td></tr><tr><td> Сульфат натрия додецилсульфата </td><td> Сигма </td><td> 71725 </td><td></td></tr><tr><td> Стерилизация сумка </td><td> VWR </td><td> 11213-035 </td><td></td></tr><tr><td> Краны (в одну сторону) </td><td> Medex </td><td> MX5061L </td><td></td></tr><tr><td> Шприц (10 мл) </td><td> BD </td><td> 309604 </td><td> Луер-Лок наконечник </td></tr><tr><td> Шприц (5 мл) </td><td> BD </td><td> 309603 </td><td> Луер-Лок наконечник </td></tr><tr><td> Голубой Трипан </td><td> Invitrogen </td><td>T10282 </td><td></td></tr><tr><td> VP5 антител </td><td> Санта-Крус </td><td> SC-13525 </td><td> ВПГ-2 антитела клона 6F10, используется в разведении 1:5000 </td></tr></tbody></table>

culturing and applications of rotating wall vessel bioreactor derived 3d epithelial cell models

Cells and tissues in the body experience environmental conditions that influence their architecture, intercellular communications, and overall functions. For in vitro cell culture models to accurately mimic the tissue of interest, the growth environment of the culture is a critical aspect to consider. Commonly used conventional cell culture systems propagate epithelial cells on flat two-dimensional (2-D) impermeable surfaces. Although much has been learned from conventional cell culture systems, many findings are not reproducible in human clinical trials or tissue explants, potentially as a result of the lack of a physiologically relevant microenvironment.
Here, we describe a culture system that overcomes many of the culture condition boundaries of 2-D cell cultures, by using the innovative rotating wall vessel (RWV) bioreactor technology. We and others have shown that organotypic RWV-derived models can recapitulate structure, function, and authentic human responses to external stimuli similarly to human explant tissues 1-6. The RWV bioreactor is a suspension culture system that allows for the growth of epithelial cells under low physiological fluid shear conditions. The bioreactors come in two different formats, a high-aspect rotating vessel (HARV) or a slow-turning lateral vessel (STLV), in which they differ by their aeration source. Epithelial cells are added to the bioreactor of choice in combination with porous, collagen-coated microcarrier beads (Figure 1A). The cells utilize the beads as a growth scaffold during the constant free fall in the bioreactor (Figure 1B). The microenvironment provided by the bioreactor allows the cells to form three-dimensional (3-D) aggregates displaying in vivo-like characteristics often not observed under standard 2-D culture conditions (Figure 1D). These characteristics include tight junctions, mucus production, apical/basal orientation, in vivo protein localization, and additional epithelial cell-type specific properties.
The progression from a monolayer of epithelial cells to a fully differentiated 3-D aggregate varies based on cell type1, 7-13. Periodic sampling from the bioreactor allows for monitoring of epithelial aggregate formation, cellular differentiation markers and viability (Figure 1D). Once cellular differentiation and aggregate formation is established, the cells are harvested from the bioreactor, and similar assays performed on 2-D cells can be applied to the 3-D aggregates with a few considerations (Figure 1E-G). In this work, we describe detailed steps of how to culture 3-D epithelial cell aggregates in the RWV bioreactor system and a variety of potential assays and analyses that can be executed with the 3-D aggregates. These analyses include, but are not limited to, structural/morphological analysis (confocal, scanning and transmission electron microscopy), cytokine/chemokine secretion and cell signaling (cytometric bead array and Western blot analysis), gene expression analysis (real-time PCR), toxicological/drug analysis and host-pathogen interactions. The utilization of these assays set the foundation for more in-depth and expansive studies such as metabolomics, transcriptomics, proteomics and other array-based applications. Our goal is to present a non-conventional means of culturing human epithelial cells to produce organotypic 3-D models that recapitulate the human in vivo tissue, in a facile and robust system to be used by researchers with diverse scientific interests.

Watch this Scientific Journal Video about Culturing and Applications of Rotating Wall Vessel Bioreactor Derived 3D Epithelial Cell Models at JoVE.com

Culturing and Applications of Rotating Wall Vessel Bioreactor Derived 3D Epithelial Cell Models

Вращающейся системе культуры клеток эпителия, который позволяет клеткам расти в физиологических условиях в результате 3-D сотовой формировании совокупного описано. Агрегаты порожденных дисплей<em&gt; В естественных условиях</em&gt;-Подобных характеристик не наблюдается в обычных моделях культуры и служить более точным органотипической модельной системой для множества научных исследований.

Культивирование и применение вращающихся стен биореактора полученных 3D эпителиальных клеточных моделях

Cells and tissues in the body experience environmental conditions that influence their architecture, intercellular communications, and overall functions. ...

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