Attayek Peter J, Hunsucker Sally A, Wang Yuli, Sims Christopher E, Armistead Paul M, Allbritton Nancy L
Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine , Chapel Hill, North Carolina 27599, United States.
Department of Chemistry, University of North Carolina , Chapel HillNorth Carolina 27599, United States.
Anal Chem. 2015 Dec 15;87(24):12281-9. doi: 10.1021/acs.analchem.5b03579. Epub 2015 Nov 23.
Microraft arrays were developed to select and separate cells based on a complex phenotype, weak intercellular adhesion, without knowledge of cell-surface markers or intracellular proteins. Since the cells were also not competent to bind to a culture surface, a method to encapsulate nonadherent cells within a gelatin plug on the concave microraft surface was developed, enabling release and collection of the cells without the need for cell attachment to the microraft surface. After microraft collection, the gelatin was liquified to release the cell(s) for culture or analysis. A semiautomated release and collection device for the microrafts demonstrated 100 ± 0% collection efficiency of the microraft while increasing throughput 5-fold relative to that of manual release and collection. Using the microraft array platform along with the gelatin encapsulation method, single cells that were not surface-attached were isolated with a 100 ± 0% efficiency and a 96 ± 4% postsort single-cell cloning efficiency. As a demonstration, Epstein-Barr virus-infected lymphoblastoid cell lines (EBV-LCL) were isolated based on their intercellular adhesive properties. The identified cell colonies were collected with a 100 ± 0% sorting efficiency and a postsort viability of 87 ± 3%. When gene expression analysis of the EBV latency-associated gene, EBNA-2, was performed, there was no difference in expression between blasting or weakly adhesive cells and nonblasting or nonadhesive cells. Microraft arrays are a versatile method enabling separation of cells based on complicated and as yet poorly understood cell phenotypes.
微筏阵列的开发目的是基于复杂表型、弱细胞间黏附来选择和分离细胞，而无需了解细胞表面标志物或细胞内蛋白质。由于这些细胞也无法黏附于培养表面，因此开发了一种将非黏附细胞封装在凹面微筏表面的明胶塞内的方法，从而能够在无需细胞黏附于微筏表面的情况下释放和收集细胞。微筏收集后，将明胶液化以释放细胞用于培养或分析。一种用于微筏的半自动释放和收集装置显示微筏的收集效率为100±0%，同时通量相对于手动释放和收集提高了5倍。使用微筏阵列平台以及明胶封装方法，未表面黏附的单细胞以100±0%的效率被分离出来，分选后单细胞克隆效率为96±4%。作为一个示例，基于细胞间黏附特性分离出了爱泼斯坦-巴尔病毒感染的淋巴母细胞系（EBV-LCL）。所鉴定的细胞集落以100±0%的分选效率和87±3%的分选后活力被收集。当对EBV潜伏相关基因EBNA-2进行基因表达分析时，增殖或弱黏附细胞与非增殖或非黏附细胞之间的表达没有差异。微筏阵列是一种通用方法，能够基于复杂且尚未完全理解的细胞表型来分离细胞。
