Capture and 3D culture of colonic crypts and colonoids in a microarray platform.
Capture and 3D culture of colonic crypts and colonoids in a microarray platform.
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DOI：
10.1039/c3lc50813g
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发表时间：
2013-12-07
期刊：
影响因子：
6.1
通讯作者：
Allbritton NL
中科院分区：
文献类型：
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作者：
Wang Y;Ahmad AA;Shah PK;Sims CE;Magness ST;Allbritton NL
隐窝是结肠上皮的基本结构和功能单位，可以从结肠中分离并在体外培养成称为“类结肠”的多细胞球体。隐窝和类结肠都是构建结肠体外组织模型的理想构件。在这里，我们提出并测试了一个微工程平台，用于捕获和体外3D培养结肠隐窝和类结肠。使用聚二甲基硅氧烷制造集成平台，该平台包含两个流体层，两个流体层由圆柱形微孔阵列（直径150 μm，深度150 μm）隔开，微孔底部穿孔（开口30 μm，深度10 μm），称为“微滤器”。当流体移动通过阵列时，隐窝或类结肠以>90%的阵列填充效率保留在微滤器中。然后将作为细胞外基质的基质胶应用于微滤器以产生包封隐窝或类结肠的分离的基质胶袋。在提供必需的生长因子、表皮生长因子、Wnt-3A、R-spondin 2和头蛋白后，在微滤器中培养48-72小时的时间段内，63±13%的隐窝和77±8%的类结肠形成有活力的3D类结肠。因此，阵列上的类集落生长与标准培养条件下的生长相似（78±5%）。此外，类集落显示出与标准培养条件下相同的形态和相似数量的干细胞和祖细胞。免疫荧光染色证实，结肠的分化细胞类型，杯状细胞，肠内分泌细胞和吸收肠上皮细胞，形成的阵列。为了证明阵列在追踪类集落命运中的效用，对暴露于诸如Wnt-3A和γ-分泌酶抑制剂LY-411575的试剂的阵列化类集落进行定量荧光分析。在微工程平台上成功形成有活力的多细胞型结肠组织代表了构建“芯片上的结肠”的第一步，其目标是为受控实验产生结肠的生理结构和器官水平功能。
Crypts are the basic structural and functional units of colonic epithelium and can be isolated from the colon and cultured in vitro into multi-cell spheroids termed “colonoids”. Both crypts and colonoids are ideal building blocks for construction of an in vitro tissue model of the colon. Here we proposed and tested a microengineered platform for capture and in vitro 3D culture of colonic crypts and colonoids. An integrated platform was fabricated from polydimethylsiloxane which contained two fluidic layers separated by an array of cylindrical microwells (150-μm diameter, 150-μm depth) with perforated bottoms (30-μm opening, 10-μm depth) termed “microstrainers”. As fluid moved through the array, crypts or colonoids were retained in the microstrainers with a >90% array-filling efficiency. Matrigel as an extracellular matrix was then applied to the microstrainers to generate isolated Matrigel pockets encapsulating the crypts or colonoids. After supplying the essential growth factors, epidermal growth factor, Wnt-3A, R-spondin 2 and noggin, 63±13% of the crypts and 77±8% of the colonoids cultured in the microstrainers over a 48–72 h period formed viable 3D colonoids. Thus colonoid growth on the array was similar to that under standard culture conditions (78±5%). Additionally the colonoids displayed the same morphology and similar numbers of stem and progenitor cells as those under standard culture conditions. Immunofluorescence staining confirmed that the differentiated cell-types of the colon, goblet cells, enteroendocrine cells and absorptive enterocytes, formed on the array. To demonstrating the utility of the array in tracking the colonoid fate, quantitative fluorescence analysis was performed on the arrayed colonoids exposed to reagents such as Wnt-3A and the γ-secretase inhibitor LY-411575. The successful formation of viable, multi-cell type colonic tissue on the microengineered platform represents a first step in the building of a “colon-on-a-chip” with the goal of producing the physiologic structure and organ-level function of the colon for controlled experiments.
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影响因子：
6.1
作者：
Grosberg A;Alford PW;McCain ML;Parker KK
通讯作者：
Parker KK
影响因子：
19
作者：
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56.9
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影响因子：
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