Microfabricated Arrays for Splitting and Assay of Clonal Colonies
Microfabricated Arrays for Splitting and Assay of Clonal Colonies
复制标题
DOI：
10.1021/ac301895t
复制
发表时间：
2012-12-18
影响因子：
7.4
通讯作者：
Allbritton, Nancy L.
中科院分区：
文献类型：
--
作者：
Gach, Philip C.;Xu, Wei;Allbritton, Nancy L.
开发了一种用于高度并行和高效的菌落挑选、分裂和克隆鉴定的微加工平台。托盘阵列提供图案化的细胞集落，其与第二印刷阵列配对，所述第二印刷阵列由支撑基底和附接的柱形成的桥接微结构组成。这些柱子使最初在托盘阵列上培养的集落中的哺乳动物细胞能够迁移到打印阵列上的相应位点。阵列的分离同时分裂菌落，产生图案化的复制品。优化的阵列元件提供的转移效率大于90%，使用30 μ m直径和100 μ m长度和3000总菌落数的桥接柱。使用五种哺乳动物细胞系的研究表明，可以培养各种贴壁细胞类型，并以78 - 92%的打印效率有效地分裂。为了证明该技术的实用性，克隆细胞系与冠状蛋白1B的siRNA敲低产生使用阵列和比较传统的流式细胞仪/蛋白质印迹为基础的方法。靶克隆的鉴定需要破坏性测定来鉴定由于干扰shRNA构建体的成功感染而导致的冠状蛋白1B缺失的细胞。凭借小型化及其并行格式，该平台能够从仅3900个细胞的起始样品中鉴定和产生12个靶克隆，并且在11天内仅需要5个工时。相比之下，传统方法需要500，000个细胞，仅产生5个靶克隆，在47天内花费34个工时。这些数据支持使用小型化平台通过破坏性测定进行克隆选择与常规方法相比在时间、人力和试剂方面的显著减少。
A microfabricated platform was developed for highly parallel and efficient colony picking, splitting, and clone identification. A pallet array provided patterned cell colonies which mated to a second printing array composed of bridging microstructures formed by a supporting base and attached post. The posts enabled mammalian cells from colonies initially cultured on the pallet array to migrate to corresponding sites on the printing array. Separation of the arrays simultaneously split the colonies, creating a patterned replica. Optimization of array elements provided transfer efficiencies greater than 90% using bridging posts of 30 mu m diameter and 100 mu m length and total colony numbers of 3000. Studies using five mammalian cell lines demonstrated that a variety of adherent cell types could be cultured and effectively split with printing efficiencies of 78-92%. To demonstrate the technique's utility, clonal cell lines with siRNA knockdown of Coronin 1B were generated using the arrays and compared to a traditional FACS/Western Blotting-based approach. Identification of target clones required a destructive assay to identify cells with an absence of Coronin 1B brought about by the successful infection of interfering shRNA construct. By virtue of miniaturization and its parallel format, the platform enabled the identification and generation of 12 target clones from a starting sample of only 3900 cells and required only S man hours over 11 days. In contrast, the traditional method required 500,000 cells and generated only 5 target clones with 34 man hours expended over 47 days. These data support the considerable reduction in time, manpower, and reagents using the miniaturized platform for clonal selection by destructive assay versus conventional approaches.