School of Medicine, Department of Medicine, Division of Hematology/Oncology, University of California, Irvine, United States.
Samueli School of Engineering, Department of Biomedical Engineering, University of California, Irvine, United States.
Sci Rep. 2017 Oct 12;7(1):13081. doi: 10.1038/s41598-017-13242-1.
Advancing knowledge of biological mechanisms has come to depend upon genetic manipulation of cells and organisms, relying upon cellular cloning methods that remain unchanged for decades, are labor and time intensive, often taking many months to come to fruition. Thus, there is a pressing need for more efficient processes. We have adapted a newly developed micropallet array platform, termed the "ferro-core micropallet array", to dramatically improve and accelerate the process of isolating clonal populations of adherent cells from heterogeneous mixtures retaining the flexibility of employing a wide range of cytometric parameters for identifying colonies and cells of interest. Using transfected (retroviral oncogene or fluorescent reporter construct) rat 208 F cells, we demonstrated the capacity to isolate and expand pure populations of genetically manipulated cells via laser release and magnetic recovery of single micropallets carrying adherent microcolonies derived from single cells. This platform can be broadly applied to biological research, across the spectrum of molecular biology to cellular biology, involving fields such as cancer, developmental, and stem cell biology. The ferro-core micropallet array platform provides significant advantages over alternative sorting and cloning methods by eliminating the necessity for repetitive purification steps and increasing throughput by dramatically shortening the time to obtain clonally expanded cell colonies.
生物机制知识的进步已经开始依赖于对细胞和生物体的基因操作，而这些操作依赖于几十年来一直没有改变的细胞克隆方法，这些方法既耗费人力又耗时，通常需要数月时间才能取得成果。因此，人们迫切需要更有效的方法。我们已经采用了一种新开发的微托盘阵列平台，称为“铁核微托盘阵列”，以极大地改进和加速从异质混合物中分离贴壁细胞克隆群体的过程，同时保持采用广泛的细胞计量学参数来鉴定感兴趣的菌落和细胞的灵活性。使用转染（逆转录病毒癌基因或荧光报告构建体）大鼠 208F 细胞，我们证明了通过激光释放和磁性回收单个微托盘来分离和扩增经基因操作的细胞的纯群体的能力，这些单个微托盘承载着源自单个细胞的贴壁微集落。该平台可以广泛应用于生物学研究，涵盖从分子生物学到细胞生物学的各个领域，涉及癌症、发育和干细胞生物学等领域。铁核微托盘阵列平台通过消除重复的纯化步骤并通过大大缩短获得克隆扩增细胞集落的时间来提高产量，从而提供了优于替代分选和克隆方法的显著优势。
