Optimization of 3-D organotypic primary colonic cultures for organ-on-chip applications.
Optimization of 3-D organotypic primary colonic cultures for organ-on-chip applications.
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DOI：
10.1186/1754-1611-8-9
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发表时间：
2014
影响因子：
5.6
通讯作者：
Allbritton NL
中科院分区：
文献类型：
--
作者：
Ahmad AA;Wang Y;Gracz AD;Sims CE;Magness ST;Allbritton NL
新的进展使结肠上皮干细胞的长期器官型培养成为可能，这些干细胞发育成称为结肠样的结构。类集落蛋白是一种主要的组织来源，可作为开发结肠体外模型的潜在起始材料。结肠隐窝分离和随后的结肠样培养的关键特征尚未被系统地优化，从而影响效率和再现性。在这里，鼠隐窝分离的产量和质量进行了优化，并在微制造的培养装置中测量结肠样培养效率。在螯合缓冲液中60分钟的最佳孵育时间从单个结肠的基质释放280，000 ± 28，000个隐窝，其中79.3%保持完整。使用平均加速度1.5 × g的机械搅拌释放了最高质量的隐窝，其中86%具有明确的管腔。在50%Matrigel中培养导致33 ± 5%的最高类集落形成效率。免疫染色表明，在这些条件下分离的类集落具有干/祖细胞和分化的细胞谱系。测试微加工基底（玻璃、聚苯乙烯、PDMS和环氧光致抗蚀剂：SU-8和1002-F）与结肠样培养物的相容性。PDMS促进含有干/祖细胞的3-D类结肠形成，而其他基质促进由分化细胞组成的2-D上皮单层的生长。改进的隐窝分离和3-D结肠样培养，沿着对微加工基质存在下结肠上皮细胞行为的理解，将支持“器官芯片”方法的发展，用于使用原代结肠上皮的研究。
New advances enable long-term organotypic culture of colonic epithelial stem cells that develop into structures known as colonoids. Colonoids represent a primary tissue source acting as a potential starting material for development of an in vitro model of the colon. Key features of colonic crypt isolation and subsequent colonoid culture have not been systematically optimized compromising efficiency and reproducibility. Here murine crypt isolation yield and quality are optimized, and colonoid culture efficiency measured in microfabricated culture devices. An optimal incubation time of 60 min in a chelating buffer released 280,000 ± 28,000 crypts from the stroma of a single colon with 79.3% remaining intact. Mechanical agitation using an average acceleration of 1.5 × g liberated the highest quality crypts with 86% possessing well-defined lumens. Culture in 50% Matrigel resulted in the highest colonoid formation efficiency of 33 ± 5%. Immunostaining demonstrated that colonoids isolated under these conditions possessed stem/progenitor cells and differentiated cell lineages. Microfabrication substrates (glass, polystyrene, PDMS, and epoxy photoresists: SU-8 and 1002-F) were tested for compatibility with colonoid culture. PDMS promoted formation of 3-D colonoids containing stem/progenitor cells, while other substrates promoted outgrowth of a 2-D epithelial monolayer composed of differentiated cells. Improved crypt isolation and 3-D colonoid culture, along with an understanding of colonic epithelial cell behavior in the presence of microfabrication substrates will support development of ‘organ-on-a-chip’ approaches for studies using primary colonic epithelium.
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影响因子：
16
作者：
Tandon, Nina;Marolt, Darja;Cimetta, Elisa;Vunjak-Novakovic, Gordana
通讯作者：
Vunjak-Novakovic, Gordana
影响因子：
19
作者：
Huh, Dongeun;Hamilton, Geraldine A.;Ingber, Donald E.
通讯作者：
Ingber, Donald E.
影响因子：
3.7
作者：
Basson, MD;Turowski, G;Emenaker, NJ
通讯作者：
Emenaker, NJ
影响因子：
6.1
作者：
Huh, Dongeun;Torisawa, Yu-suke;Ingber, Donald E.
通讯作者：
Ingber, Donald E.
影响因子：
3.7
作者：
Booth, C;O'Shea, JA;Potten, CS
通讯作者：
Potten, CS